| The selective transport of proteins between organelles is the central process in the organization of membrane compartments. This process is largely mediated by the budding of transport vesicles from a donor compartment followed by the vectoral trafficking to and fusion with an acceptor compartment. Considerable effort has been exerted to uncover the molecular mechanisms underlying this process. A set of protein families known as SNAREs is at the crux of vesicle docking and/or fusion.; My thesis involved first the cloning of a novel SNARE we called syntaxin 6, and its characterization. By raising both polyclonal and monoclonal antibodies against syntaxin 6, we determined that it was widely expressed in most rat tissues and was localized primarily to the trans-Golgi network (TGN). In addition some syntaxin 6 is present on clathrin-coated vesicles near the TGN and on clear vesicles near late endosomes. We determined several of the proteins that syntaxin 6 interacted with both in vivo and in vitro. Immunoprecipitations with anti-syntaxin 6 were greatly enriched for VPS45, the yeast ortholog of which is essential for trafficking to the lysosome, likely through the late endosome. Thus our hypothesis is that syntaxin 6 mediates TGN to late endosome trafficking.; To further our understanding of the role of SNAREs in the organization of membrane compartments, we performed an extensive search of the EST (expressed sequence tag) database, and uncovered 15 novel SNAREs. This expanded the SNARE family from 17 to 32 members. Our lab, including myself, proceeded to clone the full-length cDNA of many of these novel genes and characterize their expression pattern and subcellular localization. This data supported the hypothesis that there is a distinct set of SNAREs for each intracellular trafficking pathway. A subsequent search of the human, drosophila, and C. elegans genomes was performed to further determine the extent of the SNARE families. Interestingly while the number of SNAREs remained essentially unchanged in yeast, drosophila, and C. elegans, that number expanded significantly in humans. In addition further genomic analysis allowed us to more finely subcategorize the SNARE family than had been done previously. |
