Lecture 4PI signaling and the

puzzle of Vesicle Identity

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.

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.

Endosome

Regulation of Size, Shape, Number and Function of Each Organelle

-Organelle Systems-

The Phosphoinositide Cycle

PI

PI-Phosphatase

oo

oo

OH

OHHO

HO

HO P

PIP

PI-Kinase

oo

oo

OH

HO

HO P

P

HO

34

5

“Spatial and Temporal Control of Cell Signaling”“PIP’s as Transient Second Messengers”

phosphatidylinositol phosphoinositides

oo

oo

OH

HO P

P

HO

34

5

OH

oo

oo

OH

HO P

P

34

5

OHHO

oo

oo

OH

HO P

P34

5 HO

P

oo

oo

OH

HO P

P34

5

HO

P

oo

oo

OH

HO P

P34

5 POH

oo

oo

OH

HO P

P34

5 P

P

Effector

Phosphoinositide Cycle in Cell Signaling

Effectors

FYVE (5)PX (15)PH (30)ENTH (8)

ooo

OH

OHHO

HOHO P

o

PI

PI

PI-Kinase

PIP

PI-Phosphatase

“PIP’s Program Transport Activity via PIP Effectors”

ooo

P

o

P

PI Signaling in Membrane Trafficking Pathways

GolgiComplex

EndosomalSystem

Lysosome/Vacuole

ER

Anterograde transportRetrograde transport

PM

PI3PVps34

PI4PPik1

PI4,5P2Mss4

PI3,5P2Fab1

PI4P

PI3P

PI(4,5)P2

GFP-FYVE

FAPP-DsRed

merge

(PI4P)

(PI3P)

GFP-FYVE

FM4-64

merge

(PI3P) Sec7-GFP

FAPP-DsRed

merge

(PI4P)

(TGN)

PI4P

PI3P

PI(4,5)P2

FM4-64

merge

GFP-Atg18(PI3,5P2)

PI(4,5)P2

Nomarski

merge

FYVEDsRed

CMAC

GFP

PI3PPI(4,5)P2

PH

vacuoleChris StefanJon Audhya

Amplification of Gene Complexity from Yeast to HumanUbiquitin

E1 E2

E3

DUBs

Yeast

1 gene

13 genes

45 genes

17 genes

Humans

2 genes

> 50 genes

> 500 genes

90 genes

Kinases

PI lipid Tyr

Ser/Thr

6 genes

> 4 genes

125 genes

20 genes

> 90 genes

> 490 genesPhosphatases

PI lipid

Protein 7 genes

> 30 genes

25 genes

105 genes

Small GTPases Rab

Ras

Rho

Arf

Yeast

11 genes

4 genes

6 genes

5 genes

Humans

68 genes

30 genes

27 genes

25 genes

Membrane Traffic

Rabs

Hierarchy of Organelle Identity Codes

PIPsRabGEFPI-Kinase Global

Specific

Effectors

HumanYeast

4 7

11 63

PIPs:

Rabs:

>100 >400Effectors:

Inherited Lysosomal Storage Diseases

Disorder Deficient Hydrolase(s)

I-Cell disease Multiple enzymesTay-Sachs’ disease -HexosaminidasePompe’s disease -GlucosidaseGalactosialidosis Neuraminidase + -GalactosidaseGaucher’s disease -Glucocererosidase

I-Cell disease

Clinical defects - Severe skeletal and neurological defects. Retardationof 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: DAGPA

Yeast 2%

PEPS

Other Lipids: Sterols(10-30%) + Sphingolipids (10-25%)

5% 10% 20%

PC

40-50%

PI

10-15%

PIPs

< 0.5%

Human(brain)

25-30% 20% 5% 5-10% 1% < 0.5%

Rare SignalingLipids

5%

Core Components in Membrane Transport

Donor Acceptor

1 Coats2 Cargo3 Vesicle Fission

1 SNARE2 Tether3 Vesicle Fusion

Combinatorial Code of Organelle Surface Tags - Define Identity and Function -

Lipid Code:

Rab Code:

Compartment: PM LysosomeEndosomeGolgi

Stable TMD

Tra

nsi

ent

lab

ile

PI4P PI(4,5)P2 PI3P PI(3,5)P2

Ypt31 (Rab11)

Sec4(Rab8)

Vps21(Rab5)

Ypt7 (Rab7)

SNARE Code: Sso1/2 Pep12 Vam3Tlg2

- Effector Proteins -

Combinatorial Trafficking Code in Membrane Sorting

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

protein-protein

protein-lipid

Inputs:

Effectors

Output:

vesicle buddingmembrane fusion

Sortingeffector

proteintarget

lipidtarget localization

Conserved Codes in Membrane Trafficking

GolgiComplex

EndosomeSystem

Lysosome/Vacuole

ER

Plasma Membrane

1

2

3

4

5

TetherCoat SNARE

1

2

3

4

5

COP II TRAPP Sed5

Clathrin ?

Exocyst Sso1/2

Clathrin HOPS Tlg1/2Pep12

Clathrin EEA1 Pep12

? HOPS Vam3

Retro-mer

GARP/VTF

Tlg1/2

6

Organelle Identity: Cracking the Code

Coats,Adaptors,+ Tethers

Lipid Code GTPase Code

UbPI3P PI4P (3,5)P2 (4,5)P2 Arf Rab5 Rab7

EEA1/Vac1 √ √

HRS/Vps27 √ √

ESCRT-II √ √

Retromer √

AP-1 √ √

FAPP-1 √ √

AP-2 √

Epsin √ √

Atg18 √

En

do

so

me

Go

lgi

√PM

Va

c

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

PI(4,5)P2-PM

Yeast Mammals

PI(3,5)P2

PI4P

PI3P

PI(4,5)P2

GFP-2xPH(PLC) GFP-PH(PLC)

N

N

N

GFP-2xFYVE(EEA1)GFP-FYVE(EEA1)PI3P-

Endososmes

PI4P-Golgi

GFP-PH (FAPP1) GFP-PH (FAPP1)

(CHO cell images, De Camilli lab, 2006)

Meyer lab, 1998Varnai & Balla, 1998Emr lab, 2002

Levine & Munro, 2002Emr lab, 2002

Emr lab, 1998Stenmark lab, 1998Corvera lab, 1998

PI(4,5)P2-PM

PI4P-Golgi

PI3P-endosome

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

•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

PIP’s as Spatial Membrane-Specific Tags

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

EEA1 (FYVE)HRS (FYVE)

PI3P

Vam7 (PX)SNX (PX)

FAPP1 (PH)Osh2 (PH)

PI4P

AP-1

Atg18 (WD-40)

PI(3,5)P2

Dynamin (PH)

PI(4,5)P2

Golgi:Endosome: Lysosome: Plasma membrane:

Epsin (ENTH)HIP1 (ANTH)

AP-2AP-180 (ANTH)

Examples of Modular Lipid Binding Domains

Domain

PH

FYVE

PX

ENTH

C1

C2

Lipid Target

PI4P + PIP2 + PIP3

PI3P

PI3P + PIP2

PIP2

DAG

PIP’s + PS

Yeast

30 genes

5 genes

15 genes

8 genes

1 gene

11 genes

Humans

223 genes

30 genes

34 genes

16 genes

88 gene

200 genes

Human Diseases Linked to PI Metabolism Pathways

GenePIK3CA

hVPS34

PIKfyve

EnzymeClass I PI 3-K

Class III PI 3-K

PI3P 5-Kinase

ProductPI(3,4,5)P3

PI3P

PI(3,5)P2

DiseaseCancer

Bipolar disorder

Francois-Neetenscornea dystrophy

Kinases:

GeneMTM1

PTEN

SHIP2

OCRL!

Enzymemyotubularin

3-phosphatase

5-phosphatase

5-phosphatase

SubstratePI3P

PI(3,4,5)P3

PI(3,4,5)P3

PI(4,5)P2

DiseaseCharcot-Marie-Tooth

Cancer

Type 2 Diabetes

Lowe’s syndrome

Phosphatases:

GeneSapM

SigD/SopB

Enzyme3-phosphatase

4-Pase/PPIPase

SubstratePI3P

PI(4,5)P2

PathogenM. tuberculosis

Salmonella

Pathogenesis:

Rab GTPase Cycle in Membrane Transport

GAPP

GTP hydrolysis

GDI

Membrane Traffic

EffectorsRab

GTPRabGDP

GEF

GTP

Nucleotide exchangeGDP

Regulatory Cycles in Membrane Trafficking

Membrane Transport

PI Kinase

Phosphatase

tSN-P04

Kinase

Phosphatase

GEF

GAP

RabGDP

PI

Ub

Ub Ligase

De-Ub

RabGTPaseCycle

PI Cycle

Ubiquitin Cycle

SNARECycle

PIP

SNAREs

RabGTP

Ub-Lys

“Network of Regulation”

Molecular Shape of Lipids Influences Membrane Curvature  

QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.

(invertedcone)

(conical,cylinder)

(cone)LPA, LPC PC PA, PE

Temporal Order of Clathrin-Mediated Endocytic Intermediates

PI(4,5)P2

PIPK-

Recruitment of Clathrin Assembly Factors

AP-2*AP180A,B*Eps15Clathrin

Hip1R*Epsin*

MembraneCurvature

Amphiphysin2*Endophilin

Membrane Restriction/FissionVesicle Release

Dynamin*Actin

polymerization

PI(4,5)P2-binding Proteins*PI(4,5)P2

Conner and Schmid, Nature 2003

Adapted from Conner and Schmid, Nature 2003

PI(4,5)P2 Metabolism Controls Multiple Endocytic Intermediates

Membrane curvature generation

PI(4,5)P2

PIPK-

Stage 1Recruitment of Clathrin

Assembly Factors

AP-2*AP180A,B*Epsin*Clathrin

Eps15Hip1R*

Amphiphysin*

Stage 2MembraneCurvature

Dynamin*Endophilin

Stage 3Membrane Restriction/Fission

Vesicle Release

ActinPolymerization*

SynaptojaninAuxilinHsc70

Stage 4Vesicle

Uncoating

PI(4,5)P2 hydrolysis

?*Factors Regulated by PIP2

?

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?