A SPLICE-ISOFORM OF VESICLE-ASSOCIATED MEMBRANE PROTEIN-1 (VAMP-1) CONTAINS A MITOCHONDRIAL TARGETING SIGNAL

Screening of a library derived from primary human endothelial cells re vealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transpo rt vesicles to their target membrane. We have termed this novel isofor m VAMP-1B and designated the previously described isoform VAMP-1A. VAM P-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-LB) has recently been r eported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as ass essed by PCR. Ln contrast, brain mRNA contained VAMP-1A but no VAMP-1B . The VAMP-1B sequence encodes a protein identical to VAMP-1A except f or the carboxy-terminal five amino acids. VAMP-1 is anchored in the ve sicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. in VAMP-1B the predicted hydrophobic mem brane anchor is shortened by four amino acids, and the hydrophobic seq uence is immediately followed by three charged amino acids, arginine-a rginine-aspartic acid. Transfection of human endothelial cells with ep itope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitocho ndria whereas VAMP-1A was localized to the plasma membrane and endosom e-like structures. Analysis of C-terminal mutations of VAMP-1B demonst rated that mitochondrial targeting depends both on the addition of pos itive charge at the C terminus and a shortened hydrophobic membrane an chor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that go vern protein movement between other organelles of the cell.