Mso1p as a Sec1p regulator to vesicle fusion

Seclp family proteins are essential for intracellular transport vesicle fusion. Despite numerous studies the precise function of these proteins remains elusive. We previously identified Msolp. a Seclp binding protein, which participates in the transport vesicle fusion at the plasma membrane in yeast Saccharomyces cerevisiae (Aalto et al., 1997, PNAS 94, 7331-7336). Here we have extended those studies to elucidate the role of Msolp in regulation of Seclp function. We show that a 21 residues long N-terminal amino acid sequence in Msolp is responsible for the Seclp binding both in yeast two-hybrid assay and in in vitro binding experiments. In vivo experiments verified the role of this N-terminal sequence in vesicle fusion. Based on these experiments it can be concluded that the Seclp-binding domain is necessary, but not sufficient for Msolp in vivo function. A point mutation within the Seclp-binding domain abolishes Msolp functionality in vivo without affecting the protein stability. mso1 mutant cells containing this point mutation become synthetically lethal with sec1-1 and sec1-11 mutations but not with sec2-41 and sec4-8 mutations in contrast to mso1 deletion which forms a lethal combination with all these mutations. The GFP-tagged Mso1p localizes to the bud tip in vegetatively grown cells indicating a function of Msolp specifically in the exocytosis step. In addition to the vesicle accumulation phenotype in vegetatively grown cells. mso1 deletion causes marked vesicle accumulation at the spindle pole body and inhibition of prospore membrane formation in sporulating yeast cells. Additional results on the role of Msolp in Seclp function regulation will he present