Sorting of the v-SNARE VAMP7 in Dictyostelium discoideum: a role for more than one Adaptor Protein (AP) complex |
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Authors: | Bennett Nelly Letourneur François Ragno Michel Louwagie Mathilde |
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Institution: | a Commissariat à l'Energie Atomique, iRTSV, Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, CNRS UMR5092, Université Joseph Fourier, 38054 Grenoble, France b Institut de Biologie et Chimie des Protéines, UMR5086-CNRS/Université Lyon I, IFR 128 BioSciences Lyon-Gerland, 69367 Lyon, France c Commissariat à l'Energie Atomique, iRTSV, Laboratoire d'Etude de la Dynamique des Protéomes, INSERM U880, Université Joseph Fourier, 38054 Grenoble, France |
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Abstract: | Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptors (SNAREs) participate in the specificity of membrane fusions in the cell. The mechanisms of specific SNARE sorting are still however poorly documented. We investigated the possible role of Adaptor Protein (AP) complexes in sorting of the Dictyostelium discoideum v-SNARE VAMP7. In live cells, GFP-VAMP7 is observed in the membrane of endocytic compartments. It is also observed in the plasma membrane of a small proportion of the cells. Mutation of a potential dileucine motif dramatically increases the proportion of cells with GFP-VAMP7 in their plasma membrane, strongly supporting the participation of an AP complex in VAMP7 sorting to the endocytic pathway. A partial increase occurs in knockout cells for the medium subunits of AP-2 and AP-3 complexes, indicating a role for both AP-2 and AP-3. VAMP7, as well as its t-SNAREs partners syntaxin 8 and Vti1, are co-immunoprecipitated with each of the medium subunits of the AP-1, AP-2, AP-3 and AP-4 complexes. This result supports the conclusion that VAMP7 directly interacts with both AP-2 and AP-3. It also raises the hypothesis of an interaction with AP-1 and AP-4. GFP-VAMP7 is retrieved from the endocytic pathway at and/or before the late post-lysosomal stage through an AP-independent mechanism. |
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Keywords: | SNARE sorting VAMP7 Adaptor Protein Dileucine motif Endosomes Lysosomes Dictyostelium |
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