ASSEMBLY OF THE ER TO GOLGI SNARE COMPLEX REQUIRES USO1P

Uso1p, a Saccharomyces cerevisiae protein required for ER to Golgi tra nsport, is homologous to the mammalian intra-Golgi transport factor p1 15. We have used genetic and biochemical approaches to examine the fun ction of Uso1p. The temperature-sensitive phenotype of the uso1-1 muta nt can be suppressed by overexpression of each of the known ER to Golg i v-SNAREs (Bet1p, Bos1p, Sec22p, and Ykt6p). Overexpression of two of them, Bet1p and Sec22p, can also suppress the lethality of Delta uso1 , indicating that the SNAREs function downstream of Uso1p. In addition , overexpression of the small GTP-binding protein Ypt1p, or of a gain nf funct;nn mutant (SLY1-20) of the t-SNARE associated protein Sly1p, also confers temperature resistance. Uso1p and Yptlp appear to functio n in the same process because they have a similar set of genetic inter actions with the V-SNARE genes, they exhibit a synthetic lethal intera ction, and they are able to suppress temperature sensitive mutants of one another when overexpressed. Uso1p acts upstream of, or in conjunct ion with, Yptlp because overexpression of Ypt1p allows a Delta uso1 st rain to grow, whereas overexpression of Uso1p does not suppress a Delt a ypt1 strain. Finally, biochemical analysis indicates that Uso1p, lik e Yptlp, is required for assembly of the V-SNARE/ t-SNARE complex. The implications of these findings, with respect to the mechanism of vesi cle docking, are discussed.