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 conser ved protein modification, is catalyzed by the hetero-oligomeric protein com plex oligosaccharyltransferase (OST), So far, eight genes have been identif ied in Saccharomyces cerevisiae that are involved in this process. Enzymati cally active OST preparations from yeast were shown to be composed of four (Ost1p, Wbp1p, Ost3p, Swp1p) or six subunits (Ost2p and Ost5p in addition t o the four listed). Genetic studies have disclosed Stt3p and Ost4p as addit ional proteins needed for N-glycosylation, In this study we report the iden tification and functional characterization of a new OST gene, designated OS T6, that has homology to OST3 and in particular a strikingly similar membra ne topology. Neither gene is essential for growth of yeast. Disruption of O ST6 or OST3 causes only a minor defect in N-glycosylation, but an Delta ost 3 Delta ost6 double mutant displays a synthetic phenotype, leading to a sev ere 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 func tion, since agents affecting cell wall biogenesis rev eal different growth phenotypes in the respective null mutants. By blue nat ive electrophoresis and immunodetection, a similar to 240-kDa complex was i dentified consisting of Ost1p, Stt3p, Wbp1p, Ost3p, Ost6p, Swp1p, Ost2p, an d Ost5p, indicating that probably all so far identified OST proteins are co nstituents 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,