Sec6, Sec8, and Sec15 Are Components of a Multisubunit Complex Which Localizes to Small Bud Tips in Saccharomyces cerevisiae

• Published in: The Journal of cell biology Vol. 130; no. 2; pp. 299 - 312
• Main Authors: TerBush, Daniel R., Novick, Peter
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 01.07.1995; The Rockefeller University Press
• Subjects: Amino Acid Sequence; Antibodies; Biochemistry; Carrier Proteins; Cell membranes; Cells; Cellular biology; Chromatography, Affinity; Exocytosis; Fluorescent Antibody Technique; Fungal Proteins - analysis; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Gels; GTP-Binding Proteins - analysis; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - isolation & purification; Immunoprecipitation; Iron deficiency anemia; Molecular Sequence Data; Molecular Weight; Mutagenesis, Site-Directed; Precipitin Tests; Saccharomyces cerevisiae; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; Secretory vesicles; SNARE proteins; Vesicular Transport Proteins; Yeast; Yeasts
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.130.2.299

In the yeast Saccharomyces cerevisiae, the products of at least 14 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Two of these genes, SEC8 and SEC15, encode components of a 1-2-million D multisubunit complex that is found in the cytoplasm and associated with the plasma membrane. In this study, oligonucleotide-directed mutagenesis is used to alter the COOH-terminal portion of Sec8 with a 6-histidine tag, a 9E10 c-myc epitope, or both, to allow the isolation of the Sec8/15 complex from yeast lysates either by immobilized metal affinity chromatography or by immunoprecipitation. Sec6 cofractionates with Sec8/15 by immobilized metal affinity chromatography, gel filtration chromatography, and by sucrose velocity centrifugation. Sec6 and Sec15 coimmunoprecipitate from lysates with c-myc-tagged Sec8. These data indicate that the Sec8/15 complex contains Sec6 as a stable component. Additional proteins associated with Sec6/8/15 were identified by immunoprecipitations from radiolabeled lysates. The entire Sec6/8/15 complex contains at least eight polypeptides which range in molecular mass from 70 to 144 kD. Yeast strains containing temperature sensitive mutations in the SEC genes were also transformed with the SEC8-c-myc-6-histidine construct and analyzed by immunoprecipitation. The composition of the Sec6/8/15 complex is disrupted specifically in the sec3-2, sec5-24, and sec10-2 strain backgrounds. The c-myc-Sec8 protein is localized by immunofluorescence to small bud tips indicating that the Sec6/8/15 complex may function at sites of exocytosis.