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.

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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

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Membrane proteins in prokaryotes and eukaryotes…

#1 #2
Number of proteins

#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 N C

C C N

Intracellular

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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?

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The signal sequence…experimental evidence

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

Topology of integrated membrane proteins…

Extracellular
C

N N C

C C N

Intracellular

6
 Topology of integrated membrane protein…

Extracellular

Intracellular

Topology of integrated membrane proteins… …

Extracellular
C

Intracellular

7
 Topology of integrated membrane proteins…

C
Extracellular
N N C

C C N

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

Extracellular Extracellular

Intracellular Intracellular

C C

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The signal sequence…translocate via a
channel
N

Extracellular

Intracellular

The signal sequence…?

N

Extracellular

Intracellular

10
The signal sequence…features
N
• No precise primary sequence but
conserved general features

Extracellular
• 13-45 amino acid in length

• Several positive charged N-

terminal amino acids

Intracellular • A stretch of hydrophobic amino

acids

• Small amino acids (cys, ala, gly)

C
often at the cleavage site

The signal sequence…conservation

N
Function features and conservation

Extracellular 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).
Intracellular

2. The mechanism of recognition of

signal sequence is highly conserved as
C the signal sequence from human protein
will function in E. coli.

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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…

ER, Periplasm +
N
(mature)

[+ + + ]
Nucleus

8 a.a.
+ + + +
( )
Mitochondrial Matrix N
(mature)

Peroxisome SKL

OH OH OH OH OH
Chloraplast stroma N
(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, grp
94, 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

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…

FCYENE

KKXX (or AAXX)

ER retention
(No Rescue)
Growth Complementation Assay:
Surface Localization
4 mM K+ 100 mM K+ (Rescue)

SYG1528 Growth
No Growth

SYG1528 Growth Growth

+Kir2.1
SYG1528
No Growth Growth
+Kir2.1-KKXX
SYG1528 Growth Growth
+Kir2.1-AAXX

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Test in yeast growth…
Plate setup
KKXX – retention signal found in
Kir2.1 ER proteins
Kir2.1-RAA Kir2.1 -KKED

RKR – retention signal first found in

Kir2.1-RKR KATP potassium channel

100K 10K 7K 4K

More than just masking…?

A
LLDALTLASSRGPLRKRSVAVAKAKPKFSISPDSLS -COOH
or
RAA
CD4 EC+TM
1 420
CD4-(HA)1 N HA Kir6.2 or WBP1
11aa

CD4-(HA)3 N HA HA HA KKLETFKKTN -COOH

or
31aa AATN

CD4-(HA)5 N HA HA HA HA HA
51aa

RKR KKTN
NORMALIZED SURFACE

140
NORMALIZED SURFACE

60
120
EXPRESSION

50
EXPRESSION

100
80 40
60 30
40 20
20
10
0
(HA)1 (HA)3 (HA)5 0
(HA)1 (HA)3 (HA)5
Spacing
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

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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™”…

ER

How would a cell tells a protein in

different folding states?

What contributes the ability to

Retention
“translate” sensing into
different locations and
different level of
compartmentalization, e.g.,
cell surface expression?

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

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

High Expression Low Expression High Expression

Reduced Expression

Forward transport – “DXE”….

VSV-G

N C

-18aa-YTDIEMNRLGK

Sevier et al., 2000

Nishimura and Blach, 1997

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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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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…

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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)

# of Seq
-RXR ???
-DXE-
-FF
-FCYENE

-1 0 +1
[Surface Expression Potential]

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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

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Hypothesis….

# of Seq

RXR “Forward Trafficking Signal”

RXR retention Surface expression

-1 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
RKR (or RAA)

(no rescue) ER localization

RKR
(no rescue)
Surface Localization
(rescue)

Forward
Trafficking
(rescue)

SWTY…RKR - dependent?…

Kir2.1
85
Events
0

100 101 102 103 104

Kir2.1-RKR Kir2.1-RKR-SWTY
85
85

Events
Events

ER retention
RKR

(no rescue)
0
0

10 0 10 1 10 2 10 3 10 4
10 0 10 1 10 2 10 3 10 4
Empty
Empty

Kir2.1-RAA-SWTY
85

Kir2.1-RAA
85
Events

Events

Forward
Trafficking
0

(rescue)
0

10 0 10 1
10 2 10 3 10 4
0 10 1 10 2 10 3 10 4
Empty Empty

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

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Polytopic tetramers vs. monotopic monomer...

Kir2.1

85
Events
0

100 1
10 2
10 3
10 4
10

CD4-RKR CD4-RKR-SWTY

75

75
Kir2.1-RKR Kir2.1-RKR-SWTY

85
85

Events

Events
Events
Events

0
0

10 0
10 0 10 1 10 2 10 3 10 4 10 1 102 103 10 4
10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4

N
Empty Empty
Empty Empty

CD4-RAA CD4-RAA-SWTY
85

Kir2.1-RAA-SWTY

75

75
Kir2.1-RAA
85

Events

Events
Events
Events

0
0

10 0 10 1 10 2 10 3 10 4
0 10 1 102 103 10 4

0
Empty Empty 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4
Empty Empty

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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