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63 Characterization of the endocytic pathway of the cytokine MIF and its receptors Verena Schwartz 1 , Hongqi Lue 1 , Joanna Korbiel 1 , Jürgen Bernhagen 1 . Department of Biochemistry and Molecular Cell Biology, UKA, RWTH Aachen University 1 Abstract: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that plays a role in innate and adaptive immunity. Depending on the cellular context and disease state, MIF signaling is mediated by its receptors CXCR2, CXCR4 and/or CD74. Although it is known that MIF is endocytosed, the exact mechanism has remained unknown. In exploring the mechanism of MIF endocytosis with pathway-specific inhibitors and receptor overexpression and blockade approaches, we identified a clathrin endocytosis pathway as the main track for MIF internalization. Monodansylcadaverine (MDC) and chlorpromazine (CPZ) were used to interfere with this pathway. The role of the GTPase dynamin was analyzed in experiments with a cellpermeable inhibitor of dynamin, Dynasore, and with a dominant-negative dynamin construct. In the presence of MDC, fibroblasts and HEK293 cells showed an impaired endocytosis rate of Alexa546-MIF. Dynasore led to the largest margin in reduction. LDL endocytosis (which is clathrin-mediated) served as a control and was inhibited by MDC or Dynasore to a similar extent as MIF. Next, MIF endocytosis was compared to that of transferrin, acetylated LDL, and cholera toxin B (the latter internalized by a clathrin-independent pathway) by coloca-lization studies. MIF internalization clearly resembled that of LDL. In an attempt to identify the receptors involved in MIF endocytosis, we first focussed on CD74 and CXCR4 which form a heteromeric complex. Uptake of MIF was analyzed in HEK293 and HeLa cells which don’t express CD74. Ectopic overexpression of CD74 led to an acceleration of MIF endocytosis while blockade of CXCR4, which is endogenously expressed on these cells, with the inhibitor AMD3100 led to a 20% reduction of MIF endocytosis in HEK293-CD74 transfectants, whereas in untransfected cells, which only express CXCR4, a blockade of 40% was observed. This indicates that both CD74 and CXCR4 contribute to MIF endocytosis.
64 Identification and interaction mapping of three novel TRAPP components suggests a distinct organization of the mammalian TRAPP complex P. James Scrivens 1 , Baraa Noueihed 1 , Mihnea Bostina 2 , Isabelle Rouiller 2 , Michael Sacher 1, 2 . Concordia University, Department of Biology, Montreal, Quebec, Canada 1 , McGill University, Department of Anatomy and Cell Biology, Montreal, Quebec, Canada 2 Abstract: The multisubunit tethering complex TRAPP is conserved throughout Eukarya. Saccharomyces cerevisiae TRAPP is organized in discrete subassemblies, TRAPPI and –II, implicated in ER-to- Golgi and late Golgi trafficking, respectively. No such subassemblies have yet been reported in mammalian cells, and little is known about the architecture of mammalian TRAPP. We recently reported a novel TRAPPII-associated protein, YEL048c/TCA17, and its mammalian orthologue TrappC2L (C2L). YEL048c-p/Tca17p and C2L are ancestrally related to Trs20p and TrappC2 (C2). Given that Trs20p is found in both TRAPPI and –II, whereas YEL048c-p/Tca17p appears to be a TRAPPII component, we hypothesized that their mammalian orthologues C2 and C2L might precipitate distinct complexes. Contrary to our hypothesis, we found that TAP-C2 and – C2L complexes appeared indistinguishable by SDS-PAGE and mass spectrometry. Further, both TAP-C2 and –C2L precipitated several novel proteins which we here report as TrappC8, TrappC11, and TrappC12. TrappC8 is the mammalian orthologue of the yeast TRAPP protein Trs85p, whereas TrappC11 and –C12 have no orthologues in S. cerevisiae. RNA interference of any of the novel TRAPP proteins results in Golgi fragmentation, indicating that they are indeed involved in membrane trafficking. Depletion of C11 or C12 resulted in accumulation of VSV-G ts045-GFP in punctae colabeling with ERGIC53, but apposed to Sec31a, suggesting that exit from the ER was not impeded. In order to better understand the organization of mammalian TRAPP, we have mapped all binary interactions between the known and novel TRAPP proteins, and have performed single particle electron microscopy on TAP-C11 complexes precipitated from mammalian cells. What emerges is a portrait of mammalian TRAPP where the fundamental unit is larger than S. cerevisiae TRAPPI, and where functional diversification may be achieved by exchange of individual subunits rather than assembly of discrete subcomplexes.
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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
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4:50 p.m. - 5:00 p.m. Discussion 5:
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Poster Presentations Last Names A -
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The Role of Ceroid Lipofuscinosis N
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A novel Golgi-localized protein inv
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2 The dynamics of SNARE complexes i
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4 Significant divergence in mammali
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6 The Tumoricidal Action of the Ade
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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 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: 62 Structural Insights into Dynamin
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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