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Lecture 4
PI signaling and the
puzzle of Vesicle Identity
SMAP2, a Novel ARF GTPase-activating Protein, Interacts with
Clathrin and Clathrin Assembly Protein and Functions on the AP-1–
positive Early Endosome/Trans-Golgi Network Waka Natsume et al.
We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and
regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a
SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin
heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike
SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo. SMAP2 colocalized with the adaptor proteins for
clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2
delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde,
early endosome-to-TGN pathway in a clathrin- and AP-1–dependent manner. Thus, the SMAP gene family constitutes
an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle
trafficking.
Regulation of
Size, Shape,
Number and
Function of
Each Organelle
Endosome
-Organelle Systems-
The Phosphoinositide Cycle
phosphatidylinositol
HO
HO
HO
o
o o
OH
phosphoinositides
PI-Kinase
OH
3
POH
P
P
HO
HO
P
HO
5 HO
P
o
4
OH
HO
P
PI
PIP
o
o o
o
PI-Phosphatase
“Spatial and Temporal Control of Cell Signaling”
“PIP’s as Transient Second Messengers”
Phosphoinositide Cycle in Cell Signaling
PI-Kinase
HO
Effector
P
OH
HO
OH
HO P
o
o o
o
PI
P
PI
PIP
PI-Phosphatase
o
o o
o
Effectors
FYVE (5)
PX (15)
PH (30)
ENTH (8)
“PIP’s Program Transport Activity via PIP Effectors”
PI Signaling in Membrane Trafficking Pathways
PM
Pik1
Mss4
PI4P
PI4,5P2
PI(4,5)P
PI(4,5)P
GFP-FYVE
222
PI(4,5)P
Sec7-GFP
FAPP-DsRed
(PI3P)
(TGN)
(PI4P)
Vps34
PI3P
Golgi
Complex
ER
Anterograde transport
Retrograde transport
Endosomal
System
Fab1
PI3,5P2
Lysosome/
Vacuole
GFP-Atg18
(PI3,5P2)
PI4P
FAPP-DsRed
PI4P
FM4-64
Nomarski
GFP-FYVE
(PI4P)
(PI3P)
FM4-64
merge
merge
PI3P
PI3P
merge
merge
merge
DsRed
GFP
CMAC
PI3P
PH
PI(4,5)P2
vacuole
FYVE
Chris Stefan
Jon Audhya
Amplification of Gene Complexity from Yeast to Human
Ubiquitin
Yeast
Humans
E1
E2
1 gene
2 genes
13 genes
> 50 genes
E3
45 genes
> 500 genes
DUBs
17 genes
90 genes
Yeast
Humans
Rab
Ras
11 genes
68 genes
4 genes
30 genes
Rho
6 genes
27 genes
Arf
Kinases
5 genes
25 genes
6 genes
> 4 genes
20 genes
> 90 genes
125 genes
> 490 genes
7 genes
> 30 genes
25 genes
105 genes
Small GTPases
PI lipid
Tyr
Ser/Thr
Phosphatases
PI lipid
Protein
Hierarchy of Organelle Identity Codes
Yeast Human
PIPs:
PIPs
PI-Kinase
Global
4
7
Rabs: 11
63
RabGEF
Rabs
Specific
Effectors
Membrane Traffic
Effectors: >100
>400
Inherited Lysosomal Storage Diseases
Disorder
I-Cell disease
Tay-Sachs’ disease
Pompe’s disease
Galactosialidosis
Gaucher’s disease
Deficient Hydrolase(s)
Multiple enzymes
b-Hexosaminidase
a-Glucosidase
Neuraminidase + b -Galactosidase
b -Glucocererosidase
I-Cell disease
Clinical defects - Severe skeletal and neurological defects. Retardation
of growth and psychomotor development. Death before age 5.
Manifestations - Multiple lysosomal enzymes are secreted. Cells are
highly vacuolated and contain numerous dense inclusion bodies.
Mechanism - Deficiency in GlcNAc-phosphotransferase. Lysosomal
enzymes lack Man-6-P recognition marker.
Bulk Lipid Composition of Cell Membranes
Lipids: PA
DAG
PS
PE
PC
PIPs
PI
Yeast
2%
5%
10%
20%
40-50% 10-15% < 0.5%
Human
(brain)
1%
5%
5%
20%
25-30% 5-10%
< 0.5%
Rare Signaling
Lipids
Other Lipids: Sterols(10-30%) + Sphingolipids (10-25%)
Core Components in Membrane Transport
Donor
1 Coats
2 Cargo
3 Vesicle Fission
Acceptor
1 SNARE
2 Tether
3 Vesicle Fusion
Combinatorial Code of Organelle Surface Tags
- Define Identity and Function -
Transient labile
Compartment:
Golgi
PM
Lipid Code:
PI4P
Rab Code:
Ypt31
(Rab11)
Endosome Lysosome
PI(4,5)P2
PI3P
PI(3,5)P2
Sec4
Vps21
Ypt7
(Rab8)
(Rab5)
(Rab7)
- Effector Proteins Stable
TMD
SNARE Code: Tlg2
Sso1/2
Pep12
Vam3
Combinatorial Trafficking Code in Membrane Sorting
Inputs:
Output:
protein-protein
vesicle budding
membrane fusion
Effectors
protein-lipid
Sorting
effector
protein
target
lipid
target
AP-2
cargo
PI(4,5)P2
PM
Ent1/Epsin
Ub
PI(4,5)P2
PM
FAPPI/GPBP
Arf
PI(4)P
TGN
AP-1
cargo
PI(4)P
TGN/EE
Vac1/EEA1
Rab
PI(3)P
Endosome
Vps27/Hrs
Ub
PI(3)P
Endosome
Retromer
cargo
PI(3)P
Endosome
Vam7
SNARE
PI(3)P
Vacuole
localization
Conserved Codes in Membrane Trafficking
Plasma Membrane
Coat
Tether SNARE
1
COP II
TRAPP
2
Clathrin Exocyst
?
Sso1/2
3
Clathrin HOPS
Tlg1/2
Pep12
4
Clathrin
EEA1
Pep12
5
?
HOPS
Vam3
Retromer
GARP/
VTF
Tlg1/2
4
2
3
Golgi
Complex
6
Endosome
System
5
1
ER
Lysosome/
Vacuole
Sed5
Organelle Identity: Cracking the Code
Vac
PM
Golgi
Endosome
Coats,
Adaptors,
+ Tethers
Lipid Code
GTPase Code
PI3P PI4P (3,5)P2 (4,5)P2
Arf
Rab5 Rab7
Ub
EEA1/Vac1
√
√
HRS/Vps27
√
√
ESCRT-II
√
√
Retromer
√
AP-1
√
√
FAPP-1
√
√
AP-2
√
Epsin
√
Atg18
√
√
√
Key Roles for PIPs in Membrane Transport
Establish and maintain organelle identity
•Rapid lipid flux in secretory and endocytic pathways
•Tendency to randomize lipid & protein composition
Regulation of vesicle-mediated transport events
•Carrier vesicle formation & fission (coat proteins + dynamin)
•Vesicle targeting and fusion (SNAREs + tethers + Rabs)
•Cargo recognition and sorting (receptors and adaptors)
Localization of PIP Isoforms is Conserved
-PIPs Act as Spatial Tags in Organelle Identity
Mammals
Yeast
GFP-2xPH(PLC)
PI(3,5)P2
PI(4,5)P2PM
Meyer lab, 1998
Varnai & Balla, 1998
Emr lab, 2002
GFP-PH(PLC)
N
PI3P
PI(4,5)P2 GFP-PH (FAPP1)
PI4P
PI4PGolgi
GFP-PH (FAPP1)
N
Levine & Munro, 2002
Emr lab, 2002
GFP-FYVE(EEA1)
PI3PEndososmes
Emr lab, 1998
Stenmark lab, 1998
Corvera lab, 1998
(CHO cell images, De Camilli lab, 2006)
GFP-2xFYVE(EEA1)
N
PIP’s as Spatial Membrane-Specific Tags
PI(4,5)P2-PM
How Do PI Lipids Restrict Unique Cellular Functions
to Specific Membrane Compartments?
•Restricted localization of PI kinases leads to
compartment-specific synthesis/localization of PIP’s
PI4P-Golgi
•Membrane-restricted PIP’s program the transport activity
of membrane compartments by recruiting/activating
specific effector proteins (PH, FYVE, PX, ENTH domains)
•PI Pases inactivate/turnover PIP’s at inappropriate
membrane sites and terminate PIP signaling
PI3P-endosome
Phosphoinositides as Spatial and Temporal Regulators
of Membrane Trafficking and Organelle Identity
• Compartment specific localization of PI kinases leads to
restricted synthesis/localization of PIP’s - Spatial identity tags
• Membrane-restricted PIP’s program the transport activity of
membrane compartments by recruiting and activating specific
effector proteins - (PH, FYVE, PX, ENTH domain proteins)
• Obligate order of PI synthesis reactions regulates/balances
anterograde and retrograde membrane sorting reactions (PI3P for anterograde --> PI3,5P2 for retrograde)
• PI-Pases terminate PIP signaling and inactivate PIP’s at
inappropriate membrane sites
“Location - Location - Location”
PI-Binding Domains in Membrane Transport Proteins
Endosome:
PI3P
EEA1 (FYVE)
HRS (FYVE)
Vam7 (PX)
SNX (PX)
Golgi:
Lysosome:
PI4P
PI(3,5)P2
PI(4,5)P2
Atg18 (WD-40)
Dynamin (PH)
FAPP1 (PH)
Osh2 (PH)
AP-1
Plasma membrane:
Epsin (ENTH)
HIP1 (ANTH)
AP-2
AP-180 (ANTH)
Examples of Modular Lipid Binding Domains
Domain
Lipid Target
Yeast
Humans
PH
PI4P + PIP2 + PIP3
30 genes
223 genes
FYVE
PI3P
5 genes
30 genes
PX
PI3P + PIP2
15 genes
34 genes
ENTH
PIP2
8 genes
16 genes
C1
DAG
1 gene
88 gene
C2
PIP’s + PS
11 genes
200 genes
Human Diseases Linked to PI Metabolism Pathways
Kinases:
Gene
Enzyme
Product
Disease
PIK3CA
Class I PI 3-K
PI(3,4,5)P3
Cancer
hVPS34
Class III PI 3-K
PI3P
Bipolar disorder
PIKfyve
PI3P 5-Kinase
PI(3,5)P2
Phosphatases:
Gene
Enzyme
Francois-Neetens
cornea dystrophy
Substrate
Disease
MTM1
myotubularin
PI3P
Charcot-Marie-Tooth
PTEN
3-phosphatase
PI(3,4,5)P3
Cancer
SHIP2
5-phosphatase
Type 2 Diabetes
OCRL!
5-phosphatase
PI(3,4,5)P3
PI(4,5)P2
Pathogenesis:
Gene
Enzyme
Substrate
Pathogen
SapM
PI3P
M. tuberculosis
PI(4,5)P2
Salmonella
3-phosphatase
SigD/SopB 4-Pase/PPIPase
Lowe’s syndrome
Rab GTPase Cycle in Membrane Transport
GEF
Nucleotide exchange
GDP
GDI
GTP
GDP
Rab
Rab
GTP
Effectors
Membrane
Traffic
P
GTP hydrolysis
GAP
Regulatory Cycles in Membrane Trafficking
GEF
RabGDP
Rab
GTPase
Cycle
PI Kinase
RabGTP
PIP
GAP
PI
Phosphatase
Membrane
Transport
Kinase
tSN-P04
PI
Cycle
Ub Ligase
SNARE
Cycle SNAREs
Ub-Lys
Phosphatase
Ubiquitin
Ub
Cycle
De-Ub
“Network of Regulation”
Molecular Shape of Lipids Influences Membrane Curvature
LPA, LPC
(inverted
cone)
PC
(conical,
cylinder)
PA, PE
(cone)
QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.
Temporal Order of Clathrin-Mediated Endocytic Intermediates
Recruitment of Clathrin
Membrane
Membrane Restriction/Fission
Assembly Factors
Curvature
Vesicle Release
PIPK-g
AP-2*
Hip1R*
Amphiphysin2* Dynamin*
AP180A,B*
Epsin*
Endophilin
Actin
Eps15
polymerization
Clathrin
PI(4,5)P2
PI(4,5)P2-binding Proteins*
Conner and Schmid, Nature 2003
PI(4,5)P2 Metabolism Controls Multiple Endocytic Intermediates
Stage 1
Stage 2
Stage 3
Stage 4
Recruitment of Clathrin
Assembly Factors
Membrane
Curvature
Membrane Restriction/Fission
Vesicle Release
Vesicle
Uncoating
AP-2*
AP180A,B*
Epsin*
Clathrin
Eps15
Hip1R*
Amphiphysin*
Dynamin*
Actin
Endophilin Polymerization*
Synaptojanin
Auxilin
Hsc70
PI(4,5)P2
?
PIPK-g
*Factors Regulated by PIP2
?
PI(4,5)P2
hydrolysis
Membrane curvature generation
How are PI(4,5)P2 ‘hotspots’ locally generated to initiate clathrin coat formation?
How are PI(4,5)P2 synthesis and turnover temporally coupled with vesicle
formation and vesicle fission?
Adapted from Conner and Schmid, Nature 2003