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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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The initial targeting and insertion steps of Lep. (A) In vitro translation of nascent 32–50LepTAG3 was performed in the presence of IMVs and (Tmd)Phe-tRNAsup. After translations, samples were kept in the dark or UV irradiated and were subsequently extracted with sodium carbonate and spun down. UV-irradiated pellet fractions of 44LepTAG3 were immunoprecipitated as indicated. (B) Quantifications of SecY and YidC cross-linking to 38–50LepTAG3 (A, lanes 13–18) relative to the amount of nonirradiated carbonate-resistant nascent chains (A, lanes 4–9). Highest values for cross-linking efficiency were taken as 100%. Images in different panels represent different parts of the gel or different exposure times. *, SecY cross-link; o, YidC cross-link.
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fig2: The initial targeting and insertion steps of Lep. (A) In vitro translation of nascent 32–50LepTAG3 was performed in the presence of IMVs and (Tmd)Phe-tRNAsup. After translations, samples were kept in the dark or UV irradiated and were subsequently extracted with sodium carbonate and spun down. UV-irradiated pellet fractions of 44LepTAG3 were immunoprecipitated as indicated. (B) Quantifications of SecY and YidC cross-linking to 38–50LepTAG3 (A, lanes 13–18) relative to the amount of nonirradiated carbonate-resistant nascent chains (A, lanes 4–9). Highest values for cross-linking efficiency were taken as 100%. Images in different panels represent different parts of the gel or different exposure times. *, SecY cross-link; o, YidC cross-link.

Mentions: A previous cross-linking study has shown that nascent Lep with a length of 50 amino acids can be targeted to the membrane (Houben et al., 2002). H1 in 50Lep already cross-linked SecY and YidC, although full membrane integration was observed only with constructs that were longer than 70 amino acids (Houben et al., 2002). The remarkably early interaction of nascent Lep with the targeting factor SRP (see previous section) prompted us to reevaluate early contacts of nascent Lep with membrane components. In short, 32–50LepTAG3 were produced in the presence of inverted inner membrane vesicles (IMVs). After translation, one half of each sample was UV irradiated, whereas the other half was kept in the dark to serve as a control. Membranes were treated with sodium carbonate, and membrane-integrated material was collected by centrifugation (Fig. 2 A).


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)

The initial targeting and insertion steps of Lep. (A) In vitro translation of nascent 32–50LepTAG3 was performed in the presence of IMVs and (Tmd)Phe-tRNAsup. After translations, samples were kept in the dark or UV irradiated and were subsequently extracted with sodium carbonate and spun down. UV-irradiated pellet fractions of 44LepTAG3 were immunoprecipitated as indicated. (B) Quantifications of SecY and YidC cross-linking to 38–50LepTAG3 (A, lanes 13–18) relative to the amount of nonirradiated carbonate-resistant nascent chains (A, lanes 4–9). Highest values for cross-linking efficiency were taken as 100%. Images in different panels represent different parts of the gel or different exposure times. *, SecY cross-link; o, YidC cross-link.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171371&req=5

fig2: The initial targeting and insertion steps of Lep. (A) In vitro translation of nascent 32–50LepTAG3 was performed in the presence of IMVs and (Tmd)Phe-tRNAsup. After translations, samples were kept in the dark or UV irradiated and were subsequently extracted with sodium carbonate and spun down. UV-irradiated pellet fractions of 44LepTAG3 were immunoprecipitated as indicated. (B) Quantifications of SecY and YidC cross-linking to 38–50LepTAG3 (A, lanes 13–18) relative to the amount of nonirradiated carbonate-resistant nascent chains (A, lanes 4–9). Highest values for cross-linking efficiency were taken as 100%. Images in different panels represent different parts of the gel or different exposure times. *, SecY cross-link; o, YidC cross-link.
Mentions: A previous cross-linking study has shown that nascent Lep with a length of 50 amino acids can be targeted to the membrane (Houben et al., 2002). H1 in 50Lep already cross-linked SecY and YidC, although full membrane integration was observed only with constructs that were longer than 70 amino acids (Houben et al., 2002). The remarkably early interaction of nascent Lep with the targeting factor SRP (see previous section) prompted us to reevaluate early contacts of nascent Lep with membrane components. In short, 32–50LepTAG3 were produced in the presence of inverted inner membrane vesicles (IMVs). After translation, one half of each sample was UV irradiated, whereas the other half was kept in the dark to serve as a control. Membranes were treated with sodium carbonate, and membrane-integrated material was collected by centrifugation (Fig. 2 A).

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