Studies of yeast oligosaccharyl transferase subunits using the split-ubiquitin system: topological features and in vivo interactions.
Oligosaccharyl transferase (OT) catalyzes the cotranslational N-glycosylation of nascent polypeptides in the endoplasmic reticulum in all eukaryotic systems. Due to the inherent difficulty in characterizing this membrane protein complex, the mode of enzymatic action has not been resolved. Here, we used a membrane protein two-hybrid approach, the split-ubiquitin system, to address two aspects of the enzyme complex in yeast: the topological features, as well as the in vivo interactions of all of the components. We investigated the N- and C-terminal orientation of these proteins and the presence or the absence of a cleavable signal sequence at their N termini. We found that Ost2p and Stt3p have only their N terminus located in the cytosol, whereas Ost3p and Swp1p have only their C terminus oriented in the cytosol. In the case of Ost5p and Ost6p, both their N and C termini are present in the cytosol. These findings also suggested that Ost2p, Stt3p, Ost5p, and Ost6p do not have a cleavable N-terminal signal sequence. The pairwise analysis of in vivo interactions among all of the OT subunits demonstrated that OT subunits display specific interactions with each other in a functional complex. By comparing this interaction pattern with that detected in vitro in a nonfunctional complex, we proposed that a distinct conformation rearrangement takes place when the enzyme complex changes from the nonfunctional state to the activated functional state. This finding is consistent with earlier work by others indicating that OT exhibits allosteric properties.
Yan
Aixin
A
Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, Stony Brook University, Stony Brook, NY 11794, USA.
Wu
Elain
E
Lennarz
William J
WJ
D001483:Base Sequence; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006602:Hexosyltransferases / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011119:Polynucleotides; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014452:Ubiquitins / Q000378:metabolism*
D008565:Membrane Proteins; D011119:Polynucleotides; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D006602:Hexosyltransferases; C096554:STT3 protein, S cerevisiae; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase