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Lecture 4 PI signaling and the puzzle of Vesicle Identity

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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Lecture 4 PI signaling and the puzzle of Vesicle Identity

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  1. 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-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.

  3. Regulation of Size, Shape, Number and Function of Each Organelle Endosome -Organelle Systems-

  4. 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”

  5. 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”

  6. 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

  7. PI3P FYVE DsRed PI(4,5)P2 PH GFP vacuole CMAC Chris Stefan Jon Audhya

  8. 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

  9. 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

  10. 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.

  11. 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%)

  12. Core Components in Membrane Transport Acceptor Donor 1 SNARE 2 Tether 3 Vesicle Fusion 1 Coats 2 Cargo 3 Vesicle Fission

  13. 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:

  14. 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

  15. 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

  16. Endosome Golgi PM Organelle Identity: Cracking the Code √ Vac

  17. 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)

  18. 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)

  19. 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

  20. 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”

  21. 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)

  22. 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

  23. 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

  24. GEF GDP GDP Nucleotide exchange GDI GTP GTP Effectors Rab Rab Membrane Traffic GTP hydrolysis GAP P Rab GTPase Cycle in Membrane Transport

  25. 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”

  26. LPA, LPC PC PA, PE (inverted cone) (conical, cylinder) (cone) Molecular Shape of Lipids Influences Membrane Curvature  

  27. 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

  28. 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

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