Synaptotagmin II expression partially rescues the growth defect of the yeast sec15 secretory mutant

Abstract

Synaptotagmins are a family of calcium- and phospholipid-binding proteins implicated in the function of cell exocytosis. Synaptotagmins I and II are neurally expressed proteins thought to be involved in neurotransmitter release from neurons. We have expressed rat synaptotagmin II in several Saccharomyces cerevisiae temperature-sensitive secretory mutants that are defective in Golgi to plasma membrane vesicular transport. Synaptotagmin II expression was able to partially rescue the growth defect in one particular mutant, sec15. No suppression was observed when synaptotagmin II was expressed in sec1, sec2, sec4, sec5, sec6, sec8, sec9, sec14, sec17, or sec18. Two synaptotagmin II deletion mutants were also expressed in sec15 and screened for suppression. The expression of the cytoplasmic domain of synaptotagmin alone was not able to suppress the sec15 growth defect. In addition, the expression of a synaptotagmin II fragment lacking the second half of the cytoplasmic domain including the second C2 domain did not suppress sec15. We have isolated a membrane fraction enriched in post-Golgi vesicles from a sec15 strain expressing synaptotagmin II and found that synaptotagmin II co-purifies with this fraction, suggesting that the rat synaptotagmin II is targeted to membranes in yeast. Sec 15p forms a large multisubunit protein complex that includes Sec6p and Sec8p. This protein complex is thought to function in a late stage of exocytosis in yeast. Sec6p and Sec8p homologs have been identified in mammalian cells. Our studies suggest that synaptotagmin may be a part of this complex or regulate its function in mammalian cells.