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Early encounters of a nascent membrane protein: specificity and timing of contacts inside and outside the ribosome.

Houben EN, Zarivach R, Oudega B, Luirink J - J. Cell Biol. (2005)

Bottom Line: The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome-nascent chain complex to the Sec-YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit.The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY.Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Institute of Molecular Cell Biology, Vrije Universiteit, 1081 HV Amsterdam, Netherlands.

ABSTRACT
An unbiased photo-cross-linking approach was used to probe the "molecular path" of a growing nascent Escherichia coli inner membrane protein (IMP) from the peptidyl transferase center to the surface of the ribosome. The nascent chain was initially in proximity to the ribosomal proteins L4 and L22 and subsequently contacted L23, which is indicative of progression through the ribosome via the main ribosomal tunnel. The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome-nascent chain complex to the Sec-YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit. The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY. Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.

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Progression of nascent Lep through the ribosome. (A) Schematic representation of the 9, 24, 40, and 50Lep constructs with a cross-linking probe at position 3. The transmembrane regions (H1) and methionine tags are presented as thick gray lines and white bars, respectively. (B) In vitro translation of nascent 9–50LepTAG3 constructs was performed in the presence of (Tmd)Phe-tRNAsup. After translation, samples were irradiated with UV light to induce cross-linking, and the ribosome–nascent chain complexes were purified and analyzed by SDS-PAGE. UV-irradiated 9, 15, 24, 44, and 50LepTAG3 were immunoprecipitated with antisera as indicated. (C) Schematic representation of the cross-links observed in B. Numbers indicate the distance in amino acids of the cross-linking probe to the PTC. Images in different panels represent different parts of the gel or different exposure times. +, L4 cross-link; *, L22 cross-link; ^, L23 cross-link; o, Ffh cross-link; >, L4 and L22 cross-link to a truncated translation product.
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fig1: Progression of nascent Lep through the ribosome. (A) Schematic representation of the 9, 24, 40, and 50Lep constructs with a cross-linking probe at position 3. The transmembrane regions (H1) and methionine tags are presented as thick gray lines and white bars, respectively. (B) In vitro translation of nascent 9–50LepTAG3 constructs was performed in the presence of (Tmd)Phe-tRNAsup. After translation, samples were irradiated with UV light to induce cross-linking, and the ribosome–nascent chain complexes were purified and analyzed by SDS-PAGE. UV-irradiated 9, 15, 24, 44, and 50LepTAG3 were immunoprecipitated with antisera as indicated. (C) Schematic representation of the cross-links observed in B. Numbers indicate the distance in amino acids of the cross-linking probe to the PTC. Images in different panels represent different parts of the gel or different exposure times. +, L4 cross-link; *, L22 cross-link; ^, L23 cross-link; o, Ffh cross-link; >, L4 and L22 cross-link to a truncated translation product.

Mentions: Lep nascent chains with a length of 9–50 amino acids were prepared by in vitro translation in the presence of [35S]methionine using truncated mRNAs that included a COOH-terminal sequence containing four methionines (4meth-tag; GlySer(Met)4) to increase the labeling efficiency (Fig. 1 A). 50Lep was chosen as the longest translation intermediate because it represents the shortest intermediate known to integrate into the membrane at the Sec–YidC insertion site (Houben et al., 2002). To enable site-directed photo–cross-linking, a TAG (stop) codon was introduced at position 3 just upstream of the first TM of Lep (H1). The TAG codon was suppressed during translation by adding a suppressor tRNA that carries a phenylalanine coupled to a photoreactive cross-linking (Tmd) probe (see Materials and methods). After translation, the probe was activated by UV irradiation to covalently link nascent Lep to any molecules that are in close proximity in the E. coli translation lysate. Cross-linked ribosome–nascent chain complexes were then sedimented through a high salt sucrose cushion and were further analyzed. The Lep 48mer failed to be synthesized and was not included in these studies. It should be noted that this cross-linking technique makes use of artificially arrested nascent polypeptides. It cannot be formally excluded that these chains adopt conformations that are less likely in vivo.


Early encounters of a nascent membrane protein: specificity and timing of contacts inside and outside the ribosome.

Houben EN, Zarivach R, Oudega B, Luirink J - J. Cell Biol. (2005)

Progression of nascent Lep through the ribosome. (A) Schematic representation of the 9, 24, 40, and 50Lep constructs with a cross-linking probe at position 3. The transmembrane regions (H1) and methionine tags are presented as thick gray lines and white bars, respectively. (B) In vitro translation of nascent 9–50LepTAG3 constructs was performed in the presence of (Tmd)Phe-tRNAsup. After translation, samples were irradiated with UV light to induce cross-linking, and the ribosome–nascent chain complexes were purified and analyzed by SDS-PAGE. UV-irradiated 9, 15, 24, 44, and 50LepTAG3 were immunoprecipitated with antisera as indicated. (C) Schematic representation of the cross-links observed in B. Numbers indicate the distance in amino acids of the cross-linking probe to the PTC. Images in different panels represent different parts of the gel or different exposure times. +, L4 cross-link; *, L22 cross-link; ^, L23 cross-link; o, Ffh cross-link; >, L4 and L22 cross-link to a truncated translation product.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171371&req=5

fig1: Progression of nascent Lep through the ribosome. (A) Schematic representation of the 9, 24, 40, and 50Lep constructs with a cross-linking probe at position 3. The transmembrane regions (H1) and methionine tags are presented as thick gray lines and white bars, respectively. (B) In vitro translation of nascent 9–50LepTAG3 constructs was performed in the presence of (Tmd)Phe-tRNAsup. After translation, samples were irradiated with UV light to induce cross-linking, and the ribosome–nascent chain complexes were purified and analyzed by SDS-PAGE. UV-irradiated 9, 15, 24, 44, and 50LepTAG3 were immunoprecipitated with antisera as indicated. (C) Schematic representation of the cross-links observed in B. Numbers indicate the distance in amino acids of the cross-linking probe to the PTC. Images in different panels represent different parts of the gel or different exposure times. +, L4 cross-link; *, L22 cross-link; ^, L23 cross-link; o, Ffh cross-link; >, L4 and L22 cross-link to a truncated translation product.
Mentions: Lep nascent chains with a length of 9–50 amino acids were prepared by in vitro translation in the presence of [35S]methionine using truncated mRNAs that included a COOH-terminal sequence containing four methionines (4meth-tag; GlySer(Met)4) to increase the labeling efficiency (Fig. 1 A). 50Lep was chosen as the longest translation intermediate because it represents the shortest intermediate known to integrate into the membrane at the Sec–YidC insertion site (Houben et al., 2002). To enable site-directed photo–cross-linking, a TAG (stop) codon was introduced at position 3 just upstream of the first TM of Lep (H1). The TAG codon was suppressed during translation by adding a suppressor tRNA that carries a phenylalanine coupled to a photoreactive cross-linking (Tmd) probe (see Materials and methods). After translation, the probe was activated by UV irradiation to covalently link nascent Lep to any molecules that are in close proximity in the E. coli translation lysate. Cross-linked ribosome–nascent chain complexes were then sedimented through a high salt sucrose cushion and were further analyzed. The Lep 48mer failed to be synthesized and was not included in these studies. It should be noted that this cross-linking technique makes use of artificially arrested nascent polypeptides. It cannot be formally excluded that these chains adopt conformations that are less likely in vivo.

Bottom Line: The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome-nascent chain complex to the Sec-YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit.The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY.Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology, Institute of Molecular Cell Biology, Vrije Universiteit, 1081 HV Amsterdam, Netherlands.

ABSTRACT
An unbiased photo-cross-linking approach was used to probe the "molecular path" of a growing nascent Escherichia coli inner membrane protein (IMP) from the peptidyl transferase center to the surface of the ribosome. The nascent chain was initially in proximity to the ribosomal proteins L4 and L22 and subsequently contacted L23, which is indicative of progression through the ribosome via the main ribosomal tunnel. The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome-nascent chain complex to the Sec-YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit. The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY. Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.

Show MeSH
Related in: MedlinePlus