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J. Biol. Chem., Vol. 280, Issue 3, 1864-1871, January 21, 2005

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Studies on the N-Glycosylation of the Subunits of Oligosaccharyl Transferase in Saccharomyces cerevisiae^*

Guangtao Li, Qi Yan, Aleksandra Nita-Lazar, Robert S. Haltiwanger, and William J. Lennarz¶

From the Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794-5215

In Saccharomyces cerevisiae, oligosaccharyl transferase (OT) consists of nine different subunits. Three of the essential gene products, Ost1p, Wbp1p, and Stt3p, are N-linked glycoproteins. To study the function of the N-glycosylation of these proteins, we prepared single or multiple N-glycosylation site mutations in each of them. We established that the four potential N-glycosylation sites in Ost1p and the two potential N-glycosylation sites in Wbp1p were occupied in the mature proteins. Interestingly, none of the N-glycosylation sites in these two proteins was conserved, and no defect in growth or OT activity was observed when the N-glycosylation sites were mutated to block N-glycosylation in either subunit. However, in the third glycosylated subunit, Stt3p, there are two adjacent potential N-glycosylation sites (N⁵³⁵NTWN⁵³⁹NT) that, in contrast to the other subunits, are highly conserved in eukaryotic organisms. Mass spectrometric analysis of a tryptic digest of Stt3p showed that the peptide containing the two adjacent N-glycosylation sites was N-glycosylated at one site. Furthermore, the glycan chain identified as Man₈GlcNAc₂ is found linked only to Asn⁵³⁹. Mutation experiments were carried out at these two sites. Four single amino acid mutations blocking either N-glycosylation site (N535Q, T537A, N539Q, and T541A) resulted in strains that were either lethal or extremely temperature sensitive. However, other mutations in the two N-glycosylation sites N⁵³⁵NTWN⁵³⁹NT (N536Q, T537S, N540Q, and T541S), did not exhibit growth defects. Based on these studies, we conclude that N-glycosylation of Stt3p at Asn⁵³⁹ is essential for its function in the OT complex.

Received for publication, September 23, 2004 , and in revised form, October 18, 2004.

^* This work was supported by National Institutes of Health Grants GM33185 (to W. J. L.) and GM61126 (to R. S. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

Present address: Dept. of Molecular Biology, The Scripps Research Institute, MEM-L71, La Jolla, CA 92037.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215. Tel.: 631-632-8560; Fax: 631-632-8575; E-mail: wlennarz{at}notes.cc.sunysb.edu.

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