SEC6, SEC8, AND SEC15 ARE COMPONENTS OF A MULTISUBUNIT COMPLEX WHICH LOCALIZES TO SMALL BUD TIPS IN SACCHAROMYCES-CEREVISIAE

In the yeast Saccharomyces cerevisiae, the products of at least 14 gen es are involved specifically in vesicular transport from the Golgi app aratus to the plasma membrane. Two of these genes, SEC8 and SEC15, enc ode components of a 1-2-million D multisubunit complex that is found i n the cytoplasm and associated with the plasma membrane. In this study , oligonucleotide-directed mutagenesis is used to alter the COOH-termi nal portion of Sec8 with a 6-histidine tag, a 9E10 c-myc epitope, or b oth, to allow the isolation of the Sec8/15 complex from yeast lysates either by immobilized metal affinity chromatography or by immunoprecip itation. Sec6 cofractionates with Sec8/15 by immobilized metal affinit y chromatography, gel filtration chromatography, and by sucrose veloci ty centrifugation. Sec6 and Sec15 coimmunoprecipitate from lysates wit h c-myc-tagged Sec8. These data indicate that the Sec8/15 complex cont ains Sec6 as a stable component. Additional proteins associated with S ec6/8/15 were identified by immunoprecipitations from radiolabeled lys ates. The entire Sec6/8/15 complex contains at least eight polypeptide s which range in molecular mass from 70 to 144 kD. Yeast strains conta ining temperature sensitive mutations in the SEC genes were also trans formed with the SEC8-c-myc-6-histidine construct and analyzed by immun oprecipitation. The composition of the Sec6/8/15 complex is disrupted specifically in the sec3-2, sec5-24, and sec10-2 strain backgrounds. T he c-myc-Sec8 protein is localized by immunofluorescence to small bud tips indicating that the Sec6/8/15 complex may function at sites of ex ocytosis.