title: The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast.
abstract: Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here, we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase (OST) complex, the key enzyme of N-glycosylation. The deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity toward compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas the deletion of OST6, a functional homolog of OST3, has no effect. OST activity in vitro determined in membranes from rot1-1ost3Δ cells was found to be decreased to 45% compared with wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase Y, Gas1 or dipeptidyl aminopeptidase B, displayed an underglycosylation pattern. By affinity chromatography, a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the O-mannosylation process, as the glycosylation of distinct glycoproteins of this type were affected as well. Altogether, the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation.
authors.affiliation: Department of Cell Biology and Plant Biochemistry, University of Regensburg, Regensburg, Germany.
authors.forename: Monika
authors.initials: M
authors.lastname: Pasikowska
authors.forename: Grazyna
authors.initials: G
authors.lastname: Palamarczyk
authors.forename: Ludwig
authors.initials: L
authors.lastname: Lehle
mesh_terms: D004122:Dimethylallyltranstransferase / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006031:Glycosylation; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008364:Mannosyltransferases / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D050296:Microbial Viability; D008861:Microsomes / Q000201:enzymology; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010641:Phenotype; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D008565:Membrane Proteins; D018832:Molecular Chaperones; C523667:ROT1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D006602:Hexosyltransferases; D008364:Mannosyltransferases; C082696:protein O-mannosyltransferase; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D004122:Dimethylallyltranstransferase; D008358:Mannose