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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.

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slide1

Lecture 4

PI signaling and the

puzzle of Vesicle Identity

slide2

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.

slide3

Regulation of

Size, Shape,

Number and

Function of

Each Organelle

Endosome

-Organelle Systems-

slide4

3

4

OH

HO

OH

5

HO

P

3

3

3

3

4

4

4

4

3

HO

HO

OH

4

OH

PI-Kinase

OH

o

o

OH

OH

OH

OH

HO

HO

OH

o

5

5

5

5

o

OH

3

HO

4

P

P

P

P

P

P

P

P

P

P

P

P

HO

HO

HO

HO

5

P

P

P

P

HO

HO

P

HO

P

PIP

OH

HO

o

o

o

o

o

o

o

o

5

o

o

o

o

o

o

o

o

HO

o

o

o

o

P

o

o

o

o

o

o

o

o

PI-Phosphatase

The Phosphoinositide Cycle

phosphatidylinositol

phosphoinositides

PI

“Spatial and Temporal Control of Cell Signaling”

“PIP’s as Transient Second Messengers”

phosphoinositide cycle in cell signaling

Effector

P

HO

OH

PI-Kinase

HO

OH

HO

P

P

o

o

o

o

Effectors

o

o

o

o

O

O

O

O

PIP

FYVE (5)

PX (15)

PH (30)

ENTH (8)

HO

P

6

2

OH

1

4

5

3

OH

HO

OH

PI-Phosphatase

Phosphoinositide Cycle in Cell Signaling

PI

PI

“PIP’s Program Transport Activity via PIP Effectors”

slide6

PI(4,5)P2

(PI3P)

PI4,5P2

PI(4,5)P2

(TGN)

PI(4,5)P2

GFP-FYVE

Sec7-GFP

(PI4P)

FAPP-DsRed

Fab1

GFP-Atg18

(PI3,5P2)

PI3P

PI4P

Vps34

PI4P

(PI4P)

PI4P

Nomarski

FM4-64

(PI3P)

FAPP-DsRed

GFP-FYVE

PI3,5P2

FM4-64

Mss4

PI3P

Pik1

PI3P

merge

merge

merge

merge

merge

PI Signaling in Membrane Trafficking Pathways

PM

Endosomal

System

Golgi

Complex

ER

Lysosome/

Anterograde transport

Vacuole

Retrograde transport

slide7

PI3P

FYVE

DsRed

PI(4,5)P2

PH

GFP

vacuole

CMAC

Chris Stefan

Jon Audhya

slide8

Amplification of Gene Complexity from Yeast to Human

Ubiquitin

E1

E2

E3

DUBs

Yeast

1 gene

13 genes

45 genes

17 genes

Humans

2 genes

> 50 genes

> 500 genes

90 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

Kinases

PI lipid

Tyr

Ser/Thr

6 genes

> 4 genes

125 genes

20 genes

> 90 genes

> 490 genes

Phosphatases

PI lipid

Protein

7 genes

> 30 genes

25 genes

105 genes

slide9

Yeast

Human

PIPs:

4

7

PIPs

RabGEF

PI-Kinase

Rabs

Rabs:

11

63

Effectors

>100

>400

Effectors:

Membrane Traffic

Hierarchy of Organelle Identity Codes

Global

Specific

slide10

Inherited Lysosomal Storage Diseases

Disorder

Deficient Hydrolase(s)

I-Cell disease Multiple enzymes

Tay-Sachs’ disease b-Hexosaminidase

Pompe’s disease a-Glucosidase

Galactosialidosis Neuraminidase + b -Galactosidase

Gaucher’s disease 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
Bulk Lipid Composition of Cell Membranes

Lipids:

PA

DAG

PS

PE

PC

PIPs

PI

Yeast

2%

5%

10%

20%

40-50%

<0.5%

10-15%

1%

5%

20%

25-30%

5-10%

<0.5%

Human

(brain)

5%

Rare Signaling

Lipids

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

slide12

Core Components in Membrane Transport

Acceptor

Donor

1 SNARE

2 Tether

3 Vesicle Fusion

1 Coats

2 Cargo

3 Vesicle Fission

combinatorial code of organelle surface tags define identity and function

Golgi

PM

Endosome

Lysosome

PI4P

PI(4,5)P2

PI3P

PI(3,5)P2

Transient labile

Ypt31

(Rab11)

Sec4

(Rab8)

Vps21

(Rab5)

Ypt7

(Rab7)

Tlg2

Sso1/2

Pep12

Vam3

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

Compartment:

Lipid Code:

Rab Code:

- Effector Proteins -

Stable

TMD

SNARE Code:

combinatorial trafficking code in membrane sorting
Combinatorial Trafficking Code in Membrane Sorting

Inputs:

Output:

protein-protein

vesicle budding

membrane fusion

Effectors

protein-lipid

Sorting

effector

protein

target

lipid

target

localization

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

slide15

4

5

2

1

5

4

2

1

6

3

3

Conserved Codes in Membrane Trafficking

Plasma Membrane

Coat

Tether

SNARE

Sed5

COP II

TRAPP

Endosome

Clathrin

?

Golgi

Exocyst

Sso1/2

System

Complex

Tlg1/2

Pep12

Clathrin

HOPS

Clathrin

Pep12

EEA1

Lysosome/

ER

Vacuole

?

HOPS

Vam3

GARP/

VTF

Retro-

mer

Tlg1/2

key roles for pips in membrane transport
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)
slide18

Localization of PIP Isoforms is Conserved

-PIPs Act as Spatial Tags in Organelle Identity

Mammals

Yeast

GFP-2xPH(PLC)

GFP-PH(PLC)

PI(4,5)P2-

PM

N

Meyer lab, 1998

Varnai & Balla, 1998

Emr lab, 2002

PI(3,5)P2

PI3P

GFP-PH (FAPP1)

GFP-PH (FAPP1)

PI(4,5)P2

PI4P-

Golgi

N

PI4P

Levine & Munro, 2002

Emr lab, 2002

GFP-2xFYVE(EEA1)

GFP-FYVE(EEA1)

PI3P-

Endososmes

N

Emr lab, 1998

Stenmark lab, 1998

Corvera lab, 1998

(CHO cell images, De Camilli lab, 2006)

slide19

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)

PI3P-endosome

  • PI Pases inactivate/turnover PIP’s at inappropriate

membrane sites and terminate PIP signaling

phosphoinositides as spatial and temporal regulators of membrane trafficking and organelle identity
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
PI-Binding Domains in Membrane Transport Proteins

Lysosome:

Plasma membrane:

Endosome:

Golgi:

PI(4,5)P2

PI(3,5)P2

PI3P

PI4P

EEA1 (FYVE)

HRS (FYVE)

Dynamin (PH)

FAPP1 (PH)

Osh2 (PH)

Atg18 (WD-40)

Epsin (ENTH)

HIP1 (ANTH)

Vam7 (PX)

SNX (PX)

AP-1

AP-2

AP-180 (ANTH)

slide22

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

Examples of Modular Lipid Binding Domains

slide23

Human Diseases Linked to PI Metabolism Pathways

Kinases:

Gene

PIK3CA

hVPS34

PIKfyve

Enzyme

Class I PI 3-K

Class III PI 3-K

PI3P 5-Kinase

Product

PI(3,4,5)P3

PI3P

PI(3,5)P2

Disease

Cancer

Bipolar disorder

Francois-Neetens

cornea dystrophy

Phosphatases:

Gene

MTM1

PTEN

SHIP2

OCRL!

Enzyme

myotubularin

3-phosphatase

5-phosphatase

5-phosphatase

Substrate

PI3P

PI(3,4,5)P3

PI(3,4,5)P3

PI(4,5)P2

Disease

Charcot-Marie-Tooth

Cancer

Type 2 Diabetes

Lowe’s syndrome

Pathogenesis:

Gene

SapM

SigD/SopB

Enzyme

3-phosphatase

4-Pase/PPIPase

Substrate

PI3P

PI(4,5)P2

Pathogen

M. tuberculosis

Salmonella

rab gtpase cycle in membrane transport

GEF

GDP

GDP

Nucleotide exchange

GDI

GTP

GTP

Effectors

Rab

Rab

Membrane

Traffic

GTP hydrolysis

GAP

P

Rab GTPase Cycle in Membrane Transport
slide25

Regulatory Cycles in Membrane Trafficking

GEF

PI Kinase

Rab

GTPase

Cycle

PI

Cycle

RabGDP

RabGTP

PIP

PI

GAP

Phosphatase

Membrane

Transport

Kinase

Ub Ligase

SNARE

Cycle

Ubiquitin

Cycle

tSN-P04

SNAREs

Ub-Lys

Ub

Phosphatase

De-Ub

“Network of Regulation”

slide26

LPA, LPC

PC

PA, PE

(inverted

cone)

(conical,

cylinder)

(cone)

Molecular Shape of Lipids Influences Membrane Curvature  

slide28

Recruitment of Clathrin

Assembly Factors

Membrane

Curvature

Membrane Restriction/Fission

Vesicle Release

AP-2*

AP180A,B*

Eps15

Clathrin

Hip1R*

Epsin*

Amphiphysin2*

Endophilin

Dynamin*

Actin

polymerization

Temporal Order of Clathrin-Mediated Endocytic Intermediates

PIPK-g

PI(4,5)P2

PI(4,5)P2

PI(4,5)P2-binding Proteins*

Conner and Schmid, Nature 2003

slide29

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

Stage 1

Recruitment of Clathrin

Assembly Factors

Stage 3

Membrane Restriction/Fission

Vesicle Release

Stage 2

Membrane

Curvature

Stage 4

Vesicle

Uncoating

AP-2*

AP180A,B*

Epsin*

Clathrin

Synaptojanin

Auxilin

Hsc70

Eps15

Hip1R*

Amphiphysin*

Dynamin*

Endophilin

Actin

Polymerization*

PI(4,5)P2

?

?

PIPK-

PI(4,5)P2

hydrolysis

*Factors Regulated by PIP2

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