Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

7232019 Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

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YEASTBOOK

CELL STRUCTURE amp TRAFFICKING

Secretory Protein Biogenesis and Traf1047297c in the EarlySecretory Pathway

Charles K Barlowe and Elizabeth A Millerdagger1

Department of Biochemistry Dartmouth Medical School Hanover New Hampshire 03755 and ySchool of Biological Sciences Columbia University

New York New York 10027

ABSTRACT The secretory pathway is responsible for the synthesis folding and delivery of a diverse array of cellular proteins Secretory

protein synthesis begins in the endoplasmic reticulum (ER) which is charged with the tasks of correctly integrating nascent proteins and

ensuring correct post-translational modi1047297cation and folding Once ready for forward traf1047297c proteins are captured into ER-derived

transport vesicles that form through the action of the COPII coat COPII-coated vesicles are delivered to the early Golgi via distinct

tethering and fusion machineries Escaped ER residents and other cycling transport machinery components are returned to the ER via

COPI-coated vesicles which undergo similar tethering and fusion reactions Ultimately organelle structure function and cell

homeostasis are maintained by modulating protein and lipid 1047298ux through the early secretory pathway In the last decade structural and

mechanistic studies have added greatly to the strong foundation of yeast genetics on which this 1047297eld was built Here we discuss the key

players that mediate secretory protein biogenesis and traf1047297cking highlighting recent advances that have deepened our understanding

of the complexity of this conserved and essential process

TABLE OF CONTENTS

Abstract 383Introduction 384

Expanding Methodologies From a Parts List to Mechanisms and Back to More Parts 384

Classic screens lay the groundwork in vitro reconstitution de1047297nes mechanism 384

Dynamics and organization revealed by live cell imaging 385

New technologies yield new players and de1047297 ne interplay between pathways 385

Secretory Protein Translocation and Biogenesis 386

Polypeptide targeting and translocation 386

Maturation of secretory proteins in the ER signal sequence processing 388

Maturation of secretory proteins in the ER protein glycosylation 388

Maturation of secretory proteins in the ER glycosylphosphatidylinositol anchor addition 389

Maturation of secretory proteins in the ER disul 1047297

de bond formation 389Glucosidase mannosidase trimming and protein folding 390

Control of ER homeostasis by the Unfolded Protein Response 391

Continued

Copyright copy 2013 by the Genetics Society of Americadoi 101534genetics112142810Manuscript received June 14 2012 accepted for publication September 25 20121Corresponding author School of Biological Sciences Columbia University 1212 Amsterdam Ave MC2456 New York NY 10027 Email em2282columbiaedu

Genetics Vol 193 383ndash410 February 2013 383



CONTENTS continued

Transport From the ER Sculpting and Populating a COPII Vesicle 391

Structure and assembly of the COPII coat 392

Cargo capture stochastic sampling vs direct and indirect selection 393

Regulation of COPII function GTPase modulation coat modi 1047297 cation 394

Higher-order organization of vesicle formation 395

Vesicle Delivery to the Golgi 395

Vesicle tethering 395

SNARE protein-dependent membrane fusion 396

A concerted model for COPII vesicle tethering and fusion 397

Traf1047297c Within the Golgi 397

Transport through the Golgi complex 397

Lipid requirements for Golgi transport 398

The Return Journey Retrograde Traf1047297c via

COPI Vesicles 398

Composition and structure of the COPI coat 399

Cargo capture sorting signals cargo adaptors and coat stimulators 400

Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion 401

Perspectives 401

LIKE all eukaryotes yeast cells segregate various physio-

logical functions into distinct subcellular compartments

A key challenge is thus ensuring that appropriate proteins

are delivered to the correct subcellular destination a process

that is driven by discrete sorting signals that reside in the

proteins themselves Perhaps the most prevalent type of sort-

ing signal is that directing a protein to the secretory pathway

which handles the various proteins that are destined for the

extracellular environment or retention in the internal endo-

membrane system Approximately one-third of the yeast pro-

teome enters the secretory pathway Protein secretion is not

only essential for cellular function but also provides the

driving force for cell growth via delivery of newly synthe-

sized lipid and protein that permits cell expansion Secretory

proteins enter this set of interconnected organelles at the

endoplasmic reticulum (ER) which regulates protein trans-

lation protein translocation across the membrane protein

folding and post-translational modi1047297cation protein quality

control and forward traf 1047297c of suitable cargo molecules (both

lipid and protein) Once contained within the secretory path-

way proteins are ferried between compartments via trans-port vesicles that bud off from one donor compartment to

fuse with a downstream acceptor compartment thereby

mediating directional traf 1047297c of both lipid and protein The

forward-moving or anterograde pathway is balanced by

a reverse or retrograde pathway that returns escaped resi-

dent proteins and maintains the homeostasis of individual

organelles Early yeast screens pioneered the genetic dissec-

tion of the eukaryotic secretory pathway and were rapidly

followed by biochemical approaches that permitted the mo-

lecular dissection of individual processes of protein biogen-

esis and traf 1047297c Here we discuss the methodologies that

have yielded great insight into the conserved processes that

drive protein secretion in all eukaryotes and describe the

fundamental processes that act to ensure ef 1047297cient and ac-

curate protein secretion The reader is also referred to earlier

comprehensive reviews on these topics (Kaiser et al 1997

Lee et al 2004) as we focus our coverage on more recent

advances

Expanding Methodologies From a Parts Listto Mechanisms and Back to More Parts

Classic screens lay the groundwork in vitro reconstitutionde1047297 nes mechanism

There is no doubt that early seminal yeast genetics ap-

proaches laid the foundation upon which our understand-

ing of protein secretion is built From the original Novick

and Schekman screens that identi1047297ed a host of secretion-

defective ( sec) mutants (Novick and Schekman 1979 Novick

et al 1980) to additional more targeted approaches fromthe Schekman (more secs Deshaies and Schekman 1987

Wuestehube et al 1996) Gallwitz ( ypt Gallwitz et al 1983)

Ferro-Novick (bet Newman and Ferro-Novick 1987) Jones

( pep Jones 1977) Stevens ( vps Rothman et al 1989) and

Emr ( vps Bankaitis et al 1986) labs that expanded the rep-

ertoire of mutants with defects in secretory protein and

membrane biosynthesis the 1047297eld has been blessed with an

abundance of reagents that permitted the characterization

of each branch of the secretory pathway (Schekman and

384 C K Barlowe and E A Miller



Novick 2004) Many of these processes are essential con-

served and have direct relevance to issues of human health

yet yeast genetics approaches remain at the forefront in

deciphering molecular mechanisms unraveling cellular re-

dundancy and complexity and appreciating the cross-talk

between different branches of the pathway The strength of

yeast as a model system to probe this complexity lies in the

combination of facile genetics and robust biochemistry that

are afforded by this remarkable organism Indeed the 1047297

eldhas a long history of capitalizing on yeast mutants to inform

biochemical reconstitution approaches that in turn inform

new genetic screening approaches

The most pertinent example of the strength of this

approach is the mechanistic description of the COPII coat

proteins that drive vesicle formation from the endoplasmic

reticulum Classic epistasis analyses of the Novick and

Schekman sec mutants (Novick et al 1980) placed the early

sec genes in order within the secretory pathway sec12

sec13 sec16 and sec23 mutants blocked formation of trans-

port vesicles and induced proliferation of the ER whereas

sec17 sec18 and sec22 mutants blocked vesicle fusion and

caused accumulation of vesicles (Novick et al 1981 Kaiserand Schekman 1990) The subsequent development of

in vitro assays relied in part on the use of these mutants in

biochemical complementation assays (Baker et al 1988

Ruohola et al 1988) Recapitulation of ER ndashGolgi traf 1047297c in

permeabilized yeast cells was perturbed in sec23 mutants

but could be restored by incubation with cytosol prepared

from wild-type cells placing Sec23 as a soluble factor re-

quired for transport vesicle formation (Baker et al 1988)

Further re1047297nement of these in vitro transport assays permit-

ted the dissection of different transport stages (Rexach and

Schekman 1991) and allowed the biochemical characteriza-

tion of the COPII coat proteins (Barlowe et al 1994) that

generate transport intermediates and the membrane-bound

and cytosolic factors required for tethering and fusion steps

that consume vesicles at the Golgi membrane (Barlowe

1997 Cao et al 1998) Further mechanistic dissection came

from even more re1047297ned reconstitution systems that permit-

ted the identi1047297cation of the minimal machinery required to

generate COPII vesicles from synthetic liposomes (Matsuoka

et al 1998ab) and de1047297ned the dynamics of individual

events using real-time assays (Antonny et al 2001)

Similar reconstitution of the COPI-mediated GolgindashER

retrograde pathway in yeast lagged somewhat behind in

part due to equivalent biochemical experiments that were

under way in mammalian cells (Balch et al 1984 Waterset al 1991) Furthermore due to rapid perturbation in for-

ward (ER ndashGolgi) traf 1047297c when the retrograde pathway is

blocked for some time there was confusion over the direc-

tionality of COPI-mediated events (Gaynor and Emr 1997)

Despite these dif 1047297culties in vitro reconstitution of COPI-

coated vesicle formation was ultimately achieved (Spang

and Schekman 1998) and has been similarly dissected

in minimal systems using synthetic liposomes (Spang et al

1998)

In contrast to the genetics-informed biochemical ap-

proaches described above minimal reconstitution of the

membrane fusion events that drive vesicle consumption took

a slightly different path Armed with the knowledge that

fusion is driven by proteins known as SNAREs (soluble N-

ethylmaleimide-sensitive factor attachment protein recep-

tors) and with the full description of yeast SNAREs in hand

from computational analyses of the yeast genome Rothman

and colleagues established liposome-based assays that dem-onstrated compartment speci1047297city of different SNARE pairs

(McNew et al 2000) That this biochemical approach largely

recapitulated known pathways previously de1047297ned by ge-

netic means serves to highlight the success of mutually in-

formed genetic and biochemical approaches to fully dissect

the molecular mechanisms of budding and fusion events

Dynamics and organization revealed by live cell imaging

With budding and fusion machineries well described in

minimal systems it became apparent that there were still

pieces of the puzzle missing including the roles of some

essential proteins (eg Sec16 Espenshade et al 1995) that

remained unexplained in terms of functionality Further-more some of the more pressing mechanistic questions

could not be answered by biochemical means For example

the mode of protein and lipid traf 1047297c through the Golgi

remained controversial did COPI vesicles mediate forward

traf 1047297c or did proteins proceed through the Golgi by a process

of maturation of individual cisternae These questions were

addressed in part by the Glick and Nakano labs using high-

resolution time-lapse imaging of living yeast cells (Losev

et al 2006 Matsuura-Tokita et al 2006) Such experiments

de1047297ned discrete sites of vesicle formation known as transi-

tional ER (tER) or ER exit sites (ERES) that are dynamic in

nature can form de novo but also fuse with each other and

have clear relationships with downstream Golgi elements

(Bevis et al 2002 Shindiapina and Barlowe 2010) Further-

more imaging of distinct Golgi elements lent support for the

cisternal maturation model of protein secretion although

direct imaging of cargo molecules remains to be fully dem-

onstrated Recent advances in superresolution imaging hold

great promise in further understanding the nature of these

subdomains and their relationships with distinct protein

machineries and membrane compartments although some

limitations will still apply especially with respect to the

problem of detecting transient cargo molecules that are

in 1047298ux through the system

New technologies yield new players and de1047297 ne interplay between pathways

Since the yeast community entered the postgenomic world

a host of new tools has opened up many new approaches

the haploid deletion collection represents an accessible

large-scale analysis platform for novel screens (Tong

et al 2001) the GFP- (Huh et al 2003) and TAP-tagged

(Ghaemmaghami et al 2003) fusion databases documented

the localization and abundance of many gene products and

Early Events in Protein Secretion 385



microarray analyses of gene expression changes allow thedissection of cell-wide changes to a given perturbation

(Travers et al 2000) These new tools are being used with

remarkable imagination often capitalizing on the facile na-

ture of yeast genetics to de1047297ne the interplay between related

pathways in exciting ways For example microarray analysis

of the changes in gene expression that occur upon induction

of ER stress via the unfolded protein response (UPR) iden-

ti1047297ed upregulation of machineries involved in ER-associated

degradation (ERAD) ultimately leading to the appreciation

that these discrete pathways are intimately coordinated to

manage the burden of protein within the ER (Travers et al

2000) A second example derives from the development of

synthetic genetic array (SGA) technology which allows the

rapid generation of haploid double mutant strains (Tong

et al 2001) Although the piecemeal application of this tech-

nology was informative for individual genes the broader

application to an entire pathway was revolutionary in terms

of being able to de1047297ne novel functions based on shared

genetic 1047297ngerprints The 1047297rst so-called epistatic miniarray

pro1047297le (E-MAP) made pairwise double mutations among

almost 500 early secretory pathway components quantify-

ing the phenotypic cost of combined mutations (Schuldiner

et al 2005) Analysis of the shared patterns of genetic inter-

actions revealed (perhaps not surprisingly) that components

in common pathways shared similar pro1047297les which allowedthe assignation of novel functions to previously uncharacter-

ized and enigmatic proteins An elaboration on the E-MAP

approach made elegant use of a 1047298uorescent reporter system

to 1047297rst assess the UPR state of individual strains in the

genomic deletion collection and then to probe how UPR

activation changes in double mutant backgrounds yielding

a more subtle understanding of genetic interactions than

gross life and death dichotomies which usually form the

basis of synthetic interactions (Jonikas et al 2009) With

further development of such reporters on cell status thisarea of cross-talk between pathways will become more

and more integrated allowing a detailed picture of cellu-

lar physiology However as these new technologies yield

new functional clues to previously uncharacterized genes

we need to continue to use and develop biochemical tools

that allow true mechanistic insight Again the strength of

the yeast system is the use of both genetic and biochemical

tools to mutually inform new discoveries

Secretory Protein Translocation and Biogenesis

Polypeptide targeting and translocation

The 1047297rst step in biogenesis of most secretory proteins is

signal sequence-directed translocation of the polypeptide

into the ER Both cotranslational and post-translational

mechanisms operate in yeast to target diverse sets of soluble

and integral membrane secretory proteins to the ER (Figure

1) The cotranslational translocation process is initiated

when a hydrophobic signal sequence or transmembrane

sequence is translated and recognized by the signal-recognition

particle (SRP) for targeting to the SRP receptor at ER trans-

location sites (Figure 1a) In the case of post-translational

translocation cytosolic chaperones play a critical role in

binding hydrophobic targeting signals to maintain the na-scent secretory protein in an unfolded or loosely folded trans-

location competent state until delivery to the ER membrane

(Figure 1b) Progress on identi1047297cation and characterization

of the translocation machinery will be described in turn

below as the start of a continuum of events in biogenesis

of secretory proteins

Genetic approaches in yeast uncovered key components

in both the co- and post-translational translocation path-

ways Appending a signal sequence to the cytosolic enzyme

Figure 1 Membrane transloca-

tion of secretory proteins Three

well-characterized pathways oper-

ate to deliver secretory proteins

to the ER for membrane trans-

location (A) The signal recogni-

tion particle (SRP) recognizes a

hydrophobic signal sequence or

transmembrane segment during

protein translation followed by

targeting of the ribosomendash

nascentchain complex to the SRP receptor

for cotranslational membrane in-

sertion (B) Post-translational inser-

tion of secretory proteins depends

on cytosolic Hsp70 ATPases such

as Ssa1 to maintain the nascent

secretory protein in an unfolded

translocation competent state until delivery to the Sec63 complex formed by Sec62Sec63Sec71Sec72 The Sec61 complex forms an aqueous

channel for both post- and cotranslational polypeptide translocation Kar2 a luminal Hsp70 ATPase facilitates directed movement and folding

of nascent polypeptides (C) In GET-mediated insertion of C-terminal tail-anchored proteins the Sgt2ndashGet4ndashGet5 complex targets nascent

polypeptides to Get3 for Get1Get2 dependent translocation Tail-anchored proteins are integrated into the membrane in Sec61-independent

pathway




encoded by HIS4 targets this enzyme to the ER where it

cannot function and produces histidine auxotrophy A ge-

netic selection for mutants that are partially defective in

translocation of this signal peptide-bearing fusion protein

and therefore restore histidine prototrophy was used to

identify conditional mutations in three essential genes

SEC61 SEC62 and SEC63 (Deshaies and Schekman 1987

Rothblatt et al 1989) Sequencing indicated that all three

genes encode integral membrane proteins with the 53-kDaSec61 protein a central component that contained 10 trans-

membrane segments and striking sequence identity with the

Escherichia coli translocation protein SecY (Stirling et al

1992 Jungnickel et al 1994) Similar genetic selection

approaches using the HIS4 gene product fused to integral

membrane proteins identi1047297ed SEC65 which encodes a com-

ponent of the SRP (Stirling and Hewitt 1992 Stirling et al

1992) as well as mutations in SEC71 and SEC72 (Green

et al 1992)

Concurrent with these genetic approaches cell-free

reconstitution assays that measured post-translational

translocation of radiolabeled pre-pro-a-factor into yeast

microsomes were used to dissect molecular mechanisms inthis translocation pathway (Hansen et al 1986 Rothblatt

and Meyer 1986) Fractionation of cytosolic components re-

quired in the cell-free assay revealed that Hsp70 ATPases

stimulated post-translational translocation (Chirico et al

1988) Yeast express a partially redundant family of cyto-

solic Hsp70s encoded by the SSA1ndashSSA4 genes that are col-

lectively essential An in vivo test for Hsp70 function in

translocation was demonstrated when conditional expres-

sion of SSA1 in the background of the multiple ssa D strain

resulted in accumulation of unprocessed secretory proteins

as Ssa1 was depleted (Deshaies et al 1988) ATPase activity

of Hsp70 family members is often stimulated by a corre-

sponding Hsp40 Dna J partner and in the case of poly-

peptide translocation in yeast the YDJ1 gene encodes

a farnsylated DnaJ homolog that functions in ER transloca-

tion (Caplan et al 1992) Ydj1 has been shown to directly

regulate Ssa1 activity in vitro (Cyr et al 1992 Ziegelhoffer

et al 1995) and structural studies indicate that Ydj1 binds to

three- to four-residue hydrophobic stretches in nonnative

proteins that are then presented to Hsp70 proteins such as

Ssa1 (Li et al 2003 Fan et al 2004) Finally genetic experi-

ments connect YDJ1 to translocation components in addi-

tion to multiple other cellular pathways presumably due to

action on a subset of secretory proteins (Becker et al 1996

Tong et al 2004 Costanzo et al 2010 Hoppins et al 2011)Several lines of experimental evidence indicate that the

multispanning Sec61 forms an aqueous channel for polypep-

tide translocation into the ER Initial approaches probing

a stalled translocation intermediate in vitro revealed that

direct cross-links formed only between transiting segments

of translocation substrate and Sec61 (Musch et al 1992

Sanders et al 1992 Mothes et al 1994) Puri1047297cation of

functional Sec61 complex revealed a heterotrimeric complex

consisting of Sec61 associated with two 10-kDa proteins

identi1047297ed as Sss1 and Sbh1 (Panzner et al 1995) For ef 1047297-

cient post-translational translocation the Sec61 complex

assembles with another multimeric membrane complex

termed the Sec63 complex which consists of the genetically

identi1047297ed components Sec63 Sec62 Sec71 and Sec72

(Deshaies et al 1991 Brodsky and Schekman 1993 Panzner

et al 1995) Puri1047297cation of these complexes combined with

proteoliposome reconstitution approaches have demon-

strated that the seven polypeptides comprising the Sec61and Sec63 complexes plus the lumenal Hsp70 protein

Kar2 are suf 1047297cient for the post-translational mode of

translocation (Panzner et al 1995) Further biochemical dis-

section of this minimally reconstituted system in addition to

crystal structures of the homologous archaeal SecY complex

(Van den Berg et al 2004) have provided molecular insights

into the translocation mechanism (Rapoport 2007) Current

models for post-translational translocation suggest that the

hydrophobic N-terminal signal sequence is recognized and

bound initially by the Sec63 complex which then transmits

information through conformational changes to the Sec61

complex and to lumenally associated Kar2 (Figure 1b) In

a second step that is probably coordinated with opening of the translocation pore the signal sequence is detected at an

interface between membrane lipids and speci1047297c transmem-

brane segments in Sec61 where it binds near the cytosolic

face of the channel (Plath et al 1998) Opening of the pore

would then permit a portion of the hydrophilic polypeptide

to span the channel where association with lumenal Kar2

would capture and drive directed movement in a ratcheting

mechanism through cycles of ATP-dependent Kar2 binding

(Neupert et al 1990 Matlack et al 1999) Well-documented

genetic and biochemical interactions between Kar2 and the

lumenal DnaJ domain in Sec63 are thought to coordinate

directed movement into the ER lumen (Feldheim et al

1992 Scidmore et al 1993 Misselwitz et al 1999) The

N-terminal signal sequence is thought to remain bound

at the cytosolic face of the Sec61 complex as the nascent

polypeptide chain is threaded through the pore where at

some stage the signal sequence is cleaved by a translocon-

associated signal peptidase for release into the lumen (Antonin

et al 2000)

Of course a major pathway for delivery of nascent

secretory proteins to the ER employs the signal recognition

particle in a co-translational translocation mechanism Here

the ribosomendashnascent chainndashSRP complex is targeted to

Sec61 translocons through an initial interaction between

SRP and the ER-localized SRP receptor (SR) encoded by SRP101 and SRP102 (Ogg et al 1998) In an intricate

GTP-dependent mechanism paused SRP complexes bound

to SR transfer ribosomendashnascent chains to Sec61 tranlocons

as polypeptide translation continues in a cotranslational

translocation mode (Wild et al 2004) Genetic screens un-

covered the Sec65 subunit of SRP and puri1047297cation of native

SRP identi1047297ed the other core subunits termed Srp14 Srp21

Srp54 Srp68 and Srp72 in addition to the RNA component

encoded by SCR1 (Hann and Walter 1991 Brown et al




1994) Somewhat surprisingly deletion of the SRP compo-

nents in yeast produced yeast cells that grow slowly but

remain viable These 1047297ndings indicate that the SRP-dependent

pathway is not essential unlike the core translocation pore

components and indicates that other cytosolic machinery

can manage delivery of all essential secretory proteins to

the translocon Although yeast cells can tolerate complete

loss of the SRP pathway it became clear that certain secre-

tory proteins displayed a preference for the SRP-dependentroute whereas others were ef 1047297ciently translocated into the

ER in a post-translational mode (Hann et al 1992 Stirling

and Hewitt 1992) In general integral membrane proteins

and signal sequences of relatively high hydrophobicity pref-

erentially engage the SRP-dependent pathway whereas sol-

uble and lower hydrophobicity signal sequences depend on

a Sec63-mediated post-translational mode of translocation

(Ng et al 1996)

More recently a third post-translational translocation

pathway to the ER has been characterized in yeast and

other eukaryotes whereby short integral membrane proteins

and C-terminal tail-anchored proteins are integrated into

the membrane (Figure 1c) (Stefanovic and Hegde 2007Schuldiner et al 2008) For this class of proteins transmem-

brane segments are occluded by the ribosome until trans-

lation is completed thereby precluding SRP-dependent

targeting Bioinformatic analyses suggest that up to 5

of predicted integral membrane proteins in eukaryotic

genomes may follow this SRP-independent route including

the large class of SNARE proteins that drive intracellular

membrane fusion events and are anchored by C-terminal

membrane domains Interestingly this post-translational tar-

geting pathway operates independently of the Sec61 and

Sec63 translocon complexes (Steel et al 2002 Yabal et al

2003) and instead depends on recently de1047297ned soluble and

membrane-bound factors Large-scale genetic interaction

analyses in yeast identi1047297ed a clustered set of nonessential

genes that produced Golgi-to-ER traf 1047297cking de1047297ciencies that

were named GET genes (Schuldiner et al 2005) Get3

shares high sequence identity with the transmembrane do-

main recognition complex of 40 kDa (TRC40) that had been

identi1047297ed through biochemical strategies in mammalian

cell-free assays as a major interaction partner for newly syn-

thesized tail-anchored proteins (Stefanovic and Hegde

2007 Favaloro et al 2008) Subsequent synthetic genetic

array analyses and biochemical approaches in yeast (Jonikas

et al 2009 Battle et al 2010 Chang et al 2010 Chartron

et al 2010 Costanzo et al 2010) have implicated 1047297 ve Getproteins (Get1ndash5) and Sgt2 in this process Current models

for the GET targeting pathway in yeast suggest that a Sgt2ndash

Get4ndashGet5 subcomplex loads tail-anchored substrates onto

the targeting factor Get3 (Figure 1c) The Get3-bound

substrate then delivers these newly synthesized proteins

to an integral membrane Get1 Get2 complex In an ATP-

dependent process Get3 in association with Get1 Get2

then inserts the hydrophobic segment to span across the

ER membrane bilayer (Shao and Hegde 2011) Although

structural and biochemical studies are rapidly advancing

our understanding of the GET-dependent targeting path-

way the mechanisms by which tail-anchored proteins are

inserted into ER membrane bilayer remain to be de1047297ned

Maturation of secretory proteins in the ER signal sequence processing

For the many secretory proteins that contain an N-terminal

signal sequence the signal peptidase complex (SPC) removesthis domain by endoproteolytic cleavage at a speci1047297c cleav-

age site during translocation through the Sec61 complex

(Figure 2a) The SPC consists of four polypeptides termed

Spc1 Spc2 Spc3 and Sec11 (Bohni et al 1988 YaDeau

et al 1991) Spc3 and Sec11 are essential integral mem-

brane proteins that are required for signal sequence cleav-

age activity with the Sec11 subunit containing the protease

active site (Fang et al 1997 Meyer and Hartmann 1997)

Based on structural comparisons with E coli leader pepti-

dase the active site of SPC is thought to be located very near

the lumenal surface of the ER membrane and presumably

close to translocon exit sites The Spc1 and Spc2 subunits

are not required for viability however at elevated temper-atures the corresponding deletion strains accumulate unpro-

cessed precursors of secretory proteins in vivo (Fang et al

1996) and are required for full enzymatic activity of the SPC

in vitro (Antonin et al 2000) Interestingly Spc2 is detected

in association with the Sbh1 subunit of the Sec61 complex

and is thought to physically link the SPC and Sec61 complex

(Antonin et al 2000) Given that SEC11 was identi1047297ed in

the original SEC mutant screen as required for ER-to-Golgi

transport of secretory proteins signal sequence cleavage is

regarded as an essential step for maturation of secretory

proteins that contain N-terminal signal sequences

Maturation of secretory proteins in the ER protein glycosylation

In addition to signal sequence cleavage attachment of

asparagine-linked oligosaccharide to nascent glycopro-

teins occurs concomitantly with polypeptide translocation

through the Sec61 pore (Figure 2b) The addition of core

oligosaccharides to consensus Asn-X-SerThr sites in transit-

ing polypeptides is catalyzed by the oligosaccharyltrans-

ferase (OST) enzyme OST is composed of eight integral

membrane polypeptides (Ost1 Ost2 Ost3 or Ost6 Ost4

Ost5 Wbp1 Swp1 and Stt3) and is also detected in com-

plex with the Sec61 translocon (Kelleher and Gilmore

2006) Indeed for N-linked glycosylation sites that are nearsignal sequence cleavage sites cleavage must occur before

addition of N-linked oligosaccharide demonstrating the se-

quential stages of polypeptide translocation signal sequence

cleavage and N-linked glycosylation (Chen et al 2001) The

Stt3 subunit is critical for catalytic activity and in addition to

Stt3 most of the OST subunits are required for cell viability

indicating a critical role for N-linked glycosylation in matu-

ration of secretory proteins OST transfers a 14-residue oli-

gosaccharide core en bloc to most (but not all) Asn-X-Ser




Thr sites in transiting polypeptides The 14-residue oligosac-

charide core is assembled on the lipid-linked carrier mole-

cule dolichylpyrophosphate in a complex multistep pathway

(Burda and Aebi 1999)

The precise role(s) for N-linked glycosylation of secretory protein is not fully understood because in many instances

mutation of single and multiple sites within a given protein

produces only mild consequences Hydrophilic N-linked

glycans in1047298uence thermodynamic stability and solubility of

proteins and in the context of nascent secretory proteins

in the ER the N-linked structure is also thought to be an

integral part of a system that assists in protein folding and

quality control to manage misfolded glycoproteins (Schwarz

and Aebi 2011) This quality control process will be explored

further after covering other folding and post-translational

modi1047297cation events in secretory protein maturation

In addition to N-linked glycosylation some secretory

proteins undergo O-linked glycosylation through attach-

ment of mannose residues on SerThr amino acids by

protein O-mannosyltransferases (Pmts) Saccharomyces cer-

evisiae contains a family of seven integral membrane man-

nosyltranferases (Pmt1ndashPmt7) that covalently link mannose

residues to SerThr residues using dolichol phosphate man-

nose as the mannosyl donor (Orlean 1990 Willer et al

2003) Both O-linked mannose residues and N-linked core

oligosaccharides added in the ER are extended in the Golgi

complex by the nine-membered KRE2 MNT1 family of man-

nosyltranferases that use GDP-mannose in these polymeri-

zation reactions (Lussier et al 1997ab) O-linked mannosyl

modi1047297cation of secretory proteins in the ER is essential inyeast (Gentzsch and Tanner 1996) and required for cell wall

integrity as well as normal morphogenesis (Strahl-Bolsinger

et al 1999) The role of O-linked glycosylation in ER quality

control processes remains unclear although investigators

have reported in1047298uences of speci1047297c pmt mutations on turn-

over rates of misfolded glycoproteins (Harty et al 2001

Vashist et al 2001 Hirayama et al 2008 Goder and Melero

2011) and the PMT genes are upregulated by activation of

the UPR (Travers et al 2000)

Maturation of secretory proteins in the ERglycosylphosphatidylinositol anchor addition

Approximately 15 of proteins that enter the secretory

pathway are post-translationally modi1047297ed on their C termi-

nus by addition of a lipid-anchored glycosylphosphatidyli-

nositol (GPI) moiety The synthesis and attachment of GPI

anchors occur in the ER through a multistep pathway that

depends on 20 gene products (Orlean and Menon 2007)

GPI synthesis and attachment are essential processes in

yeast and GPI anchored proteins on the cell surface are

thought to play critical roles in cell wall structure and cell

morphology (Leidich et al 1994 Pittet and Conzelmann

2007) As with assembly of the N-linked core oligosaccha-

ride the GPI anchor is fully synthesized as a lipid anchored

precursor and then transferred to target proteins en bloc by

the GPI transamidase complex (Fraering et al 2001) The

GPI-anchoring machinery recognizes features and signalsin the C terminus of target proteins that result in covalent

linkage to what becomes the terminal amino acid (termed the

v residue) and removal of the 30-amino-acid C-terminal

GPI signal sequence (Udenfriend and Kodukula 1995) Bio-

informatic approaches are now reasonably effective in pre-

dicting GPI anchored proteins These algorithms scan for

open reading frames that contain an N-terminal signal se-

quence and a C terminus that consists of an v residue

bracketed by 10 residues of moderate polarity plus a hy-

drophobic stretch near the C terminus of suf 1047297cient length

to span a membrane bilayer (Eisenhaber et al 2004) GPI

precursor proteins that do not receive GPI-anchor addition

and removal of their C-terminal hydrophobic signal arenot exported from the ER (Nuoffer et al 1993 Doering

and Schekman 1996) and are probably retained through an

ER quality control mechanism

Maturation of secretory proteins in the ER disul 1047297 debond formation

Most secretory proteins contain disul1047297de bonds that form

when nascent polypeptides are translocated into the oxidiz-

ing environment of the ER lumen A family of protein-

Figure 2 Folding and matura-

tion of secretory proteins A se-

ries of covalent modi1047297cations

and folding events accompany

secretory protein biogenesis in

the ER (A) Signal peptidase com-

plex consisting of Spc1Spc2

Spc3Sec11 cleaves hydrophobic

signal sequences during polypep-

tide translocation (B) Coincident

with polypeptide translocationand signal sequence cleavage

N-linked core-oligosaccharide is

attached to consensus N-X-ST

sites within the transiting poly-

peptide by the multisubunit oligosaccharyl transferase complex (C) In the oxidizing environment of the ER lumen disul1047297de bond formation is reversibly

catalyzed by protein disul1047297de isomerases (such as Pdi1) with Ero1 providing oxidizing equivalents (D) Trimming of individual glucose and mannose

residues from the attached core-oligosaccharide assists protein folding and quality control processes which involve the calnexin family member Cne1

For terminally misfolded glycoproteins sequential trimming of mannose residues by Mns1 and Htm1 generates a signal for ER-associated degradation




disul1047297de isomerases that contain thioredoxin-like domains

catalyze the formation reduction and isomerization of

disul1047297de bonds to facilitate correct protein folding in the

ER lumen (Figure 2c) In yeast Pdi1 is an essential pro-

tein disul1047297de isomerase that is required for formation of

correct disul1047297de bonds in secretory and cell surface proteins

(Farquhar et al 1991 Laboissiere et al 1995) Pdi1 obtains

oxidizing equivalents for disul1047297de formation from the es-

sential 1047298

avoenzyme Ero1 which is bound to the luminalface of the ER membrane (Sevier et al 2007) Ero1 and

Pdi1 form the major pathway for protein disul1047297de bond

formation by shuttling electrons between Ero1 Pdi1 and

substrate proteins (Tu and Weissman 2002 Gross et al

2006) In reconstituted cell-free reactions FAD-linked Ero1

can use molecular oxygen as the electron acceptor to drive

Pdi1 and substrate protein oxidation The electron acceptor(s)

used by Ero1 in vivo remain to be fully characterized (Hatahet

and Ruddock 2009)

In addition to Pdi1 yeast express four other nonessential

ER-localized protein disul1047297de isomerase homologs Mpd1

Mpd2 Eug1 and Eps1 Overexpression of Mpd1 or mutant

forms of Eug1 can partially compensate for loss of Pdi1(Norgaard et al 2001 Norgaard and Winther 2001) In

addition to oxidoreductase activity Pdi1 can act as a molec-

ular chaperone in protein folding even for proteins that lack

disul1047297de bonds (Wang and Tsou 1993 Cai et al 1994)

More recently Pdi1 and other members of this family were

reported to interact with components of the ER folding ma-

chinery including calnexin (Cne1) and Kar2 (Kimura et al

2005) as well as the quality control mannosidase enzyme

Htm1 (Gauss et al 2011) Growing evidence indicates that

this family of protein disul1047297de isomerases contains different

domain architectures (Vitu et al 2008) to dictate interac-

tions with speci1047297c ER-chaperone proteins and thus shepherd

a broad range of client proteins into folded forms or into ER-

associated degradation pathways (Figure 2d)

Glucosidase mannosidase trimming and protein folding

The initial 14-residue N-linked core oligosaccharide that is

attached en bloc to nascent polypeptides is subsequently

processed by glycosylhydrolases in a sequential and protein

conformation-dependent manner to assist protein folding

and quality control in the ER lumen (Helenius and Aebi

2004) The Glc3Man9GlcNAc2 glycan which comprises the

N-linked core is rapidly processed by glucosidase I (Gls1

Cwh41) and glucosidase II (Gls2 Rot2) enzymes to remove

the three terminal glucose residues and generate Man9-

GlcNAc2 Molecular chaperones collaborate in protein fold-

ing during these glucose-trimming events and Rot1 alone

has been shown to possess a general chaperone activity

(Takeuchi et al 2008) In many cell types a calnexin-

dependent folding cycle operates to iteratively fold and

monitor polypeptide status through the coordinated activi-

ties of glucosidase I glucosidase II UDP-glucoseglycopro-

tein glucosyltransferase (UGGT) and calnexin (Cne1) After

removal of terminal glucose residues by the glucosidase

enzymes UGGT can add back a terminal glucose to the

glycan if the polypeptide is not fully folded to generate the

Glc1Man9GlcNAc2 structure This Glc1Man9GlcNAc2 form of

an unfolded protein binds to calnexin which keeps the na-

scent polypeptide in an iterative folding cycle Once fully

folded UGGT does not act after glucosidase II and the na-

scent protein exits the cycle (Helenius and Aebi 2004) This

calnexin cycle operates in many eukaryotes but it is cur-

rently unclear how or if the cycle works in yeast since de-letion of Cne1 Gls1 Gls2 or Kre5 (potential UGGT-like

protein) do not produce strong delays in biogenesis of se-

cretory proteins but are known to produce defects in bio-

synthesis of cell wall b-16-glucan (Shahinian and Bussey

2000) Although a precise molecular understanding of the

calnexin cycle components in yeast folding remains to be

determined there are clear genetic (Takeuchi et al 2006

Costanzo et al 2010) and biochemical (Xu et al 2004

Kimura et al 2005) interactions that indicate a coordinated

role for these factors in protein folding

In addition to the glucose trimming of core oligosaccha-

ride two additional ER-localized mannosidase enzymes

termed Mns1 and Htm1 remove terminal mannose residuesfrom the Man9GlcNAc2 glycan-linked structure (Figure 2d)

Mns1 and Htm1 are related enzymes with distinct speci1047297c-

ities Mns1 removes the terminal mannosyl residue of the B

branch of Man9GlcNAc2 and it is typically the Man8GlcNAc2processed form of fully folded glycoproteins that is exported

from the ER (Jakob et al 1998) Htm1 is thought to act after

Mns1 on terminally misfolded proteins (or misfolded pro-

teins that have lingered in the ER folding cycle for too long)

to remove the outermost mannosyl residue from the C

branch of the glycan to generate Man7GlcNAc2 (Clerc

et al 2009) This form of the glycan is then recognized by

the ER lectin Yos9 and targets misfolded proteins for ER-

associated degradation (Carvalho et al 2006 Denic et al

2006) Although Mns1- and Htm1-de1047297cient cells appear to

transport folded secretory proteins at normal rates both

display signi1047297cant delays in turnover of terminally misfolded

glycoproteins (Jakob et al 1998 2001) which serves to

highlight an important role for mannosidase activity in ER

quality control

Folding of nascent polypeptides throughout transloca-

tion and within the ER is also managed by Hsp70 ATPase

systems which handle partially folded intermediates In

general Hsp70 proteins hydrolyze ATP when binding to

exposed hydrophobic stretches in unfolded polypeptides

to facilitate protein folding The Hsp70 remains bound tounfolded substrates until ADP is released with this Hsp70

ATPase cycle governed by speci1047297c DnaJ-like proteins that

stimulate ATP hydrolysis and nucleotide exchange factors that

drive ADP release (Hartl 1996 Bukau and Horwich 1998) In

yeast the Hsp70 Kar2 plays a prominent role in ER folding in

concert with the related Hsp70 protein Lhs1 (Rose et al

1989 Baxter et al 1996 Brodsky et al 1999 Steel et al

2004) For Kar2 the known DnaJ-like stimulating factors

include Sec63 Scj1 and Jem1 (Schlenstedt et al 1995




Nishikawa and Endo 1997) whereas the GrpE family mem-

ber Sil1 and surprisingly the unrelated ATPase Lhs1 serve as

nucleotide exchange factors (Hale et al 2010) Complexity in

regulating the Kar2 ATPase cycle probably re1047298ects the range of

unfolded substrates that Kar2 must handle in maintaining ER

homeostasis and there are likely to be additional factors that

couple Kar2 activity to other speci1047297c ER processes As mentioned

above Kar2 chaperone activity is tightly linked with the PDI

calnexin and glycan trimming pathways (Figure 2d) FinallyKar2 also plays a prominent role in ER-associated degradation

(ERAD) pathways to dispose of terminally misfolded proteins

(Nishikawa et al 2001) Although our understanding of Kar2

biochemical activity is advanced the coordinated control of

Kar2-dependent folding and modi1047297cation cycles in the context

of an ER lumenal environment remains a challenging area

ERAD of misfolded and unassembled proteins proceeds

through a series of pathways that remove targeted proteins

from the ER for ubiquitin- and proteasome-dependent deg-

radation in the cytoplasm ERAD is thought to play a key

role in ER homeostasis and cellular physiology Since these

pathways divert misfolded secretory proteins from their

routes of biogenesis this important topic is beyond thescope of this current review and the reader is referred to

excellent recent reviews (Vembar and Brodsky 2008 Smith

et al 2011)

Control of ER homeostasis by the Unfolded Protein Response

Much of the folding and biogenesis machinery in the ER is

under a global transcriptional control program referred to

as the UPR The yeast UPR is activated by an increase in

the level of unfolded proteins in the ER which can be

experimentally induced by treatment with inhibitors of

ER protein folding (eg tunicamycin dithiothreitol) or by

overexpression of terminally misfolded proteins (Bernales

et al 2006) Regulation of the UPR was initially examined

through identi1047297cation of a 22-nucleotide segment in the

KAR2 promoter region termed the unfolded protein re-

sponse element (UPRE) which was required for UPR ac-

tivation of Kar2 expression Fusion of this KAR2 promoter

element to a lacZ reporter provided an elegant screen for

gene mutations that blunted UPR reporter expression (Cox

et al 1993 Mori et al 1993) Genetic screening led to the

discovery that IRE1 HAC1 and RLG1 were required for

a robust UPR under ER stress conditions (Cox and Walter

1996 Sidrauski et al 1996) Further studies revealed that

IRE1 encodes an ER transmembrane protein with cytosolickinaseribonuclease domains and a lumenal sensor domain

that together are thought to serve as readout on unfolded

protein levels HAC1 encodes a basic leucine zipper tran-

scription factor that binds to UPRE-containing segments of

DNA and induces their expression (Cox and Walter 1996)

Surprisingly RLG1 encodes a tRNA ligase that is required for

the nonconventional splicing of HAC1 pre-mRNA Structural

and mechanistic dissection of these core components is now

advanced Current models indicate that the Ire1 lumenal

domain interacts with Kar2 and unfolded proteins to sense

protein folding status (Bertolotti et al 2000 Pincus et al

2010 Gardner and Walter 2011) When unfolded proteins

accumulate in the ER Ire1 forms oligomers that activate the

cytoplasmic kinase and ribonuclease domains Activated

Ire1 ribonuclease then acts on HAC1 pre-mRNA to remove

a nonconventional intron and this splicing intermediate is

then ligated by the Rlg1 ligase to produce mature HAC1

mRNA Translation of HAC1 message produces Hac1 pro-tein which is a potent transcriptional activator of UPR target

genes (Bernales et al 2006)

In addition to Kar2 the UPR was known to induce other

ER folding components including Pdi1 and Eug1 (Cox et al

1993 Mori et al 1993) To comprehensively assess the tran-

scriptional pro1047297le of the yeast UPR DNA microarray analysis

was powerfully applied to monitor mRNA levels under ER

stress conditions (Travers et al 2000) Comparing transcrip-

tion pro1047297les in wild-type ire1 D and hac1 D strains after UPR

induction revealed 381 genes that passed stringent criteria

as UPR targets Not surprisingly 10 genes involved in ER

protein folding were identi1047297ed as UPR targets and included

JEM1 LHS1 SCJ1 and ERO1 In addition dozens of genesinvolved in ER polypeptide translocation protein glycosyla-

tion and ER-associated degradation were induced Perhaps

more surprisingly 19 genes involved in lipid and inositol

metabolism as well as 16 genes encoding proteins that func-

tion in vesicle traf 1047297cking between the ER and Golgi were

upregulated by the UPR These 1047297ndings highlight a global

role for the UPR in regulating ER homeostasis through bal-

ancing ER lipid and protein biosynthetic rates In the context

of cellular physiology the UPR is now thought to serve a cen-

tral role in sensing and integrating secretory pathway func-

tion to 1047297nely tune ER capacity in response to cellular

demands (Walter and Ron 2011)

Transport From the ER Sculpting and Populatinga COPII Vesicle

Once secretory proteins have completed their synthesis and

modi1047297cation regimes they become competent for forward

traf 1047297c through the secretory pathway a process mediated

by a series of transport vesicles that bud off from one

compartment traverse the cytoplasm and fuse with a down-

stream organelle (Figure 3) ER-derived vesicles are created

by the COPII coat that like other coat protein complexes is

charged with the dual tasks of creating a spherical transport

vesicle from a planar donor membrane and populating thenascent vesicle with the appropriate cargoes Biochemical

characterization of this process 1047297rst from complex mi-

crosomal membranes using puri1047297ed COPII coat proteins

(Barlowe et al 1994) then in more reduced form from syn-

thetic liposomes (Matsuoka et al 1998b) and subsequently

at the structural level through cryo-EM (Stagg et al 2006)

and X-ray crystallography (Bi et al 2002 Fath et al 2007)

has been remarkably fruitful in de1047297ning the molecular basis

of these events What has emerged is an elegant mechanism




whereby the minimal COPII machinery composed of 1047297 ve

proteins (Sar1 Sec23 Sec24 Sec13 and Sec31) suf 1047297ces

to ful1047297ll these multiple functions However recent insights

into how this process is regulated suggest there is still much

to learn about coat dynamics in the cell and the precise

physical basis for various steps including membrane scission

during vesicle release vesicle uncoating and the formation

of large transport carriers capable of shuttling large cargoes

Structure and assembly of the COPII coat

COPII coat assembly (Figure 3) is initiated by the local re-

cruitment and activation of the small G protein Sar1

(Nakano and Muramatsu 1989 Barlowe et al 1993) upon

exchange of GDP for GTP catalyzed by an ER membrane

protein the guanine nucleotide exchange factor (GEF)

Sec12 (Nakano et al 1988 drsquoEnfert et al 1991) GTP load-

ing on Sar1 exposes an amphipathic a-helix that likely

induces initial membrane curvature by locally expanding

the cytoplasmic lea1047298et relative to the lumenal lea1047298et (Lee

et al 2005) GTP-bound membrane-associated Sar1 sub-

sequently recruits the heterodimeric complex of Sec23

and Sec24 (Matsuoka et al 1998b) Sec23 is the GTPase-

activating protein (GAP) for Sar1 (Yoshihisa et al 1993)

contributing a catalytic arginine residue analogous to GAP

stimulation in many Ras-related G proteins (Bi et al 2002)Sec24 provides the cargo-binding function of the coat con-

taining multiple independent domains that interact directly

with speci1047297c sorting signals on various cargo proteins (Miller

et al 2002 2003 Mossessova et al 2003) The Sar1 Sec23

Sec24 ldquoprebuddingrdquo complex in turn recruits the hetero-

tetrameric complex of Sec13 and Sec31 (Matsuoka et al

1998b) Sec31 also contributes to the GTPase activity of

the coat by stimulating the GAP activity of Sec23 (Antonny

et al 2001 Bi et al 2007) Thus the fully assembled coat is

composed of two distinct layers the ldquoinnerrdquo membrane

proximal layer of Sar1 Sec23 Sec24 that intimately asso-

ciates with lipid headgroups (Matsuoka et al 2001) and

contributes cargo-binding function and the ldquoouterrdquo mem-

brane distal layer composed of Sec13 Sec31 Both layers

contribute to the catalytic cycle of Sar1 and endowing

maximal GTPase activity when the coat is fully assembled

(Antonny et al 2001)

Our mechanistic understanding of COPII coat action has

been signi1047297cantly enhanced by the structural characteriza-

tion of the different coat components A structure of the

Sec23 Sec24 dimer showed a bow-tie shaped assembly with

a concave face that is presumed to lie proximal to the mem-

brane and is enriched in basic amino acids (Bi et al 2002)

These charged residues may facilitate association with the

acidic phospholipid headgroups of the ER membrane Sub-

sequent structural genetic and biochemical analyses of

Sec24 revealed multiple discrete sites of cargo interaction

dispersed around the perimeter of the protein (Miller et al

2003 Mossessova et al 2003) Structural analysis of the

outer coat was facilitated by the observation that under

some conditions the puri1047297ed coat proteins can self-assemble

into ldquocagesrdquo of the approximate size of a COPII vesicle

(Antonny et al 2003) Further experiments using mamma-

lian Sec13 Sec31 recapitulated this self-assembly reactionand led to a cryoelectron microscopy structure of the COPII

cage which forms a lattice-like structure with geometry dis-

tinct from that of the clathrin coat (Stagg et al 2006) Het-

erotetrameric Sec13 Sec31 complexes form straight rods

known as ldquoedgerdquo elements four of which come together at

ldquo vertexrdquo regions to drive cage assembly (Figure 3) Subse-

quent crystal structures of Sec13 and a portion of Sec31

revealed an unexpected domain arrangement within the

edge element whereby Sec31 forms both the dimerization

Figure 3 Coat assembly drives

vesicle formation Both the COPII

(left) and COPI (right) coats are

directed in their assembly by

small GTPases of the ArfSar1

family In the COPII coat Sar1

is activated by its guanine nu-

cleotide exchange factor (GEF)

Sec12 which localizes to the ER

membrane Activated Sar1ndashGTP

recruits the Sec23Sec24 dimerwhich corresponds to the ldquoin-

ner coatrdquo layer and provides the

cargo-binding function A heter-

otetramer of Sec13Sec31 is sub-

sequently recruited forming the

ldquoouter coatrdquo and polymerizing

into a lattice-like structure that

drives membrane curvature In

the COPII cage formed by Sec13

Sec31 four molecules of Sec31

assemble head-to-head via b-propeller domains to form the ldquovertexrdquo of the cage (inset) The COPI coat assembles upon activation of Arf1 which is

driven by either of the redundant GEFs Gea1 or Gea2 Arf1 in turn recruits the inner coat complex of Sec21Sec26Ret2Ret3 which has homology

to the clathrin AP-2 adaptor complex The COPI outer coat is formed by Sec27Ret1Sec28 which assembles in a triskelion structure via interactions

of three b-propeller domains of Sec27 (inset)




interface along the edge element and the vertex assembly

unit with Sec13 sandwiched between these structural ele-

ments (Fath et al 2007) However the fragment of Sec31

that 1047297ts well into the density of the cryo-EM structure

represents only about half of the protein an additional

proline-rich domain contains the GAP-stimulatory activity of Sec31 Again the crystal structure of this region bound

to Sar1 Sec23 has yielded great insight into the mecha-

nism of GAP activity whereby the active fragment of Sec31

lies along the membrane-distal surface of Sec23 Sar1 and

optimizes the orientation of the catalytic histidine of Sar1

(Bi et al 2007)

The ability of Sec13 Sec31 to assemble into a spherical

structure that matches closely the size of a COPII vesicle

suggests that the primary membrane bending force may

come from the scaffolding effect of this structure on the

ER membrane Indeed when the curvature-inducing amphi-

pathic helix of Sar1 is replaced with an N-terminal histidine

tag to drive recruitment to Ni-containing liposomes subse-

quent recruitment of Sec23 Sec24 and Sec13 Sec31 is suf-

1047297cient to drive the generation of spherical buds that remain

attached to the donor liposome (Lee et al 2005) Thus an

additional function of the Sar1 helix is to drive vesicle scis-

sion a model supported by experiments that link GTPase

activity to vesicle release in a manner analogous to that

proposed for dynamin (Pucadyil and Schmid 2009 Kung

et al 2012) Although the concave face of Sec23 Sec24

may also contribute to membrane curvature it has been

suggested that the relatively paltry dimer interface between

these two molecules is not robust enough to impart curva-

ture despite an intimate interaction with the lipid bilayer(Zimmerberg and Kozlov 2006) Thus although Sar1 and

Sec23 Sec24 may participate in membrane curvature the

majority of membrane bending force likely comes from

Sec13 Sec31 Indeed recent genetic and biochemical

experiments support this model Sec31 likely forms all the

contacts needed to make the COPII cage (Fath et al 2007)

with Sec13 providing structural rigidity to the cage edge

element to overcome the membrane bending energy of

a cargo-rich membrane (Copic et al 2012)

Cargo capture stochastic sampling vs direct and indirect selection

The fundamental function of vesicles is to ensure directional

traf 1047297c of protein cargoes making cargo capture an in-

tegral part of coat action To some extent cargo can enter

into vesicles in a nonspeci1047297c manner known as bulk 1047298ow

whereby stochastic sampling of the ER membrane and

lumen occurs during vesicle formation capturing local

molecules by chance Although this mode of transport could

traf 1047297c some abundant cargoes the random nature of this

process cannot explain the ef 1047297ciency with which some ER

export occurs In particular some cargoes are dramatically

enriched in vesicles above their prevailing concentration in

the ER suggesting a more ef 1047297cient and selective packaging

process Although the concentrative mode of cargo selection

has gained favor in the last decade recent experiments

reevaluating the potential for bulk 1047298ow to explain forward

traf 1047297c of some proteins warrants a more detailed analysis of the potential prevalence of this nonspeci1047297c pathway espe-

cially with respect to abundant nonessential proteins where

the ef 1047297ciency of secretion may not be central to cellular

viability (Thor et al 2009)

Selective enrichment of cargo in transport vesicles via

speci1047297c sorting signals is a common paradigm in intracellu-

lar protein traf 1047297cking 1047297rst characterized in endocytosis

Deciphering a similar mode of transport for the entire

spectrum of cargoes handled by the COPII coat however

has been hindered by the absence of a single common signal

used by the entire secretome Instead multiple signals seem

to drive selective capture meaning the COPII coat mustrecognize various signals employed by structurally diverse

cargoes Such signals range from simple acidic peptides

(Malkus et al 2002) to folded epitopes (Mancias and Goldberg

2007) and can act either by interacting directly with the

COPII coat or by binding to a cargo adaptor that links them

to the coat indirectly (Figure 4) (Dancourt and Barlowe

2010)

Genetic biochemical and structural data support Sec24

as the cargo binding adaptor for the COPII coat forming

Figure 4 Cargo selection can be direct or indirect Selec-

tive cargo capture during vesicle formation can occur via

direct interaction of cargo molecules with the COPI and

COPII coats ER export signals (eg DxE LxxLE and

YxxNPF) interact directly with Sec24 to facilitate capture

into COPII vesicles Similarly dilysine and diaromatic sig-

nals mediate interaction with the COPI coat to direct ret-

rograde traf1047297c back to the ER Soluble secretory proteins

may be captured indirectly via speci1047297c cargo receptors that

serve to recognize the transport-competent cargo and link

it to the coat Erv29 is the cargo receptor for many soluble

secretory proteins Soluble ER residents are returned back

to the ER via a similar cargo receptor system driven by

Erd2 which recognizes HDEL signals Membrane proteins

may also require cargo adaptor proteins such as Erv14 and

Rer1 although the basis for cargo recognition is not as

well de1047297ned




a relatively static platform that has multiple binding sites for

interaction with distinct sorting signals The so-called A site

binds the SNARE Sed5 via a NPF motif (Mossessova et al

2003 Miller et al 2005) the B site is most diverse recog-

nizing acidic sorting signals such as those found on the

SNARE Bet1 the Golgi membrane protein Sys1 and un-

known signals on additional cargoes (Miller et al 2003

Mossessova et al 2003) the C site binds a folded epitope

formed by the longin domain of the SNARE Sec22 (Milleret al 2003 Mancias and Goldberg 2007) The repertoire of

binding sites is further expanded by the presence of addi-

tional Sec24 isoforms the nonessential Iss1 and Lst1 pro-

teins (Roberg et al 1999 Kurihara et al 2000 Peng et al

2000) Sec24ndashcargo interactions are in general fairly low

af 1047297nity (Mossessova et al 2003) which is compatible with

the transient nature of the association of cargo with coat

proteins must bind during vesicle formation but must also be

released prior to vesicle fusion to allow coat recycling and

exposure of fusogenic domains The possibility remains that

additional layers of regulation impact coat dissociation from

cargo molecules after vesicle release Sec23 is both ubiquiti-

nated (Cohen et al 2003) and phosphorylated (Lord et al2011) and similar activity on Sec24 may promote uncou-

pling of coat from cargo

Some cargoes by topology or preference do not interact

directly with Sec24 but instead use adaptorreceptor pro-

teins to link them to the coat indirectly (Dancourt and

Barlowe 2010) Some of these adaptors likely function as

canonical receptors binding to their ligands in one compart-

ment and simultaneously interacting with Sec24 to couple

cargo with coat then releasing their ligand in another com-

partment perhaps as the result of a change in ionic strength

or pH of the acceptor organelle (Figure 3) Although their

precise mechanisms of ligand binding and release remain to

be fully explored such receptors include Erv29 which medi-

ates traf 1047297c of soluble secretory proteins like pro-a-factor and

CPY (Belden and Barlowe 2001) and Emp46 Emp47 which

are homologous to the mammalian ERGIC-53 family of pro-

teins that mediate traf 1047297c of coagulation factors (Sato and

Nakano 2002) Other receptors function to enrich vesicles

with membrane protein cargoes The p24 proteins Emp24

Erv25 Erp1 and Erp2 are required for ef 1047297cient ER ex-

port of GPI-anchored proteins whose lumenal orientation

precludes direct coupling to the COPII coat (Belden and

Barlowe 1996 Muniz et al 2000 Belden 2001) Others like

Erv26 (Bue et al 2006 Bue and Barlowe 2009) and Erv14

(Powers and Barlowe 1998 Powers and Barlowe 2002Herzig et al 2012) mediate ef 1047297cient export of transmem-

brane proteins that have cytoplasmically oriented regions

but either do not contain ER export signals or require addi-

tional af 1047297nity or organization to achieve ef 1047297cient capture

The requirement for receptors for such transmembrane car-

goes remains unexplained but may derive from the ancestral

history of the cargoes whereby previously soluble proteins

became membrane anchored as a result of gene fusion events

(Dancourt and Barlowe 2010) Alternatively the receptor

proteins may provide additional functionality required for

ef 1047297cient ER egress like a chaperoning function that would

protect the long transmembrane domains of plasma mem-

brane proteins from the relatively thinner lipid bilayer char-

acteristic of the ER (Sharpe et al 2010) Indeed some cargo

proteins have speci1047297c chaperoning needs with ER resi-

dent proteins that are not themselves captured into COPII

vesicles likely functioning to promote assembly and folding

of polytopic membrane proteins For example the aminoacid permeases all depend on an ER resident Shr3 for cor-

rect folding and quaternary assembly which is itself a pre-

requisite for COPII capture (Ljungdahl et al 1992 Kuehn

et al 1996 Gilstring et al 1999 Kota et al 2007)

Regulation of COPII function GTPase modulationcoat modi 1047297 cation

The GTPase activity of the coat is the primary mode of

regulation known to govern initiation of coat assembly

disassembly through canonical GEF and GAP activities of

Sec12 (drsquoEnfert et al 1991) and Sec23 (Yoshihisa et al

1993) respectively but also contributing to additional func-

tions like discrimination of relevant cargo proteins (Satoand Nakano 2005) and vesicle scission (Bielli et al 2005

Lee et al 2005) Unlike other coat systems the COPII coat

uses a combinatorial GAP activity that is provided by com-

ponents of the coat themselves Sec23 (Yoshihisa et al

1993) and Sec31 (Antonny et al 2001) The effect of this

autonomous GAP in minimal systems is that as soon as the

coat fully assembles GTP is hydrolyzed and the coat is rap-

idly released (Antonny et al 2001) creating a paradox as to

how coat assembly might be sustained for a suf 1047297cient length

of time to generate vesicles One solution to this conundrum

is that constant Sec12 GEF activity feeds new coat elements

into a nascent bud (Futai et al 2004 Sato and Nakano

2005) coat release from the membrane might also be

delayed by the increased af 1047297nity afforded by cargo proteins

(Sato and Nakano 2005) However recent 1047297ndings suggest

that a GAP inhibitory function contributed by the peripheral

ER protein Sec16 also modulates the activity of the coat

(Kung et al 2012 Yorimitsu and Sato 2012) Sec16 is

a large essential protein that associates with the cytoplas-

mic face of the ER membrane at ERES (Espenshade et al

1995 Connerly et al 2005) It interacts with all of the COPII

coat proteins (Gimeno et al 1996 Shaywitz et al 1997) and

is thus thought to scaffold andor organize coat assembly at

these discrete domains (Supek et al 2002 Shindiapina and

Barlowe 2010) In addition to this recruitment functiona fragment of Sec16 dampens the GAP-stimulatory effect

of Sec31 probably by preventing Sec31 recruitment to

Sar1 Sec23 Sec24 (Kung et al 2012) The GAP-inhibitory

effect of Sec16 was diminished in the context of a point muta-

tion in Sec24 (Kung et al 2012) raising the tantalizing possi-

bility that cargo engagement by Sec24 could trigger interaction

with Sec16 to inhibit the full GTPase activity of the coat in such

a manner that a vesicle is initiated around a cargo-bound com-

plex of Sar1 Sec23 Sec24 Sec16 (Springer et al 1999)




Another poorly explored aspect of COPII regulation is

post-translational modi1047297cation of the coat Sec23 is a target

for ubiquitination and is seemingly rescued from degrada-

tion by the action of the ubiqutin protease complex Bre5

Ubp3 (Cohen et al 2003) Whether this activity only con-

trols expression levels of the protein or contributes more

subtly to regulate proteinndashprotein interactions remains to

be tested Furthermore the potential ubiquitination of other

COPII coat components also warrants investigation recentexperiments in mammalian cells identi1047297ed Sec31 as a target

for a speci1047297c monoubiquitination event that is important for

ER export of collagen 1047297bers (Jin et al 2012) Whether yeast

Sec31 is similarly modi1047297ed by the equivalent E3 ubiquitin

ligases and how such a modi1047297cation might in1047298uence coat

action perhaps by contributing to the structural integrity

of the coat to drive membrane bending around rigid car-

goes remains to be tested Like ubiquitination the role of

coat phosphorylation is only starting to be explored It has

long been known that Sec31 is a phosphoprotein and that

dephosphorylation speci1047297cally impacted vesicle release

(Salama et al 1997) However despite the many sites of

Sec31 phosphorylation being revealed by high throughputphosphoproteomics the precise function of these modi1047297-

cations remains unclear In contrast progress has recently

been made in understanding phosphorylation of Sec23

and how this event probably in1047298uences the directionality

of vesicle traf 1047297c by controlling sequential interactions with

different Sec23 partners (Lord et al 2011) It is tempting to

speculate that similar phosphorylation of Sec24 might also

regulate coat displacement from cargo molecules to further

promote coat release and expose the fusogenic SNARE pro-

teins that would otherwise be occluded by their interaction

with the coat Indeed at least partial uncoating of COPII

vesicles is required for fusion to ensue since when GTP hy-

drolysis is prevented vesicles fail to fuse (Barlowe et al

1994) Whether additional proteinndashprotein interactions or

post-translational modi1047297cations contribute to coat shedding

remains to be seen

Higher-order organization of vesicle formation

Although the minimal COPII coat can drive vesicle forma-

tion from naked liposomes (Matsuoka et al 1998b) this

process in vivo is likely tightly regulated to enable both ef-

1047297cient vesicle production and adaptability to suit the secre-

tory burden of the cell (Farhan et al 2008) In part this

regulation occurs at the level of the subdivision of the ER

into discrete ERES from which vesicles form These smalldomains are marked by both the COPII coat proteins them-

selves and accessory proteins such as Sec16 and in some

cells Sec12 (Rossanese et al 1999 Connerly et al 2005

Watson et al 2006) ERES are located throughout the ER

with a seemingly random distribution that may in fact cor-

respond to regions of high local curvature induced by the ER

membrane proteins Rtn1 Rtn2 and Yop1 (Okamoto et al

2012) In related yeasts these sites are dynamic with the

ability to form de novo fuse and divide (Bevis et al 2002)

Although the precise mechanisms that regulate the steady

state distribution and size of these domains remain unclear

activity of both Sec12 and Sec16 seems to play a role

(Connerly et al 2005) as does the lipid composition of

the ER (Shindiapina and Barlowe 2010) In mammalian

cells misfolded proteins that are incompetent for forward

traf 1047297c are excluded from ERES (Mezzacasa and Helenius

2002) and this also seems to be true for some proteins

in yeast most notably GPI-anchored proteins with lipidanchors that have not been adequately remodeled which

are not concentrated at ERES but instead remain dispersed

within the bulk ER (Castillon et al 2009)

Vesicle Delivery to the Golgi

After release of COPII vesicles from ER membranes tethering

and fusion machineries guide ER-derived vesicles to Golgi

acceptor membranes through the action of over a dozen

gene products (Figure 5) Although ER ndashGolgi transport

can be separated into biochemically distinct stages using

cell-free assays evidence suggests that these events may

be organized in a manner that couples the budding andfusion stages In general budded vesicles become tethered

to Golgi membranes through the action of the Ypt1 GTPase

and tethering proteins Uso1 and the transport protein par-

ticle I (TRAPPI) complex Membrane fusion between vesicle

and Golgi acceptor membranes is then catalyzed through

assembly of SNARE protein complexes from the apposed

membrane compartments How the budding tethering

and fusion events are coordinated in cells remains an open

question although genetic biochemical and structural

studies have advanced our understanding of underlying

molecular mechanisms in vesicle tethering and membrane

fusion described below

Vesicle tethering

Initial cell free transport assays coupled with genetic ap-

proaches placed ER ndashGolgi transport requirements into

distinct vesicle budding and vesicle consumptionfusion

stages (Kaiser and Schekman 1990 Rexach and Schekman

1991) Ypt1 identi1047297ed as a founding member of the Rab

family of GTPases was implicated in the vesicle targeting

stage in the ER ndashGolgi transport pathway (Schmitt et al

1988 Segev et al 1988 Baker et al 1990) In reconstituted

vesicle fusion reactions Ypt1 was found to act in concert

with the extended coil-coiled domain protein Uso1 to tether

COPII vesicles to Golgi acceptor membranes (Nakajima et al1991 Barlowe 1997) In these assays freely diffusible COPII

vesicles could be tethered to and sedimented with washed

Golgi acceptor membranes upon addition of puri1047297ed Uso1

Interestingly the Uso1- and Ypt1-dependent tethering stage

does not appear to require the downstream SNARE protein

fusion machinery (Sapperstein et al 1996 Cao et al 1998)

In addition to the extended structure of Uso1 which is

predicted to span a distance of 180 nm (Yamakawa et al

1996) the multisubunit TRAPPI complex is required for




COPII-dependent transport to Golgi acceptor membranes(Rossi et al 1995 Sacher et al 1998) In vitro assays

revealed that TRAPPI can also function to physically link

COPII vesicles to Golgi membranes (Sacher et al 2001)

Structural analyses show that TRAPPI is a 170-kDa particle

consisting of six subunits (Bet3 Bet5 Trs20 Trs23 Trs31

and Trs33) that assemble into a 1047298at bilobed arrangement

with dimensions of 18 nm middot 6 nm middot 5 nm (Kim et al

2006) Bet3 can bind directly to Sec23 and with TRAPPI

peripherally bound to membranes this activity is thought

to link partially coated COPII vesicles to Golgi acceptor

membranes (Cai et al 2007) In a recent study the Golgi-

associated Hrr25 kinase was reported to phosphorylate

Sec23 Sec24 and regulate interactions between Sec23 and

TRAPPI to control directionality of anterograde transport (Lord

et al 2011) Moreover TRAPPI functions as a GEF for Ypt1

in a manner that is thought to generate activated Ypt1 on

the surface of Golgi acceptor membranes andor COPII

vesicles (Jones et al 2000 Wang et al 2000 Lord et al

2011) A subassembly of TRAPPI consisting of Bet3 Bet5

Trs23 and Trs31 binds Ypt1p and catalyzes nucleotide ex-

change by stabilizing an open form of this GTPase (Cai et al

2008) TRAPPI does not appear to interact directly with

Uso1 although Ypt1 activation could serve to coordinate

the long-distance tethering mediated by Uso1 with a closer

TRAPPI-dependent tethering event The precise orientationof TRAPPI on Golgi and vesicle membranes is not known

but current models suggest that this multisubunit complex

links COPII vesicles to the cis-Golgi surface and serves as a

central hub in coordinating vesicle tethering with SNARE-

mediated membrane fusion

Genetic and biochemical evidence indicate that other

coiled-coil domain proteins also act in COPII vesicle tether-

ing andor organization of the early Golgi compartment in

yeast The GRASP65 homolog Grh1 is anchored to cis-Golgi

membranes through N-terminal acetylation and formsa complex with another coiled-coil domain protein termed

Bug1 (Behnia et al 2007) Grh1 and Bug1 are not essential

but deletion of either protein reduces COPII vesicle tether-

ing and transport levels in cell-free assays and the grh1 D

and bug1 D mutants display negative genetic interactions

with thermosensitive ypt1 and uso1 mutants (Behnia et al

2007) These 1047297ndings suggest a redundant network of

coiled-coil proteins that act in tethering vesicles and orga-

nizing the cis-Golgi compartment Indeed additional coiled-

coil proteins including Rud3 and Coy1 localize to cis-Golgi

membranes and are implicated in organization of the cis-

Golgi and interface with COPII vesicles (VanRheenen et al

1999 Gillingham et al 2002 2004) Although some double

deletion analyses have been performed with these genes

multiple deletions may be required to severely impact this

redundant network

SNARE protein-dependent membrane fusion

Fusion of tethered COPII vesicles with cis-Golgi membranes

depends on a set of membrane-bound SNARE proteins Sev-

eral lines of evidence indicate that the SNARE proteins

Sed5 Bos1 Bet1 and Sec22 catalyze this membrane fusion

event in yeast (Newman et al 1990 Hardwick and Pelham

1992 Sogaard et al 1994 Cao and Barlowe 2000) The

SNARE protein family is de1047297ned by a conserved 70-amino-acid heptad repeat sequence termed the SNARE mo-

tif which is typically adjacent to a C-terminal tail-anchored

membrane segment (Rothman 1994 Fasshauer et al 1998)

Cognate sets of SNARE proteins form stable complexes

through assembly of their SNARE motifs into parallel four-

helix coiled-coil structures (Hanson et al 1997 Sutton et al

1998) The close apposition of membranes that follows as-

sembly of SNARE complexes in trans is thought to drive

membrane bilayer fusion (Weber et al 1998) Structural

Figure 5 Vesicle tethering and fu-

sion Anterograde delivery of COPII-

coated vesicles is mediated by a

variety of tethering and fusion com-

plexes The TRAPP complex binds to

Sec23 on the surface of a COPII ves-

icle and mediates local activation of

the Rab family member Ypt1 Yptndash

GTP recruits downstream effectors

such as the long coiled-coil tether

Uso1 A Golgi-localized kinase Hrr25phosphorylates Sec23 and displa-

ces TRAPP perhaps contributing to

coat shedding Removal of the coat

exposes the fusogenic SNARE pro-

teins which assemble to drive

membrane mixing In the retrograde

pathway COPI-coated vesicles em-

ploy the DSL1 complex composed

of Dsl1Sec39Tip20 to recognize

the incoming vesicle and coordinate

coat release and SNARE pairing




studies of the four-helix bundle reveal that the central or

ldquozero layerrdquo consists of ionic residues such that three of the

SNARE proteins contribute a glutamine residue and are

thus termed Q-SNARES whereas the fourth helix contains

an arginine residue and is known as the R-SNARE (Fasshauer

et al 1998 Sutton et al 1998) Further re1047297nement of the

Q-SNARE proteins based on sequence conservation iden-

ti1047297es each as a member of the Qa Qb or Qc subfamily

(Kloepper et al 2007) SNARE-dependent membrane fusionis though to proceed through a conserved mechanism in

which three Q-SNARES (Qa Qb and Qc) and one R-SNARE

zipper together from the N-terminal side of the SNARE motif

toward the membrane (Sudhof and Rothman 2009) In

the case of COPII vesicle fusion with Golgi membranes

Sed5 serves as the Qa-SNARE Bos1 the Qb-SNARE Bet1

the Qc-SNARE and Sec22 the R-SNARE Furthermore this

SNARE set is suf 1047297cient to catalyze membrane fusion when

reconstituted into synthetic proteoliposomes (Parlati et al

2000)

In addition to Sed5 Bos1 Bet1 and Sec22 other regu-

latory factors are required to control fusion speci1047297city and

govern SNARE complex assemblydisassembly Members of the Sec1 Munc18-1 (SM) family of SNARE-binding proteins

regulate distinct SNARE-dependent fusion events (Sudhof

and Rothman 2009) The SM family member Sly1 is re-

quired for fusion of COPII vesicles with Golgi membrane

in yeast (Ossig et al 1991 Cao et al 1998) SLY1 was ini-

tially identi1047297ed as a suppressor of loss of YPT1 function

when the gain-of-function SLY1-20 allele was isolated in

a selection for mutations that permit growth in the absence

of YPT1 (Dascher et al 1991) Sly1 binds directly to Sed5

and increases the 1047297delity of SNARE complex assembly be-

tween Sed5 Bos1 Bet1 and Sec22 compared to noncognate

SNARE complexes (Peng and Gallwitz 2002) Crystallo-

graphic studies of Sly1 reveal a three-domain arch-shaped

architecture that binds a 45-amino-acid N-terminal domain

of Sed5 as observed for other SM protein interactions with

Qa-SNAREs (Bracher and Weissenhorn 2002) Working

models for Sly1 and SM protein function in general are

based on multiple binding modes wherein Sly1 initially

bound to the N terminus of Sed5 would subsequently bind

to other cognate SNARE proteins to regulate assembly and

ultimately to act as a clamp in stabilizing a trans-SNARE

complex (Furgason et al 2009 Sudhof and Rothman 2009)

After SNARE-mediated membrane fusion is complete

stable four-helix bundles of cis-SNARE complexes are now

present on the acceptor membrane compartment To recycleassembled Sed5ndashBos1ndashBet1ndashSec22 complexes for use in ad-

ditional rounds of membrane fusion the general fusion fac-

tors Sec17 and Sec18 catalyze SNARE complex disassembly

(Sogaard et al 1994 Bonifacino and Glick 2004) Sec18

belongs to the AAA family of ATPase chaperones and uses

the energy of ATP hydrolysis to separate stable cis-SNARE

complexes Sec17 is thought to recruit Sec18 to SNARE pro-

tein complexes and couples ATPase dependent disassembly

of cis-SNARE complexes (Bonifacino and Glick 2004) How

Sec17 Sec18-mediated disassembly is coordinated with

coat-dependent capture of SNARE proteins into vesicles

and Sly1-dependent assembly of trans-SNARE complexes

during fusion remain open questions

A concerted model for COPII vesicle tethering and fusion

Although distinct stages in vesicle tethering and fusion can

be de1047297ned through biochemical and genetic analyses these

are likely concerted reactions in a continuum of eventsthrough the early secretory pathway (Figure 5) The multi-

subunit TRAPPI may serve as an organizational hub on cis-

Golgi membranes or vesicles to coordinate vesicle tethering

and fusion events TRAPPI interactions with the COPII

subunit Sec23 with the Ypt1 GTPase and potentially with

SNARE proteins (Jang et al 2002 Kim et al 2006) could

link tethering and fusion stages TRAPPI-activated Ypt1

could recruit Uso1 to Golgi membranes and as COPII

vesicles emerge from the ER Uso1 could forge a long-

distance link between newly formed vesicles and acceptor

membranes With tethered vesicles aligned to fusion sites

TRAPPI interactions with vesicle-associated Sec23 and Golgi

SNARE machinery would then position vesicles in closerproximity to acceptor membranes TRAPPI-bound vesicles

could transmit signals to the SNARE machinery by direct

contact or perhaps through generation of elevated levels of

activated Ypt1 The result of such a signal may be to disas-

semble cis-SNARE complexes or to generate a Sly1ndashSed5

conformation that promotes assembly of fusogeneic SNARE

complexes Assembly of trans-SNARE complexes would then

presumably lead to rapid hemifusion followed by bilayer

fusion and compartment mixing

Traf1047297c Within the Golgi

Transport through the Golgi complex

Newly synthesized secretory proteins arrive at the cis-Golgi

in COPII vesicles and after membrane fusion progress

through the Golgi complex Secretory cargo may receive

outer-chain carbohydrate modi1047297cations and proteolytic pro-

cessing in a sequential manner as cargo advances through

distinct Golgi compartments For glycoproteins the N-linked

core carbohydrate is extended by addition of a-16-mannose

residues in the cis-Golgi and by addition of a-12- and

a-13-mannose residues in the medial compartment Kex2-

dependent proteolytic processing of certain secretory cargo

occurs in the trans-Golgi compartment Each of these eventscan be resolved by blocking membrane fusion through in-

activation of the thermosensitive sec18-1 allele (Graham and

Emr 1991 Brigance et al 2000) In support of this sequen-

tial organization distinct Golgi compartments can be visu-

alized through 1047298uorescence microscopy or immuno-EM

by monitoring components of the glycosylation and pro-

cessing machinery (Franzusoff et al 1991 Preuss et al 1992

Wooding and Pelham 1998 Rossanese et al 1999) However

genetic and morphological approaches have not uncovered




a vesicle-mediated anterograde transport pathway through

distinct compartments of the yeast Golgi complex Instead

a model of cisternal maturation in which Golgi cisternae are

the anterograde carriers of secretory cargo is most consis-

tent with a range of experimental observations (Bonifacino

and Glick 2004) In the cisternal maturation model Golgi

cisterna containing nascent secretory cargo are formed at

the cis-face of the Golgi and mature into a medial and then

trans-compartment as resident Golgi glycosylation and pro-cessing proteins are dynamically retrieved in retrograde

vesicles to preceding cisternae Indeed the dispersed orga-

nization of Golgi compartments in S cerevisiae are resolv-

able by 1047298uorescence microscopy and provided a powerful

test of the maturation model through live cell imaging of

cis- and trans-Golgi proteins labeled with different 1047298uores-

cent tags In such a dual labeled strain a cis-compartment

should be observed to change color to a trans-compartment

over the time period required for secretory cargo to transit

the Golgi complex Strikingly two independent research

groups using time resolved high resolution microscopy docu-

mented individual cisterna transitioning from early to late

compartments in accord with the cisternal maturationmodel (Losev et al 2006 Matsuura-Tokita et al 2006)

In addition to retrograde transport from cis-Golgi to ER

(discussed below) the COPI coat is thought to mediate ret-

rograde transport within the Golgi complex to retrieve recy-

cling Golgi machinery to earlier compartments as Golgi

cisternae mature (Bonifacino and Glick 2004) In current

working models anterograde-directed COPI vesicles are tar-

geted to preceding Golgi compartments by the conserved

oligomeric Golgi (COG) complex a large multisubunit teth-

ering complex identi1047297ed through a combination of genetic

and biochemical approaches (Miller and Ungar 2012) COG

consists of eight subunits and belongs to the larger CATCHR

(complex associated with tethering containing helical rods)

family of tethering factors that includes the exocyst and

GARP complexes (Yu and Hughson 2010) In intra-Golgi

retrograde transport the COG complex appears to operate

as a tethering and fusion hub with multiple interactions that

link COG to the g-COPI subunit to Ypt1 and to Golgi SNARE

proteins (Suvorova et al 2002) More speci1047297cally fusion

of retrograde-directed COPI vesicles with cis-Golgi mem-

branes is thought to depend on COG complex interactions

with a distinct SNARE complex consisting of Sed5 (Qa)

Gos1 (Qb) Sft1 (Qc) and Ykt6 or Sec22 as the R-SNARE

(Shestakova et al 2007) Mutations in COG complex subu-

nits disrupt Golgi transport and glycosylation of secretory cargo fully consistent with this model However at this

stage there are no cell-free assays to measure COG-dependent

fusion of COPI vesicles to fully dissect underlying molecular

mechanisms (Miller and Ungar 2012)

Lipid requirements for Golgi transport

While the protein machinery underlying Golgi transport has

received much attention the role of speci1047297c lipid biosyn-

thetic and transfer pathways in Golgi traf 1047297cking remain

relatively understudied One of the 1047297rst connections for

a lipid requirement in transport through the Golgi complex

was the identi1047297cation and characterization of Sec14 as an

essential phosphatidylinositolphosphatidylcholine (PIPC)

transfer protein in yeast (Novick et al 1981 Bankaitis

et al 1989 Cleves et al 1991) The traf 1047297cking blocks asso-

ciated with Sec14 de1047297ciencies lead to an accumulation of

Golgi membranes and Golgi forms of secretory cargo Sec14

probably does not play a major role in transporting bulk phospholipids but rather is thought to function in regulating

phospholipid homeostasis through presentation of PIs to

modifying activities such as the PI4 kinases (Schaaf et al

2008) Interestingly PI4P levels in the Golgi complex also

play a critical role in Golgi structure and function as dem-

onstrated by mutations in the essential PI4 kinase Pik1

which block transport through the Golgi (Walch-Solimena

and Novick 1999 Audhya et al 2000) More recently a di-

rect requirement for PI4P levels on Golgi organization has

been documented through characterization of the Golgi-

localized PI4P binding protein encoded by VPS74 (Schmitz

et al 2008 Tu et al 2008) Loss of Vps74 function results

in mislocalization of Golgi mannosyltransferases from early Golgi compartments to the vacuole Vps74 appears to bind

to cytoplasmic sorting signals contained on Golgi resident

enzymes and to the COPI coat in addition to PI4P in sorting

Golgi-localized proteins into retrograde-directed vesicles In

this manner PI4P levels and Vps74 may function together

in dynamic recycling of Golgi modi1047297cation enzymes as cis-

terna containing nascent secretory cargo mature in accord

with Golgi maturation models Indeed the polarized dis-

tribution of PI4P across the Golgi with increasing concen-

trations from cis- to trans-compartments appears to play

several important roles in organization and transport through

the Golgi complex (Graham and Burd 2011)

The Return Journey Retrograde Traf1047297c viaCOPI Vesicles

Although it remains to this day somewhat controversial as to

the precise function (and thus direction) of COPI-mediated

vesicular traf 1047297c within the Golgi (Emr et al 2009) the role

of these vesicles in retrograde GolgindashER transport is well

established This is despite the original confusion in the 1047297eld

as to the directionality of COPI-mediated traf 1047297c yeast COPI

mutants generally have anterograde traf 1047297cking defects that

probably stem from indirect effects of blocking retrograde

transport rather than impacting forward traf 1047297c directly (Gaynor and Emr 1997) Although one COPI component

Sec21 was identi1047297ed in the original sec mutant screen

(Novick et al 1980) advances in understanding this step of

the secretory pathway largely lagged behind and was informed

by the biochemical advances made in mammalian systems

(Sera1047297ni et al 1991) Once Sec21 was cloned and realized

to be an ortholog of the mammalian coatomer complex

(Hosobuchi et al 1992) biochemical analyses allowed the

identi1047297cation of all equivalent yeast subunits which were




in turn also subsequently identi1047297ed in a variety of genetic

screens as additional sec ret cop mutants (Duden et al

1994 Cosson et al 1996) The major advances in dissecting

the mechanisms of retrograde traf 1047297c have continued to be

led by biochemical approaches (Spang et al 1998 Spang

and Schekman 1998) with many recent high resolution

structures of the relevant coat (Lee and Goldberg 2010

Faini et al 2012 Yu et al 2012) and tether proteins (Ren

et al 2009 Tripathi et al 2009) Given the strong homology between the mammalian and yeast proteins it seems likely

that the global structure of the yeast COPI coat is broadly

similar to that of mammals (Yip and Walz 2011) Indeed

current approaches make good use of yeast genetics ap-

proaches to test functional relevance of the structural data

yielding insight into areas including cargo selection (Michelsen

et al 2007) directionality of vesicle delivery (Kamena and

Spang 2004) and coattether in1047298uences on vesicle fusion

(Zink et al 2009)

Composition and structure of the COPI coat

Originally characterized from mammalian cells as a single

coat protomer or coatomer (Waters et al 1991) the COPIcoat is composed of seven subunits a- b- b9- g- d- e- and

z-COP that correspond to the yeast proteins Cop1 Sec33

Ret1 Sec26 Sec27 Sec21 Ret2 Sec28 and Ret3 respec-

tively Although found as a large cytosolic complex it is now

appreciated that like the COPII coat COPI comprises two

separable layers an inner layer that functions in cargo bind-

ing composed of g- d- z- and b-COP and an outer layer

formed by a- b9- and e-COP (Figure 3) Furthermore sig-

ni1047297cant sequence homology was apparent between the inner

COPI coat and the adaptor subunits of the clathrin coat

system Indeed a recent structural analysis of the g z sub-

complex of the inner COPI coat shows clear homology with

the a s subunits of the AP2 clathrin adaptor with Arf1

bound at a site that corresponds spatially to the PI(45)P2

binding site on AP2 (Yu et al 2012) Although the structure

of the b d subcomplex remains to be determined homology

modeling suggests that it adopts a conformation very similar

to the b2ndash AP2 subunit and biochemical analyses suggest

that a second Arf1 molecule can bind to the PI(45)P2 bind-

ing site on b2ndash AP2 (Yu et al 2012) Unlike the inner coat

which is most similar to the clathrin coat adaptors the outer

COPI coat shows homology with both clathrin and COPII

coats with b-propeller and a-solenoid domains forming

the building blocks of the putative cage Structural analysis

of stable fragments of the a-b9-COPI subcomplex supportsthe concept that the global architecture of the COPI coat is

intermediate between that of the COPII and clathrin coats

the individual b-barrel and a-solenoid structures most

closely resemble the Sec13 Sec31 structure of the COPII

cage but they assemble in a clathrin-like triskelion (Lee

and Goldberg 2010) It remains unclear exactly how the

inner and outer layers come together either in solution

prior to assembly on the membrane or during vesicle forma-

tion although puri1047297ed yeast coatomer examined by single

particle electron microscopy suggests a somewhat 1047298exible

con1047297guration that would need to stabilize during poly-

merization or oligomerization on the surface of the mem-

brane (Yip and Walz 2011) This concept of structural

1047298exibility for the COPI coat is supported by recent EM anal-

ysis of COPI vesicles budded from synthetic liposomes

which showed striking structural diversity of coat arrange-

ment on the surface of the budded vesicles (Faini et al

2012) Although all the crystallographic and much of thebiochemical analysis of the COPI coat has employed mam-

malian proteins the yeast orthologs are highly likely to

adopt similar conformations Indeed the known structures

are consistent with the nonessential nature of Sec28 its

ortholog e-COP is a helical structure that interacts with

a-COPI but likely does not form part of the cage (Hsia and

Hoelz 2010 Lee and Goldberg 2010) probably rendering

it dispensable in vivo despite some destabilization of Cop1

(a-COP) in the sec28 mutant (Duden et al 1998)

Like the COPII coat COPI assembly on the membrane is

initiated by a small GTPase Arf1 which in addition to the N-

terminal amphipathic a-helix also contains a myristoyl

group that facilitates membrane anchorage (Antonny et al1997a) GDPndashGTP exchange on Arf1 and its paralogs makes

use of a common structural motif the Sec7 domain named

for the late Golgi GEF that is the target of the fungal me-

tabolite Brefeldin A (Sata et al 1998 1999) In GolgindashER

retrograde traf 1047297c two redundant GEFs Gea1 and Gea2

each with a Sec7 domain likely initiate coat assembly by

triggering local recruitment of Arf1 (Peyroche et al 1996

Spang et al 2001) Unlike the COPII system the GAP activ-

ity for the COPI coat is not an integral part of the coat itself

but is instead contributed by a separate protein known (not

surprisingly) as ArfGAP1 in mammalian cells In yeast Arf ndash

GAP activity derives from two distinct proteins Gcs1 and

Glo3 with partially overlapping roles (Poon et al 1996

1999) Mammalian ArfGAP1 employs a lipid-packing sensor

domain to regulate its activity according to membrane cur-

vature becoming active on highly curved membranes likely

after vesicle formation has completed or at least progressed

enough as to permit Arf release without destabilizing the

coat (Bigay et al 2003 2005) Yeast Gcs1 also showed

a binding preference for conical lipids suggesting a similar

mechanism could regulate GTPase activity of the yeast COPI

coat (Antonny et al 1997b) However curvature-responsive

activity may not be the only mode of regulation of the COPI

GTPase cycle Coatomer itself also seems to in1047298uence Arf-

GAP activity (Goldberg 1999) although the mechanismremains to be fully de1047297ned (Luo and Randazzo 2008) Fur-

thermore the ability of some sorting signals on cargo pro-

teins to inhibit the coatomer-stimulated GAP activity directly

links coat recruitment to cargo selection (Springer et al

1999 Goldberg 2000) an appealing model whereby the

coat stably associates with the membrane only when bound

to cargo proteins (Springer et al 1999) Further complicat-

ing the problem is evidence that implicate ArfGAP proteins

as positive regulators of the COPI coat rather than negative




regulators overexpression of any of the four yeast ArfGAPs

suppressed the lethality of an arf1 mutant (Zhang et al

1998 2003) Further yeast experiments also support an

active role for Gcs1 and Glo3 in cargo selection acting

on SNARE proteins prior to incorporation into vesicles to

promote Arf1 and coatomer interaction (Rein et al 2002

Schindler and Spang 2007 Schindler et al 2009) Clearly

the precise role of the GAP in the COPI system remains

to be fully understood complicated by con1047298

icting resultsfrom different labs andor systems and may in fact be mul-

tifaceted by serving both positive and negative roles at dif-

ferent stages during the vesicle formation process (Spang

et al 2010)

Cargo capture sorting signals cargo adaptorsand coat stimulators

Like other vesicle traf 1047297cking events retrieval of ER resident

proteins via COPI vesicles employs sorting signals most

notably the canonical retrieval motifs HDEL for soluble

lumenal cargoes and K(X)KXX for membrane proteins

(Figure 4) Soluble proteins bind to a retrieval receptor

Erd2 (Semenza et al 1990) which couples them to the COPIcoat to facilitate retrograde traf 1047297c The COPI coat can dis-

criminate between similar but distinct motifs including the

canonical K(X)KXX which must be located at the C terminus

of the cargo and membrane-proximal to ensure ef 1047297cient

retrieval R-based motifs that only function when spaced

some distance from the membrane surface and other basic

motifs that remain to be fully dissected (Cosson et al

1998 Shikano and Li 2003) Yeast two-hybrid experi-

ments and subsequent mutagenesis analyses suggest that

the R-based motif binds at the interface between the b- and

d-COP subunits (Sec26 and Ret2 respectively) in a manner

that is distinct from KKXX binding to the coat (Michelsen

et al 2007) The site of KKXX recognition remains some-

what unclear Multiple lines of evidence support a role for

the a-b9-e-COP complex in KKXX binding (Cosson and

Letourneur 1994 Letourneur et al 1994 Fiedler et al 1996)

whereas direct cross-linking studies implicate the g-COP

subunit in KKXX binding (Harter et al 1996 Harter and

Wieland 1998)

In addition to retrieval motifs based on basic residues

diaromatic retrieval signals have also been identi1047297ed per-

haps best characterized for the p24 family of proteins albeit

largely using the mammalian family members (Strating

and Martens 2009) This class of signal likely binds to

the inner COPI coat via the g-COP subunit causing a con-formational change that may open up the cargo adaptor

platform to become receptive to additional cargo clients

(Beacutethune et al 2006 Strating and Martens 2009) Yet an-

other mode of cargo binding is represented by the SNARE

proteins that drive membrane fusion Unlike SNARE inter-

action with the COPII coat direct binding of SNARE sorting

signals with COPI components has not been observed In-

stead SNARE incorporation into COPI vesicles depends

on the activity of the Arf ndashGAP Glo3 although the precise

function of Glo3 in promoting a SNARE con1047297guration that

is favorable for vesicle capture remains to be fully dissected

(Rein et al 2002)

As with the COPII coat capture of cargo proteins into

retrograde COPI vesicles sometimes requires the action of

cargo adaptors The 1047297rst of these described was the HDEL

receptor Erd2 described above where the lumenal domain

likely provides ligand-binding function (Scheel and Pelham

1998) with changing pH conditions likely driving bindingand release in the appropriate compartments (Wilson et al

1993) Another well-described cargo adaptor is the mem-

brane protein Rer1 (Nishikawa and Nakano 1993 Sato

et al 1995) which is important for the ef 1047297cient retrieval

and thus steady-state ER localization of some ER resident

proteins including the COPII GEF Sec12 and the translo-

con components Sec63 and Sec71 (Sato et al 1997) The

reason these proteins would require an escort back to the ER

rather than employing their own retrieval motifs is unclear

but Rer1 seems to bind these clients within their transmem-

brane domains via polar residues embedded within the hy-

drophobic environment (Sato et al 1996 2001) Sec12 and

Sec71 appear to use different sites on Rer1 to facilitate ret-rograde traf 1047297c since mutation of the Sec12-binding site had

no effect on Sec71 retrieval suggesting that Rer1 forms

a multivalent cargo receptor that has the capacity to bind

multiple cargo clients simultaneously (Sato et al 2003)

Yet another important player in COPI vesicle formation

is the class of proteins that seem to serve as coat nucleators

increasing or stabilizing the recruitment of the COPI coat

on the Golgi to stimulate retrograde traf 1047297c Although the

mechanistic details remain to be fully understood two

classes of protein seem to stimulate retrograde traf 1047297c by

modulating the ability of the COPI coat to form vesicles The

1047297rst description of this function was for a membrane protein

Mst27 which suppresses the lethality of a sec21-1 mutant

when overexpressed (Sandmann et al 2003) Mst27 and its

related binding partner Mst28 both bind to yeast coatomer

via KKXX motifs and this function is required for the sec21-1

suppression Although the endogenous function of Mst27

Mst28 is unclear the ability of these cargo proteins to stim-

ulate vesicle production was one of the 1047297rst concrete pieces

of evidence that cargo abundance can directly in1047298uence

vesicle format ion More recently a similar role has been

postulated for the abundant class of p24 proteins genetic

interactions between EMP24 and various COPI components

including SEC21 and the Arf ndashGAP GLO3 are suggestive

of a functional relationship and membranes isolated fromemp24 D cells are diminished in their ability to form COPI

vesicles in vitro (Aguilera-Romero et al 2008) Since some

of the mammalian p24 proteins showed a capacity to mod-

ulate the GTPase activity of the COPI coat (Goldberg 2000)

it is tempting to link these observations by slowing the

GTPase activity of Arf1 the COPI coat might be stabilized

on the membrane prolonging the cargo-engagement step

and perhaps stimulating coat oligomerization to enhance

vesicle production




Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion

Like other vesicle traf 1047297cking steps the 1047297nal stages of

delivery of COPI vesicles employ a long-distance tether to

bring the vesicle into proximity of the acceptor membrane

and SNARE proteins to drive membrane fusion (Spang

2012) The ER-localized tethering complex the Dsl1 com-

plex performs the tethering function recognizing COPI

vesicles via their intact coat and also participates in thefusion event by proofreading the SNARE pairing that occurs

prior to fusion (Figure 5) Originally identi1047297ed as a mutant

that was dependent on the presence of the dominant sly1-20

allele dsl1 mutants showed accumulation of vesicles at

restrictive temperature and were suppressed by overex-

pression of SEC21 although they also showed ER ndashGolgi

transport defects making a precise function dif 1047297cult to dis-

cern (VanRheenen et al 2001) Dsl1 forms a complex with

Dsl3 Sec39 and Tip20 to form the Dsl1 complex another

member of the CATCHR family of tethering complexes noted

for their extended helical rod structures (Lees et al 2010)

Further genetic and biochemical dissection of these proteinsconverged on a role in retrograde transport from the Golgi

to the ER tip20 and dsl1 mutants showed genetic interac-

tions with a variety of ER ndashGolgi SNAREs (Sweet and Pelham

1993 Andag et al 2001 Kraynack et al 2005) tip20 mutants

showed defects in fusion of COPI vesicles (Kamena and Spang

2004) the Dsl1 complex was localized to the ER (Kraynack

et al 2005) and Dsl1 interacts directly with multiple compo-

nents of the COPI coat (Andag and Schmitt 2003)

Recent structural analyses have generated an appealing

mechanistic model by which the extended Dsl1 complex

performs three functions by virtue of its ability to interact

with both the COPI coat and the fusogenic SNAREs (Ren

et al 2009 Tripathi et al 2009 Zink et al 2009) A com-posite crystal structure suggests that a long stalk formed

largely by Sec39 extends away from the ER membrane

with Dsl1 located at the membrane-distal end to ldquocatchrdquo

incoming COPI vesicles via an unstructured loop that would

interact directly with the coat via an a-helical structure

formed by a- and e-COPI (Ren et al 2009 Hsia and Hoelz

2010) Sec39 itself binds to the N-terminal domain of the ER

resident SNARE Use1 via a region that likely lies proximal

to the membrane (Tripathi et al 2009) and Tip20 contains

a second SNARE-binding site interacting with the N-terminal

domain of Sec20 (Ren et al 2009) In addition to bind-

ing individual SNAREs the Dsl1 complex also promotesSNARE assembly and thus may serve two roles in fusion

maintaining individual SNAREs in an unpaired receptive

state and scaffolding assembly of the fusogenic SNARE

complex to promote fusion (Kraynack et al 2005 Ren

et al 2009) An additional role in vesicle uncoating is sug-

gested by the tendency of vesicles to accumulate en masse

under conditions of Dsl1 depletion (Zink et al 2009) COPI

shedding might be assisted by a Dsl1ndashCOPI interaction that

would prevent repolymerization of disassembled coat sub-

units or could be driven by conformational changes in the

Dsl1 complex that would capitalize on the ability of Dsl1 to

interact with both the outer a-e-COPI domain and a second

site on the inner d-COP subunit to prize the coat from the

membrane (Ren et al 2009 Zink et al 2009) Indeed neg-

ative stain EM images of the Dsl1 complex suggest a variety

of possible con1047297gurations although the mechanistic impact

of the different conformations with respect to coat and

SNARE binding remain to be tested (Ren et al 2009)Clearly the Dsl1 complex is a multifunctional tether that

may serve as a useful paradigm for other vesicle ldquotetheringrdquo

systems that may contribute to multiple layers of vesicle

uncoating docking and fusion in addition to their canonical

long-distance vesicle trapping function

Perspectives

Having moved from the ldquoparts listrdquo generated by numerous

genetic screens to molecular mechanisms de1047297ned by in vitro

assays where is the 1047297eld currently heading Emerging ques-

tions currently center on how the varied processes that drive

protein secretion are coordinated and regulated both at themolecular level and at the higher-order organizational level

The biosynthesis of secretory proteins can be thought of as

a series of simple events (translationtranslocation post-

translational modi1047297cation chaperone binding forward

transport) but are these events more closely entwined than

we currently appreciate How are protein quality control

decisions made are they a simple outcome of a tug of war

between the ER-associated degradation machinery and the

forward transport machinery Adding a dominant ER export

signal to a misfolded protein could drive forward traf 1047297c

(Kincaid and Cooper 2007) but the converse experiment

of blocking ERAD of a different misfolded substrate did

not lead to its secretion (Pagant et al 2007) Understanding

the interplay between the folding degradation and export

machineries will be key in appreciating the intricate regula-

tion of secretory protein production and how the different

machineries might be coregulated to cope with the changing

secretory burden of the cell under different environmental

conditions

Additional questions stem from our relatively poor un-

derstanding of how the early secretory pathway is organized

and how this organization is maintained Although it is clear

that ER exit sites form discrete subdomains of the ER

(Rossanese et al 1999 Shindiapina and Barlowe 2010)

what is the functional signi1047297cance of this organization Isthe segregation of cargo molecules into different ER exit

sites (Muniz et al 2001) driven by active processes or does

it re1047298ect the passive in1047298uence of speci1047297c lipid and protein

requirements for subsets of cargo molecules Similarly do all

secretory cargo proteins follow the same route through the

Golgi or are speci1047297c itineraries devised for distinct cargoes

that might also be driven by speci1047297c lipid microenvironments

andor post-translational modi1047297cation needs Larger-scale

questions also remain How is the cis-Golgi founded through




homotypic fusion of COPII vesicles by heterotypic fusion of

COPII and COPI vesicles or by templating from an existing

cis-Golgi fragment that expands through delivery of COPII

and COPI vesicles Electron tomography of yeast cells show

distinct transport vesicles and Golgi cisternae but no apparent

intermediates (West et al 2011) How are vesicles targeted to

the correct destination Is there a role for the cytoskeleton in

vesicle delivery and how do COPI vesicles that bud from the

Golgi 1047297

nd the proper acceptor compartment Indeed arethere multiple types of COPI vesicles that drive different

transport events between different Golgi cisternae and do

tubular elements play a role in lipid and protein traf 1047297c as

they appear to do in mammalian cells Finally how are the

protein and lipid needs of the cell sensed and maintained to

ensure ef 1047297cient protein secretion which lies at the heart of

cell growth to permit cell division and how are the rates of

anterograde and retrograde traf 1047297c balanced to maintain the

correct morphology and distribution of the various secretory

organelles As in the past the facile genetics and accessible

biochemistry of the yeast system still hold promise in answer-

ing these questions with the development of new tools serv-

ing to strengthen the 1047297eld and provide new avenues forfurther exploration

Literature Cited

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Andag U and H D Schmitt 2003 Dsl1p an essential componentof the Golgi-endoplasmic reticulum retrieval system in yeast usesthe same sequence motif to interact with different subunits of theCOPI vesicle coat J Biol Chem 278 51722ndash51734

Andag U T Neumann and H D Schmitt 2001 The coatomer-interacting protein Dsl1p is required for Golgi-to-endoplasmicreticulum retrieval in yeast J Biol Chem 276 39150ndash39160

Antonin W H A Meyer and E Hartmann 2000 Interactionsbetween Spc2p and other components of the endoplasmic re-ticulum translocation sites of the yeast Saccharomyces cerevi-siae J Biol Chem 275 34068ndash34072

Antonny B S Beraud-Dufour P Chardin and M Chabre1997a N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipidsupon GDP to GTP exchange Biochemistry 36 4675ndash4684

Antonny B I Huber S Paris M Chabre and D Cassel1997b Activation of ADP-ribosylation factor 1 GTPase-activatingprotein by phosphatidylcholine-derived diacylglycerols J BiolChem 272 30848ndash30851

Antonny B D Madden S Hamamoto L Orci and R Schekman2001 Dynamics of the COPII coat with GTP and stable ana-logues Nat Cell Biol 3 531ndash537

Antonny B P Gounon R Schekman and L Orci 2003 Self-assembly of minimal COPII cages EMBO Rep 4 419ndash424

Audhya A M Foti and S D Emr 2000 Distinct roles for theyeast phosphatidylinositol 4-kinases Stt4p and Pik1p in secre-tion cell growth and organelle membrane dynamics Mol BiolCell 11 2673ndash2689

Baker D L Hicke M Rexach M Schleyer and R Schekman1988 Reconstitution of SEC gene product-dependent inter-compartmental protein transport Cell 54 335ndash344

Baker D L Wuestehube R Schekman D Botstein and N Segev1990 GTP-binding Ypt1 protein and Ca2+ function indepen-dently in a cell-free protein transport reaction Proc Natl AcadSci USA 87 355ndash359

Balch W E W G Dunphy W A Braell and J E Rothman1984 Reconstitution of the transport of protein between suc-cessive compartments of the Golgi measured by the coupledincorporation of N-acetylglucosamine Cell 39 405ndash416

Bankaitis V A L M Johnson and S D Emr 1986 Isolation of yeast mutants defective in protein targeting to the vacuole Proc

Natl Acad Sci USA 83 9075ndash

9079Bankaitis V A D E Malehorn S D Emr and R Greene

1989 The Saccharomyces cerevisiae SEC14 gene encodes a cy-tosolic factor that is required for transport of secretory proteinsfrom the yeast Golgi complex J Cell Biol 108 1271ndash1281

Barlowe C 1997 Coupled ER to Golgi transport reconstituted with puri1047297ed cytosolic proteins J Cell Biol 139 1097ndash1108

Barlowe C C drsquoEnfert and R Schekman 1993 Puri1047297cation andcharacterization of SAR1p a small GTP-binding protein re-quired for transport vesicle formation from the endoplasmic re-ticulum J Biol Chem 268 873ndash879

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Battle A M C Jonikas P Walter J S Weissman and D Koller2010 Automated identi1047297cation of pathways from quantitativegenetic interaction data Mol Syst Biol 6 379

Baxter B K P James T Evans and E A Craig 1996 SSI1encodes a novel Hsp70 of the Saccharomyces cerevisiae endo-plasmic reticulum Mol Cell Biol 16 6444ndash6456

Becker J W Walter W Yan and E A Craig 1996 Functionalinteraction of cytosolic hsp70 and a DnaJ-related protein Ydj1pin protein translocation in vivo Mol Cell Biol 16 4378ndash4386

Behnia R F A Barr J J Flanagan C Barlowe and S Munro2007 The yeast orthologue of GRASP65 forms a complex witha coiled-coil protein that contributes to ER to Golgi traf 1047297c J CellBiol 176 255ndash261

Belden W J 2001 Distinct roles for the cytoplasmic tail sequencesof Emp24p and Erv25p in transport between the endoplasmic re-

ticulum and Golgi complex J Biol Chem 276 43040ndash

43048Belden W J and C Barlowe 1996 Erv25p a component of

COPII-coated vesicles forms a complex with Emp24p that isrequired for ef 1047297cient endoplasmic reticulum to Golgi transportJ Biol Chem 271 26939ndash26946

Belden W J and C Barlowe 2001 Role of Erv29p in collectingsoluble secretory proteins into ER-derived transport vesiclesScience 294 1528ndash1531

Bernales S F R Papa and P Walter 2006 Intracellular signal-ing by the unfolded protein response Annu Rev Cell Dev Biol22 487ndash508

Bertolotti A Y Zhang L M Hendershot H P Harding and D Ron2000 Dynamic interaction of BiP and ER stress transducers inthe unfolded-protein response Nat Cell Biol 2 326ndash332

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2006 Coatomer the coat protein of COPI transport vesiclesdiscriminates endoplasmic reticulum residents from p24 pro-teins Mol Cell Biol 26 8011ndash8021

Bevis B A Hammond C Reinke and B Glick 2002 De novoformation of transitional ER sites and Golgi structures in Pichiapastoris Nat Cell Biol 4 750ndash756

Bi X R A Corpina and J Goldberg 2002 Structure of theSec2324-Sar1 pre-budding complex of the COPII vesicle coatNature 419 271ndash277

Bi X J D Mancias and J Goldberg 2007 Insights into COPIIcoat nucleation from the structure of Sec23Sar1 complexed with the active fragment of Sec31 Dev Cell 13 635ndash645




Bielli A C J Haney G Gabreski S C Watkins S I Bannykhet al 2005 Regulation of Sar1 NH2 terminus by GTP bindingand hydrolysis promotes membrane deformation to controlCOPII vesicle 1047297ssion J Cell Biol 171 919ndash924

Bigay J P Gounon S Robineau and B Antonny 2003 Lipidpacking sensed by ArfGAP1 couples COPI coat disassembly tomembrane bilayer curvature Nature 426 563ndash566

Bigay J J Casella G Drin B Mesmin and B Antonny2005 ArfGAP1 responds to membrane curvature through thefolding of a lipid packing sensor motif EMBO J 24 2244ndash2253

Bohni P C R J Deshaies and R W Schekman 1988 SEC11 isrequired for signal peptide processing and yeast cell growth JCell Biol 106 1035ndash1042

Bonifacino J and B Glick 2004 The mechanisms of vesicle bud-ding and fusion Cell 116 153ndash166

Bracher A and W Weissenhorn 2002 Structural basis for the Golgimembrane recruitment of Sly1p by Sed5p EMBO J 21 6114ndash6124

Brigance W T C Barlowe and T R Graham 2000 Organizationof the yeast Golgi complex into at least four functionally distinctcompartments Mol Biol Cell 11 171ndash182

Brodsky J L and R Schekman 1993 A Sec63p-BiP complexfrom yeast is required for protein translocation in a reconstitutedproteoliposome J Cell Biol 123 1355ndash1363

Brodsky J L E D Werner M E Dubas J L Goeckeler K B Kruseet al 1999 The requirement for molecular chaperones during

endoplasmic reticulum-associated protein degradation demon-strates that protein export and import are mechanistically dis-tinct J Biol Chem 274 3453ndash3460

Brown J D B C Hann K F Medzihradszky M Niwa A LBurlingame et al 1994 Subunits of the Saccharomyces cerevisiae signal recognition particle required for its functional ex-pression EMBO J 13 4390ndash4400

Bue C A and C Barlowe 2009 Molecular dissection of erv26pidenti1047297es separable cargo binding and coat protein sorting ac-tivities J Biol Chem 284 24049ndash24060

Bue C A C M Bentivoglio and C Barlowe 2006 Erv26p di-rects pro-alkaline phosphatase into endoplasmic reticulum-derived coat protein complex II transport vesicles Mol BiolCell 17 4780ndash4789

Bukau B and A L Horwich 1998 The Hsp70 and Hsp60 chap-

erone machines Cell 92 351ndash

366Burda P and M Aebi 1999 The dolichol pathway of N-linked

glycosylation Biochim Biophys Acta 1426 239ndash257Cai H C C Wang and C L Tsou 1994 Chaperone-like activity

of protein disul1047297de isomerase in the refolding of a protein withno disul1047297de bonds J Biol Chem 269 24550ndash24552

Cai H S Yu S Menon Y Cai D Lazarova et al 2007 TRAPPItethers COPII vesicles by binding the coat subunit Sec23 Nature445 941ndash944

Cai Y H F Chin D Lazarova S Menon C Fu et al 2008 Thestructural basis for activation of the Rab Ypt1p by the TRAPPmembrane-tethering complexes Cell 133 1202ndash1213

Cao X and C Barlowe 2000 Asymmetric requirements for a RabGTPase and SNARE proteins in fusion of COPII vesicles withacceptor membranes J Cell Biol 149 55ndash66

Cao X N Ballew and C Barlowe 1998 Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins EMBO J 17 2156ndash2165

Caplan A J D M Cyr and M G Douglas 1992 YDJ1p facili-tates polypeptide translocation across different intracellularmembranes by a conserved mechanism Cell 71 1143ndash1155

Carvalho P V Goder and T Rapoport 2006 Distinct ubiquitin-ligase complexes de1047297ne convergent pathways for the degrada-tion of ER proteins Cell 126 361ndash373

Castillon G A R Watanabe M Taylor T M E Schwabe and HRiezman 2009 Concentration of GPI-anchored proteins uponER exit in yeast Traf 1047297c 10 186ndash200

Chang Y W Y C Chuang Y C Ho M Y Cheng Y J Sun

et al 2010 Crystal structure of Get4-Get5 complex and its

interactions with Sgt2 Get3 and Ydj1 J Biol Chem 2859962ndash9970

Chartron J W C J Suloway M Zaslaver and W M Clemons Jr

2010 Structural characterization of the Get4Get5 complexand its interaction with Get3 Proc Natl Acad Sci USA 10712127ndash12132

Chen X C VanValkenburgh H Liang H Fang and N Green

2001 Signal peptidase and oligosaccharyltransferase interact

in a sequential and dependent manner within the endoplasmicreticulum J Biol Chem 276 2411ndash2416

Chirico W J M G Waters and G Blobel 1988 70K heat shock related proteins stimulate protein translocation into micro-somes Nature 332 805ndash810

Clerc S C Hirsch D M Oggier P Deprez C Jakob et al 2009 Htm1protein generates the N-glycan signal for glycoprotein degradation

in the endoplasmic reticulum J Cell Biol 184 159ndash172Cleves A E T P McGee E A Whitters K M Champion J R

Aitken et al 1991 Mutations in the CDP-choline pathway forphospholipid biosynthesis bypass the requirement for an essen-

tial phospholipid transfer protein Cell 64 789ndash800Cohen M F Stutz N Belgareh R Haguenauer-Tsapis and C

Dargemont 2003 Ubp3 requires a cofactor Bre5 to speci1047297-

cally de-ubiquitinate the COPII protein Sec23 Nat Cell Biol

5 661ndash

667Connerly P L M Esaki E A Montegna D E Strongin S Levi

et al 2005 Sec16 is a determinant of transitional ER organi-zation Curr Biol 15 1439ndash1447

Copic A C F Latham M A Horlbeck J G Drsquo Arcangelo and E A

Miller 2012 ER cargo properties specify a requirement for COPII

coat rigidity mediated by Sec13p Science 335 1359ndash1362Cosson P and F Letourneur 1994 Coatomer interaction with di-

lysine endoplasmic reticulum retention motifs Science 2631629ndash1631

Cosson P C Demolliere S Hennecke R Duden and F Letourneur1996 Delta- and zeta-COP two coatomer subunits homologousto clathrin-associated proteins are involved in ER retrievalEMBO J 15 1792ndash1798

Cosson P Y Lefkir C Demolliere and F Letourneur 1998 NewCOP1-binding motifs involved in ER retrieval EMBO J 176863ndash6870

Costanzo M A Baryshnikova J Bellay Y Kim E D Spear et al2010 The genetic landscape of a cell Science 327 425ndash431

Cox J C Shamu and P Walter 1993 Transcriptional inductionof genes encoding endoplasmic reticulum resident proteins re-quires a transmembrane protein kinase Cell 73 1197ndash1206

Cox J S and P Walter 1996 A novel mechanism for regulatingactivity of a transcription factor that controls the unfolded pro-tein response Cell 87 391ndash404

Cyr D M X Lu and M G Douglas 1992 Regulation of Hsp70function by a eukaryotic DnaJ homolog J Biol Chem 26720927ndash20931

Dancourt J and C Barlowe 2010 Protein sorting receptors inthe early secretory pathway Annu Rev Biochem 79 777ndash802

Dascher C R Ossig D Gallwitz and H D Schmitt1991 Identi1047297cation and structure of four yeast genes (SLY)that are able to suppress the functional loss of YPT1 a memberof the RAS superfamily Mol Cell Biol 11 872ndash885

drsquoEnfert C L J Wuestehube T Lila and R Schekman1991 Sec12p-dependent membrane binding of the smallGTP-binding protein Sar1p promotes formation of transport

vesicles from the ER J Cell Biol 114 663ndash670Denic V E M Quan and J S Weissman 2006 A luminal

surveillance complex that selects misfolded glycoproteins for

ER-associated degradation Cell 126 349ndash359




Deshaies R J and R Schekman 1987 A yeast mutant defectiveat an early stage in import of secretory protein precursors intothe endoplasmic reticulum J Cell Biol 105 633ndash645

Deshaies R J B D Koch M Werner-Washburne E A Craig andR Schekman 1988 A subfamily of stress proteins facilitatestranslocation of secretory and mitochondrial precursor polypep-tides Nature 332 800ndash805

Deshaies R J S L Sanders D A Feldheim and R Schekman1991 Assembly of yeast Sec proteins involved in translocationinto the endoplasmic reticulum into a membrane-bound multi-

subunit complex Nature 349 806ndash

808Doering T L and R Schekman 1996 GPI anchor attachment is

required for Gas1p transport from the endoplasmic reticulum inCOP II vesicles EMBO J 15 182ndash191

Duden R M Hosobuchi S Hamamoto M Winey B Byers et al1994 Yeast beta- and betarsquo-coat proteins (COP) Two coatomersubunits essential for endoplasmic reticulum-to-Golgi proteintraf 1047297c J Biol Chem 269 24486ndash24495

Duden R L Kajikawa L Wuestehube and R Schekman1998 epsilon-COP is a structural component of coatomer thatfunctions to stabilize alpha-COP EMBO J 17 985ndash995

Eisenhaber B G Schneider M Wildpaner and F Eisenhaber2004 A sensitive predictor for potential GPI lipid modi1047297cationsites in fungal protein sequences and its application to genome- wide studies for Aspergillus nidulans Candida albicans Neuros-

pora crassa Saccharomyces cerevisiae and Schizosaccharomycespombe J Mol Biol 337 243ndash253

Emr S B S Glick A D Linstedt J Lippincott-Schwartz A Luiniet al 2009 Journeys through the Golgindashtaking stock in a newera J Cell Biol 187 449ndash453

Espenshade P R E Gimeno E Holzmacher P Teung and C AKaiser 1995 Yeast SEC16 gene encodes a multidomain vesiclecoat protein that interacts with Sec23p J Cell Biol 131 311ndash324

Faini M S Prinz R Beck M Schorb J D Riches et al 2012 Thestructures of COPI-coated vesicles reveal alternate coatomer con-formations and interactions Science 336 1451ndash1454

Fan C Y S Lee H Y Ren and D M Cyr 2004 Exchangeablechaperone modules contribute to speci1047297cation of type I and typeII Hsp40 cellular function Mol Biol Cell 15 761ndash773

Fang H S Panzner C Mullins E Hartmann and N Green

1996 The homologue of mammalian SPC12 is important foref 1047297cient signal peptidase activity in Saccharomyces cerevisiae JBiol Chem 271 16460ndash16465

Fang H C Mullins and N Green 1997 In addition to SEC11a newly identi1047297ed gene SPC3 is essential for signal peptidaseactivity in the yeast endoplasmic reticulum J Biol Chem 27213152ndash13158

Farhan H M Weiss K Tani R J Kaufman and H-P Hauri2008 Adaptation of endoplasmic reticulum exit sites to acuteand chronic increases in cargo load EMBO J 27 2043ndash2054

Farquhar R N Honey S J Murant P Bossier L Schultz et al1991 Protein disul1047297de isomerase is essential for viability inSaccharomyces cerevisiae Gene 108 81ndash89

Fasshauer D R B Sutton A T Brunger and R Jahn1998 Conserved structural features of the synaptic fusion

complex SNARE proteins reclassi1047297

ed as Q- and R-SNAREsProc Natl Acad Sci USA 95 15781ndash15786Fath S J D Mancias X Bi and J Goldberg 2007 Structure

and organization of coat proteins in the COPII cage Cell 1291325ndash1336

Favaloro V M Spasic B Schwappach and B Dobberstein2008 Distinct targeting pathways for the membrane insertionof tail-anchored (TA) proteins J Cell Sci 121 1832ndash1840

Feldheim D J Rothblatt and R Schekman 1992 Topology andfunctional domains of Sec63p an endoplasmic reticulum mem-brane protein required for secretory protein translocation MolCell Biol 12 3288ndash3296

Fiedler K M Veit M Stamnes and J Rothman 1996 Bimodalinteraction of coatomer with the p24 family of putative cargoreceptors Science 273 1396ndash1399

Fraering P I Imhof U Meyer J M Strub A van Dorsselaer et al2001 The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p Gpi8p and Gpi16p Mol Biol Cell 123295ndash3306

Franzusoff A K Redding J Crosby R S Fuller and R Schekman1991 Localization of components involved in protein transportand processing through the yeast Golgi apparatus J Cell Biol

112 27ndash

37Furgason M L C MacDonald S G Shanks S P Ryder N J

Bryant et al 2009 The N-terminal peptide of the syntaxinTlg2p modulates binding of its closed conformation to Vps45pProc Natl Acad Sci USA 106 14303ndash14308

Futai E S Hamamoto L Orci and R Schekman 2004 GTPGDP exchange by Sec12p enables COPII vesicle bud formationon synthetic liposomes EMBO J 23 4146ndash4155

Gallwitz D C Donath and C Sander 1983 A yeast gene en-coding a protein homologous to the human c-hasbas proto-oncogene product Nature 306 704ndash707

Gardner B M and P Walter 2011 Unfolded proteins are Ire1-activating ligands that directly induce the unfolded proteinresponse Science 333 1891ndash1894

Gauss R K Kanehara P Carvalho D T Ng and M Aebi

2011 A complex of Pdi1p and the mannosidase Htm1p ini-tiates clearance of unfolded glycoproteins from the endoplasmicreticulum Mol Cell 42 782ndash793

Gaynor E C and S D Emr 1997 COPI-independent anterogradetransport cargo-selective ER to Golgi protein transport in yeastCOPI mutants J Cell Biol 136 789ndash802

Gentzsch M and W Tanner 1996 The PMT gene family proteinO-glycosylation in Saccharomyces cerevisiae is vital EMBO J15 5752ndash5759

Ghaemmaghami S W Huh K Bower R Howson A Belle et al2003 Global analysis of protein expression in yeast Nature425 737ndash741

Gillingham A K A C Pfeifer and S Munro 2002 CASP thealternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor is a Golgi membrane

protein related to giantin Mol Biol Cell 13 3761ndash

3774Gillingham A K A H Y Tong C Boone and S Munro

2004 The GTPase Arf1p and the ER to Golgi cargo receptorErv14p cooperate to recruit the golgin Rud3p to the cis-Golgi JCell Biol 167 281ndash292

Gilstring C F M Melin-Larsson and P O Ljungdahl1999 Shr3p mediates speci1047297c COPII coatomer-cargo interac-tions required for the packaging of amino acid permeases intoER-derived transport vesicles Mol Biol Cell 10 3549ndash3565

Gimeno R E P Espenshade and C A Kaiser 1996 COPII coatsubunit interactions Sec24p and Sec23p bind to adjacent re-gions of Sec16p Mol Biol Cell 7 1815ndash1823

Goder V and A Melero 2011 Protein O-mannosyltransferasesparticipate in ER protein quality control J Cell Sci 124 144ndash153

Goldberg J 1999 Structural and functional analysis of the ARF1-

ARFGAP complex reveals a role for coatomer in GTP hydrolysisCell 96 893ndash902Goldberg J 2000 Decoding of sorting signals by coatomer through

a GTPase switch in the COPI coat complex Cell 100 671ndash679Graham T R and C G Burd 2011 Coordination of Golgi functions

by phosphatidylinositol 4-kinases Trends Cell Biol 21 113ndash121Graham T R and S D Emr 1991 Compartmental organization

of Golgi-speci1047297c protein modi1047297cation and vacuolar protein sort-ing events de1047297ned in a yeast sec18 (NSF) mutant J Cell Biol114 207ndash218

Green N H Fang and P Walter 1992 Mutants in three novelcomplementation groups inhibit membrane protein insertion




into and soluble protein translocation across the endoplasmicreticulum membrane of Saccharomyces cerevisiae J Cell Biol116 597ndash604

Gross E C S Sevier N Heldman E Vitu M Bentzur et al2006 Generating disul1047297des enzymatically reaction productsand electron acceptors of the endoplasmic reticulum thiol oxi-dase Ero1p Proc Natl Acad Sci USA 103 299ndash304

Hale S J S C Lovell J de Keyzer and C J Stirling2010 Interactions between Kar2p and its nucleotide exchangefactors Sil1p and Lhs1p are mechanistically distinct J Biol

Chem 285 21600ndash

21606Hann B C and P Walter 1991 The signal recognition particle in

S cerevisiae Cell 67 131ndash144Hann B C C J Stirling and P Walter 1992 SEC65 gene prod-

uct is a subunit of the yeast signal recognition particle requiredfor its integrity Nature 356 532ndash533

Hansen W P D Garcia and P Walter 1986 In vitro proteintranslocation across the yeast endoplasmic reticulum ATP-dependent posttranslational translocation of the prepro-alpha-factor Cell 45 397ndash406

Hanson P I R Roth H Morisaki R Jahn and J E Heuser1997 Structure and conformational changes in NSF and itsmembrane receptor complexes visualized by quick-freezedeep-etch electron microscopy Cell 90 523ndash535

Hardwick K G and H R Pelham 1992 SED5 encodes a 39-kD

integral membrane protein required for vesicular transport be-tween the ER and the Golgi complex J Cell Biol 119 513ndash521

Harter C and F Wieland 1998 A single binding site for dilysineretrieval motifs and p23 within the gamma subunit of coatomerProc Natl Acad Sci USA 95 11649ndash11654

Harter C J Pavel F Coccia E Draken S Wegehingel et al1996 Nonclathrin coat protein gamma a subunit of coatomerbinds to the cytoplasmic dilysine motif of membrane proteins of theearly secretory pathway Proc Natl Acad Sci USA 93 1902ndash1906

Hartl F U 1996 Molecular chaperones in cellular protein fold-ing Nature 381 571ndash579

Harty C S Strahl and K Romisch 2001 O-mannosylation pro-tects mutant alpha-factor precursor from endoplasmic reticu-lum-associated degradation Mol Biol Cell 12 1093ndash1101

Hatahet F and L W Ruddock 2009 Protein disul1047297de isomerase

a critical evaluation of its function in disul1047297de bond formation Antioxid Redox Signal 11 2807ndash2850

Helenius A and M Aebi 2004 Roles of N-linked glycans in theendoplasmic reticulum Annu Rev Biochem 73 1019ndash1049

Herzig Y H J Sharpe Y Elbaz S Munro and M Schuldiner2012 A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14 PLoS Biol 10 e1001329

Hirayama H M Fujita T Yoko-o and Y Jigami 2008 O-mannosylation is required for degradation of the endoplasmicreticulum-associated degradation substrate Gas1p via the ubiqui-tinproteasome pathway in Saccharomyces cerevisiae J Biochem143 555ndash567

Hoppins S S R Collins A Cassidy-Stone E Hummel R MDevay et al 2011 A mitochondrial-focused genetic interaction

map reveals a scaffold-like complex required for inner mem-brane organization in mitochondria J Cell Biol 195 323ndash340Hosobuchi M T Kreis and R Schekman 1992 SEC21 is a gene

required for ER to Golgi protein transport that encodes a subunitof a yeast coatomer Nature 360 603ndash605

Hsia K C and A Hoelz 2010 Crystal structure of alpha-COP incomplex with epsilon-COP provides insight into the architectureof the COPI vesicular coat Proc Natl Acad Sci USA 10711271ndash11276

Huh W J Falvo L Gerke A Carroll R Howson et al2003 Global analysis of protein localization in budding yeastNature 425 686ndash691

Jakob C A P Burda J Roth and M Aebi 1998 Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomy-ces cerevisiae is determined by a speci1047297c oligosaccharide struc-ture J Cell Biol 142 1223ndash1233

Jakob C A D Bodmer U Spirig P Battig A Marcil et al2001 Htm1p a mannosidase-like protein is involved in glyco-protein degradation in yeast EMBO Rep 2 423ndash430

Jang S B Y G Kim Y S Cho P G Suh K H Kim et al2002 Crystal structure of SEDL and its implications for a ge-netic disease spondyloepiphyseal dysplasia tarda J Biol Chem

277 49863ndash

49869Jin L K B Pahuja K E Wickliffe A Gorur C Baumgartel et al

2012 Ubiquitin-dependent regulation of COPII coat size andfunction Nature 482 495ndash500

Jones E W 1977 Proteinase mutants of Saccharomyces cerevi-siae Genetics 85 23ndash33

Jones S C Newman F Liu and N Segev 2000 The TRAPPcomplex is a nucleotide exchanger for Ypt1 and Ypt3132Mol Biol Cell 11 4403ndash4411

Jonikas M S Collins V Denic E Oh E Quan et al2009 Comprehensive characterization of genes required for pro-tein folding in the endoplasmic reticulum Science 323 1693ndash1697

Jungnickel B T A Rapoport and E Hartmann 1994 Proteintranslocation common themes from bacteria to man FEBS Lett346 73ndash77

Kaiser C and R Schekman 1990 Distinct sets of SEC genesgovern transport vesicle formation and fusion early in the secre-tory pathway Cell 61 723ndash733

Kaiser C R E Gimeno and D A Shaywitz 1997 Protein secretionmembrane biogenesis and endocytosis pp 91ndash227 in The Molec-ular and Cellular Biology of the Yeast Saccharomyces cerevisiaeCold Spring Harbor Laboratory Press Cold Spring Harbor NY

Kamena F and A Spang 2004 Tip20p prohibits back-fusion of COPII vesicles with the endoplasmic reticulum Science 304286ndash289

Kelleher D J and R Gilmore 2006 An evolving view of the eu-karyotic oligosaccharyltransferase Glycobiology 16 47R ndash62R

Kim Y S Raunser C Munger J Wagner Y Song et al2006 The architecture of the multisubunit TRAPP I complexsuggests a model for vesicle tethering Cell 127 817ndash830

Kimura T Y Hosoda Y Sato Y Kitamura T Ikeda et al2005 Interactions among yeast protein-disul1047297de isomeraseproteins and endoplasmic reticulum chaperone proteins in1047298u-ence their activities J Biol Chem 280 31438ndash31441

Kincaid M and A Cooper 2007 Misfolded proteins traf 1047297c fromthe endoplasmic reticulum (ER) due to ER export signals MolBiol Cell 18 455ndash463

Kloepper T H C N Kienle and D Fasshauer 2007 An elaborateclassi1047297cation of SNARE proteins sheds light on the conservationof the eukaryotic endomembrane system Mol Biol Cell 183463ndash3471

Kota J C Gilstring and P Ljungdahl 2007 Membrane chaper-one Shr3 assists in folding amino acid permeases preventingprecocious ERAD J Cell Biol 176 617ndash628

Kraynack B A A Chan E Rosenthal M Essid B Umansky et al

2005 Dsl1p Tip20p and the novel Dsl3(Sec39) protein arerequired for the stability of the Qt-SNARE complex at the en-doplasmic reticulum in yeast Mol Biol Cell 16 3963ndash3977

Kuehn M J R Schekman and P O Ljungdahl 1996 Aminoacid permeases require COPII components and the ER residentmembrane protein Shr3p for packaging into transport vesiclesin vitro J Cell Biol 135 585ndash595

Kung L F S Pagant E Futai J G D rsquo Arcangelo R Buchananet al 2012 Sec24p and Sec16p cooperate to regulate theGTP cycle of the COPII coat EMBO J 31 1014ndash1027

Kurihara T S Hamamoto R E Gimeno C A Kaiser R Schekmanet al 2000 Sec24p and Iss1p function interchangeably in




transport vesicle formation from the endoplasmic reticulumin Saccharomyces cerevisiae Mol Biol Cell 11 983ndash998

Laboissiere M C S L Sturley and R T Raines 1995 The es-sential function of protein-disul1047297de isomerase is to unscramblenon-native disul1047297de bonds J Biol Chem 270 28006ndash28009

Lee C and J Goldberg 2010 Structure of coatomer cage pro-teins and the relationship among COPI COPII and clathrin vesicle coats Cell 142 123ndash132

Lee M C S E A Miller J Goldberg L Orci and R Schekman2004 Bi-directional protein transport between the ER and

Golgi Annu Rev Cell Dev Biol 20 87ndash

123Lee M C S L Orci S Hamamoto E Futai M Ravazzola et al

2005 Sar1p N-terminal helix initiates membrane curvatureand completes the 1047297ssion of a COPII vesicle Cell 122 605ndash617

Lees J A C K Yip T Walz and F M Hughson 2010 Molecularorganization of the COG vesicle tethering complex Nat StructMol Biol 17 1292ndash1297

Leidich S D D A Drapp and P Orlean 1994 A conditionally lethal yeast mutant blocked at the 1047297rst step in glycosyl phospha-tidylinositol anchor synthesis J Biol Chem 269 10193ndash10196

Letourneur F E Gaynor S Hennecke C Demolliere R Dudenet al 1994 Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum Cell 79 1199ndash1207

Li J X Qian and B Sha 2003 The crystal structure of the yeastHsp40 Ydj1 complexed with its peptide substrate Structure 111475ndash1483

Ljungdahl P O C J Gimeno C A Styles and G R Fink1992 SHR3 a novel component of the secretory pathway spe-ci1047297cally required for localization of amino acid permeases inyeast Cell 71 463ndash478

Lord C D Bhandari S Menon M Ghassemian D Nycz et al2011 Sequential interactions with Sec23 control the directionof vesicle traf 1047297c Nature 473 181ndash186

Losev E C A Reinke J Jellen D E Strongin B J Bevis et al2006 Golgi maturation visualized in living yeast Nature 4411002ndash1006

Luo R and P A Randazzo 2008 Kinetic analysis of Arf GAP1indicates a regulatory role for coatomer J Biol Chem 283

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1997a The Ktr1p Ktr3p and Kre2pMnt1p mannosyltrans-ferases participate in the elaboration of yeast O- and N-linkedcarbohydrate chains J Biol Chem 272 15527ndash15531

Lussier M A M Sdicu E Winnett D H Vo J Sheraton et al1997b Completion of the Saccharomyces cerevisiae genomesequence allows identi1047297cation of KTR5 KTR6 and KTR7 andde1047297nition of the nine-membered KRE2MNT1 mannosyltrans-ferase gene family in this organism Yeast 13 267ndash274

Malkus P F Jiang and R Schekman 2002 Concentrative sort-ing of secretory cargo proteins into COPII-coated vesicles J CellBiol 159 915ndash921

Mancias J D and J Goldberg 2007 The transport signal onSec22 for packaging into COPII-coated vesicles is a conforma-

tional epitope Mol Cell 26 403ndash

414Matlack K E B Misselwitz K Plath and T A Rapoport1999 BiP acts as a molecular ratchet during posttranslationaltransport of prepro-alpha factor across the ER membrane Cell97 553ndash564

Matsuoka K Y Morimitsu K Uchida and R Schekman1998a Coat assembly directs v-SNARE concentration into syn-thetic COPII vesicles Mol Cell 2 703ndash708

Matsuoka K L Orci M Amherdt S Y Bednarek S Hamamotoet al 1998b COPII-coated vesicle formation reconstituted with puri1047297ed coat proteins and chemically de1047297ned liposomesCell 93 263ndash275

Matsuoka K R Schekman L Orci and J E Heuser2001 Surface structure of the COPII-coated vesicle Proc Natl Acad Sci USA 98 13705ndash13709

Matsuura-Tokita K M Takeuchi A Ichihara K Mikuriya and ANakano 2006 Live imaging of yeast Golgi cisternal matura-tion Nature 441 1007ndash1010

McNew J F Parlati R Fukuda R Johnston K Paz et al2000 Compartmental speci1047297city of cellular membrane fusionencoded in SNARE proteins Nature 407 153ndash159

Meyer H A and E Hartmann 1997 The yeast SPC2223 homo-

log Spc3p is essential for signal peptidase activity J Biol Chem272 13159ndash13164

Mezzacasa A and A Helenius 2002 The transitional ER de1047297nesa boundary for quality control in the secretion of tsO45 VSV glycoprotein Traf 1047297c 3 833ndash849

Michelsen K V Schmid J Metz K Heusser U Liebel et al2007 Novel cargo-binding site in the beta and delta subunitsof coatomer J Cell Biol 179 209ndash217

Miller E B Antonny S Hamamoto and R Schekman2002 Cargo selection into COPII vesicles is driven by theSec24p subunit EMBO J 21 6105ndash6113

Miller E A T H Beilharz P N Malkus M C S Lee S Hamamotoet al 2003 Multiple cargo binding sites on the COPII sub-unit Sec24p ensure capture of diverse membrane proteins intotransport vesicles Cell 114 497ndash509

Miller E A Y Liu C Barlowe and R Schekman 2005 ER-Golgitransport defects are associated with mutations in the Sed5p-binding domain of the COPII coat subunit Sec24p Mol BiolCell 16 3719ndash3726

Miller V J and D Ungar 2012 RersquoCOGrsquonition at the Golgi Traf-1047297c 13 891ndash897

Misselwitz B O Staeck K E Matlack and T A Rapoport1999 Interaction of BiP with the J-domain of the Sec63p com-ponent of the endoplasmic reticulum protein translocation com-plex J Biol Chem 274 20110ndash20115

Mori K W Ma M J Gething and J Sambrook 1993 A trans-membrane protein with a cdc2+CDC28-related kinase activity is required for signaling from the ER to the nucleus Cell 74743ndash756

Mossessova E L C Bickford and J Goldberg 2003 SNARE

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ing of the environment of a translocating secretory protein dur-ing translocation through the ER membrane EMBO J 133973ndash3982

Muniz M C Nuoffer H Hauri and H Riezman 2000 TheEmp24 complex recruits a speci1047297c cargo molecule into endo-plasmic reticulum-derived vesicles J Cell Biol 148 925ndash930

Muniz M P Morsomme and H Riezman 2001 Protein sortingupon exit from the endoplasmic reticulum Cell 104 313ndash320

Musch A M Wiedmann and T A Rapoport 1992 Yeast Secproteins interact with polypeptides traversing the endoplasmicreticulum membrane Cell 69 343ndash352

Nakajima H A Hirata Y Ogawa T Yonehara K Yoda et al1991 A cytoskeleton-related gene uso1 is required for intra-

cellular protein transport in Saccharomyces cerevisiae J CellBiol 113 245ndash260Nakano A and M Muramatsu 1989 A novel GTP-binding pro-

tein Sar1p is involved in transport from the endoplasmic re-ticulum to the Golgi apparatus J Cell Biol 109 2677ndash2691

Nakano A D Brada and R Schekman 1988 A membrane gly-coprotein Sec12p required for protein transport from the en-doplasmic reticulum to the Golgi apparatus in yeast J Cell Biol107 851ndash863

Neupert W F U Hartl E A Craig and N Pfanner 1990 Howdo polypeptides cross the mitochondrial membranes Cell 63447ndash450




Newman A P and S Ferro-Novick 1987 Characterization of new mutants in the early part of the yeast secretory pathway isolated by a [3H]mannose suicide selection J Cell Biol 1051587ndash1594

Newman A P J Shim and S Ferro-Novick 1990 BET1 BOS1and SEC22 are members of a group of interacting yeast genesrequired for transport from the endoplasmic reticulum to theGolgi complex Mol Cell Biol 10 3405ndash3414

Ng D T J D Brown and P Walter 1996 Signal sequencesspecify the targeting route to the endoplasmic reticulum mem-

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278Nishikawa S and T Endo 1997 The yeast JEM1p is a DnaJ-like

protein of the endoplasmic reticulum membrane required fornuclear fusion J Biol Chem 272 12889ndash12892

Nishikawa S and A Nakano 1993 Identi1047297cation of a gene re-quired for membrane protein retention in the early secretory pathway Proc Natl Acad Sci USA 90 8179ndash8183

Nishikawa S I S W Fewell Y Kato J L Brodsky and T Endo2001 Molecular chaperones in the yeast endoplasmic reticu-lum maintain the solubility of proteins for retrotranslocationand degradation J Cell Biol 153 1061ndash1070

Norgaard P and J R Winther 2001 Mutation of yeast Eug1pCXXS active sites to CXXC results in a dramatic increase in pro-tein disulphide isomerase activity Biochem J 358 269ndash274

Norgaard P V Westphal C Tachibana L Alsoe B Holst et al

2001 Functional differences in yeast protein disul1047297de iso-merases J Cell Biol 152 553ndash562

Novick P and R Schekman 1979 Secretion and cell-surfacegrowth are blocked in a temperature-sensitive mutant of Saccha-romyces cerevisiae Proc Natl Acad Sci USA 76 1858ndash1862

Novick P C Field and R Schekman 1980 Identi1047297cation of 23complementation groups required for post-translational eventsin the yeast secretory pathway Cell 21 205ndash215

Novick P S Ferro and R Schekman 1981 Order of events inthe yeast secretory pathway Cell 25 461ndash469

Nuoffer C A Horvath and H Riezman 1993 Analysis of thesequence requirements for glycosylphosphatidylinositol anchor-ing of Saccharomyces cerevisiae Gas1 protein J Biol Chem268 10558ndash10563

Ogg S C W P Barz and P Walter 1998 A functional GTPase

domain but not its transmembrane domain is required forfunction of the SRP receptor beta-subunit J Cell Biol 142341ndash354

Okamoto M K Kurokawa K Matsuura-Tokita C Saito R Hirataet al 2012 High-curvature domains of the ER are importantfor the organization of ER exit sites in Saccharomyces cerevisiaeJ Cell Sci 125(Pt 14) 3412ndash3420

Orlean P 1990 Dolichol phosphate mannose synthase is re-quired in vivo for glycosyl phosphatidylinositol membrane an-choring O mannosylation and N glycosylation of protein inSaccharomyces cerevisiae Mol Cell Biol 10 5796ndash5805

Orlean P and A Menon 2007 Thematic review series lipidposttranslational modi1047297cations GPI anchoring of protein inyeast and mammalian cells or how we learned to stop worry-ing and love glycophospholipids J Lipid Res 48 993ndash1011

Ossig R C Dascher H H Trepte H D Schmitt and D Gallwitz1991 The yeast SLY gene products suppressors of defects inthe essential GTP-binding Ypt1 protein may act in endoplasmicreticulum-to-Golgi transport Mol Cell Biol 11 2980ndash2993

Pagant S L Kung M Dorrington M C S Lee and E A Miller2007 Inhibiting endoplasmic reticulum (ER)-associated degrada-tion of misfolded Yor1p does not permit ER export despite thepresence of a diacidic sorting signal Mol Biol Cell 18 3398ndash3413

Panzner S L Dreier E Hartmann S Kostka and T A Rapoport1995 Posttranslational protein transport in yeast reconsti-tuted with a puri1047297ed complex of Sec proteins and Kar2p Cell81 561ndash570

Parlati F J McNew R Fukuda R Miller T Sollner et al2000 Topological restriction of SNARE-dependent membranefusion Nature 407 194ndash198

Peng R and D Gallwitz 2002 Sly1 protein bound to Golgi syn-taxin Sed5p allows assembly and contributes to speci1047297city of SNARE fusion complexes J Cell Biol 157 645ndash655

Peng R A De Antoni and D Gallwitz 2000 Evidence foroverlapping and distinct functions in protein transport of coat protein Sec24p family members J Biol Chem 27511521ndash11528

Peyroche A S Paris and C Jackson 1996 Nucleotide exchangeon ARF mediated by yeast Gea1 protein Nature 384 479ndash481

Pincus D M W Chevalier T Aragon E van Anken S E Vidalet al 2010 BiP binding to the ER-stress sensor Ire1 tunes thehomeostatic behavior of the unfolded protein response PLoSBiol 8 e1000415

Pittet M and A Conzelmann 2007 Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae BiochimBiophys Acta 1771 405ndash420

Plath K W Mothes B M Wilkinson C J Stirling and T ARapoport 1998 Signal sequence recognition in posttransla-tional protein transport across the yeast ER membrane Cell94 795ndash807

Poon P D Cassel A Spang M Rotman E Pick et al1999 Retrograde transport from the yeast Golgi is mediated

by two ARF GAP proteins with overlapping function EMBO J18 555ndash564

Poon P P X Wang M Rotman I Huber E Cukierman et al1996 Saccharomyces cerevisiae Gcs1 is an ADP-ribosylationfactor GTPase-activating protein Proc Natl Acad Sci USA 93 10074ndash10077

Powers J and C Barlowe 1998 Transport of axl2p depends onerv14p an ER-vesicle protein related to the Drosophila corni-chon gene product J Cell Biol 142 1209ndash1222

Powers J and C Barlowe 2002 Erv14p directs a transmembranesecretory protein into COPII-coated transport vesicles Mol BiolCell 13 880ndash891

Preuss D J Mulholland A Franzusoff N Segev and D Botstein1992 Characterization of the Saccharomyces Golgi complexthrough the cell cycle by immunoelectron microscopy Mol Biol

Cell 3 789ndash

803Pucadyil T J and S L Schmid 2009 Conserved functions of

membrane active GTPases in coated vesicle formation Science325 1217ndash1220

Rapoport T A 2007 Protein translocation across the eukaryoticendoplasmic reticulum and bacterial plasma membranes Na-ture 450 663ndash669

Rein U U Andag R Duden H D Schmitt and A Spang2002 ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat J Cell Biol 157 395ndash404

Ren Y C K Yip A Tripathi D Huie P D Jeffrey et al 2009 A structure-based mechanism for vesicle capture by the multisu-bunit tethering complex Dsl1 Cell 139 1119ndash1129

Rexach M F and R W Schekman 1991 Distinct biochemicalrequirements for the budding targeting and fusion of ER-

derived transport vesicles J Cell Biol 114 219ndash

229Roberg K J M Crotwell P Espenshade R Gimeno and C AKaiser 1999 LST1 is a SEC24 homologue used for selectiveexport of the plasma membrane ATPase from the endoplasmicreticulum J Cell Biol 145 659ndash672

Rose M D L M Misra and J P Vogel 1989 KAR2 a karyogamy gene is the yeast homolog of the mammalian BiPGRP78 geneCell 57 1211ndash1221

Rossanese O W J Soderholm B J Bevis I B Sears J O rsquoConnoret al 1999 Golgi structure correlates with transitional endo-plasmic reticulum organization in Pichia pastoris and Saccharo-myces cerevisiae J Cell Biol 145 69ndash81




Rossi G K Kolstad S Stone F Palluault and S Ferro-Novick1995 BET3 encodes a novel hydrophilic protein that acts inconjunction with yeast SNAREs Mol Biol Cell 6 1769ndash1780

Rothblatt J A and D I Meyer 1986 Secretion in yeast recon-stitution of the translocation and glycosylation of alpha-factorand invertase in a homologous cell-free system Cell 44 619ndash628

Rothblatt J A R J Deshaies S L Sanders G Daum and RSchekman 1989 Multiple genes are required for proper inser-tion of secretory proteins into the endoplasmic reticulum in

yeast J Cell Biol 109 2641ndash

2652Rothman J E 1994 Mechanisms of intracellular protein trans-

port Nature 372 55ndash63Rothman J H I Howald and T H Stevens 1989 Characterization

of genes required for protein sorting and vacuolar function inthe yeast Saccharomyces cerevisiae EMBO J 8 2057ndash2065

Ruohola H A K Kabcenell and S Ferro-Novick 1988 Re-constitution of protein transport from the endoplasmic re-ticulum to the Golgi complex in yeast the acceptor Golgicompartment is defective in the sec23 mutant J Cell Biol107 1465ndash1476

Sacher M Y Jiang J Barrowman A Scarpa J Burston et al1998 TRAPP a highly conserved novel complex on the cis-Golgi that mediates vesicle docking and fusion EMBO J 172494ndash2503

Sacher M J Barrowman W Wang J Horecka Y Zhang et al2001 TRAPP I implicated in the speci1047297city of tethering inER-to-Golgi transport Mol Cell 7 433ndash442

Salama N R J S Chuang and R W Schekman 1997 Sec31encodes an essential component of the COPII coat required fortransport vesicle budding from the endoplasmic reticulum MolBiol Cell 8 205ndash217

Sanders S K Whit1047297eld J Vogel M Rose and R Schekman1992 Sec61p and BiP directly facilitate polypeptide transloca-tion into the ER Cell 69 353ndash365

Sandmann T J M Herrmann J Dengjel H Schwarz and ASpang 2003 Suppression of coatomer mutants by a new pro-tein family with COPI and COPII binding motifs in Saccharomy-ces cerevisiae Mol Biol Cell 14 3097ndash3113

Sapperstein S V Lupashin H Schmitt and M Waters1996 Assembly of the ER to Golgi SNARE complex requiresUso1p J Cell Biol 132 755ndash767

Sata M J G Donaldson J Moss and M Vaughan1998 Brefeldin A-inhibited guanine nucleotide-exchange ac-tivity of Sec7 domain from yeast Sec7 with yeast and mamma-lian ADP ribosylation factors Proc Natl Acad Sci USA 954204ndash4208

Sata M J Moss and M Vaughan 1999 Structural basis for theinhibitory effect of brefeldin A on guanine nucleotide-exchangeproteins for ADP-ribosylation factors Proc Natl Acad Sci USA

96 2752ndash2757Sato K and A Nakano 2002 Emp47p and its close homolog

Emp46p have a tyrosine-containing endoplasmic reticulum exitsignal and function in glycoprotein secretion in Saccharomycescerevisiae Mol Biol Cell 13 2518ndash2532

Sato K and A Nakano 2005 Dissection of COPII subunit-cargoassembly and disassembly kinetics during Sar1p-GTP hydrolysisNat Struct Mol Biol 12 167ndash174

Sato K S Nishikawa and A Nakano 1995 Membrane proteinretrieval from the Golgi apparatus to the endoplasmic reticulum(ER) characterization of the RER1 gene product as a componentinvolved in ER localization of Sec12p Mol Biol Cell 6 1459ndash1477

Sato M K Sato and A Nakano 1996 Endoplasmic reticulumlocalization of Sec12p is achieved by two mechanisms Rer1p-

dependent retrieval that requires the transmembrane domain

and Rer1p-independent retention that involves the cytoplasmicdomain J Cell Biol 134 279ndash293

Sato K M Sato and A Nakano 1997 Rer1p as common ma-chinery for the endoplasmic reticulum localization of membraneproteins Proc Natl Acad Sci USA 94 9693ndash9698

Sato K M Sato and A Nakano 2001 Rer1p a retrieval receptorfor endoplasmic reticulum membrane proteins is dynamically localized to the Golgi apparatus by coatomer J Cell Biol 152935ndash944

Sato K M Sato and A Nakano 2003 Rer1p a retrieval receptor

for ER membrane proteins recognizes transmembrane domainsin multiple modes Mol Biol Cell 14 3605ndash3616

Schaaf G E A Ortlund K R Tyeryar C J Mousley K E Ile et al2008 Functional anatomy of phospholipid binding and regu-lation of phosphoinositide homeostasis by proteins of the sec14superfamily Mol Cell 29 191ndash206

Scheel A and H Pelham 1998 Identi1047297cation of amino acids inthe binding pocket of the human KDEL receptor J Biol Chem273 2467ndash2472

Schekman R and P Novick 2004 23 genes 23 years later Cell116 S13ndashS15

Schindler C and A Spang 2007 Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18pNSF Mol Biol Cell18 2852ndash2863

Schindler C F Rodriguez P P Poon R A Singer G C Johnston

et al 2009 The GAP domain and the SNARE coatomer andcargo interaction region of the ArfGAP23 Glo3 are suf 1047297cient forGlo3 function Traf 1047297c 10 1362ndash1375

Schlenstedt G S Harris B Risse R Lill and P A Silver 1995 A yeast DnaJ homologue Scj1p can function in the endoplasmicreticulum with BiPKar2p via a conserved domain that speci1047297esinteractions with Hsp70s J Cell Biol 129 979ndash988

Schmitt H D M Puzicha and D Gallwitz 1988 Study of a tem-perature-sensitive mutant of the ras-related YPT1 gene productin yeast suggests a role in the regulation of intracellular calciumCell 53 635ndash647

Schmitz K R J Liu S Li T G Setty C S Wood et al2008 Golgi localization of glycosyltransferases requiresa Vps74p oligomer Dev Cell 14 523ndash534

Schuldiner M S Collins N Thompson V Denic A Bhamidipati

et al 2005 Exploration of the function and organization of theyeast early secretory pathway through an epistatic miniarray pro1047297le Cell 123 507ndash519

Schuldiner M J Metz V Schmid V Denic M Rakwalska et al2008 The GET complex mediates insertion of tail-anchoredproteins into the ER membrane Cell 134 634ndash645

Schwarz F and M Aebi 2011 Mechanisms and principles of N-linked protein glycosylation Curr Opin Struct Biol 21 576ndash582

Scidmore M A H H Okamura and M D Rose 1993 Geneticinteractions between KAR2 and SEC63 encoding eukaryotichomologues of DnaK and DnaJ in the endoplasmic reticulumMol Biol Cell 4 1145ndash1159

Segev N J Mulholland and D Botstein 1988 The yeast GTP-binding YPT1 protein and a mammalian counterpart are associ-ated with the secretion machinery Cell 52 915ndash924

Semenza J K Hardwick N Dean and H Pelham 1990 ERD2a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway Cell 611349ndash1357

Sera1047297ni T L Orci M Amherdt M Brunner R A Kahn et al1991 ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles a novel role for a GTP-bind-ing protein Cell 67 239ndash253

Sevier C S H Qu N Heldman E Gross D Fass et al2007 Modulation of cellular disul1047297de-bond formation andthe ER redox environment by feedback regulation of Ero1 Cell129 333ndash344




Shahinian S and H Bussey 2000 beta-16-Glucan synthesis inSaccharomyces cerevisiae Mol Microbiol 35 477ndash489

Shao S and R S Hegde 2011 Membrane protein insertionat the endoplasmic reticulum Annu Rev Cell Dev Biol 2725ndash56

Sharpe H J T J Stevens and S Munro 2010 A comprehensivecomparison of transmembrane domains reveals organelle-speci1047297c properties Cell 142 158ndash169

Shaywitz D A P J Espenshade R E Gimeno and C A Kaiser1997 COPII subunit interactions in the assembly of the vesicle

coat J Biol Chem 272 25413ndash

25416Shestakova A E Suvorova O Pavliv G Khaidakova and V Lupashin

2007 Interaction of the conserved oligomeric Golgi complex with t-SNARE Syntaxin5aSed5 enhances intra-Golgi SNAREcomplex stability J Cell Biol 179 1179ndash1192

Shikano S and M Li 2003 Membrane receptor traf 1047297ckingevidence of proximal and distal zones conferred by two in-dependent endoplasmic reticulum localization signals ProcNatl Acad Sci USA 100 5783ndash5788

Shindiapina P and C Barlowe 2010 Requirements for transi-tional endoplasmic reticulum site structure and function inSaccharomyces cerevisiae Mol Biol Cell 21 1530ndash1545

Sidrauski C J S Cox and P Walter 1996 tRNA ligase is re-quired for regulated mRNA splicing in the unfolded proteinresponse Cell 87 405ndash413

Smith M H H L Ploegh and J S Weissman 2011 Road toruin targeting proteins for degradation in the endoplasmic re-ticulum Science 334 1086ndash1090

Sogaard M K Tani R R Ye S Geromanos P Tempst et al1994 A rab protein is required for the assembly of SNARE com-plexes in the docking of transport vesicles Cell 78 937ndash948

Spang A 2012 The DSL1 complex the smallest but not the leastCATCHR Traf 1047297c 13 908ndash913

Spang A and R Schekman 1998 Reconstitution of retrogradetransport from the Golgi to the ER in vitro J Cell Biol 143589ndash599

Spang A K Matsuoka S Hamamoto R Schekman and L Orci1998 Coatomer Arf1p and nucleotide are required to budcoat protein complex I-coated vesicles from large syntheticliposomes Proc Natl Acad Sci USA 95 11199ndash11204

Spang A J Herrmann S Hamamoto and R Schekman2001 The ADP ribosylation factor-nucleotide exchange factorsGea1p and Gea2p have overlapping but not redundant func-tions in retrograde transport from the Golgi to the endoplasmicreticulum Mol Biol Cell 12 1035ndash1045

Spang A Y Shiba and P A Randazzo 2010 Arf GAPs gate-keepers of vesicle generation FEBS Lett 584 2646ndash2651

Springer S A Spang and R Schekman 1999 A primer on ves-icle budding Cell 97 145ndash148

Stagg S M C Guumlrkan D M Fowler P LaPointe T R Foss et al2006 Structure of the Sec1331 COPII coat cage Nature 439234ndash238

Steel G J J Brownsword and C J Stirling 2002 Tail-anchoredprotein insertion into yeast ER requires a novel posttranslationalmechanism which is independent of the SEC machinery Bio-

chemistry 41 11914ndash

11920Steel G J D M Fullerton J R Tyson and C J Stirling2004 Coordinated activation of Hsp70 chaperones Science303 98ndash101

Stefanovic S and R Hegde 2007 Identi1047297cation of a targetingfactor for posttranslational membrane protein insertion into theER Cell 128 1147ndash1159

Stirling C J and E W Hewitt 1992 The S cerevisiae SEC65gene encodes a component of yeast signal recognition particle with homology to human SRP19 Nature 356 534ndash537

Stirling C J J Rothblatt M Hosobuchi R Deshaies and RSchekman 1992 Protein translocation mutants defective in

the insertion of integral membrane proteins into the endoplas-mic reticulum Mol Biol Cell 3 129ndash142

Strahl-Bolsinger S M Gentzsch and W Tanner 1999 Protein O-mannosylation Biochim Biophys Acta 1426 297ndash307

Strating J R and G J Martens 2009 The p24 family and se-lective transport processes at the ER-Golgi interface Biol Cell101 495ndash509

Sudhof T C and J E Rothman 2009 Membrane fusion grap-pling with SNARE and SM proteins Science 323 474ndash477

Supek F D T Madden S Hamamoto L Orci and R Schekman

2002 Sec16p potentiates the action of COPII proteins to budtransport vesicles J Cell Biol 158 1029ndash1038

Sutton R B D Fasshauer R Jahn and A T Brunger1998 Crystal structure of a SNARE complex involved in syn-aptic exocytosis at 24 A resolution Nature 395 347ndash353

Suvorova E S R Duden and V V Lupashin 2002 The Sec34Sec35p complex a Ypt1p effector required for retrograde intra-Golgi traf 1047297cking interacts with Golgi SNAREs and COPI vesiclecoat proteins J Cell Biol 157 631ndash643

Sweet D J and H R Pelham 1993 The TIP1 gene of Saccha-romyces cerevisiae encodes an 80 kDa cytoplasmic protein thatinteracts with the cytoplasmic domain of Sec20p EMBO J 122831ndash2840

Takeuchi M Y Kimata A Hirata M Oka and K Kohno2006 Saccharomyces cerevisiae Rot1p is an ER-localized mem-

brane protein that may function with BiPKar2p in protein fold-ing J Biochem 139 597ndash605

Takeuchi M Y Kimata and K Kohno 2008 Saccharomyces cer-evisiae Rot1 is an essential molecular chaperone in the endo-plasmic reticulum Mol Biol Cell 19 3514ndash3525

Thor F M Gautschi R Geiger and A Helenius 2009 Bulk 1047298owrevisited transport of a soluble protein in the secretory pathwayTraf 1047297c 10 1819ndash1830

Tong A H M Evangelista A B Parsons H Xu G D Bader et al2001 Systematic genetic analysis with ordered arrays of yeastdeletion mutants Science 294 2364ndash2368

Tong A H G Lesage G D Bader H Ding H Xu et al2004 Global mapping of the yeast genetic interaction networkScience 303 808ndash813

Travers K C Patil L Wodicka D Lockhart J Weissman et al

2000 Functional and genomic analyses reveal an essentialcoordination between the unfolded protein response andER-associated degradation Cell 101 249ndash258

Tripathi A Y Ren P D Jeffrey and F M Hughson2009 Structural characterization of Tip20p and Dsl1p subu-nits of the Dsl1p vesicle tethering complex Nat Struct MolBiol 16 114ndash123

Tu B P and J S Weissman 2002 The FAD- and O(2)-dependentreaction cycle of Ero1-mediated oxidative protein folding in theendoplasmic reticulum Mol Cell 10 983ndash994

Tu L W C Tai L Chen and D K Ban1047297eld 2008 Signal-mediated dynamic retention of glycosyltransferases in the GolgiScience 321 404ndash407

Udenfriend S and K Kodukula 1995 How glycosylphosphatidy-linositol-anchored membrane proteins are made Annu Rev Bi-

ochem 64 563ndash

591 Van den Berg B W M Clemons Jr I Collinson Y Modis EHartmann et al 2004 X-ray structure of a protein-conductingchannel Nature 427 36ndash44

VanRheenen S M X Cao S K Sapperstein E C Chiang V VLupashin et al 1999 Sec34p a protein required for vesicletethering to the yeast Golgi apparatus is in a complex withSec35p J Cell Biol 147 729ndash742

VanRheenen S M B A Reilly S J Chamberlain and M GWaters 2001 Dsl1p an essential protein required for mem-brane traf 1047297c at the endoplasmic reticulumGolgi interface inyeast Traf 1047297c 2 212ndash231




Vashist S W Kim W J Belden E D Spear C Barlowe et al2001 Distinct retrieval and retention mechanisms are requiredfor the quality control of endoplasmic reticulum protein foldingJ Cell Biol 155 355ndash368

Vembar S S and J L Brodsky 2008 One step at a time endo-plasmic reticulum-associated degradation Nat Rev Mol CellBiol 9 944ndash957

Vitu E E Gross H M Greenblatt C S Sevier C A Kaiser et al2008 Yeast Mpd1p reveals the structural diversity of the pro-tein disul1047297de isomerase family J Mol Biol 384 631ndash640

Walch-Solimena C and P Novick 1999 The yeast phosphatidy-linositol-4-OH kinase pik1 regulates secretion at the Golgi NatCell Biol 1 523ndash525

Walter P and D Ron 2011 The unfolded protein response fromstress pathway to homeostatic regulation Science 334 1081ndash1086

Wang C C and C L Tsou 1993 Protein disul1047297de isomerase isboth an enzyme and a chaperone FASEB J 7 1515ndash1517

Wang W M Sacher and S Ferro-Novick 2000 TRAPP stimu-lates guanine nucleotide exchange on Ypt1p J Cell Biol 151289ndash296

Waters M G T Sera1047297ni and J E Rothman 1991 lsquoCoatomerrsquoa cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles Nature 349 248ndash251

Watson P A K Townley P Koka K J Palmer and D J Stephens2006 Sec16 de1047297nes endoplasmic reticulum exit sites and is

required for secretory cargo export in mammalian cells Traf 1047297c7 1678ndash1687

Weber T B V Zemelman J A McNew B Westermann MGmachl et al 1998 SNAREpins minimal machinery for mem-brane fusion Cell 92 759ndash772

West M N Zurek A Hoenger and G K Voeltz 2011 A 3Danalysis of yeast ER structure reveals how ER domains are or-ganized by membrane curvature J Cell Biol 193 333ndash346

Wild K M Halic I Sinning and R Beckmann 2004 SRP meetsthe ribosome Nat Struct Mol Biol 11 1049ndash1053

Willer T M C Valero W Tanner J Cruces and S Strahl2003 O-mannosyl glycans from yeast to novel associations with human disease Curr Opin Struct Biol 13 621ndash630

Wilson D M Lewis and H Pelham 1993 pH-dependent bindingof KDEL to its receptor in vitro J Biol Chem 268 7465ndash7468

Wooding S and H R Pelham 1998 The dynamics of golgi pro-tein traf 1047297c visualized in living yeast cells Mol Biol Cell 92667ndash2680

Wuestehube L J R Duden A Eun S Hamamoto P Korn et al1996 New mutants of Saccharomyces cerevisiae affected inthe transport of proteins from the endoplasmic reticulum tothe Golgi complex Genetics 142 393ndash406

Xu X K Kanbara H Azakami and A Kato 2004 Expression andcharacterization of Saccharomyces cerevisiae Cne1p a calnexinhomologue J Biochem 135 615ndash618

Yabal M S Brambillasca P Sof 1047297entini E Pedrazzini N Borgeseet al 2003 Translocation of the C terminus of a tail-anchoredprotein across the endoplasmic reticulum membrane in yeastmutants defective in signal peptide-driven translocation J BiolChem 278 3489ndash3496

YaDeau J T C Klein and G Blobel 1991 Yeast signal peptidasecontains a glycoprotein and the Sec11 gene product Proc Natl

Acad Sci USA 88 517ndash

521 Yamakawa H D Seog K Yoda M Yamasaki and T Wakabayashi

1996 Uso1 protein is a dimer with two globular heads anda long coiled-coil tail J Struct Biol 116 356ndash365

Yip C K and T Walz 2011 Molecular structure and 1047298exibility of the yeast coatomer as revealed by electron microscopyJ Mol Biol 408 825ndash831

Yorimitsu T and K Sato 2012 Insights into structural and reg-ulatory roles of Sec16 in COPII vesicle formation at ER exit sitesMol Biol Cell 23 2930ndash2942

Yoshihisa T C Barlowe and R Schekman 1993 Requirementfor a GTPase-activating protein in vesicle budding from the en-doplasmic reticulum Science 259 1466ndash1468

Yu I M and F M Hughson 2010 Tethering factors as organ-izers of intracellular vesicular traf 1047297c Annu Rev Cell Dev Biol

26 137ndash

156 Yu X M Breitman and J Goldberg 2012 A structure-based

mechanism for Arf1-dependent recruitment of coatomer tomembranes Cell 148 530ndash542

Zhang C J M M Cavenagh and R A Kahn 1998 A family of Arf effectors de1047297ned as suppressors of the loss of Arf function inthe yeast Saccharomyces cerevisiae J Biol Chem 273 19792ndash19796

Zhang C J J B Bowzard A Anido and R A Kahn 2003 Four ARF GAPs in Saccharomyces cerevisiae have both overlappingand distinct functions Yeast 20 315ndash330

Ziegelhoffer T P Lopez-Buesa and E A Craig 1995 The disso-ciation of ATP from hsp70 of Saccharomyces cerevisiae is stim-ulated by both Ydj1p and peptide substrates J Biol Chem 27010412ndash10419

Zimmerberg J and M Kozlov 2006 How proteins produce cel-lular membrane curvature Nat Rev Mol Cell Biol 7 9ndash19Zink S D Wenzel C A Wurm and H D Schmitt 2009 A link

between ER tethering and COP-I vesicle uncoating Dev Cell 17403ndash416

Communicating editor T Davis



CONTENTS continued

Transport From the ER Sculpting and Populating a COPII Vesicle 391

Structure and assembly of the COPII coat 392

Cargo capture stochastic sampling vs direct and indirect selection 393

Regulation of COPII function GTPase modulation coat modi 1047297 cation 394

Higher-order organization of vesicle formation 395

Vesicle Delivery to the Golgi 395

Vesicle tethering 395

SNARE protein-dependent membrane fusion 396

A concerted model for COPII vesicle tethering and fusion 397

Traf1047297c Within the Golgi 397

Transport through the Golgi complex 397

Lipid requirements for Golgi transport 398

The Return Journey Retrograde Traf1047297c via

COPI Vesicles 398

Composition and structure of the COPI coat 399

Cargo capture sorting signals cargo adaptors and coat stimulators 400

Vesicle delivery DSL-mediated tethering and SNARE-mediated fusion 401

Perspectives 401

LIKE all eukaryotes yeast cells segregate various physio-

logical functions into distinct subcellular compartments

A key challenge is thus ensuring that appropriate proteins

are delivered to the correct subcellular destination a process

that is driven by discrete sorting signals that reside in the

proteins themselves Perhaps the most prevalent type of sort-

ing signal is that directing a protein to the secretory pathway

which handles the various proteins that are destined for the

extracellular environment or retention in the internal endo-

membrane system Approximately one-third of the yeast pro-

teome enters the secretory pathway Protein secretion is not

only essential for cellular function but also provides the

driving force for cell growth via delivery of newly synthe-

sized lipid and protein that permits cell expansion Secretory

proteins enter this set of interconnected organelles at the

endoplasmic reticulum (ER) which regulates protein trans-

lation protein translocation across the membrane protein

folding and post-translational modi1047297cation protein quality

control and forward traf 1047297c of suitable cargo molecules (both

lipid and protein) Once contained within the secretory path-

way proteins are ferried between compartments via trans-port vesicles that bud off from one donor compartment to

fuse with a downstream acceptor compartment thereby

mediating directional traf 1047297c of both lipid and protein The

forward-moving or anterograde pathway is balanced by

a reverse or retrograde pathway that returns escaped resi-

dent proteins and maintains the homeostasis of individual

organelles Early yeast screens pioneered the genetic dissec-

tion of the eukaryotic secretory pathway and were rapidly

followed by biochemical approaches that permitted the mo-

lecular dissection of individual processes of protein biogen-

esis and traf 1047297c Here we discuss the methodologies that

have yielded great insight into the conserved processes that

drive protein secretion in all eukaryotes and describe the

fundamental processes that act to ensure ef 1047297cient and ac-

curate protein secretion The reader is also referred to earlier

comprehensive reviews on these topics (Kaiser et al 1997

Lee et al 2004) as we focus our coverage on more recent

advances

Expanding Methodologies From a Parts Listto Mechanisms and Back to More Parts

Classic screens lay the groundwork in vitro reconstitutionde1047297 nes mechanism

There is no doubt that early seminal yeast genetics ap-

proaches laid the foundation upon which our understand-

ing of protein secretion is built From the original Novick

and Schekman screens that identi1047297ed a host of secretion-

defective ( sec) mutants (Novick and Schekman 1979 Novick

et al 1980) to additional more targeted approaches fromthe Schekman (more secs Deshaies and Schekman 1987

Wuestehube et al 1996) Gallwitz ( ypt Gallwitz et al 1983)

Ferro-Novick (bet Newman and Ferro-Novick 1987) Jones

( pep Jones 1977) Stevens ( vps Rothman et al 1989) and

Emr ( vps Bankaitis et al 1986) labs that expanded the rep-

ertoire of mutants with defects in secretory protein and

membrane biosynthesis the 1047297eld has been blessed with an

abundance of reagents that permitted the characterization

of each branch of the secretory pathway (Schekman and

























































1998)












































































































































pathway




















et al 1992)











































































et al 2000)

































(Ng et al 1996)






























































































































































































sential 1047298









and Ruddock 2009)










































































quality control








































et al 2011)
























































































































































































(Bi et al 2007)





























































2010)



















well de1047297ned


















































































































































remains to be seen















































Vesicle tethering







































































redundant network


























































2000)

















































































































































































































(Zink et al 2009)








































































































et al 2010)

























Wieland 1998)

















(Rein et al 2002)


















































vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































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ochem 64 563ndash


































26 137ndash
































































1998)












































































































































pathway




















et al 1992)











































































et al 2000)

































(Ng et al 1996)






























































































































































































sential 1047298









and Ruddock 2009)










































































quality control








































et al 2011)
























































































































































































(Bi et al 2007)





























































2010)



















well de1047297ned


















































































































































remains to be seen















































Vesicle tethering







































































redundant network


























































2000)

















































































































































































































(Zink et al 2009)








































































































et al 2010)

























Wieland 1998)

















(Rein et al 2002)


















































vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































112 27ndash






























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ochem 64 563ndash


































26 137ndash
































































































pathway




















et al 1992)











































































et al 2000)

































(Ng et al 1996)






























































































































































































sential 1047298









and Ruddock 2009)










































































quality control








































et al 2011)
























































































































































































(Bi et al 2007)





























































2010)



















well de1047297ned


















































































































































remains to be seen















































Vesicle tethering







































































redundant network


























































2000)

















































































































































































































(Zink et al 2009)








































































































et al 2010)

























Wieland 1998)

















(Rein et al 2002)


















































vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































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et al 1992)











































































et al 2000)

































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sential 1047298









and Ruddock 2009)










































































quality control








































et al 2011)
























































































































































































(Bi et al 2007)





























































2010)



















well de1047297ned


















































































































































remains to be seen















































Vesicle tethering







































































redundant network


























































2000)

















































































































































































































(Zink et al 2009)








































































































et al 2010)

























Wieland 1998)

















(Rein et al 2002)


















































vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































112 27ndash






























Chem 285 21600ndash

























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sential 1047298









and Ruddock 2009)










































































quality control








































et al 2011)
























































































































































































(Bi et al 2007)





























































2010)



















well de1047297ned


















































































































































remains to be seen















































Vesicle tethering







































































redundant network


























































2000)

















































































































































































































(Zink et al 2009)








































































































et al 2010)

























Wieland 1998)

















(Rein et al 2002)


















































vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































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sential 1047298









and Ruddock 2009)










































































quality control








































et al 2011)
























































































































































































(Bi et al 2007)





























































2010)



















well de1047297ned


















































































































































remains to be seen















































Vesicle tethering







































































redundant network


























































2000)

















































































































































































































(Zink et al 2009)








































































































et al 2010)

























Wieland 1998)

















(Rein et al 2002)


















































vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































112 27ndash






























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et al 2011)
























































































































































































(Bi et al 2007)





























































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well de1047297ned


















































































































































remains to be seen















































Vesicle tethering







































































redundant network


























































2000)

















































































































































































































(Zink et al 2009)








































































































et al 2010)

























Wieland 1998)

















(Rein et al 2002)


















































vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































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Chem 285 21600ndash

























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(Bi et al 2007)





























































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remains to be seen















































Vesicle tethering







































































redundant network


























































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(Zink et al 2009)








































































































et al 2010)

























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(Rein et al 2002)


















































vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































112 27ndash






























Chem 285 21600ndash

























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remains to be seen















































Vesicle tethering







































































redundant network


























































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et al 2010)

























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vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































112 27ndash






























Chem 285 21600ndash

























277 49863ndash







































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(Zink et al 2009)








































































































et al 2010)

























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(Rein et al 2002)


















































vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































112 27ndash






























Chem 285 21600ndash

























277 49863ndash







































21965ndash










































































Cell 3 789ndash












































































































ochem 64 563ndash


































26 137ndash




























2000)

















































































































































































































(Zink et al 2009)








































































































et al 2010)

























Wieland 1998)

















(Rein et al 2002)


















































vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































112 27ndash






























Chem 285 21600ndash

























277 49863ndash







































21965ndash










































































Cell 3 789ndash












































































































ochem 64 563ndash


































26 137ndash




































































































































(Zink et al 2009)








































































































et al 2010)

























Wieland 1998)

















(Rein et al 2002)


















































vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































112 27ndash






























Chem 285 21600ndash

























277 49863ndash







































21965ndash










































































Cell 3 789ndash












































































































ochem 64 563ndash


































26 137ndash























et al 2010)

























Wieland 1998)

















(Rein et al 2002)


















































vesicle production



































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































112 27ndash






























Chem 285 21600ndash

























277 49863ndash







































21965ndash










































































Cell 3 789ndash












































































































ochem 64 563ndash


































26 137ndash










































































Perspectives























conditions


























Golgi 1047297














Literature Cited















































































5 661ndash


















































112 27ndash






























Chem 285 21600ndash

























277 49863ndash







































21965ndash










































































Cell 3 789ndash












































































































ochem 64 563ndash


































26 137ndash



















Golgi 1047297














Literature Cited















































































5 661ndash


















































112 27ndash






























Chem 285 21600ndash

























277 49863ndash







































21965ndash










































































Cell 3 789ndash












































































































ochem 64 563ndash


































26 137ndash



















































5 661ndash


















































112 27ndash






























Chem 285 21600ndash

























277 49863ndash







































21965ndash










































































Cell 3 789ndash












































































































ochem 64 563ndash


































26 137ndash







































112 27ndash






























Chem 285 21600ndash

























277 49863ndash







































21965ndash










































































Cell 3 789ndash












































































































ochem 64 563ndash


































26 137ndash














Chem 285 21600ndash

























277 49863ndash







































21965ndash










































































Cell 3 789ndash












































































































ochem 64 563ndash


































26 137ndash


























21965ndash










































































Cell 3 789ndash












































































































ochem 64 563ndash


































26 137ndash















































Cell 3 789ndash












































































































ochem 64 563ndash


































26 137ndash









































































































ochem 64 563ndash


































26 137ndash






































26 137ndash









Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

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Transcript of Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway