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

Nilsson I, Kelleher DJ, Miao Y, Shao Y, Kreibich G, Gilmore R, von Heijne G, Johnson AE - J. Cell Biol. (2003)

Bottom Line: This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, TX 77843, USA.

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, 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.

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A competitive peptide substrate of OST greatly reduces nascent chain photocross-linking to STT3. Translocation intermediates containing a cryptic QKT sequence were photocross-linked in the presence (+) or absence (−) of a peptide substrate, N-benzoyl-Asn-Leu-Thr-methylamide. This peptide functions as a competitive inhibitor of N glycosylation. Bands are identified as in Fig. 1.
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fig5: A competitive peptide substrate of OST greatly reduces nascent chain photocross-linking to STT3. Translocation intermediates containing a cryptic QKT sequence were photocross-linked in the presence (+) or absence (−) of a peptide substrate, N-benzoyl-Asn-Leu-Thr-methylamide. This peptide functions as a competitive inhibitor of N glycosylation. Bands are identified as in Fig. 1.

Mentions: To assess whether nascent chain photocross-linking to STT3 was occurring at the active site of OST, we prepared translocation intermediates in the presence of an acceptor peptide, N-benzoyl-Asn-Leu-Thr-methylamide, that functions as a competitive inhibitor of glycosylation. When these samples were photolyzed, photocross-linking to STT3 was greatly reduced (Fig. 5). As the acceptor peptide is a competitive inhibitor of both glycosylation and photocross-linking, the observed photocross-linking of a nascent chain to STT3 must require nascent chain binding to the OST active site or its immediate vicinity.


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

Nilsson I, Kelleher DJ, Miao Y, Shao Y, Kreibich G, Gilmore R, von Heijne G, Johnson AE - J. Cell Biol. (2003)

A competitive peptide substrate of OST greatly reduces nascent chain photocross-linking to STT3. Translocation intermediates containing a cryptic QKT sequence were photocross-linked in the presence (+) or absence (−) of a peptide substrate, N-benzoyl-Asn-Leu-Thr-methylamide. This peptide functions as a competitive inhibitor of N glycosylation. Bands are identified as in Fig. 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199356&req=5

fig5: A competitive peptide substrate of OST greatly reduces nascent chain photocross-linking to STT3. Translocation intermediates containing a cryptic QKT sequence were photocross-linked in the presence (+) or absence (−) of a peptide substrate, N-benzoyl-Asn-Leu-Thr-methylamide. This peptide functions as a competitive inhibitor of N glycosylation. Bands are identified as in Fig. 1.
Mentions: To assess whether nascent chain photocross-linking to STT3 was occurring at the active site of OST, we prepared translocation intermediates in the presence of an acceptor peptide, N-benzoyl-Asn-Leu-Thr-methylamide, that functions as a competitive inhibitor of glycosylation. When these samples were photolyzed, photocross-linking to STT3 was greatly reduced (Fig. 5). As the acceptor peptide is a competitive inhibitor of both glycosylation and photocross-linking, the observed photocross-linking of a nascent chain to STT3 must require nascent chain binding to the OST active site or its immediate vicinity.

Bottom Line: This modification is effected cotranslationally by the multimeric oligosaccharyltransferase (OST) enzyme.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, TX 77843, USA.

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, 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.

Show MeSH
Related in: MedlinePlus