Functional characterization of Ost3p. Loss of the 34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates.
Within the lumen of the rough endoplasmic reticulum, oligosaccharyltransferase catalyzes the en bloc transfer of a high mannose oligosaccharide moiety from the lipid-linked oligosaccharide donor to asparagine acceptor sites in nascent polypeptides. The Saccharomyces cerevisiae oligosaccharyltransferase was purified as a heteroligomeric complex consisting of six subunits (alpha-zeta) having apparent molecular masses of 64 kD (Ost1p), 45 kD (Wbp1p), 34 kD, 30 kD (Swp1p), 16 kD, and 9 kD. Here we report a structural and functional characterization of Ost3p which corresponds to the 34-kD gamma-subunit of the oligosaccharyltransferase. Unlike Ost1p, Wbp1p, and Swp1p, expression of Ost3p is not essential for viability of yeast. Instead, ost3 null mutant yeast grow at wild-type rates on solid or in liquid media irrespective of culture temperature. Nonetheless, detergent extracts prepared from ost3 null mutant membranes are twofold less active than extracts prepared from wild-type membranes in an in vitro oligosaccharyltransferase assay. Furthermore, loss of Ost3p is accompanied by significant underglycosylation of soluble and membrane-bound glycoproteins in vivo. Compared to the previously characterized ost1-1 mutant in the oligosaccharyltransferase, and the alg5 mutant in the oligosaccharide assembly pathway, ost3 null mutant yeast appear to be selectively impaired in the glycosylation of several membrane glycoproteins. The latter observation suggests that Ost3p may enhance oligosaccharide transfer in vivo to a subset of acceptor substrates.
Karaoglu
D
D
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01655-0103, USA.
Kelleher
D J
DJ
Gilmore
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003433:Crosses, Genetic; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D005800:Genes, Fungal / Q000235:genetics; D015698:Genomic Library; D006031:Glycosylation; D006602:Hexosyltransferases; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009844:Oligosaccharides / Q000378:metabolism*; D011233:Precipitin Tests; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D017421:Sequence Analysis; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009844:Oligosaccharides; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase