PI signaling and the

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Lecture 4 PI signaling and the puzzle of Vesicle
Identity
2
SMAP2, a Novel ARF GTPase-activating Protein,
Interacts with Clathrin and Clathrin Assembly
Protein and Functions on the AP-1positive 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-1dependent manner. Thus, the
SMAP gene family constitutes an important ArfGAP
subfamily, with each SMAP member exerting both
common and distinct functions in vesicle
trafficking.
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Regulation of Size, Shape, Number and Function
of Each Organelle
Endosome
-Organelle Systems-
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The Phosphoinositide Cycle
phosphatidylinositol
phosphoinositides
PI
Spatial and Temporal Control of Cell
Signaling PIPs as Transient Second Messengers
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Phosphoinositide Cycle in Cell Signaling
PI
PI
PIPs Program Transport Activity via PIP
Effectors
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PI Signaling in Membrane Trafficking Pathways
PM
Endosomal
System
Golgi
Complex
ER
Lysosome/
Anterograde transport
Vacuole
Retrograde transport
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(No Transcript)
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Amplification of Gene Complexity from Yeast to
Human
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Hierarchy of Organelle Identity Codes
Global
Specific
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Inherited Lysosomal Storage Diseases
Disorder
Deficient Hydrolase(s)
I-Cell disease Multiple enzymes Tay-Sachs
disease b-Hexosaminidase Pompes
disease a-Glucosidase Galactosialidosis Neuram
inidase b -Galactosidase Gauchers 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.
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Bulk Lipid Composition of Cell Membranes
Lipids
DAG
PA
PE
PS
PC
PIPs
PI
Yeast
2
5
10
20
40-50
lt 0.5
10-15
25-30
20
5
5-10
1
lt 0.5
Human (brain)
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Rare Signaling Lipids
Other Lipids Sterols(10-30) Sphingolipids
(10-25)
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Core Components in Membrane Transport
Acceptor
Donor
1 SNARE 2 Tether 3 Vesicle Fusion
1 Coats 2 Cargo 3 Vesicle Fission
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Combinatorial Code of Organelle Surface Tags -
Define Identity and Function -
Compartment
Lipid Code
Rab Code
- Effector Proteins -
Stable TMD
SNARE Code
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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 P
I(4)P TGN/EE Vac1/EEA1 Rab PI(3)P Endosome Vps
27/Hrs Ub PI(3)P Endosome Retromer cargo PI(3
)P Endosome Vam7 SNARE PI(3)P Vacuole
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Conserved Codes in Membrane Trafficking
Plasma Membrane
Tether
Coat
SNARE
Sed5
COP II
TRAPP
Endosome
Clathrin ?
Golgi
System
Exocyst
Sso1/2
Complex
Tlg1/2 Pep12
Clathrin
HOPS
Clathrin
Pep12
EEA1
Lysosome/
ER
Vacuole
?
HOPS
Vam3
GARP/ VTF
Retro- mer
Tlg1/2
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Organelle Identity Cracking the Code
Coats, Adaptors, Tethers Lipid Code Lipid Code Lipid Code Lipid Code GTPase Code GTPase Code GTPase Code Ub
Coats, Adaptors, Tethers PI3P PI4P (3,5)P2 (4,5)P2 Arf Rab5 Rab7 Ub
EEA1/Vac1 v v
HRS/Vps27 v v
ESCRT-II v v
Retromer v
AP-1 v v
FAPP-1 v v
AP-2 v
Epsin v v
Atg18 v
v
Vac
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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)

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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)
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PIPs 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
  PIPs

PI4P-Golgi

• Membrane-restricted PIPs program the transport
  activity
• of membrane compartments by recruiting/activating
  
• specific effector proteins (PH, FYVE, PX, ENTH
  domains)

PI3P-endosome

• PI Pases inactivate/turnover PIPs at
  inappropriate
• membrane sites and terminate PIP signaling

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Phosphoinositides as Spatial and Temporal
Regulators of Membrane Trafficking and Organelle
Identity

• Compartment specific localization of PI kinases
  leads to
• restricted synthesis/localization of PIPs -
  Spatial identity tags

• Membrane-restricted PIPs 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 --gt PI3,5P2 for
  retrograde)

• PI-Pases terminate PIP signaling and inactivate
  PIPs at
• inappropriate membrane sites

Location - Location - Location
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Phosphoinositide Cycle in Membrane Trafficking
PI
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PI-Binding Domains in Membrane Transport Proteins
Lysosome
Plasma membrane
Golgi
Endosome
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)
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Examples of Modular Lipid Binding Domains
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Human Diseases Linked to PI Metabolism Pathways
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Rab GTPase Cycle in Membrane Transport
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PI Lipids and Rabs Program Transport Activity
PI3P
PI Kinase
Rab GEF
Rab5
EEA1
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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
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LPAT
cytosolic leaflet
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Molecular Shape of Lipids Influences Membrane
Curvature  
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Molecular Shape of Lipids Influences Membrane
Curvature  
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Multiple Pathways for Endocytic Uptake in
Mammalian Cells
Conner and Schmid, Nature 2003
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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
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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