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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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Interactions of nascent LepKO, pre-PhoE, and RpoB with ribosomal proteins and cytosolic chaperones. (A) Schematic representation of LepTAG3 and LepKOTAG3. The sequences of wild-type and mutated H1 are depicted. (B and C) 15–44LepKOTAG3 (B) and 15/44pre-PhoETAG3 and 15/44RpoBTAG3 (C) were produced, cross-linked, purified, and immunoprecipitated as described in Fig. 1. Images in different panels represent different parts of the gel or different exposure times. +, L4 cross-link; *, L22 cross-link; ^, L23 cross-link; >, TF cross-link.
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fig3: Interactions of nascent LepKO, pre-PhoE, and RpoB with ribosomal proteins and cytosolic chaperones. (A) Schematic representation of LepTAG3 and LepKOTAG3. The sequences of wild-type and mutated H1 are depicted. (B and C) 15–44LepKOTAG3 (B) and 15/44pre-PhoETAG3 and 15/44RpoBTAG3 (C) were produced, cross-linked, purified, and immunoprecipitated as described in Fig. 1. Images in different panels represent different parts of the gel or different exposure times. +, L4 cross-link; *, L22 cross-link; ^, L23 cross-link; >, TF cross-link.

Mentions: Recruitment of SRP to the ribosomal exit site might be indirectly induced by specific interactions of nascent Lep within the ribosomal tunnel. In this scenario, H1 of Lep that is still buried inside the tunnel would be expected to play a decisive discriminatory role. Because we now have a detailed map of the interactions of nascent Lep with L4, L22, and L23 as it moves through the ribosomal tunnel, we decided to examine if these interactions depend on a functional H1 domain. H1 in LepTAG3 constructs was “knocked out” by replacing four hydrophobic Leu residues with charged residues (Glu, Arg, and Lys) and a helix-breaking residue (Pro; Fig. 3 A). 15, 24, 34, and 44mers of the resulting Lep knockout (LepKOTAG3) were synthesized, cross-linked, and analyzed (Fig. 3 B).


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)

Interactions of nascent LepKO, pre-PhoE, and RpoB with ribosomal proteins and cytosolic chaperones. (A) Schematic representation of LepTAG3 and LepKOTAG3. The sequences of wild-type and mutated H1 are depicted. (B and C) 15–44LepKOTAG3 (B) and 15/44pre-PhoETAG3 and 15/44RpoBTAG3 (C) were produced, cross-linked, purified, and immunoprecipitated as described in Fig. 1. Images in different panels represent different parts of the gel or different exposure times. +, L4 cross-link; *, L22 cross-link; ^, L23 cross-link; >, TF cross-link.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171371&req=5

fig3: Interactions of nascent LepKO, pre-PhoE, and RpoB with ribosomal proteins and cytosolic chaperones. (A) Schematic representation of LepTAG3 and LepKOTAG3. The sequences of wild-type and mutated H1 are depicted. (B and C) 15–44LepKOTAG3 (B) and 15/44pre-PhoETAG3 and 15/44RpoBTAG3 (C) were produced, cross-linked, purified, and immunoprecipitated as described in Fig. 1. Images in different panels represent different parts of the gel or different exposure times. +, L4 cross-link; *, L22 cross-link; ^, L23 cross-link; >, TF cross-link.
Mentions: Recruitment of SRP to the ribosomal exit site might be indirectly induced by specific interactions of nascent Lep within the ribosomal tunnel. In this scenario, H1 of Lep that is still buried inside the tunnel would be expected to play a decisive discriminatory role. Because we now have a detailed map of the interactions of nascent Lep with L4, L22, and L23 as it moves through the ribosomal tunnel, we decided to examine if these interactions depend on a functional H1 domain. H1 in LepTAG3 constructs was “knocked out” by replacing four hydrophobic Leu residues with charged residues (Glu, Arg, and Lys) and a helix-breaking residue (Pro; Fig. 3 A). 15, 24, 34, and 44mers of the resulting Lep knockout (LepKOTAG3) were synthesized, cross-linked, and analyzed (Fig. 3 B).

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