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Characterization of Drosophila SNAREs, alpha-SNAP and NSFs

Mohtashami, M. (Program in Cell Biology, Hospital for Sick Children, University of Toronto, Canada)
Peng, X.R. (Program in Cell Biology, Hospital for Sick Children, University of Toronto, Canada)
Trimble, W.S. (Program in Cell Biology, Hospital for Sick Children, University of Toronto, Canada)

Contact Person: Mahmood Mohtashami (moody@zoo.utoronto.ca)


SNARE proteins are integral membrane proteins that mediate vesicle and target membrane fusion through specific and stable interactions. In order for membrane fusion to occur, it is hypothesized that pre-existing stable complexes of SNARE proteins within vesicle or target membranes first be uncoupled in order to permit the formation of new SNARE complexes between the two membranes. This uncoupling process is thought to be mediated through the enzymatic activity of the protein NSF that binds to the SNARE complex via the adaptor protein alpha-SNAP. Both NSF and alpha-SNAP were previously shown to be required for membrane fusion to occur. To investigate this mechanism fully, and to apply powerful genetic approaches to its analysis, we decided to characterize the involvement of SNARE proteins in mechanisms controlling membrane fusion in the fruitfly Drosophila melanogaster. We first examined the SNARE complexes and discovered that, similar to their mammalian counterparts, Drosophila SNARE complexes are stable in the presence of 1% SDS. We then cloned the Drosophila homologs of alpha-SNAP and NSF. a-SNAP was found to have 62% identity with mammalian alpha-SNAP. This surprising degree of homology led us to test whether the Drosophila alpha-SNAP (d-alpha-SNAP) would interact with the mammalian SNARE protein, syntaxin-1A, which is also highly homologous to the Drosophila syntaxin. However, unlike its mammalian counterpart, d-alpha-SNAP failed to bind the mammalian syntaxin. NSF was found to be encoded by two highly homologous isoforms, dNSF-1 and -2. This is the first observation of presence of two isoforms of NSF in a single species. Interestingly, dNSF-1 maps to the locus of a paralytic mutation, called comatose. We sequenced the cDNA of a temperature-sensitive allele of comatose (TP7) and identified a single point mutation responsible for the phenotype. Since the presence of dNSF-2 does not compensate for the functional loss of dNSF-1, it suggests that each dNSF isoform may have specialized properties. We are c urrently exploring this possibility by studying the biochemical properties of dNSF-1 and -2 and their interaction with other proteins involved in membrane fusion.

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Presentation Number SAmohtashami0771
Keywords: SNARE, Drosophila, alpha-SNAP, NSF, exocytosis

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Mohtashami, M.; Peng, X.R.; Trimble, W.S.; (1998). Characterization of Drosophila SNAREs, alpha-SNAP and NSFs. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/klip/mohtashami0771/index.html
© 1998 Author(s) Hold Copyright