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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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Photocross-linking of nascent chains containing a cryptic glycosylation sequence to the translocon and a new protein. Translation intermediates containing pPL-sK(QKT) nascent chains with an ɛANB-Lys probe positioned k residues from the tRNA were prepared as in Fig. 1. (A) Photocross-linking of the nascent chains to translocon components when k is 67 or less. Radioactive species in the total sample are shown in lanes 1–6, whereas lanes 7–9 and 10–12 show the material immunoprecipitated by antibodies specific for Sec61α and TRAM, respectively. (B) Photocross-linking to a new protein with an apparent molecular mass of 60–70 kD as the nascent chain lengthens and the cryptic glycosylation sequence emerges from the translocon. (C) Cross-linking to both the translocon proteins and the new 60–70-kD protein target is light dependent.
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fig2: Photocross-linking of nascent chains containing a cryptic glycosylation sequence to the translocon and a new protein. Translation intermediates containing pPL-sK(QKT) nascent chains with an ɛANB-Lys probe positioned k residues from the tRNA were prepared as in Fig. 1. (A) Photocross-linking of the nascent chains to translocon components when k is 67 or less. Radioactive species in the total sample are shown in lanes 1–6, whereas lanes 7–9 and 10–12 show the material immunoprecipitated by antibodies specific for Sec61α and TRAM, respectively. (B) Photocross-linking to a new protein with an apparent molecular mass of 60–70 kD as the nascent chain lengthens and the cryptic glycosylation sequence emerges from the translocon. (C) Cross-linking to both the translocon proteins and the new 60–70-kD protein target is light dependent.

Mentions: When the authentic NKT glycosylation sequence was replaced by a cryptic QKT glycosylation sequence, the nascent chains were no longer glycosylated (Fig. 2). Thus, as expected, the QKT sequence cannot substitute for NKT in the glycosylation reaction. Photocross-linking to translocon proteins was again observed when the probe was inside or close to the translocon (k ≤ 67) (Fig. 2 A; Fig. 2 B, lane 1). A major new photoadduct appeared when the nascent chain became long enough for the probe to emerge from the translocon (k > 67) (Fig. 2 B, lanes 2–4). This broad photoadduct band contained, in addition to the nascent chain, a protein that migrated with an apparent molecular mass of 60–70 kD. Interestingly, the appearance of this photoadduct coincided with the disappearance of the photocross-links to Sec61α and TRAM (Fig. 2 B, compare lanes 1 and 2). The presence of the cryptic QKT glycosylation sequence in the nascent chain therefore appears to have positioned the nascent chain adjacent to a protein not previously identified in cross-linking studies.


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)

Photocross-linking of nascent chains containing a cryptic glycosylation sequence to the translocon and a new protein. Translation intermediates containing pPL-sK(QKT) nascent chains with an ɛANB-Lys probe positioned k residues from the tRNA were prepared as in Fig. 1. (A) Photocross-linking of the nascent chains to translocon components when k is 67 or less. Radioactive species in the total sample are shown in lanes 1–6, whereas lanes 7–9 and 10–12 show the material immunoprecipitated by antibodies specific for Sec61α and TRAM, respectively. (B) Photocross-linking to a new protein with an apparent molecular mass of 60–70 kD as the nascent chain lengthens and the cryptic glycosylation sequence emerges from the translocon. (C) Cross-linking to both the translocon proteins and the new 60–70-kD protein target is light dependent.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199356&req=5

fig2: Photocross-linking of nascent chains containing a cryptic glycosylation sequence to the translocon and a new protein. Translation intermediates containing pPL-sK(QKT) nascent chains with an ɛANB-Lys probe positioned k residues from the tRNA were prepared as in Fig. 1. (A) Photocross-linking of the nascent chains to translocon components when k is 67 or less. Radioactive species in the total sample are shown in lanes 1–6, whereas lanes 7–9 and 10–12 show the material immunoprecipitated by antibodies specific for Sec61α and TRAM, respectively. (B) Photocross-linking to a new protein with an apparent molecular mass of 60–70 kD as the nascent chain lengthens and the cryptic glycosylation sequence emerges from the translocon. (C) Cross-linking to both the translocon proteins and the new 60–70-kD protein target is light dependent.
Mentions: When the authentic NKT glycosylation sequence was replaced by a cryptic QKT glycosylation sequence, the nascent chains were no longer glycosylated (Fig. 2). Thus, as expected, the QKT sequence cannot substitute for NKT in the glycosylation reaction. Photocross-linking to translocon proteins was again observed when the probe was inside or close to the translocon (k ≤ 67) (Fig. 2 A; Fig. 2 B, lane 1). A major new photoadduct appeared when the nascent chain became long enough for the probe to emerge from the translocon (k > 67) (Fig. 2 B, lanes 2–4). This broad photoadduct band contained, in addition to the nascent chain, a protein that migrated with an apparent molecular mass of 60–70 kD. Interestingly, the appearance of this photoadduct coincided with the disappearance of the photocross-links to Sec61α and TRAM (Fig. 2 B, compare lanes 1 and 2). The presence of the cryptic QKT glycosylation sequence in the nascent chain therefore appears to have positioned the nascent chain adjacent to a protein not previously identified in cross-linking studies.

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