Department of Biochemistry and Molecular Pharmacology, Department of Neurobiology University of Massachusetts Medical School
Amino Acid Sequence; Animals; Binding Sites; Endoplasmic Reticulum; *Hexosyltransferases; Light; Membrane Proteins; Molecular Weight; Protein Binding; *Protein Biosynthesis; Protein Subunits; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity; Transferases
Biochemistry | Biochemistry, Biophysics, and Structural Biology | Cell Biology | Molecular Biology
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, Sec61alpha 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.
N glycosylation, oligosaccharyltransferase, STT3, photocross-linking, nascent protein chain
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DOI of Published Version
J Cell Biol. 2003 May 26;161(4):715-25. Epub 2003 May 19. Link to article on publisher's site
The Journal of cell biology
Nilsson, IngMarie; Kelleher, Daniel J.; Miao, Yiwei; Shao, Yuanlong; Kreibich, Gert; Gilmore, Reid; von Heijne, Gunnar; and Johnson, Arthur E., "Photocross-linking of nascent chains to the STT3 subunit of the oligosaccharyltransferase complex" (2003). Biochemistry and Molecular Pharmacology Publications and Presentations. 186.