Protein Trafficking 041209
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Transcript of Protein Trafficking 041209
1
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?
6
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
7
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
11
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
12
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)
13
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.)
14
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
15
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:
16
Retention… soluble ER proteins
ER retention – localized biological activities
Jackson et al., 1990
N
C
N
C
17
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)
18
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
19
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
20
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
21
“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
22
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
23
Protein machinery in vesicular pathway… How to identify them?…
Protein machinery in vesicular pathway… How to identify them?…
24
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…
25
Incorporation into COPII vesicles…
Sorting in polarized cells…
26
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
27
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
28
[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
29
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.
30
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.
31
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 …