Yan Qi, Lennarz William J
Department of Biochemistry and Cell Biology and the Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, New York 11794-5215, USA.
J Biol Chem. 2002 Dec 6;277(49):47692-700. doi: 10.1074/jbc.M208136200. Epub 2002 Sep 30.
In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT) is composed of nine different transmembrane proteins. Using a glycosylatable peptide containing a photoprobe, we previously found that only one essential subunit, Ost1p, was specifically labeled by the photoprobe and recently have shown that it does not contain the recognition domain for the glycosylatable sequence Asn-Xaa-Thr/Ser. In this study we utilized additional glycosylatable peptides containing two photoreactive groups and found that these were linked to Stt3p and Ost3p. Stt3p is the most conserved subunit in the OT complex, and therefore 21 block mutants in the lumenal region were prepared. Of the 14 lethal mutant proteins only two, as well as one temperature-sensitive mutant protein, were incorporated into the OT complex. However, using microsomes prepared from these three strains, the labeling of Ost1p was markedly decreased upon photoactivation with the Asn-Bpa-Thr photoprobe. Based on the block mutants single amino acid mutations were prepared and analyzed. From all of these results, we conclude that the sequence from residues 516 to 520, WWDYG in Stt3p, plays a central role in glycosylatable peptide recognition and/or the catalytic glycosylation process.
在酿酒酵母中，寡糖基转移酶（OT）由九种不同的跨膜蛋白组成。我们之前使用含有光探针的可糖基化肽发现，只有一个必需亚基Ost1p被光探针特异性标记，并且最近已表明它不包含可糖基化序列Asn-Xaa-Thr/Ser的识别结构域。在本研究中，我们使用了含有两个光反应性基团的其他可糖基化肽，发现它们与Stt3p和Ost3p相连。Stt3p是OT复合物中最保守的亚基，因此在腔区制备了21个阻断突变体。在14个致死突变蛋白中，只有两个以及一个温度敏感突变蛋白被整合到OT复合物中。然而，使用从这三个菌株制备的微粒体，在用Asn-Bpa-Thr光探针进行光激活后，Ost1p的标记明显减少。基于阻断突变体，制备并分析了单氨基酸突变。从所有这些结果中，我们得出结论，Stt3p中516至520位残基的序列WWDYG在可糖基化肽识别和/或催化糖基化过程中起核心作用。
