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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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Conversion of the pPL-sK secretory protein into a membrane protein. (A) By introducing an NST sequence into the nascent chain behind the putative TM sequence, the extent of nascent chain integration into the ER membrane can be assessed (Sääf et al., 1998). If the potential TM segment does not function as a stop-transfer sequence (light gray rectangle), the entire protein will be translocated into the ER lumen and will be glycosylated at two sites. But if the TM segment is inserted into the bilayer (dark gray rectangle), only one of its glycosylation sites will be modified (black Y). (B) Full-length polypeptides containing zero (pPL-sK[NKT], which is the same as pPL-sK[N0]), two (pPL-sK[N2]), or four (pPL-sK[N4]) leucine substitutions in addition to an extra glycosylation site in the COOH-terminal portion of the protein were translated in reticulocyte lysate in the absence (−) or presence (+) of rough microsomes (RM). Signal sequence–cleaved diglycosylated, monoglycosylated, and nonglycosylated molecules are indicated by two white dots, one white dot, and one black dot, respectively. Very little cleaved, nonglycosylated product is seen in this experiment (black dot), but translation of the same construct lacking both glycosylation sites confirms the identity of this band (not depicted).
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fig6: Conversion of the pPL-sK secretory protein into a membrane protein. (A) By introducing an NST sequence into the nascent chain behind the putative TM sequence, the extent of nascent chain integration into the ER membrane can be assessed (Sääf et al., 1998). If the potential TM segment does not function as a stop-transfer sequence (light gray rectangle), the entire protein will be translocated into the ER lumen and will be glycosylated at two sites. But if the TM segment is inserted into the bilayer (dark gray rectangle), only one of its glycosylation sites will be modified (black Y). (B) Full-length polypeptides containing zero (pPL-sK[NKT], which is the same as pPL-sK[N0]), two (pPL-sK[N2]), or four (pPL-sK[N4]) leucine substitutions in addition to an extra glycosylation site in the COOH-terminal portion of the protein were translated in reticulocyte lysate in the absence (−) or presence (+) of rough microsomes (RM). Signal sequence–cleaved diglycosylated, monoglycosylated, and nonglycosylated molecules are indicated by two white dots, one white dot, and one black dot, respectively. Very little cleaved, nonglycosylated product is seen in this experiment (black dot), but translation of the same construct lacking both glycosylation sites confirms the identity of this band (not depicted).

Mentions: As OST glycosylates both secretory and membrane proteins in vivo, we wished to determine whether a photoreactive nascent membrane protein containing a QKT sequence would react covalently with STT3. We therefore introduced leucines into the nascent chain sequence to convert the secretory pPL-sK protein into a membrane protein. This approach has been used previously to show that a stretch of as few as eight leucines can function as a stop-transfer sequence and anchor a polypeptide in the ER membrane (Kuroiwa et al., 1991; Nilsson et al., 1994). Here, we substituted two, four, or six leucines into the mature pPL sequence adjacent to a naturally occurring stretch of 10 nonpolar residues that were located COOH terminal of the NKT or QKT sequences in the nascent chain. To assess the stop-transfer efficiency of these constructs, we positioned a second glycosylation site COOH terminal of the potential transmembrane (TM) segment. The ratio of polypeptides translocated across the ER membrane to polypeptides integrated into the membrane is then given by the ratio of diglycosylated to monoglycosylated proteins (Fig. 6 A), an approach that we have used successfully before to evaluate TM insertion efficiency (Sääf et al., 1998). As is evident from the data of Fig. 6 B, the lengthening of the nonpolar stretch in pPL-sK by only four leucines converts the polypeptide into a membrane protein by creating a TM sequence with sufficient hydrophobicity and length to insert into the ER membrane.


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)

Conversion of the pPL-sK secretory protein into a membrane protein. (A) By introducing an NST sequence into the nascent chain behind the putative TM sequence, the extent of nascent chain integration into the ER membrane can be assessed (Sääf et al., 1998). If the potential TM segment does not function as a stop-transfer sequence (light gray rectangle), the entire protein will be translocated into the ER lumen and will be glycosylated at two sites. But if the TM segment is inserted into the bilayer (dark gray rectangle), only one of its glycosylation sites will be modified (black Y). (B) Full-length polypeptides containing zero (pPL-sK[NKT], which is the same as pPL-sK[N0]), two (pPL-sK[N2]), or four (pPL-sK[N4]) leucine substitutions in addition to an extra glycosylation site in the COOH-terminal portion of the protein were translated in reticulocyte lysate in the absence (−) or presence (+) of rough microsomes (RM). Signal sequence–cleaved diglycosylated, monoglycosylated, and nonglycosylated molecules are indicated by two white dots, one white dot, and one black dot, respectively. Very little cleaved, nonglycosylated product is seen in this experiment (black dot), but translation of the same construct lacking both glycosylation sites confirms the identity of this band (not depicted).
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Related In: Results  -  Collection

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fig6: Conversion of the pPL-sK secretory protein into a membrane protein. (A) By introducing an NST sequence into the nascent chain behind the putative TM sequence, the extent of nascent chain integration into the ER membrane can be assessed (Sääf et al., 1998). If the potential TM segment does not function as a stop-transfer sequence (light gray rectangle), the entire protein will be translocated into the ER lumen and will be glycosylated at two sites. But if the TM segment is inserted into the bilayer (dark gray rectangle), only one of its glycosylation sites will be modified (black Y). (B) Full-length polypeptides containing zero (pPL-sK[NKT], which is the same as pPL-sK[N0]), two (pPL-sK[N2]), or four (pPL-sK[N4]) leucine substitutions in addition to an extra glycosylation site in the COOH-terminal portion of the protein were translated in reticulocyte lysate in the absence (−) or presence (+) of rough microsomes (RM). Signal sequence–cleaved diglycosylated, monoglycosylated, and nonglycosylated molecules are indicated by two white dots, one white dot, and one black dot, respectively. Very little cleaved, nonglycosylated product is seen in this experiment (black dot), but translation of the same construct lacking both glycosylation sites confirms the identity of this band (not depicted).
Mentions: As OST glycosylates both secretory and membrane proteins in vivo, we wished to determine whether a photoreactive nascent membrane protein containing a QKT sequence would react covalently with STT3. We therefore introduced leucines into the nascent chain sequence to convert the secretory pPL-sK protein into a membrane protein. This approach has been used previously to show that a stretch of as few as eight leucines can function as a stop-transfer sequence and anchor a polypeptide in the ER membrane (Kuroiwa et al., 1991; Nilsson et al., 1994). Here, we substituted two, four, or six leucines into the mature pPL sequence adjacent to a naturally occurring stretch of 10 nonpolar residues that were located COOH terminal of the NKT or QKT sequences in the nascent chain. To assess the stop-transfer efficiency of these constructs, we positioned a second glycosylation site COOH terminal of the potential transmembrane (TM) segment. The ratio of polypeptides translocated across the ER membrane to polypeptides integrated into the membrane is then given by the ratio of diglycosylated to monoglycosylated proteins (Fig. 6 A), an approach that we have used successfully before to evaluate TM insertion efficiency (Sääf et al., 1998). As is evident from the data of Fig. 6 B, the lengthening of the nonpolar stretch in pPL-sK by only four leucines converts the polypeptide into a membrane protein by creating a TM sequence with sufficient hydrophobicity and length to insert into the ER membrane.

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