The VPS16 gene product associates with a sedimentable protein complex and is essential for vacuolar protein sorting in yeast

• Published in: The Journal of biological chemistry 1993-03, Vol.268 (7), p.4953-4962
• Main Authors: Horazdovsky, B.F. (California Institute of Technology, Pasadena, CA), Emr, S.D
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Cloning, Molecular; DNA, Fungal; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; Fungal Proteins - metabolism; GENE; gene products; GENES; Genes, Fungal; Genes. Genome; Membrane Proteins - metabolism; METABOLISME DES PROTEINES; METABOLISMO PROTEICO; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; nucleotide sequence; PEPTIDASAS; PEPTIDASE; PROTEINAS; PROTEINE; purification; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; SECRECION; SECRETION; SECUENCIA NUCLEICA; SEQUENCE NUCLEIQUE; VACUOLA; VACUOLE; Vacuoles - metabolism; Vesicular Transport Proteins; vps16 gene
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)53488-8

The Saccharomyces cerevisiae vacuolar protein sorting mutant, vps16, exhibits pleiotropic defects in vacuolar protein targeting and vacuole morphology. To understand the role of the VPS16 gene in vacuolar protein sorting and organelle assembly, a vps16 ts mutant was used to clone the wild-type gene. DNA sequence analysis identified a single open reading frame within a vps16 complementing DNA fragment, capable of encoding a protein of 92,000 Da. Hydrophobicity analysis indicates that the Vps16 protein (Vps16p) is hydrophilic and contains no obvious signal sequence or membrane spanning domains. Gene disruption experiments have shown that VPS16 is not essential. delta vps16 cells exhibit, 1) a severe defect in vacuolar protein sorting; 2) a ts growth defect; 3) a grossly abnormal vacuole morphology, no normal vacuole compartment(s) is present; and 4) a defect in alpha-factor processing. A trpE-vps16 fusion protein has been used to generate polyclonal antiserum. This antiserum detects an unglycosylated protein of 90,000 Da. Subcellular fractionation studies indicate that the vast majority of the VPS16 gene product is associated with a particulate cell fraction. This association is resistant to detergent and salt extractions, but Vps16p can be extracted with 6 M urea and alkali buffer. In addition, overexpression of Vps16p appears to saturate the association sites available in this sedimentable structure. These data indicate that Vps16p may be specifically associated with a large protein complex, or with a limited number of sites on cytoskeletal elements of the cell