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Protein Trafficking: Lecture II

Min Li

Solutions of protein partition…1. Eukaryotic cells have an elaborate system of internal

membrane-bound structures called organelles.

2. Each organelle has a unique composition of (glyco)proteinsand (glyco)lipids that carry out a particular set of functions.

3. An organelle comprises one or more membrane-bound compartments.

4. Organelles may act autonomously or in cooperation to accomplish a given function.

5. In the endocytic and exocytic pathways, cargo proteins are transferred between compartments by transport vesiclesthat form by budding from an organelle's surface.

6. Transport vesicles can selectively include material destined for transfer and exclude material that must remain in the organelle from which they bud.

7. Selective inclusion into transport vesicles is ensured by signals in a protein's amino acid sequence or carbohydrate structures.

8. Transport vesicles contain proteins that target them specifically to their intended destinations.

2

Solutions of protein partition…

Spatial distribution of macromolecules

Nuclear Transport

Transmembranetransport

Vesicular transport

Membrane proteins in prokaryotes and eukaryotes…

Wallin and von Heijne, 1998

3

Membrane proteins in prokaryotes and eukaryotes…

Num

ber o

f pro

tein

s

Number of transmembrane segments

#1 #2

#3

Membrane proteins in prokaryotes and eukaryotes…

Wallin and von Heijne, 1998

4

Transmembrane transport…

• Process – Soluble or membrane-bound proteins into an organelle.

• Specificity – Which and how to engage a correct organelle prior to the entry.

• Topology – how an integral protein establishes its topology in membrane?

Topology of integrated membrane proteins…

N

C

N

C

C

N

N

CExtracellular

Intracellular

5

The signal sequence… a discovery originated from a discrepancy

Cell-free synthesis of IgG light chain:

a. Microsomesb. Microsome-derived polysomes

Milstein et al., Nature New Biology, 239: 117 (1972)

The signal sequence…experimental evidence

What would the additional experiments be supportive (or necessary) for the hypothesis?

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The signal sequence…experimental evidence

Milstein et al., Nature New Biology, 239: 117 (1972)

Topology of integrated membrane proteins…

N

C

N

C

C

N

N

CExtracellular

Intracellular

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Topology of integrated membrane protein…

Extracellular

Intracellular

N

C

Topology of integrated membrane proteins… …

C

N

Extracellular

Intracellular

8

Topology of integrated membrane proteins…

N

C

N

C

C

N

N

CExtracellular

Intracellular

The signal sequence…

9

The signal sequence…N

C

Extracellular

Intracellular

Composition of SRP54:

• The G domain, which binds guanosine triphosphate (GTP) and hydrolyzes it to guanosine diphosphate (GDP)

• The N domain, an N-terminal domain that interacts with the G domain; and

• The M domain, which is a C-terminal domain containing a large number of methionine residues

The signal sequence…

N

C

Extracellular

Intracellular

N

C

Extracellular

Intracellular

10

The signal sequence…translocate via a channel

N

C

Extracellular

Intracellular

The signal sequence…?N

C

Extracellular

Intracellular

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The signal sequence…features• No precise primary sequence but

conserved general features

• 13-45 amino acid in length

• Several positive charged N-terminal amino acids

• A stretch of hydrophobic amino acids

• Small amino acids (cys, ala, gly) often at the cleavage site

N

C

Extracellular

Intracellular

The signal sequence…conservation

Function features and conservation

1. Placement of a signal sequence at the N-terminus of a normally non-secreted protein can result in proper targeting of the protein to the ER (or inner membrane in bacteria).

2. The mechanism of recognition of signal sequence is highly conserved as the signal sequence from human protein will function in E. coli.

N

C

Extracellular

Intracellular

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Questions…

What would be the potential physiological implication concerning the conserved features but lack of precise sequence identity?

How wound you test your hypothesis experimentally?

Positions of targeting signals…+

N

+N

+ + + ( )

8 a.a.

+ + +[ ]

SKL

NOH OH OH OH OH

ER, Periplasm

Nucleus

Mitochondrial Matrix

Peroxisome

Chloraplast stroma

(mature)

(mature)

(mature)

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Methods to determine protein topology…

Tag: immuno-epitope, toxin epitope, and enzyme

Vesicular trafficking…

Key issues (questions):

• Entry of ER

• Exit of ER

• Where to be transported (or should be “ where to go.)

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Retention and forward trafficking

Vesicular transport - retention

• Conformation – dependent but not function – dependent

• Retention takes place in ER

• Essential for both health and disease states

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Retention and forward trafficking

• Biology– Retain ER-specific proteins– Quality control for protein folding, posttranslational modifications– Discriminate macromolecular assembly

• Diseases– Toxins use ER – associated degradation (ERAD) components

for transport to the cytoplasm.– Viruses evade immune detection using ERAD to destroy

components of the immune system.– Many human diseases (e.g., cystic fibrosis) develop because of

gaining sensitivity to ER quality control system.– Porin diseases develop on the basis of escape from the ER

quality control

A C-Terminal Signal Prevents Secretion of Lumenal ER proteins

Munro S. and Pelham H. have noted that three soluble ER proteins whose sequences were known (grp 78, grp94, and protein disulphide isomerase) share a common carboxyl terminal tetrapeptide sequence, KDEL.

Mutagenesis analysis of grp78:

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Retention… soluble ER proteins

ER retention – localized biological activities

Jackson et al., 1990

N

C

N

C

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ER retention – localized activities

Jackson et al., 1990

Can we conclude…

-KK is position-specific (?).

-KKXX is necessary & sufficient for the ER retention (?).

-KKXX retention activity is dominant (?).

SYG1528+Kir2.1-AAXX

Growth

GrowthGrowth

No GrowthSYG1528+Kir2.1-KKXX

Design of a screening system…

FCYE

NE KKXX (or AAXX)

ER retention

Surface Localization

SYG1528

SYG1528+Kir2.1

4 mM K+ 100 mM K+

Growth Complementation Assay:

Growth

GrowthGrowth

No Growth

(No Rescue)

(Rescue)

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Test in yeast growth…

Kir2.1 -KKED

Kir2.1

Kir2.1-RAA

Plate setup

100K 10K 7K 4K

Kir2.1-RKR

KKXX – retention signal found in ER proteins

RKR – retention signal first found in KATP potassium channel

More than just masking…?

LLDALTLASSRGPLRKRSVAVAKAKPKFSISPDSLS -COOH

CD4 EC+TM

HA Kir6.2NCD4-(HA)1

HAN HA HA

HAN

11aa

31aa

51aa

HA HA HA HA

KKLETFKKTN -COOH

or WBP14201

orRAA

orAATN

CD4-(HA)3

CD4-(HA)5

A

010

2030

40

5060

(HA)1 (HA)3 (HA)5

NO

RM

ALI

ZED

SU

RFA

CE

EXPR

ESSI

ON

KKTN

Spacing

020406080

100120140

(HA)1 (HA)3 (HA)5

NO

RM

ALI

ZED

SU

RFA

CE

EXPR

ESSI

ON

RKR

Spacing

Shikano & Li, 2003

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KKXX

RXR

Extracellular Intracellular

KKXX zone

RXR zone

Differential retention zones….

Shikano & Li, 2003

Retention and forward trafficking

• Biology– Retain ER-specific proteins– Quality control for protein folding, posttranslational modifications– Discriminate macromolecular assembly

• Diseases– Toxins use ER – associated degradation (ERAD) components

for transport to the cytoplasm.– Viruses evade immune detection using ERAD to destroy

components of the immune system.– Many human diseases (e.g., cystic fibrosis) develop because of

gaining sensitivity to ER quality control system.– Porin diseases develop on the basis of escape from the ER

quality control

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Retention – quality control? How…

“Sensors” to detect misfolding…• Classical chaperons

– ER lumenal: BiP (GRP78)/Kar2p, GRP94, Sec63p – Cytosolic: Hsp70 & Hsp90, Ssa1p, Hsc70, Hdj2 and CHIP

• Disulfide modifying proteins– PDI (ERp59), Eug1p, ERp57, ERp72 and oxidase of PDI (Ero1p)

• Peptidyl prolyl isomerases– FK506 binding protein, cyclophilins

• Lectin-like chaperons– Calnexin (CNX/Cne1p/Cnx1), Calreticulin (CRT)

• N-glycan modifying proteins– Mannosidase and glucosidase 1 & 2 (GLS1/2), glycoprotein

glycosyltransferase

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“Sensing and sensitivity™”…

How would a cell tells a protein in different folding states?

What contributes the ability to “translate” sensing into different locations and different level of compartmentalization, e.g., cell surface expression?

ER

Cell Surface

Retention

ER retention – quality controlConferring detection, retention, and redirection of misfolding proteins – on the basis of structural rather than functional criteria.

In health (Kir6.1 and SUR)In disease (CFTR, long QT. etc )

ER and Golgi Compartments Exit to Cell Surface

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Forward transport (trafficking)…

Reduced ExpressionHigh Expression Low Expression High Expression

… motifs and machinery which potentiate surface expression

Forward transport – “DXE”….

Nishimura and Blach, 1997

Sevier et al., 2000

N C

-18aa-YTDIEMNRLGK

VSV-G

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Protein machinery in vesicular pathway… How to identify them?…

Protein machinery in vesicular pathway… How to identify them?…

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Yeast strain secretingInvertase

Random mutagenesisUsing mutagens

Fractionation of mutatedCells according density

Imaging or assay invertaseStrains with increased density

Genetic isolation of genes important in secretory pathways

COPII – machinery…

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Incorporation into COPII vesicles…

Sorting in polarized cells…

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Posttranslational ER translocation machinery …

• In vivo experiments in yeast indicate that genes encoding components of SRP can be eliminated and cell still survive.

• In vitro experiment with microsomes show intact proteins can translocate across microsomal membranes. This reaction requires cytosolic proteins. Further purification showed that the essential factors were Hsp70 and ATP. Later, additional proteins have been shown to be required.

Surface expression potential (SEP)

[Surface Expression Potential]

# of Seq

-RXR-DXE--FF-FCYENE

???

0 +1-1

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Subcellular distribution of proteins …

Andrews et al., Nature Biotech. 21:1297Andrews et al Nature Biotech 21:1297

Subcellular distribution of proteins …

• Understand the basic concepts

• Appreciate importance and richness of biological questions and the classic experiments

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[Surface expression potential]

# of Seq

“Forward Trafficking Signal”

Surface expression

RXR

RXR retention

0 +1-1

Hypothesis….

Design a screening system…

FCYE

NE

RK

RFC

YEN

E

?

RK

RFC

YEN

E

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Design of a screening system……..

RK

R (or R

AA)

ER Localization(no rescue)

Surface Localization(rescue)

RK

R

ER localization(no rescue)

A B

Forward Trafficking(rescue)

SWTY…RKR - dependent?…

100 101 102 103 104

085

Eve

nts

10 0 10 1 10 2 10 3 10 4

Empty

085

Even

ts

10 0 10 1 10 2 10 3 10 4

Empty

085

Even

ts

10 0 10 1 10 2 10 3 10 4Empty

085

Eve

nts

Kir2.1-RKR

Empty

085

0 10 1 10 2 10 3 10 4

Eve

nts

Kir2.1

Kir2.1-RKR-SWTY

Kir2.1-RAA Kir2.1-RAA-SWTY

RK

R

ER retention(no rescue)

Forward Trafficking(rescue)

SWTY motif confers a “gain of function” activity compared to wildtype.

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Polytopic tetramers vs. monotopic monomer...

100 101 102 103 104

085

Eve

nts

10 0 10 1 10 2 10 3 10 4

Empty

085

Eve

nts

10 0 10 1 10 2 10 3 10 4

Empty

085

Eve

nts

10 0 10 1 102 103 10 4Empty

085

Eve

nts

Kir2.1-RKR

Empty

085

0 10 1 102 103 10 4

Eve

nts

Kir2.1

Kir2.1-RKR-SWTY

Kir2.1-RAA Kir2.1-RAA-SWTY

100 101 102 103 104

Empty

075

Eve

nts

100 101 102 103 104

Empty

075

Eve

nts

100 101 102 103 104

Empty

075

Eve

nts

100 101 102 103 104

Empty

075

Eve

nts

CD4-RKR CD4-RKR-SWTY

CD4-RAA CD4-RAA-SWTY

N

N

Exam questions - 2003

Using genetic linkage analysis, you have studied a large group of patients who have a specific defect in liver function. The disease phenotype is autosomal dominant (i.e., one mutated copy of chromosome is sufficient to cause the disease). You were able to identify the locus that harbors mutations. This has allowed you to isolate a cDNA that encodes a novel protein from hepatocytes (liver cells).

1. (20%) Suggest two sequence criteria which you may use to predict whether the protein might be an ER resident protein.

2. (20%) Suggest two sequence criteria with which you may use to predict whether the protein might be a membrane protein.

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Exam questions - 2003

Using genetic linkage analysis, you have studied a large group of patients who have a specific defect in liver function. The disease phenotype is autosomaldominant (i.e., one mutated copy of chromosome is sufficient to cause the disease). You were able to identify the locus that harbors mutations. This has allowed you to isolate a cDNA that encodes a novel protein from hepatocytes (liver cells).

3. (30%) Based on the deduced amino acid sequence, you were able to develop antibodies which allowed you to localize the native protein and found it was on cell surface. When you expressed the cDNA in cultured human embryonic kidney (HEK) cells, you found no protein on cell surface. Using immunoblot, you were able to confirm the protein expression. (1) Propose a mechanism that may account for the lack of surface expression. (2) Suggest an experimental strategy to test the proposed mechanism.

4. (30%) Suppose that the wild-type protein when expressed is found on cell surface, but a mutant protein from a patient when expressed is found in ER. (1) Propose a mechanism for the autosomal dominant phenotype. (2) Suggest an experimental strategy to test the proposed mechanism.

Critical steps controlling the membrane protein expression …