Determination of the membrane topology of Ost4p and its subunit interactions in the oligosaccharyltransferase complex in Saccharomyces cerevisiae.
Ost4p is a minimembrane protein containing only 36 amino acids and is a subunit of oligosaccharyltransferase (OT) in Saccharomyces cerevisiae. It was found previously when amino acid residues 18-25 of Ost4p were mutated to ionizable amino acids and defects were observed in the interaction between Ost4p and either Stt3p or Ost3p, two other components of OT. The transmembrane segment of Ost4p is likely to extend from residues 10-25. This is consistent with the finding that alpha-helicity is estimated to be 36% by CD analysis of synthetic Ost4p in liposomes. This value is in reasonable agreement with the assumption that amino acids 10-25 (16 of 36 or 44%) are transmembrane. Therefore, the mutation-sensitive region (residues 18-25) is localized to only one half of the putative transmembrane domain of Ost4p. To learn where this region of Ost4p is situated in relation to the faces of endoplasmic reticulum (ER) membrane, we determined the membrane topology of Ost4p using an in vivo method and established that it is an Nlumen-Ccyto, type I membrane protein. These results indicate that the mutation-sensitive region of Ost4p is localized in the cytoplasmic leaflet of the ER membrane. In the current study, we also observed a loss of direct interaction between Ost3p and Stt3p in the presence of ost4 temperature-sensitive mutants, which indicates Ost4p, via interactions with amino acid residues in the cytosolic leaflet of the ER membrane, functions to bind these two proteins together in a subcomplex of OT.
Kim
Hyun
H
Department of Biochemistry and Cell Biology and the Institute for Cell and Developmental Biology, Stony Brook University, Stony Brook, NY 11794-5215, USA.
Yan
Qi
Q
Von Heijne
Gunnar
G
Caputo
Gregory A
GA
Lennarz
William J
WJ
D002455:Cell Division; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002942:Circular Dichroism; D003600:Cytosol / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006602:Hexosyltransferases; D008239:Lysine / Q000737:chemistry; D008565:Membrane Proteins / Q000235:genetics; D008954:Models, Biological; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D006602:Hexosyltransferases; C096554:STT3 protein, S cerevisiae; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D006026:Glycoside Hydrolases; D008239:Lysine