THE ESSENTIAL OST2 GENE ENCODES THE 16-KD SUBUNIT OF THE YEAST OLIGOSACCHARYLTRANSFERASE, A HIGHLY CONSERVED PROTEIN EXPRESSED IN DIVERSE EUKARYOTIC ORGANISMS

Oligosaccharyltransferase catalyzes the transfer of a preassembled hig h mannose oligosaccharide from a dolichol-oligosaccharide donor to con sensus glycosylation acceptor sites in newly synthesized proteins in t he lumen of the rough endoplasmic reticulum. The Saccharomyces cerevis iae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta). The alpha, beta, gamma, and delta subunits of the oligosaccharyltransferase are encoded by the OST1, WB P1, OST3, and SWP1 genes, respectively. Here we describe the functiona l characterization of the OST2 gene that encodes the epsilon-subunit o f the oligosaccharyltransferase. Genomic disruption of the OST2 locus was lethal in haploid yeast showing that expression of the Ost2 protei n is essential for viability. Overexpression of the Ost2 protein suppr esses the temperature-sensitive phenotype of the wbp1-2 allele and inc reases in vivo and in vitro oligosaccharyltransferase activity in a wb p1-2 strain. An analysis of a series of conditional ost2 mutants demon strated that defects in the Ost2 protein cause pleiotropic underglycos ylation of soluble and membrane-bound glycoproteins. Microsomal membra nes isolated from ost2 mutant yeast show marked reductions in the in v itro transfer of high mannose oligosaccharide from exogenous lipid-lin ked oligosaccharide to a glycosylation site acceptor tripeptide. Surpr isingly, the Ost2 protein was found to be 40% identical to the DAD1 pr otein (defender against apoptotic cell death), a highly conserved prot ein initially identified in vertebrate organisms. The protein sequence of ost2 mutant alleles revealed mutations at highly conserved residue s in the Ost2p/DAD1 protein sequence.