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© The Rockefeller University Press, 0021-9525/2003/5/715 $5.00
The Journal of Cell Biology, Volume 161, Number 4, 715-725

Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex

¹ Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center
² Department of Chemistry and Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843
³ Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden
⁴ Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655
⁵ Department of Cell Biology, New York University Medical Center, New York, NY 10016

Address correspondence to Arthur E. Johnson, Texas A&M University System Health Science Center, 116 Reynolds Medical Bldg., 1114 TAMU, College Station, TX 77843-1114. Tel.: (979) 862-3188. Fax: (979) 862-3339. E-mail: aejohnson{at}tamu.edu

In eukaryotic cells, polypeptides are N glycosylated after passing through the membrane of the ER into the ER lumen. This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme. Here, we report the first cross-linking of an OST subunit to a nascent chain that is undergoing translocation through, or integration into, the ER membrane. A photoreactive probe was incorporated into a nascent chain using a modified Lys-tRNA and was positioned in a cryptic glycosylation site (-Q-K-T- instead of -N-K-T-) in the nascent chain. When translocation intermediates with nascent chains of increasing length were irradiated, nascent chain photocross-linking to translocon components, Sec61 and TRAM, was replaced by efficient photocross-linking solely to a protein identified by immunoprecipitation as the STT3 subunit of the OST. No cross-linking was observed in the absence of a cryptic sequence or in the presence of a competitive peptide substrate of the OST. As no significant nascent chain photocross-linking to other OST subunits was detected in these fully assembled translocation and integration intermediates, our results strongly indicate that the nascent chain portion of the OST active site is located in STT3.

Key Words: N glycosylation; oligosaccharyltransferase; STT3; photocross-linking; nascent protein chain

^* Abbreviations used in this paper: ANB-Lys, N-(5-azido-2-nitrobenzoyl)-lysine; CRM, column-washed rough microsome; OST, oligosaccharyltransferase; pPL, preprolactin; RI and -II, ribophorin I and II; RNC, ribosome–nascent chain complex; SRP, signal recognition particle; TM, transmembrane.

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