Protein Trafficking 041209

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)proteins and (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 vesicles that 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.

1

Solutions of protein partition… Spatial distribution of macromolecules Nuclear Transport Transmembrane transport Vesicular transport

Membrane proteins in prokaryotes and eukaryotes…

Wallin and von Heijne, 1998

2

Number of proteins

Membrane proteins in prokaryotes and eukaryotes…

#1

#2 #3

Number of transmembrane segments

Membrane proteins in prokaryotes and eukaryotes…

Wallin and von Heijne, 1998

3

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… Extracellular C N

C

C

N

N N C

Intracellular

4

The signal sequence… a discovery originated from a discrepancy

Cell-free synthesis of IgG light chain: a. Microsomes b. 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?

5

The signal sequence…experimental evidence

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

Topology of integrated membrane proteins… Extracellular C N

C

C

N

N N C

Intracellular

6

Topology of integrated membrane protein…

Extracellular

Intracellular N C

Topology of integrated membrane proteins… … Extracellular C

N

Intracellular

7

Topology of integrated membrane proteins… C

Extracellular

N

C

C

N

N N C

Intracellular

The signal sequence…

8

The signal sequence… N

Extracellular

Intracellular

Composition of SRP54:

C

•

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

N

C

Extracellular

Extracellular

Intracellular

Intracellular

C

9

The signal sequence…translocate via a channel N

Extracellular

Intracellular

C

The signal sequence…? N

Extracellular

Intracellular

C

10

The signal sequence…features • No precise primary sequence but conserved general features

N

Extracellular

• 13-45 amino acid in length • Several positive charged Nterminal amino acids

Intracellular

• A stretch of hydrophobic amino acids • Small amino acids (cys, ala, gly) often at the cleavage site

C

The signal sequence…conservation N

Function features and conservation Extracellular

Intracellular

C

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.

11

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… ER, Periplasm

N

+ (mature) [+

Nucleus

+ + ]

+ + + + Mitochondrial Matrix

N

8 a.a. (

) (mature)

Peroxisome

Chloraplast stroma

SKL

N

OH OH OH OH OH (mature)

12

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.)

13

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

14

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, grp 94, and protein disulphide isomerase) share a common carboxyl terminal tetrapeptide sequence, KDEL.

Mutagenesis analysis of grp78:

15

Retention… soluble ER proteins

ER retention – localized biological activities N

N

C C Jackson et al., 1990

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ER retention – localized activities

Can we conclude… -KK is position-specific (?). -KKXX is necessary & sufficient for the ER retention (?). -KKXX retention activity is dominant (?). Jackson et al., 1990

Design of a screening system…

4 mM K+

100 mM K+

No Growth

Growth

Growth

Growth

SYG1528 +Kir2.1-KKXX

No Growth

Growth

SYG1528 +Kir2.1-AAXX

Growth

Growth

SYG1528 SYG1528 +Kir2.1

KKXX (or AAXX)

FCYENE

Growth Complementation Assay:

ER retention (No Rescue) Surface Localization (Rescue)

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Test in yeast growth… Plate setup KKXX – retention signal found in ER proteins

Kir2.1 Kir2.1-RAA

Kir2.1 -KKED

RKR – retention signal first found in KATP potassium channel

Kir2.1-RKR

100K

10K

7K

4K

More than just masking…? A LLDALTLASSRGPLRKRSVAVAKAKPKFSISPDSLS -COOH or RAA CD4 EC+TM 420

1

CD4-(HA)1 N

HA

Kir6.2

or

WBP1

11aa

CD4-(HA)3 N

HA

KKLETFKKTN -COOH or AATN

HA HA 31aa

CD4-(HA)5 N

HA

HA HA

HA HA

RKR

140 120 100 80 60 40 20 0 (HA)1

(HA)3 (HA)5

Spacing

NORMALIZED SURFACE EXPRESSION

NORMALIZED SURFACE EXPRESSION

51aa

KKTN 60 50 40 30 20 10 0 (HA)1 (HA)3 (HA)5

Spacing Shikano & Li, 2003

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Differential retention zones…. Extracellular

Intracellular

KKXX

RXR

KKXX zone RXR zone

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

19

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™”…

ER

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?

Retention

Cell Surface

ER retention – quality control Conferring 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

21

Forward transport (trafficking)… … motifs and machinery which potentiate surface expression

High Expression

Reduced Expression

Low Expression

High Expression

Forward transport – “DXE”….

VSV-G

N

C

-18aa-YTDIEMNRLGK Sevier et al., 2000

Nishimura and Blach, 1997

22

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

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

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Genetic isolation of genes important in secretory pathways Yeast strain secreting Invertase

Random mutagenesis Using mutagens

Fractionation of mutated Cells according density

Imaging or assay invertase Strains with increased density

COPII – machinery…

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Incorporation into COPII vesicles…

Sorting in polarized cells…

25

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) # of Seq -RXR

??? -DXE-FF -FCYENE

-1

0

+1

[Surface Expression Potential]

26

Subcellular distribution of proteins …

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

27

Hypothesis…. # of Seq RXR

“Forward Trafficking Signal”

RXR retention

-1

Surface expression

0

+1

[Surface expression potential]

Design a screening system…

FCYENE

FCYENE

FCYENE

RKR

RKR ?

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Design of a screening system……..

A

B

ER localization (no rescue)

RKR

RKR (or RAA)

ER Localization (no rescue) Surface Localization (rescue)

Forward Trafficking (rescue)

SWTY…RKR - dependent?…

0

Events

85

Kir2.1

100

101

102

103

104

85 0

Events 0

10 1

10 2 Empty

10 3

10 0

10 4

10 2 Empty

Kir2.1-RAA

10 4

Kir2.1-RAA-SWTY

0

Events

Events

10 3

0

Forward Trafficking (rescue)

10 1

85

10 0

85

RKR

ER retention (no rescue)

Kir2.1-RKR-SWTY

Events

85

Kir2.1-RKR

10 0

10

1

10 2 Empty

10 3

10

4

0

10 1

10 2 Empty

10 3

10 4

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

29

Polytopic tetramers vs. monotopic monomer... N

0

Events

85

Kir2.1

4 10

10 2 Empty

10 3

10 0

10 4

10 1

102 Empty

103

10 4

10 1

10 3

10 4

10 0

10 1

10 2 Empty

10 3

10 4

CD4-RAA-SWTY

10

1

10 2 Empty

10 3

10 4

0

10 1

102 Empty

103

10 4

10 0

10 1

10 2 Empty

10 3

10 4

0

10 0

0

0

0

Events

Events

Events

85 Events

10 2 Empty

CD4-RAA

75

Kir2.1-RAA-SWTY

Kir2.1-RAA

75 Events

10 0

85

10 1

CD4-RKR-SWTY

0

0

Events 0

10 0

N

CD4-RKR

75

Kir2.1-RKR-SWTY

Events

85

Kir2.1-RKR

75

3 10

Events

2 10

0

1 10

85

100

10 0

10 1

10 2 Empty

10 3

10 4

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 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). 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 …

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