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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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Glycosylation and photocross-linking of nascent chains containing an authentic glycosylation sequence. Translocation intermediates containing pPL-sK(NKT) or pPL-sK(NST) nascent chains of various lengths were prepared, photolyzed, and analyzed as detailed in the Materials and methods. (A) Approximate location of photoreactive probe (small black circle) in a translocation intermediate in which the probe is located 70–90 residues from the COOH-terminal end of the nascent chain (the ribosomal P site). (B) SDS-PAGE analysis of translocation intermediates after photolysis. The number of nascent chain residues between the probe and the tRNA are given by k, and the number of nascent chain amino acids between the asparagine residue and the tRNA are given by n. The asterisk indicates the size range expected for photoadducts between these nascent chains and STT3, RI, or RII, while o indicates the size range expected for photocross-links of these nascent chains to TRAM and Sec61α. The bands indicated by np are present in samples lacking the ɛANB probe (lanes 5 and 6) and in samples that have not been photolyzed (not depicted), so these radioactive species are not nascent chain photoadducts. The glycosylated nascent chains are indicated by G, while the nonglycosylated nascent chains are indicated by nc.
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fig1: Glycosylation and photocross-linking of nascent chains containing an authentic glycosylation sequence. Translocation intermediates containing pPL-sK(NKT) or pPL-sK(NST) nascent chains of various lengths were prepared, photolyzed, and analyzed as detailed in the Materials and methods. (A) Approximate location of photoreactive probe (small black circle) in a translocation intermediate in which the probe is located 70–90 residues from the COOH-terminal end of the nascent chain (the ribosomal P site). (B) SDS-PAGE analysis of translocation intermediates after photolysis. The number of nascent chain residues between the probe and the tRNA are given by k, and the number of nascent chain amino acids between the asparagine residue and the tRNA are given by n. The asterisk indicates the size range expected for photoadducts between these nascent chains and STT3, RI, or RII, while o indicates the size range expected for photocross-links of these nascent chains to TRAM and Sec61α. The bands indicated by np are present in samples lacking the ɛANB probe (lanes 5 and 6) and in samples that have not been photolyzed (not depicted), so these radioactive species are not nascent chain photoadducts. The glycosylated nascent chains are indicated by G, while the nonglycosylated nascent chains are indicated by nc.

Mentions: A homogeneous population of ribosome–nascent chain complexes (RNCs) can be prepared by the translation of truncated mRNAs that lack a stop codon and encode a specific length of a secretory protein. Ribosomes initiate normally on such mRNAs and translation continues until the ribosome reaches the end of the truncated mRNA. As the mRNA lacks a stop codon, the ribosome does not dissociate from the mRNA but instead remains bound to the truncated mRNA and peptidyl-tRNA to yield RNCs in which the length of the nascent chain is dictated by the length of the truncated mRNA. When such nascent chains with signal sequences are translated in the presence of signal recognition particle (SRP) and ER microsomes, a homogeneous sample of membrane-bound RNCs is created (Fig. 1 A). All of the nascent chains examined here were long enough to be targeted efficiently to the translocon in the microsomal membrane and to be processed by signal peptidase.


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)

Glycosylation and photocross-linking of nascent chains containing an authentic glycosylation sequence. Translocation intermediates containing pPL-sK(NKT) or pPL-sK(NST) nascent chains of various lengths were prepared, photolyzed, and analyzed as detailed in the Materials and methods. (A) Approximate location of photoreactive probe (small black circle) in a translocation intermediate in which the probe is located 70–90 residues from the COOH-terminal end of the nascent chain (the ribosomal P site). (B) SDS-PAGE analysis of translocation intermediates after photolysis. The number of nascent chain residues between the probe and the tRNA are given by k, and the number of nascent chain amino acids between the asparagine residue and the tRNA are given by n. The asterisk indicates the size range expected for photoadducts between these nascent chains and STT3, RI, or RII, while o indicates the size range expected for photocross-links of these nascent chains to TRAM and Sec61α. The bands indicated by np are present in samples lacking the ɛANB probe (lanes 5 and 6) and in samples that have not been photolyzed (not depicted), so these radioactive species are not nascent chain photoadducts. The glycosylated nascent chains are indicated by G, while the nonglycosylated nascent chains are indicated by nc.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199356&req=5

fig1: Glycosylation and photocross-linking of nascent chains containing an authentic glycosylation sequence. Translocation intermediates containing pPL-sK(NKT) or pPL-sK(NST) nascent chains of various lengths were prepared, photolyzed, and analyzed as detailed in the Materials and methods. (A) Approximate location of photoreactive probe (small black circle) in a translocation intermediate in which the probe is located 70–90 residues from the COOH-terminal end of the nascent chain (the ribosomal P site). (B) SDS-PAGE analysis of translocation intermediates after photolysis. The number of nascent chain residues between the probe and the tRNA are given by k, and the number of nascent chain amino acids between the asparagine residue and the tRNA are given by n. The asterisk indicates the size range expected for photoadducts between these nascent chains and STT3, RI, or RII, while o indicates the size range expected for photocross-links of these nascent chains to TRAM and Sec61α. The bands indicated by np are present in samples lacking the ɛANB probe (lanes 5 and 6) and in samples that have not been photolyzed (not depicted), so these radioactive species are not nascent chain photoadducts. The glycosylated nascent chains are indicated by G, while the nonglycosylated nascent chains are indicated by nc.
Mentions: A homogeneous population of ribosome–nascent chain complexes (RNCs) can be prepared by the translation of truncated mRNAs that lack a stop codon and encode a specific length of a secretory protein. Ribosomes initiate normally on such mRNAs and translation continues until the ribosome reaches the end of the truncated mRNA. As the mRNA lacks a stop codon, the ribosome does not dissociate from the mRNA but instead remains bound to the truncated mRNA and peptidyl-tRNA to yield RNCs in which the length of the nascent chain is dictated by the length of the truncated mRNA. When such nascent chains with signal sequences are translated in the presence of signal recognition particle (SRP) and ER microsomes, a homogeneous sample of membrane-bound RNCs is created (Fig. 1 A). All of the nascent chains examined here were long enough to be targeted efficiently to the translocon in the microsomal membrane and to be processed by signal peptidase.

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