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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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Immunoprecipitation of the photoadduct containing the 60–70-kD protein. (A) A large sample of photoreactive pPL-sK(QKT) translocation intermediates was prepared as in Fig. 1 (lane 1). Five equal aliquots of this sample were then immunoprecipitated with antisera specific for STT3-A (lane 2), RI (lane 3), RII (lane 4), OST48 (lane 5), or Dad1 (lane 6) before SDS-PAGE. (B) Translation interme- diates with nascent chains of different lengths were photolyzed and immunoprecipitated with STT3-A antiserum. Bands are identified as in the legend to Fig. 1.
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fig4: Immunoprecipitation of the photoadduct containing the 60–70-kD protein. (A) A large sample of photoreactive pPL-sK(QKT) translocation intermediates was prepared as in Fig. 1 (lane 1). Five equal aliquots of this sample were then immunoprecipitated with antisera specific for STT3-A (lane 2), RI (lane 3), RII (lane 4), OST48 (lane 5), or Dad1 (lane 6) before SDS-PAGE. (B) Translation interme- diates with nascent chains of different lengths were photolyzed and immunoprecipitated with STT3-A antiserum. Bands are identified as in the legend to Fig. 1.

Mentions: The identity of the 60–70-kD protein that photocross-links to ribosome-bound nascent chains with appropriately positioned cryptic glycosylation sequences was determined by immunoprecipitation with specific antibodies. A large sample containing pPL-sK(QKT) nascent chains with the probe 81 residues from the ribosomal P site was photolyzed and split into five equal aliquots that were then immunoprecipitated with antibodies specific for STT3-A, RI, RII, OST48, or Dad1 (Fig. 4 A). It is clear from lanes 2–6 of Fig. 4 A that the prominent photocross-linked product is almost exclusively derived from STT3-A. There is also a very small amount of photocross-linking to RI in lane 3 that becomes evident only upon prolonged exposure of the gel to the phosphorimager plate. When translocation intermediates containing a range of nascent chain lengths were photolyzed and analyzed, photoadducts containing STT3-A were found as soon as the cryptic glycosylation sequence emerged from the translocon (Fig. 4 B). Thus, the cryptic glycosylation sequence in the nascent chain appears to be recognized by, and interact solely with, STT3-A. Antibodies specific for the less-abundant STT3-B were not tested in this experiment because the 94-kD STT3-B protein would yield less rapidly migrating cross-linked products in a molecular mass range where no photoadducts were seen in our experiments.


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)

Immunoprecipitation of the photoadduct containing the 60–70-kD protein. (A) A large sample of photoreactive pPL-sK(QKT) translocation intermediates was prepared as in Fig. 1 (lane 1). Five equal aliquots of this sample were then immunoprecipitated with antisera specific for STT3-A (lane 2), RI (lane 3), RII (lane 4), OST48 (lane 5), or Dad1 (lane 6) before SDS-PAGE. (B) Translation interme- diates with nascent chains of different lengths were photolyzed and immunoprecipitated with STT3-A antiserum. Bands are identified as in the legend to Fig. 1.
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fig4: Immunoprecipitation of the photoadduct containing the 60–70-kD protein. (A) A large sample of photoreactive pPL-sK(QKT) translocation intermediates was prepared as in Fig. 1 (lane 1). Five equal aliquots of this sample were then immunoprecipitated with antisera specific for STT3-A (lane 2), RI (lane 3), RII (lane 4), OST48 (lane 5), or Dad1 (lane 6) before SDS-PAGE. (B) Translation interme- diates with nascent chains of different lengths were photolyzed and immunoprecipitated with STT3-A antiserum. Bands are identified as in the legend to Fig. 1.
Mentions: The identity of the 60–70-kD protein that photocross-links to ribosome-bound nascent chains with appropriately positioned cryptic glycosylation sequences was determined by immunoprecipitation with specific antibodies. A large sample containing pPL-sK(QKT) nascent chains with the probe 81 residues from the ribosomal P site was photolyzed and split into five equal aliquots that were then immunoprecipitated with antibodies specific for STT3-A, RI, RII, OST48, or Dad1 (Fig. 4 A). It is clear from lanes 2–6 of Fig. 4 A that the prominent photocross-linked product is almost exclusively derived from STT3-A. There is also a very small amount of photocross-linking to RI in lane 3 that becomes evident only upon prolonged exposure of the gel to the phosphorimager plate. When translocation intermediates containing a range of nascent chain lengths were photolyzed and analyzed, photoadducts containing STT3-A were found as soon as the cryptic glycosylation sequence emerged from the translocon (Fig. 4 B). Thus, the cryptic glycosylation sequence in the nascent chain appears to be recognized by, and interact solely with, STT3-A. Antibodies specific for the less-abundant STT3-B were not tested in this experiment because the 94-kD STT3-B protein would yield less rapidly migrating cross-linked products in a molecular mass range where no photoadducts were seen in our experiments.

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