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47 Isoform specific properties of Dynamin 2 Sylvia Neumann 1 , Ya Wen Liu 1 , Thomas Pucadyil 1 , Rajesh Ramachandran 2 , Sharmistha Acharya 1 , Vasyl Lukiyanchuk 1 , Sandra Schmid 1 . The Scripps Research Institute, La Jolla, CA 92037 1 , Case Western University, Cleveland, OH 44106 2 Abstract: Dynamin is best studied for its role in clathrin-mediated endocytosis (CME) and most of the studies have employed the neuronal isoform, dynamin-1 (Dyn1). However, there is a second major, ubiquitously expressed isoform Dyn2 in mammals. Contrary to previous assumptions, findings in knockout mice and cell lines have suggested that Dyn1 and 2 are not functionally redundant for CME either at the synapse (Ferguson et al., 2007) or in non-neuronal cells (Liu et al., 2008). We have performed comparative analyses of dynamin’s in vitro activities to define the biochemical differences that account for these differential in vivo activities. Using an in vitro fission assay (Pucadyil et al., 2008) we found that Dyn1 could efficiently catalyze membrane fission and vesicle formation from planar membranes, while Dyn2 could not. However if applied to membrane tethers, which resemble highly curved membranes formed at the necks of clathrin coated pits, both isoforms were capable of scission. While basal GTPase activities were identical, liposome stimulated GTPase activities were distinct. Dyn2 GTPase activity was only stimulated in the presence of highly curved membranes, whereas the stimulated GTPase activity of Dyn1 was less sensitive to membrane curvature. These findings suggest that Dyn2 is less effective in membrane binding and/or curvature generation than Dyn1, which is a powerful curvature generator (Ramachandran et al., 2009). This was confirmed by direct measurements of these activities. Using Dyn1/Dyn2 chimeras, we identified the PH domain as being responsible for the differential biochemical properties of the two isoforms. The relative abilities of these chimeras to reconstitute CME are currently being tested in non-neuronal cells. We speculate that these biochemical differences reflect the differential requirements for Dyn1 in mediating rapid endocytosis at the synapse vs. the role of Dyn2 in regulating slower, clathrin-mediated endocytic events in non-neuronal cells.
48 Phosphatidylinositol 4-phosphate controls both membrane recruitment and a regulatory switch of the Rab GEF, Sec2p Peter Novick 1 , Emi Mizuno-Yamasaki 1 , Martina Medkova 2 , Jeff Coleman 2 . Department of Cellular and Molecular Medicine, UCSD, La Jolla, CA 1 , Department of Cell Biology, Yale University School of Medicine, New Haven, CT 2 Abstract: Sec2p is the guanine nucleotide exchange factor (GEF) that activates the Rab GTPase Sec4p on secretory vesicles. Sec2p also binds a Rab acting earlier in the secretory pathway, Ypt32p-GTP, forming a RabGEF cascade. Ypt32p and the Sec4p effector Sec15p (a component of the exocyst complex) compete for binding to Sec2p. Indeed Ypt32p initially recruits Sec2p, but subsequently allows a handoff of active Sec2p/Sec4p to Sec15p. Intriguingly, Golgi-associated phosphatidylinositol 4-phosphate (PI4P) works together with Ypt32-GTP in this context. PI4P inhibits Sec2p-Sec15p interactions, promoting recruitment of Sec2p by Ypt32p as secretory vesicles form. However, PI4P levels appear to decline as vesicles reach secretory sites, allowing Sec15p to replace Ypt32p as vesicles mature. In this way, the regulation of PI4P levels may switch Sec2p/Sec4p function during vesicle maturation, from a RabGEF recruitment cascade involving Ypt32p, to an effector positive feedback loop involving Sec15p.
Page 1 and 2:
October 28 - October 31, 2010 Bioch
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Symposium Agenda Thursday, October
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12:10 p.m. - 4:00 p.m. Lunch and Fr
Page 7 and 8: 4:50 p.m. - 5:00 p.m. Discussion 5:
Page 9 and 10: Poster Presentations Last Names A -
Page 11 and 12: The Role of Ceroid Lipofuscinosis N
Page 13 and 14: A novel Golgi-localized protein inv
Page 15 and 16: 2 The dynamics of SNARE complexes i
Page 17 and 18: 4 Significant divergence in mammali
Page 19 and 20: 6 The Tumoricidal Action of the Ade
Page 21 and 22: 8 LMTK2 Regulates CFTR Recycling in
Page 23 and 24: 10 Sorting in the Golgi Apparatus C
Page 25 and 26: 12 Negative regulation of the endoc
Page 27 and 28: 14 Mechanistic details of sorting n
Page 29 and 30: 16 An ESCRTs View of Receptor Endoc
Page 31 and 32: 18 GOLPH3 Bridges PtdIns(4)P and My
Page 33 and 34: 20 Novel fluorescent probes to stud
Page 35 and 36: 22 Regulation of Ligand-Independent
Page 37 and 38: 24 A Fifth Adaptor Protein Complex
Page 39 and 40: 26 CATCHR-Family Tethering Complexe
Page 41 and 42: 28 Rab7 localization and activity a
Page 43 and 44: 30 The Drosophila Golgi transmembra
Page 45 and 46: 33 Cbl Amino Acids at a Putative Di
Page 47 and 48: 35 The role of COG subcomplexes in
Page 49 and 50: 37 Mechanism of secretory cargo sor
Page 51 and 52: 39 Understanding the mechanism of G
Page 53 and 54: 41 Mechanisms for selective activat
Page 55 and 56: 44 A novel coat complex traffics me
Page 57: 46 Membrane trafficking in cytokine
Page 61 and 62: 50 Control of Golgi function by Rab
Page 63 and 64: 52 Adenosine mediated modulation of
Page 65 and 66: 54 Syp1 regulation of actin polymer
Page 67 and 68: 56 Rap1A: A Novel Interactor Involv
Page 69 and 70: 58 Ctage5 is involved in the endopl
Page 71 and 72: 60 A vesicle carrier that mediates
Page 73 and 74: 62 Structural Insights into Dynamin
Page 75 and 76: 64 Identification and interaction m
Page 77 and 78: 66 hRME-6, a rab5GEF that integrate
Page 79 and 80: 68 Assembly of caveolin-1 and cavin
Page 81 and 82: 70 Opposing roles of Annexin A1 and
Page 83 and 84: 72 The role of SNX-BAR proteins in
Page 85 and 86: 74 TRAPP is required for ER exit of
Page 87 and 88: 76 A structural analysis of the ER
Page 89 and 90: 78 Cell cycle-regulated Golgi stack
Page 91 and 92: 80 Rab 14 regulates tight junction
Page 93 and 94: 82 Dissecting the roles of O-glycos
Page 95 and 96: Author Index - A- Abay, S., 33 Acha
Page 97 and 98: Marsico, G., 83 Mattera, R., 11 Max
Page 99: 2011 ASBMB Special Symposia Series
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