The highly conserved Stt3 protein is a subunit of the yeast oligosaccharyltransferase and forms a subcomplex with Ost3p and Ost4p.
The oligosaccharyltransferase has been purified from Saccharomyces cerevisiae as an hetero-oligomeric complex composed of four or six subunits. Here, the in vivo subunit composition and stoichiometry of the oligosaccharyltransferase were investigated by attaching an epitope coding sequence to a previously characterized subunit gene, OST3. Five (Ost1p, Wbp1p, Swp1p, Ost2p, and Ost5p) of the seven polypeptides that were coimmunoprecipitated with the epitope-tagged Ost3p were identical to those obtained by the conventional purification procedure. Two additional coprecipitating polypeptides with apparent molecular masses of 60 and 3.6 kDa were identified as the 78-kDa Stt3 protein and the 36-residue Ost4 protein, respectively. Stt3p and Ost4p were previously identified in screens for gene products involved in N-linked glycosylation. Quantification of the in vivo radiolabeled subunits and the radioiodinated purified enzyme shows that the yeast oligosaccharyltransferase is composed of equimolar amounts of eight subunits. Exposure of the immunoprecipitated oligosaccharyltransferase to mild protein denaturants yielded a subcomplex comprised of Stt3p, Ost3p, and Ost4p. These experiments, taken together with genetic and biochemical evidence for subunit interactions, suggest that the enzyme is composed of the following three subcomplexes: (a) Stt3p-Ost4p-Ost3p, (b) Swp1p-Wbp1p-Ost2p, and (c) Ost1p-Ost5p.
Karaoglu
D
D
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0103, USA.
Kelleher
D J
DJ
Gilmore
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R
D005656:Fungal Proteins / Q000378:metabolism; D006602:Hexosyltransferases; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D011233:Precipitin Tests; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases / Q000737:chemistry / Q000378:metabolism*
D005656:Fungal Proteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D006602:Hexosyltransferases; C096554:STT3 protein, S cerevisiae; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase