New findings on interactions among the yeast oligosaccharyl transferase subunits using a chemical cross-linker.
At present, there is very limited knowledge about the structural organization of the yeast oligosaccharyl transferase (OT) complex and the function of each of its nine subunits. Because of the failure of the yeast two-hybrid system to reveal interactions between luminal domains of these subunits, we utilized a membrane permeable, thiocleavable cross-linking reagent dithiobis-succinimidyl propionate to biochemically study the interactions of various OT subunits. Four essential gene products, Ost1p, Wbp1p, Swp1p, and Stt3p were shown to be cross-linked to each other in a pairwise fashion. In addition, Ost1p was found to be cross-linked to all other eight OT subunits individually. This led us to propose that Ost1p may reside in the core of the OT complex and could play an important role in its assembly. Ost4p and Ost5p were found to only interact with specific components of the OT complex and may function as an additional anchor for optimal stability of Stt3p and Ost1p in the membrane, respectively. Interestingly, Ost3p and Ost6p subunits exhibited a surprisingly identical pattern of cross-linking to other subunits, which is consistent with their proposed redundant function. Based on these findings, we analyzed the distribution of the lysine residues that are likely to be involved in cross-linking of OT subunits and propose that the OT subunits interact with each other through either their transmembrane domains and/or a region proximal to it, rather than through their luminal or cytoplasmic domains.
Yan
Aixin
A
Department of Biochemistry and Cell Biology and the Institute for Cell and Developmental Biology, State University of New York at Stony Brook, New York 11794-5215, USA.
Ahmed
Eilaf
E
Yan
Qi
Q
Lennarz
William J
WJ
D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006602:Hexosyltransferases; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000737:chemistry; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000378:metabolism; D014166:Transferases / Q000737:chemistry* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D003432:Cross-Linking Reagents; D003902:Detergents; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D006602:Hexosyltransferases; C096554:STT3 protein, S cerevisiae; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase