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17 A potential role for Rab10 in ER network assembly Amber English 1 , Gia Voeltz 1 . University of Colorado, Boulder, CO 80303 1 Abstract: The Endoplasmic Reticulum (ER) is a membrane bound organelle that includes the nuclear envelope and the peripheral ER. The structure of the ER is quite dynamic; throughout the cell cycle the ER membrane network is constantly rearranging in the cytoplasm using a fusion mechanism that has not been characterized. We are interested in identifying the fusion machinery required for ER network formation and maintenance. To identify the fusion machinery, we are using an ER formation assay derived by fractionation of Xenopus laevis eggs. In this system, ER vesicles fuse to form tubular networks in a GTP-dependent manner. To isolate the GTP-dependent fusion machinery, we purified GTP binding proteins from the ER extract on a GTP-agarose column and then identified the bound proteins by Mass Spectrometry. Several Rab proteins were identified; Rab proteins are known GTPases that are involved in membrane fusion of vesicles. Previous studies have shown that in vitro ER formation is disrupted by the addition of a Rab GDP-dissociation inhibitor (GDI); indicating a Rab could be involved in ER fusion. Of the several Rab GTPases identified only Rab8/10 has no previously characterized function. We demonstrate that Rab 8/10 is tightly associated with the Xenopus ER membrane vesicles but can be dissociated by the addition of recombinant RabGDI. Current in vitro studies are focused on determining if Rab8/10 depletion from ER extracts affects ER network formation. We find that a transient transfection of mCherry-Rab10 (human Rab8/10 homolog) into COS-7 cells co-localizes with an ER marker throughout the ER, consistent with a potential role in ER fusion. Furthermore, transfection of a dominant negative form of Rab10, mCherry-Rab10 (T23N), into COS-7 cells disrupts the ER in a manner consistent with a fusion defect. Current studies are aimed at measuring the effects of Rab10 depletion and overexpression on ER morphology to confirm a role for Rab10 in ER fusion.
18 GOLPH3 Bridges PtdIns(4)P and Myosin18A to Stretch and Shape the Golgi to Promote Vesicle Budding Seth Field 1 , Holly Dippold 1 , Michelle Ng 1 , Suzette Farber-Katz 1 , Sun-Kyung Lee 1 , Monica Kerr 2 , Marshall Peterman 1 , Ronald Sim 1 , Patricia Wiharto 1 , Kenneth Galbraith 1 , Swetha Madhavarapu 2 , Greg Fuchs 3, 4 , Timo Meerloo 3 , Marilyn Farquhar 3 , Huilin Zhou 3, 4 . Endocrinology and Metabolism, University of California, San Diego, La Jolla, CA 92093 1 , Signal Transduction, Beth Israel-Deaconess Medical Center, Boston, MA 02215 2 , Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093 3 , Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA 92093 4 Abstract: Golgi membranes, from yeast to humans, are uniquely enriched in PtdIns(4)P, although the role of this lipid has been poorly understood. Using a proteomic lipid binding screen we discovered that GOLPH3 (yeast VPS74p) is a major direct effector of PtdIns(4)P at the Golgi. Furthermore, we provide overwhelming evidence that GOLPH3 also interacts with MYO18A, providing a link from the Golgi to F-actin. We demonstrate that this linkage is required for normal Golgi morphology, ultrastructure, and vesicle budding for anterograde trafficking. These results can be explained simply by a role for this motor-containing, membrane-binding complex to deliver a tensile force to the Golgi membrane to assist in vesicle abstraction, and that this force consequently helps to flatten the stacks and to stretch the ribbon around the nucleus. The model makes testable predictions that have been experimentally confirmed. GOLPH3 is also the first example of a Golgi protein that is an oncogene, frequently overexpressed in human solid tumors. Our new data addresses the regulation of GOLPH3 function and its role in promoting oncogenic transformation.
Page 1 and 2: October 28 - October 31, 2010 Bioch
Page 3 and 4: Symposium Agenda Thursday, October
Page 5 and 6: 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: 16 An ESCRTs View of Receptor Endoc
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 and 58: 46 Membrane trafficking in cytokine
Page 59 and 60: 48 Phosphatidylinositol 4-phosphate
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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