YEAST SEC16 GENE ENCODES A MULTIDOMAIN VESICLE COAT PROTEIN THAT INTERACTS WITH SEC23P

Temperature-sensitive mutations in the SEC16 gene of Saccharomyces cer evisiae block budding of transport vesicles from the ER. SEC16 was clo ned by complementation of the sec16-1 mutation and encodes a 240-kD pr otein located in the insoluble, particulate component of cell lysates. Sec16p is released from this particulate fraction by high salt, but n ot by nonionic detergents or urea, Some Sec16p is localized to the ER by immunofluorescence microscopy. Membrane-associated Sec16p is incorp orated into transport vesicles derived from the ER that are formed in an in vitro vesicle budding reaction, Sec16p binds to Sec23p, a COPII vesicle coat protein, as shown by the two-hybrid interaction assay and affinity studies in cell extracts. These findings indicate that Sec16 p associates with Sec23p as part of the transport vesicle coat structu re. Genetic analysis of SEC16 identifies three functionally distinguis hable domains, One domain is defined by the five temperature-sensitive mutations clustered in the middle of SEC16. Each of these mutations c an be complemented by the central domain of SEC16 expressed alone, The stoichiometry of Sec16p is critical for secretory function since over expression of Sec16p causes a lethal secretion defect, This lethal fun ction maps to the NH2-terminus of the protein, defining a second funct ional domain, A separate function for the COOH-terminal domain of Sec1 6p is shown by its ability to bind Sec23p. Together, these results sug gest that Sec16p engages in multiple protein-protein interactions both on the ER membrane and as part of the coat of a completed vesicle.