The OST4 gene of Saccharomyces cerevisiae encodes an unusually small protein required for normal levels of oligosaccharyltransferase activity.
Sodium vanadate is an effective drug for the enrichment of yeast mutants defective in glycosylation reactions that are carried out in the Golgi complex. We have isolated vanadate-resistant, hygromycin B-sensitive mutants that act at very early steps of N-linked glycosylation, occurring in the endoplasmic reticulum. Here we describe the phenotypic characterization of ost4, a vanadate-resistant mutant that is defective in oligosaccharyltransferase (OTase) activity both in vivo and in vitro. The OST4 open reading frame is unusual in that it predicts a protein of only 36 amino acids. We demonstrate that the OST4 gene product is, in fact, an unusually small protein of approximately 3.6 kDa, predicted to lie almost entirely in the hydrophobic environment of the membrane. Strains carrying a disruption of the OST4 gene are viable but grow poorly at 25 degrees C. The null mutant is inviable at 37 degrees C, demonstrating that the OST4 gene product is essential for growth at high temperatures. Deletion of the OST4 gene greatly diminishes OTase activity but does not abolish it. These results suggest that the OST4 gene encodes a subunit or accessory component of OTase that is essential at high temperature.
Chi
J H
JH
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794-5215, USA.
Roos
J
J
Dean
N
N
D000465:Algorithms; D000595:Amino Acid Sequence; D001483:Base Sequence; D002268:Carboxypeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D043402:Cathepsin A; D003545:Cysteine / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D005838:Genotype; D006031:Glycosylation; D006602:Hexosyltransferases; D008565:Membrane Proteins; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D014176:Protein Biosynthesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014166:Transferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D005656:Fungal Proteins; D008565:Membrane Proteins; D013462:Sulfur Radioisotopes; D014638:Vanadates; D008715:Methionine; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D002268:Carboxypeptidases; D043402:Cathepsin A; D003545:Cysteine