Limits...
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.

Show MeSH

Related in: MedlinePlus

SRP is not oriented toward the ribosomal exit before it is able to interact with H1. (A) Schematic representation of 30 and 44Lep with a cross-linking probe at position 3 and 48Pf3Lep and 48Pf3LepKO with a cross-linking probe at position seven. H1 and the Pf3 extension are depicted as a thick gray line and a thin white bar, respectively. The four mutations in H1 to obtain the Pf3LepKO construct are the same as in Fig. 3 and are indicated here with four asterisks. (B) The constructs shown in A were translated in vitro with and without the addition of 350 nM of purified SRP. After translation, samples were cross-linked, purified, and immunoprecipitated as described in Fig. 1. Images in different panels represent different parts of the gel or different exposure times. *, L22 cross-link; ^, L23 cross-link; o, Ffh cross-link; >, TF cross-link.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171371&req=5

fig4: SRP is not oriented toward the ribosomal exit before it is able to interact with H1. (A) Schematic representation of 30 and 44Lep with a cross-linking probe at position 3 and 48Pf3Lep and 48Pf3LepKO with a cross-linking probe at position seven. H1 and the Pf3 extension are depicted as a thick gray line and a thin white bar, respectively. The four mutations in H1 to obtain the Pf3LepKO construct are the same as in Fig. 3 and are indicated here with four asterisks. (B) The constructs shown in A were translated in vitro with and without the addition of 350 nM of purified SRP. After translation, samples were cross-linked, purified, and immunoprecipitated as described in Fig. 1. Images in different panels represent different parts of the gel or different exposure times. *, L22 cross-link; ^, L23 cross-link; o, Ffh cross-link; >, TF cross-link.

Mentions: It has been suggested that structural information can be sensed in the exit tunnel and transduced to the ribosomal surface to influence the binding of SRP and TF near the exit site (Gu et al., 2003; Ullers et al., 2003). Although differences in intraribosomal cross-linking between nascent Lep derivatives with a functional or compromised TM were not detected (see the previous section), subtle changes in ribosomal contacts and conformation in response to a functional TM cannot be excluded. To more directly examine a possible recruitment of SRP at the nascent chain exit site by Lep H1 present in the ribosome interior, we designed and executed the following experiment. 30Lep, in which H1 is completely buried in the ribosomal tunnel, was extended at its NH2 terminus with an arbitrary hydrophilic peptide, including a cross-link probe to monitor a possible recruitment of SRP near the ribosomal exit site. The extension comprises the 18 NH2-terminal residues of the phage coat protein Pf3 with a TAG codon at position 7 (Fig. 4 A, 48Pf3LepTAG7). This peptide does not interfere with the targeting and insertion of full-length Lep in vivo (Lee et al., 1992) and is translocated across the inner membrane like the natural Lep NH2-terminal three amino acids. The position of the TAG is chosen such that it has the same spacing (41 amino acids) to the PTC as TAG3 in the 44Lep construct that was shown to cross-link strongly to Ffh (Fig. 4 A). As a control, H1 in 48Pf3LepTAG7 was subjected to knockout mutagenesis (described in previous section) to abolish its hydrophobic character without altering the spacing between the PTC and the cross-linking probe.


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)

SRP is not oriented toward the ribosomal exit before it is able to interact with H1. (A) Schematic representation of 30 and 44Lep with a cross-linking probe at position 3 and 48Pf3Lep and 48Pf3LepKO with a cross-linking probe at position seven. H1 and the Pf3 extension are depicted as a thick gray line and a thin white bar, respectively. The four mutations in H1 to obtain the Pf3LepKO construct are the same as in Fig. 3 and are indicated here with four asterisks. (B) The constructs shown in A were translated in vitro with and without the addition of 350 nM of purified SRP. After translation, samples were cross-linked, purified, and immunoprecipitated as described in Fig. 1. Images in different panels represent different parts of the gel or different exposure times. *, L22 cross-link; ^, L23 cross-link; o, Ffh cross-link; >, TF cross-link.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: SRP is not oriented toward the ribosomal exit before it is able to interact with H1. (A) Schematic representation of 30 and 44Lep with a cross-linking probe at position 3 and 48Pf3Lep and 48Pf3LepKO with a cross-linking probe at position seven. H1 and the Pf3 extension are depicted as a thick gray line and a thin white bar, respectively. The four mutations in H1 to obtain the Pf3LepKO construct are the same as in Fig. 3 and are indicated here with four asterisks. (B) The constructs shown in A were translated in vitro with and without the addition of 350 nM of purified SRP. After translation, samples were cross-linked, purified, and immunoprecipitated as described in Fig. 1. Images in different panels represent different parts of the gel or different exposure times. *, L22 cross-link; ^, L23 cross-link; o, Ffh cross-link; >, TF cross-link.
Mentions: It has been suggested that structural information can be sensed in the exit tunnel and transduced to the ribosomal surface to influence the binding of SRP and TF near the exit site (Gu et al., 2003; Ullers et al., 2003). Although differences in intraribosomal cross-linking between nascent Lep derivatives with a functional or compromised TM were not detected (see the previous section), subtle changes in ribosomal contacts and conformation in response to a functional TM cannot be excluded. To more directly examine a possible recruitment of SRP at the nascent chain exit site by Lep H1 present in the ribosome interior, we designed and executed the following experiment. 30Lep, in which H1 is completely buried in the ribosomal tunnel, was extended at its NH2 terminus with an arbitrary hydrophilic peptide, including a cross-link probe to monitor a possible recruitment of SRP near the ribosomal exit site. The extension comprises the 18 NH2-terminal residues of the phage coat protein Pf3 with a TAG codon at position 7 (Fig. 4 A, 48Pf3LepTAG7). This peptide does not interfere with the targeting and insertion of full-length Lep in vivo (Lee et al., 1992) and is translocated across the inner membrane like the natural Lep NH2-terminal three amino acids. The position of the TAG is chosen such that it has the same spacing (41 amino acids) to the PTC as TAG3 in the 44Lep construct that was shown to cross-link strongly to Ffh (Fig. 4 A). As a control, H1 in 48Pf3LepTAG7 was subjected to knockout mutagenesis (described in previous section) to abolish its hydrophobic character without altering the spacing between the PTC and the cross-linking probe.

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