Limits...
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.

Show MeSH

Related in: MedlinePlus

Photoadduct formation requires ribosome-bound nascent chains. Translocation intermediates containing a cryptic glycosylation sequence in their nascent chains were incubated without (lanes 1–4) or with (lanes 5–8) puromycin before photolysis. The asterisk identifies photoadducts containing the 60–70-kD protein. Radioactive species are identified as in Fig. 1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199356&req=5

fig3: Photoadduct formation requires ribosome-bound nascent chains. Translocation intermediates containing a cryptic glycosylation sequence in their nascent chains were incubated without (lanes 1–4) or with (lanes 5–8) puromycin before photolysis. The asterisk identifies photoadducts containing the 60–70-kD protein. Radioactive species are identified as in Fig. 1.

Mentions: To further characterize the photocross-linking between nascent chains containing a cryptic glycosylation sequence and the 60–70-kD protein, we treated the membrane-bound translocation intermediates with puromycin to release the nascent chains from the ribosomes before photolysis. No photocross-linking was observed after the nascent chain was released from the ribosome into the ER lumen by puromycin (Fig. 3). As the nascent chain is no longer adjacent to either the translocon proteins or to the 60–70-kD protein after being released from the ribosome and the membrane, it appears that the nascent chain interacts with the 60–70-kD protein cotranslationally, in the context of the translocon and its associated proteins.


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)

Photoadduct formation requires ribosome-bound nascent chains. Translocation intermediates containing a cryptic glycosylation sequence in their nascent chains were incubated without (lanes 1–4) or with (lanes 5–8) puromycin before photolysis. The asterisk identifies photoadducts containing the 60–70-kD protein. Radioactive species are identified as in Fig. 1.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Photoadduct formation requires ribosome-bound nascent chains. Translocation intermediates containing a cryptic glycosylation sequence in their nascent chains were incubated without (lanes 1–4) or with (lanes 5–8) puromycin before photolysis. The asterisk identifies photoadducts containing the 60–70-kD protein. Radioactive species are identified as in Fig. 1.
Mentions: To further characterize the photocross-linking between nascent chains containing a cryptic glycosylation sequence and the 60–70-kD protein, we treated the membrane-bound translocation intermediates with puromycin to release the nascent chains from the ribosomes before photolysis. No photocross-linking was observed after the nascent chain was released from the ribosome into the ER lumen by puromycin (Fig. 3). As the nascent chain is no longer adjacent to either the translocon proteins or to the 60–70-kD protein after being released from the ribosome and the membrane, it appears that the nascent chain interacts with the 60–70-kD protein cotranslationally, in the context of the translocon and its associated proteins.

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