Journal of Biological Chemistry
Volume 274, Issue 8, 19 February 1999, Pages 5021-5025
Journal home page for Journal of Biological Chemistry

CELL BIOLOGY AND METABOLISM
The Ost1p Subunit of Yeast Oligosaccharyl Transferase Recognizes the Peptide Glycosylation Site Sequence, -Asn-X-Ser/Thr-*

https://doi.org/10.1074/jbc.274.8.5021Get rights and content
Under a Creative Commons license
open access

Other laboratories have established that oligosaccharyl transferase (OST) from Saccharomyces cerevisiae can be purified as a protein complex containing eight different subunits. To identify the OST subunit that recognizes the peptide sites that can be glycosylated, we developed photoaffinity probes containing a photoreactive benzophenone derivative,p-benzoylphenylalanine (Bpa), as part of an125I-labeled peptide that could be expected to be glycosylated. We found that Asn-Bpa-Thr peptides served as substrates for OST and that photoactivation of these probes in the presence of microsomes abolished the OST activity. Photoactivation of125I-labeled Asn-Bpa-Thr in the presence of microsomes resulted in specific covalent labeling of a protein doublet of molecular mass 62 and 64 kDa. By carrying out the photoactivation of the probe using microsomes containing epitope-tagged Ost1p, we demonstrated that the 125I-labeled protein was Ost1p. Radiolabeling of this protein was dependent on irradiation at 350 nm. No labeling was detected using a probe containing Ala instead of Thr as the third amino acid residue. We conclude that Ost1p is the subunit of the OST complex that recognizes the peptide sites in the nascent chains that are destined to be glycosylated.

Cited by (0)

*

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