Protein Processing in the Endoplasmic Reticulum

Andreas Herrlich MD PhDDepartment of Medicine,

Department of Cell Biology,

McDonnell Science Rm 863

aherrlich@wustl.edu

Thank you to Phylis Hanson!

Outline

•  ER morphology•  Protein folding•  What happens when protein folding is

successful (Secretory pathway)– ER exit via COPII vesicles- Retrieval of ER residents from Golgi

Endoplasmic reticulumThe ER is a compartment with a single continuous membrane and luminal space that can occupy up to 30% of cell volume

Subdomains of the ER•  Rough ER (mostly ER sheets or cisternae)

–  Protein translocation–  Protein folding and oligomerization–  Carbohydrate addition–  ER degradation

•  Smooth ER (mostly ER tubules)–  Lipid metabolism–  Calcium release–  Detoxification

•  ER exit sites (ERES, a.k.a. ribosome-free transitional ER) - export of proteins and lipids into the secretory pathway, marked by COPII coat

•  ER contact zones - transport of lipids, contact with other organelles

•  Nuclear envelope–  Nuclear pores–  Chromatin anchoring

]About 1/3 of cellular proteintransits through the ER

ER subdomains asdefined by proximityto other structures

Examples from cells expressingfluorescently taggedorganelle markers

Voeltz lab, UC Boulder

Posttranslational modificationsProtein folding

Protein Processing and Quality Control in the Endoplasmic Reticulum

Unfolded Native

Unfolded protein response

ERAD: ER-associated degradationExit from the ER

Protein Modifications and Folding in the ER

•  Folding challenging in setting of ~400 mg/ml protein concentration

•  “Hydrogel”•  “Proteostasis”•  In this unique milieu, proteins are continuously

challenged and “massaged” by a relentless folding machinery

Heterogeneous mixture of diverse proteins in different states of conformation, modification, oligomerization, and aggregation

Protein Modifications and Folding in the ER

•  Chaperones interact with unfolded proteins but not with properly folded proteins/protein complexes

•  Compartimentalization of reactions within the lumen of the ER

Role of classical chaperones•  ER contains abundant Hsp70 and Hsp90 chaperones•  Chaperones help other proteins acquire native

conformation, but do not form stable complexes

Hsp70s & Hsp90s bind exposed hydrophobic segments

BiP is main ER Hsp70, GRP94 is main ER Hsp90, interactions with clients are regulated by ATP status

Peptidyl-prolyl isomerases (PPIs) catalyse !cis–trans isomerisation of peptide bonds N-terminal to proline

Participation ofmany co-factorsin this regulation

N-linked glycosylation: Asn - X - Ser/Thr

Oligosaccharide additioncontaining a total of 14 sugars

En bloc addition to protein; subsequent trimming and additions as protein progresses through the secretory pathway;five core residues are retained in all glycoproteins

Role of glycosylation dependent chaperones in ER folding

Fate of newly synthesized glycoproteins in the ER I

•  Path when nascent protein folds efficiently (green arrows)

•  Players–  OST = oligosaccharyl transferase–  GI, GII = glucosidase I and II–  Cnx/Crt = Calnexin and

Calreticulin, lectin chaperones–  ERp57 = oxidoreductase–  ERMan1 = ER mannosidase 1–  ERGIC53, ERGL, VIP36 = lectins

that facilitate ER exit (cargo capture “receptors”

Increases solubility

glucose

mannose

Domain structure and interactions of calnexin

binds sugar

binds other proteins

Williams, 2006 J Cell Sci 119:615

Model showing interaction of a foldingglycoprotein with calnexin and ERp57Calnexin

Fate of newly synthesized glycoproteins in the ER II

•  Path when nascent protein goes through folding intermediates (orange arrows)

•  Players–  UGT1 (a.k.a. UGGT) = UDP-

glucose–glycoprotein glucosyltransferase, recognizes “nearly native” proteins, acting as conformational sensor

–  Reglucosylated protein goes through Cnx/Crt cycle for another round

–  GII removes glucose to try again and pass QC of UGT1

–  BiP = hsc70 chaperone that recognizes exposed hydrophobic sequences on misfolded proteins

0

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0 0.2 0.4 0.6 0.8RNAse (mg)

cpm

Intact RNAse

Denatured RNAse

UDP-glucose glycoprotein glucosyltransferase (UGT1 a.k.a. UGGT or GT) is an ER folding sensor

Best substrates in vitro are “nearly folded glycoprotein intermediates”not the native, compact structure or a terminally misfolded protein

In vitro UGT1 reaction usingRNAse as glycoprotein substrateMeasure incorporation of [14C] glucoseinto the oligosaccharide attached to �RNAse, compare native vs. denaturedRNAseResult: Only denatured RNAse is asubstrate.

Fate of newly synthesized glycoproteins in the ER III

•  Folding-defective proteins need to be degraded - transported out of the ER for degradation (red arrows)

•  How do proteins avoid futile cycles?–  UGT1 does not recognize

fatally misfolded proteins and won’t reglucosylate them for binding to Cnx/Crt

–  Resident mannosidases will trim mannose residues - protein can no longer be glucosylated and bind to Cnx/Crt

–  BiP binds hydrophobic regions–  Mannosidase trimmed glycans

recognized by OS9 associated with ubiquitination machinery

•  Leads to kinetic competition between folding and degradation of newly synthesized glycoproteins

Slow

Posttranslational modificationsProtein folding

Protein Processing and Quality Control in the Endoplasmic Reticulum

Unfolded

Unfolded protein response

Native

ERAD: ER-associated degradation

Exit from the ER

VTC = Vesicular Tubular Cluster

TGN = Trans Golgi Network

ER exit sites defined as sites of COPII vesicle formation

Overview of COPII vesicle biogenesis

Minimal COPII machinery

Five proteins added to liposomes or in vitro reactions form vesicles:

Sar1p, Sec23p, Sec24p, Sec13p, Sec31p

How is cargo packaged into vesicles leaving the ER?Selective Nonselective

Four core concepts of the early secretory pathway

•  CARGO CAPTURE (selective)•  BULK FLOW (nonselective)•  RETENTION•  RETRIEVAL

ER!Vesicles!Vesicular Tubular clusters!cis Golgi

Specific amino acid signals mediate selective transport

Requirement of two acidic residues in the cytoplasmic tail of VSV-G for efficient export from the ER. Nishimura & Balch, Science 1997

Diacidic motifs are common theme in efficiently secreted proteins

VSV-G TM-18aa -YTDIEMNRLGKCFTR TM-212aa-YKDADLYLLD-287aaTMGLUT4 TM-36aa -YLGPDENDLDLR TM-17aa -YQKTTEDEVHICH-20aaCI-M6PR TM-26aa -YSKVSKEEETDENE-127aaE-cadherin TM-95aa -YDSLLVFDYEGSGS-42aaEGFR TM-58aa -YKGLWIPEGEKVKIP-467aaASGPR H1 MTKEYQDLQHLDNEES-24aaTMNGFR TM-65aa -YSSLPPAKREEVEKLLNG-74aaTfR -19aa -YTRFSLARQVDGDNSHV-26aaTM

COPII cargo binding site(s)

•  Cargo binding sites recognize ER export signals in cytoplasmic domains of cargo

•  Best studied are the diacidic motifs in exiting membrane proteins, but there are others that bind to alternate sites in Sec24

(GAP)

(Cargo binding)

What about lumenal cargo?

Two possibilities: -bulk flow, with specific retention of ER resident proteins-receptor mediated exit via binding to secreted TM protein

-Measure secretion of soluble model protein derived from Semliki Forest Virus capsid protein

-Protein folds rapidly, no need for chaperones

-Use pulse-chase analysis to follow newly synthesized protein

-First molecule secreted 15 min after synthesis

-Rate constant of secretion is 1.2% per minute,corresponding to bulk flow rate of 155 COPIIvesicles per second

! Soluble proteins are efficiently secreted by bulk flow

And what about large cargo?

Malhotra and ErlmannEMBO J 201130: 3475

Cargo Capture + Bulk FlowLarge Cargo

How is ER volume and content maintained?

Retrograde traffic from Golgi to ER •  Includes receptor-mediated

mechanism for retrieving ER resident proteins

•  HDEL receptor identified in yeast - ERD2; multispanning transmembrane protein

•  KDEL receptor in higher eukaryotes.

•  Dilysine motif in C-terminal tail of receptor binds to COPI coat, lumenal domain binds HDEL/KDEL motif in pH dependent manner

http://www.ergito.com/lookup.jsp?expt=pelham

Thank you!