The oligosaccharyltransferase complex from Saccharomyces cerevisiae. Isolation of the OST6 gene, its synthetic interaction with OST3, and analysis of the native complex.
The key step of N-glycosylation of proteins, an essential and highly conserved protein modification, is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). So far, eight genes have been identified in Saccharomyces cerevisiae that are involved in this process. Enzymatically active OST preparations from yeast were shown to be composed of four (Ost1p, Wbp1p, Ost3p, Swp1p) or six subunits (Ost2p and Ost5p in addition to the four listed). Genetic studies have disclosed Stt3p and Ost4p as additional proteins needed for N-glycosylation. In this study we report the identification and functional characterization of a new OST gene, designated OST6, that has homology to OST3 and in particular a strikingly similar membrane topology. Neither gene is essential for growth of yeast. Disruption of OST6 or OST3 causes only a minor defect in N-glycosylation, but an Deltaost3Deltaost6 double mutant displays a synthetic phenotype, leading to a severe underglycosylation of soluble and membrane-bound glycoproteins in vivo and to a reduced OST activity in vitro. Moreover, each of the two genes has also a specific function, since agents affecting cell wall biogenesis reveal different growth phenotypes in the respective null mutants. By blue native electrophoresis and immunodetection, a approximately 240-kDa complex was identified consisting of Ost1p, Stt3p, Wbp1p, Ost3p, Ost6p, Swp1p, Ost2p, and Ost5p, indicating that probably all so far identified OST proteins are constituents of the OST complex. It is also shown that disruption of OST3 and OST6 leads to a defect in the assembly of the complex. Hence, the function of these genes seems to be essential for recruiting a fully active complex necessary for efficient N-glycosylation.
Knauer
R
R
Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.
Lehle
L
L
D000595:Amino Acid Sequence; D018628:Gene Dosage; D005800:Genes, Fungal; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013489:Suppression, Genetic; D014166:Transferases / Q000235:genetics* / Q000378:metabolism
D006023:Glycoproteins; D008565:Membrane Proteins; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase