Abstract
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, Sec61α 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.
Highlights
N-linked glycosylation is one of the most common types of eukaryotic protein modification
We show that photocross-linking between nascent chains and OST occurs on the lumenal side of the ER membrane after the probe emerges from the translocon, and this cross-linking is dependent on the presence of a cryptic glycosylation sequence in the nascent chain
As nascent chains are glycosylated cotranslationally by the OST, one would predict that a nascent polypeptide could be photocross-linked to one or more of the OST subunits after the photoreactive amino acid emerges from the lumenal side of the translocon
Summary
N-linked glycosylation is one of the most common types of eukaryotic protein modification. The transfer of high mannose oligosaccharides from a dolichol carrier to -Asn-X-Thr/Ser- acceptor sites in the nascent chain, with X being any amino acid except proline, is catalyzed by the oligosaccharyltransferase (OST)* enzyme (Silberstein and Gilmore, 1996; Knauer and Lehle, 1999; Yan and Lennarz, 1999). The yeast OST is composed of eight subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Swp1p, Wbp1p, and Stt3p), five of which (Ost1p, Ost2p, Wbp1p, Swp1p, and Stt3p) are encoded by essential genes (Silberstein and Gilmore, 1996, Karaoglu et al, 1997; Knauer and Lehle, 1999). Homologues of Stt3p (STT3-A and STT3-B), Ost3p/Ost6p (N33 and IAP), and Ost4p are expressed in mammalian organisms and are assembled together with RI, RII, OST48, The Rockefeller University Press, 0021-9525/2003/05/715/11 $8.00
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