SEC35P, A NOVEL PERIPHERAL MEMBRANE-PROTEIN, IS REQUIRED FOR ER TO GOLGI VESICLE DOCKING

SEC35 was identified in a novel screen for temperature-sensitive mutan ts in the secretory pathway of the yeast Sacclaromyces cerevisiae (Wue stehube et al., 1996, Genetics. 142:393-406). At the restrictive tempe rature, the sec35-1 strain exhibits a transport block between the ER a nd the Golgi apparatus and accumulates numerous vesicles. SEC35 encode s a novel cytosolic protein of 32 kD, peripherally associated with mem branes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early i n the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target-SNARE-associated protein Sly1p. Weaker suppr ession is evident upon overexpression of genes encoding the vesicle-SN AREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results f rom deleting SEC35 is suppressed by YPT1 or SLY1-20, These genetic rel ationships suggest that Sec35p acts upstream of, or in conjunction wit h, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-fre e assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage c atalyzed by Uso1p. These genetic and biochemical results suggest Sec35 p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.