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• W2005518105 abstract "In Escherichia coli, translocation of exported proteins across the cytoplasmic membrane is dependent on the motor protein SecA and typically begins only after synthesis of the substrate has already been completed (i.e., posttranslationally). Thus, it has generally been assumed that the translocation machinery also recognizes its protein substrates posttranslationally. Here we report a specific interaction between SecA and the ribosome at a site near the polypeptide exit channel. This interaction is mediated by conserved motifs in SecA and ribosomal protein L23, and partial disruption of this interaction in vivo by introducing mutations into the genes encoding SecA or L23 affects the efficiency of translocation by the posttranslational pathway. Based on these findings, we propose that SecA could interact with its nascent substrates during translation in order to efficiently channel them into the “posttranslational” translocation pathway. In Escherichia coli, translocation of exported proteins across the cytoplasmic membrane is dependent on the motor protein SecA and typically begins only after synthesis of the substrate has already been completed (i.e., posttranslationally). Thus, it has generally been assumed that the translocation machinery also recognizes its protein substrates posttranslationally. Here we report a specific interaction between SecA and the ribosome at a site near the polypeptide exit channel. This interaction is mediated by conserved motifs in SecA and ribosomal protein L23, and partial disruption of this interaction in vivo by introducing mutations into the genes encoding SecA or L23 affects the efficiency of translocation by the posttranslational pathway. Based on these findings, we propose that SecA could interact with its nascent substrates during translation in order to efficiently channel them into the “posttranslational” translocation pathway. SecA binds directly to ribosomes near the polypeptide exit channel Binding involves conserved residues in ribosomal protein L23 and SecA Disruption of the SecA-ribosome interaction causes a protein translocation defect SecA binds with increased affinity specifically to ribosomes containing substrates In E. coli, the translocation of most soluble periplasmic and outer-membrane proteins across the cytoplasmic membrane is carried out by the Sec machinery and begins only after all or a significant portion of the substrate protein has already been synthesized (i.e., posttranslationally) (Driessen and Nouwen, 2008Driessen A.J. Nouwen N. Protein translocation across the bacterial cytoplasmic membrane.Annu. Rev. Biochem. 2008; 77: 643-667Crossref PubMed Scopus (446) Google Scholar, Rapoport, 2007Rapoport T.A. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes.Nature. 2007; 450: 663-669Crossref PubMed Scopus (639) Google Scholar). The posttranslational translocation machinery consists minimally of two components: the SecYEG translocon and the ATPase SecA. SecYEG forms an hourglass-shaped hydrophilic pore in the cytoplasmic membrane through which proteins must pass in an unfolded conformation in transit across the membrane (Van den Berg et al., 2004Van den Berg B. Clemons Jr., W.M. Collinson I. Modis Y. Hartmann E. Harrison S.C. Rapoport T.A. X-ray structure of a protein-conducting channel.Nature. 2004; 427: 36-44Crossref PubMed Scopus (921) Google Scholar) and the motor protein SecA drives posttranslational translocation by pushing protein substrates through SecYEG through repeated rounds of ATP binding and hydrolysis (Brundage et al., 1990Brundage L. Hendrick J.P. Schiebel E. Driessen A.J. Wickner W. The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation.Cell. 1990; 62: 649-657Abstract Full Text PDF PubMed Scopus (374) Google Scholar, Erlandson et al., 2008Erlandson K.J. Miller S.B. Nam Y. Osborne A.R. Zimmer J. Rapoport T.A. A role for the two-helix finger of the SecA ATPase in protein translocation.Nature. 2008; 455: 984-987Crossref PubMed Scopus (100) Google Scholar). Periplasmic and outer-membrane proteins are targeted to the Sec translocation machinery by an N-terminal signal sequence, which is proteolytically removed from the protein during translocation (Rapoport, 2007Rapoport T.A. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes.Nature. 2007; 450: 663-669Crossref PubMed Scopus (639) Google Scholar). Alternatively, a small subset of proteins with highly hydrophobic signal sequences target proteins for efficient, cotranslational translocation, which is dependent on the signal recognition particle (SRP) (Huber et al., 2005Huber D. Boyd D. Xia Y. Olma M.H. Gerstein M. Beckwith J. Use of thioredoxin as a reporter to identify a subset of Escherichia coli signal sequences that promote signal recognition particle-dependent translocation.J. Bacteriol. 2005; 187: 2983-2991Crossref PubMed Scopus (100) Google Scholar). Although much is known about the mechanism of posttranslational translocation, substrate recognition by this pathway is much less well understood. One possibility is that SecA directly recognizes substrates by interacting with their N-terminal signal sequences (Gelis et al., 2007Gelis I. Bonvin A.M. Keramisanou D. Koukaki M. Gouridis G. Karamanou S. Economou A. Kalodimos C.G. Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR.Cell. 2007; 131: 756-769Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Alternatively, it has been suggested that the dedicated Sec chaperone SecB recognizes posttranslational substrates by specifically interacting with low-affinity sites in the unfolded protein and passing it to SecA (Fekkes et al., 1998Fekkes P. de Wit J.G. van der Wolk J.P. Kimsey H.H. Kumamoto C.A. Driessen A.J. Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA.Mol. Microbiol. 1998; 29: 1179-1190Crossref PubMed Scopus (99) Google Scholar, Hartl et al., 1990Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane.Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (433) Google Scholar). Regardless of which component is responsible for recognizing substrate proteins, all current models for posttranslational translocation agree that recognition of substrate proteins occurs posttranslationally without involvement of the ribosome (Driessen and Nouwen, 2008Driessen A.J. Nouwen N. Protein translocation across the bacterial cytoplasmic membrane.Annu. Rev. Biochem. 2008; 77: 643-667Crossref PubMed Scopus (446) Google Scholar, Rapoport, 2007Rapoport T.A. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes.Nature. 2007; 450: 663-669Crossref PubMed Scopus (639) Google Scholar). However, a number of recent genetic studies indirectly suggest that substrate recognition could occur cotranslationally. For example, the rate of signal sequence processing is faster in strains lacking the ribosome-associated chaperone trigger factor (TF) and translocation defects caused by mutations in the Sec machinery can be suppressed in strains expressing ribosome-binding-deficient TF (Lee and Bernstein, 2002Lee H.C. Bernstein H.D. Trigger factor retards protein export in Escherichia coli.J. Biol. Chem. 2002; 277: 43527-43535Crossref PubMed Scopus (64) Google Scholar, Ullers et al., 2007Ullers R.S. Ang D. Schwager F. Georgopoulos C. Genevaux P. Trigger Factor can antagonize both SecB and DnaK/DnaJ chaperone functions in Escherichia coli.Proc. Natl. Acad. Sci. USA. 2007; 104: 3101-3106Crossref PubMed Scopus (61) Google Scholar). Likewise, increasing overexpression of wild-type (WT) TF slows the rate of protein translocation (Lee and Bernstein, 2002Lee H.C. Bernstein H.D. Trigger factor retards protein export in Escherichia coli.J. Biol. Chem. 2002; 277: 43527-43535Crossref PubMed Scopus (64) Google Scholar). In the present study, we report that SecA binds to ribosomes specifically in a 1:1 stoichiometry. SecA binds to a site on the ribosome that includes ribosomal protein L23, which is situated near the opening of the polypeptide exit channel on the large subunit of the ribosome. In addition, we identified two conserved lysine residues in a region of SecA that are important for ribosome binding. Finally, we present evidence that the interaction between SecA and the ribosome facilitates translocation by the posttranslational pathway in vivo. Our findings suggest that SecA could cotranslationally interact with its substrates on the ribosome in order to channel them into the posttranslational translocation pathway. In order to test if SecA can bind to ribosomes, we incubated purified SecA with ribosomes at equimolar concentrations and separated ribosome-bound SecA from unbound SecA by pelleting ribosomes through a 30% sucrose cushion by ultracentrifugation. The majority of the SecA present in the binding reaction cosedimented with the ribosomes, indicating that SecA can bind to ribosomes (Figure 1A ). Increasing the concentration of potassium chloride in the binding reaction to 500 mM completely disrupted ribosome binding (Figure 1A), suggesting that the binding interface is dominated by hydrophilic interactions. In addition, increasing the concentration of SecA in the binding reaction resulted in a saturation of binding at an approximately 1:1 stoichiometry of SecA to ribosomes as determined by Coomassie staining and quantitative western blotting (Figure S1A, available online), which suggests that there is a single SecA-binding site on the ribosome. Serial dilutions of binding reactions containing equimolar concentrations of SecA and ribosomes suggested a KD for the SecA-ribosome complex in the submicromolar range (Figure S1B). We analyzed ribosome binding in greater detail by fluorescence anisotropy with SecA labeled with the fluorophore Ru(bpy)2(dcbpy) (den Blaauwen et al., 1997den Blaauwen T. Terpetschnig E. Lakowicz J.R. Driessen A.J. Interaction of SecB with soluble SecA.FEBS Lett. 1997; 416: 35-38Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). When 700 nM Ru(bpy)2(dcbpy)-labeled SecA was incubated with ribosomes, we observed an increase in anisotropy that equilibrated with increasing concentrations of ribosomes. We calculated a KD of around 900 nM by fitting the anisotropy data to the quadratic equation and assuming a 1:1 binding stoichiometry (Figure 1B). Scatchard analysis revealed a similar KD and confirmed that the binding stoichiometry was 1:1. Because the cellular concentrations of ribosomes and SecA are ∼20 μM and ∼8 μM, respectively (Akita et al., 1991Akita M. Shinkai A. Matsuyama S. Mizushima S. SecA, an essential component of the secretory machinery of Escherichia coli, exists as homodimer.Biochem. Biophys. Res. Commun. 1991; 174: 211-216Crossref PubMed Scopus (97) Google Scholar, Lill et al., 1988Lill R. Crooke E. Guthrie B. Wickner W. The “trigger factor cycle” includes ribosomes, presecretory proteins, and the plasma membrane.Cell. 1988; 54: 1013-1018Abstract Full Text PDF PubMed Scopus (114) Google Scholar), our results suggest that SecA binds to ribosomes in a physiologically relevant fashion. SecA normally forms dimers in solution that are in dynamic equilibrium with a KD of around 1 μM (Akita et al., 1991Akita M. Shinkai A. Matsuyama S. Mizushima S. SecA, an essential component of the secretory machinery of Escherichia coli, exists as homodimer.Biochem. Biophys. Res. Commun. 1991; 174: 211-216Crossref PubMed Scopus (97) Google Scholar, Doyle et al., 2000Doyle S.M. Braswell E.H. Teschke C.M. SecA folds via a dimeric intermediate.Biochemistry. 2000; 39: 11667-11676Crossref PubMed Scopus (61) Google Scholar, Or et al., 2002Or E. Navon A. Rapoport T. Dissociation of the dimeric SecA ATPase during protein translocation across the bacterial membrane.EMBO J. 2002; 21: 4470-4479Crossref PubMed Scopus (134) Google Scholar, Woodbury et al., 2002Woodbury R.L. Hardy S.J. Randall L.L. Complex behavior in solution of homodimeric SecA.Protein Sci. 2002; 11: 875-882Crossref PubMed Scopus (123) Google Scholar). The 1:1 stoichiometry for ribosome binding therefore suggested that SecA might interact with the ribosome along its dimer interface. Several different dimer interfaces have been suggested by different structural models. However, when we compared the interfaces in three structural models of the SecA dimer (PDB files 2FSF, 2IBM, and 2IPC) (Papanikolau et al., 2007Papanikolau Y. Papadovasilaki M. Ravelli R.B. McCarthy A.A. Cusack S. Economou A. Petratos K. Structure of dimeric SecA, the Escherichia coli preprotein translocase motor.J. Mol. Biol. 2007; 366: 1545-1557Crossref PubMed Scopus (111) Google Scholar, Vassylyev et al., 2006Vassylyev D.G. Mori H. Vassylyeva M.N. Tsukazaki T. Kimura Y. Tahirov T.H. Ito K. Crystal structure of the translocation ATPase SecA from Thermus thermophilus reveals a parallel, head-to-head dimer.J. Mol. Biol. 2006; 364: 248-258Crossref PubMed Scopus (75) Google Scholar, Zimmer et al., 2006Zimmer J. Li W. Rapoport T.A. A novel dimer interface and conformational changes revealed by an X-ray structure of B. subtilis SecA.J. Mol. Biol. 2006; 364: 259-265Crossref PubMed Scopus (68) Google Scholar), we noticed that many of the residues participating in hydrophilic bonds between the two SecA subunits were located in or near nucleotide binding domain-2 (NBD2; ∼ residues 419–615). Moreover, in two of the structural models (2IBM and 2IPC), these residues cluster to the C-terminal region of NBD2 and the long α-helical linker domain immediately C-terminal to NBD2 (residues 616–669; Figure 2A and Table S1). We therefore purified a fragment of SecA consisting of amino acid residues 418–668 (SecA418–668) in order to test if it could bind to ribosomes. This protein fragment includes all of NBD2 and the α-helical linker domain and is nearly identical to the fragment of SecA used by Gelis et al., 2007Gelis I. Bonvin A.M. Keramisanou D. Koukaki M. Gouridis G. Karamanou S. Economou A. Kalodimos C.G. Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR.Cell. 2007; 131: 756-769Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar for structural studies of SecA. Purification of SecA418–668 yielded a series of truncation products, which were the result of in vivo proteolytic processing from the C terminus because the protein was purified by using an N-terminal affinity tag that was cleaved from the protein during purification. Full-length SecA418–668 and the largest truncation product bound ribosomes in cosedimentation experiments (Figure 2B). However, the smaller truncation products could not bind ribosomes. Because the N-termini of these truncation products were identical, we could determine the C-termini of the fragments by mass spectrometry. The smallest truncation product that retained ribosome-binding activity terminated with glutamine-644 (Figure 2C), and the largest fragment that could not bind to ribosomes terminated with alanine-626. Because both of these residues are located in the N-terminal region of the α-helical linker domain, these results suggested that this region is important for ribosome binding. In order to determine if the α-helical linker domain alone was sufficient to direct ribosome binding, we tested whether a fragment of SecA consisting of residues 616–668 could target the small ubiquitin-like modifier (SUMO) of Saccharomyces cerevisiae, which has no detectable ribosome-binding activity, to the ribosome. Similar to SecA418–668, a fusion protein consisting of SecA616–668 fused to the C terminus of a Strep-tagged SUMO protein (Strep-SUMO-SecA616–668) purified as a series of C-terminally truncated fragments. Although the truncation products could not bind to the ribosome, full-length Strep-SUMO-SecA616–668 cosedimented with ribosomes, albeit weakly, through a 30% sucrose cushion (Figure 2D), suggesting that the linker domain is both necessary and sufficient to target SecA to the ribosome. Two residues in the α-helical linker domain, lysine-625 and lysine-633, raised our interest as being potentially involved in ribosome binding based on the following characteristics: (1) both residues are located in the region of SecA418–668 that appears to be important for ribosome binding; (2) the side chains of these residues are completely solvent exposed and located on the same face of an α helix; (3) despite the general sequence variability in this region of SecA (Papanikolau et al., 2007Papanikolau Y. Papadovasilaki M. Ravelli R.B. McCarthy A.A. Cusack S. Economou A. Petratos K. Structure of dimeric SecA, the Escherichia coli preprotein translocase motor.J. Mol. Biol. 2007; 366: 1545-1557Crossref PubMed Scopus (111) Google Scholar), all of the orthologs of SecA (but not SecA2) from widely diverged bacterial species in the COG database (http://www.ncbi.nlm.nih.gov/COG/) contain a positively charged residue at either position 625 or 633 or both, which could interact with the negatively charged ribosome; (4) lysine-625 and lysine-633 participate in hydrophilic bonds between subunits in one or more of the structural models of the SecA dimer (Table S1); (5) to our knowledge, these residues have not previously been identified as being important for any known activity of SecA. When we repeated the ribosome sedimentation experiments by using SecA variants containing alanine substitutions at one (SecA[K625A] or SecA[K633A]) or both (SecA[K625A/K633A]) of these positions, we found that the individual K625A and K633A substitutions greatly interfered with ribosome binding and the double K625A/K633A substitution even further reduced the amount of SecA that copelleted with ribosomes (Figure 2E). These substitutions do not appear to significantly affect the SecA structure because the CD spectra and the basal ATPase rates of the purified SecA variants were indistinguishable from those of the wild-type protein (Figure S2A and Table S2). In addition, analytical size-exclusion chromatography indicated that SecA(K625A/K633A) dimerized to a similar extent as the wild-type protein (Figure S2B). We sought to identify the SecA-binding site on the ribosome by using nonspecific crosslinking. Incubation of SecA with ribosomes in the presence of EDC, which nonspecifically catalyzes the formation of a peptide bond between carboxyl groups and primary amino groups, resulted in the appearance of a single high-molecular weight crosslinking adduct that was visible by Coomassie staining (Figure 3A ) and could be recognized by anti-SecA antibodies. The crosslinking adduct was not present when either SecA alone or ribosomes alone were incubated with EDC, indicating that the band was an adduct between SecA and a component of the ribosome. In order to facilitate purification of the crosslinking adduct, we fused a short (15-amino acid) biotin-attachment peptide (Beckett et al., 1999Beckett D. Kovaleva E. Schatz P.J. A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation.Protein Sci. 1999; 8: 921-929Crossref PubMed Scopus (518) Google Scholar) to the C terminus of SecA (SecA-biotin). SecA-biotin competed with wild-type SecA for ribosome binding in vitro (Figure S3) and could complement a ΔsecA mutation in vivo, indicating that it behaves like wild-type SecA. Using SecA-biotin, we purified the SecA-containing crosslinking adduct by using streptavidin-coupled magnetic beads (Figure 3B). When we analyzed the purified crosslinking adduct by western blotting against each of nearly all of the ribosomal proteins, we found that it cross-reacted only with the anti-L23 and anti-SecA antibodies (Figure 3C), and we could confirm the presence of L23 in the crosslinking adduct by LC-MS/MS (Table S3). These data suggested that SecA binds to the ribosome near L23, which is located at the polypeptide exit channel on the large subunit of the ribosome. In order to identify which amino acid residues in L23 might be involved in SecA binding, we devised a genetic screen for mutations in the L23 gene (rplW) that cause a dominant protein translocation defect. We expressed different L23 variants from an IPTG-inducible promoter on a pTrc99b plasmid in a strain containing the malE-lacZ reporter gene, which encodes a fusion protein between maltose binding protein (MBP) and β-galactosidase (MBP-LacZ) and has been used previously to screen for sec mutants (Gannon and Kumamoto, 1993Gannon P.M. Kumamoto C.A. Mutations of the molecular chaperone protein SecB which alter the interaction between SecB and maltose-binding protein.J. Biol. Chem. 1993; 268: 1590-1595Abstract Full Text PDF PubMed Google Scholar, Kumamoto and Beckwith, 1983Kumamoto C.A. Beckwith J. Mutations in a new gene, secB, cause defective protein localization in Escherichia coli.J. Bacteriol. 1983; 154: 253-260Crossref PubMed Google Scholar, Oliver and Beckwith, 1981Oliver D.B. Beckwith J. E. coli mutant pleiotropically defective in the export of secreted proteins.Cell. 1981; 25: 765-772Abstract Full Text PDF PubMed Scopus (276) Google Scholar). Because the MBP-LacZ fusion protein is translocated across the cytoplasmic membrane by virtue of the N-terminal signal sequence of MBP, and because β-galactosidase is not functional in the periplasm, otherwise wild-type cells are normally phenotypically Lac-. However, the expression of mutant L23 proteins that cause a defect in protein translocation should result in the accumulation of β-galactosidase in the cytoplasm and a Lac+ phenotype. As expected, expression of wild-type L23 from a plasmid did not affect protein translocation in N48 as indicated by the low β-galactosidase activity in these cells. However, expression of L23 variants containing the following substitutions in conserved surface residues caused a significant increase in β-galactosidase activity (Figure 4A ): E42A, E89A, F51AAA, S78A/D79A/W80A (L23[SDW]), E42A/I43A/K44A (L23[EIK]), F51A/E52A/E54A/E56A (L23[FEVEVE]), F51A/E52A/E54A/E56A/E89A (L23[FEVEVE/E89A]), E42A/F51A/E52A/E54A/E56A (L23[E42A/FEVEVE]), and E42A/I43A/K44A/F51A/E52A/E54A/E56 (L23[EIK/FEVEVE]). In addition, partial (K66-S78) or complete (V63-K81) deletion of the loop domain of L23 (Bornemann et al., 2008Bornemann T. Jöckel J. Rodnina M.V. Wintermeyer W. Signal sequence-independent membrane targeting of ribosomes containing short nascent peptides within the exit tunnel.Nat. Struct. Mol. Biol. 2008; 15: 494-499Crossref PubMed Scopus (121) Google Scholar), which extends into the interior of the ribosome and forms a portion of the wall of the polypeptide exit channel, caused increased β-galactosidase activities in N48. These mutations cluster around three conserved regions in L23 (Figure 4B): (1) a previously identified patch of mostly acidic surface residues of unknown function beginning with phenylalanine-51 and consisting of the glutamates in the sequence 51FEVEVE (Kramer et al., 2004Kramer G. Rutkowska A. Wegrzyn R.D. Patzelt H. Kurz T.A. Merz F. Rauch T. Vorderwülbecke S. Deuerling E. Bukau B. Functional dissection of Escherichia coli trigger factor: unraveling the function of individual domains.J. Bacteriol. 2004; 186: 3777-3784Crossref PubMed Scopus (70) Google Scholar). We considered glutamate-89 to be a member of this cluster because it appears to continue the ridge of conserved acidic residues formed by this sequence (Schuwirth et al., 2005Schuwirth B.S. Borovinskaya M.A. Hau C.W. Zhang W. Vila-Sanjurjo A. Holton J.M. Cate J.H. Structures of the bacterial ribosome at 3.5 A resolution.Science. 2005; 310: 827-834Crossref PubMed Scopus (1042) Google Scholar); (2) a cluster of three conserved partially surface-exposed residues on the same face of L23 as 51FEVEVE with the sequence 42EIK; and (3) the loop domain of L23 (residues 63–81). In contrast, expression of L23 variants that were defective for TF binding (that is, substitutions in the sequence 16VSEKAS, which is located on the opposite face of L23 from the above-described motifs) had no effect on β-galactosidase activity (Figure 4A), suggesting that the translocation defects were not the result of a defect in TF binding. Because a number of other factors involved in protein translocation (such as the SRP, SecYEG, and YidC) also bind to the ribosome at or near L23 (Gu et al., 2003Gu S.Q. Peske F. Wieden H.J. Rodnina M.V. Wintermeyer W. The signal recognition particle binds to protein L23 at the peptide exit of the Escherichia coli ribosome.RNA. 2003; 9: 566-573Crossref PubMed Scopus (123) Google Scholar, Kohler et al., 2009Kohler R. Boehringer D. Greber B. Bingel-Erlenmeyer R. Collinson I. Schaffitzel C. Ban N. YidC and Oxa1 form dimeric insertion pores on the translating ribosome.Mol. Cell. 2009; 34: 344-353Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, Mitra et al., 2005Mitra K. Schaffitzel C. Shaikh T. Tama F. Jenni S. Brooks 3rd, C.L. Ban N. Frank J. Structure of the E. coli protein-conducting channel bound to a translating ribosome.Nature. 2005; 438: 318-324Crossref PubMed Scopus (214) Google Scholar, Schaffitzel et al., 2006Schaffitzel C. Oswald M. Berger I. Ishikawa T. Abrahams J.P. Koerten H.K. Koning R.I. Ban N. Structure of the E. coli signal recognition particle bound to a translating ribosome.Nature. 2006; 444: 503-506Crossref PubMed Scopus (103) Google Scholar), we wished to confirm that the residues we identified in our genetic screen were important for SecA binding. To this end, we purified L23(FEVEVE)-, L23(EIK)-, L23(E42A/FEVEVE)-, and L23(FEVEVE/E89A)-containing ribosomes from strains whose sole copy of rplW was expressed from an IPTG-inducible promoter on a plasmid. Cells expressing the mutant L23 proteins grew slightly faster than those expressing wild-type L23 at 37°C with the exception of those expressing L23(E42A/FEVEVE), which grew slightly slower. We could not obtain ΔrplW transductants of strains expressing L23(EIK/FEVEVE), suggesting that this variant was not functional enough to support growth. The affinity of SecA for ribosomes containing the L23 variants was decreased, demonstrated by a 2–3-fold increase in the KD as determined by fluorescence anisotropy experiments (Figure 4C). In line with these results, 20%–60% less SecA cosedimented with the mutant ribosomes through a sucrose cushion when incubated at an equimolar concentration of 1 μM (Figure 4D). In addition, the Strep-SUMO-SecA616–668 construct, which we used to identify the binding domain in SecA, could not bind to ribosomes containing L23(E42A) (Figure 2D). Ribosomes containing L23 variants that caused a SecA-binding defect were not defective for SRP binding in ribosome sedimentation experiments, suggesting that the mutations do not interfere with SRP binding (data not shown). These data suggest that L23 makes up part of the SecA-binding site on the ribosome. In order to test whether ribosome binding by SecA was important for protein translocation in vivo, we examined the translocation of two well-studied posttranslational substrates, MBP and β-lactamase, in strains whose sole copy of the secA or rplW gene was under the control of an IPTG-inducible promoter. The rate of protein translocation, as measured by signal sequence processing in pulse-chase experiments, in strains expressing ribosome-binding-deficient variants of SecA was retarded with respect to strains expressing wild-type SecA, respectively (Figure 5A ). A fit of the disappearance of the precursor MBP band in strains expressing SecA-binding-deficient L23 variants revealed that although the increase in the half-life of precursor MBP is small, the calculated amplitude as a fraction of the total signal change was significantly larger compared to strains expressing wild-type L23 (Figure 5B and Figure S5). This result suggests that these strains accumulate more full-length precursor MBP at early time points and that more protein escapes the cotranslational processing observed by Randall and colleagues for many posttranslational translocation substrates (Josefsson and Randall, 1983Josefsson L.G. Randall L.L. Analysis of cotranslational proteolytic processing of nascent chains using two-dimensional gel electrophoresis.Methods Enzymol. 1983; 97: 77-85Crossref PubMed Scopus (16) Google Scholar, Randall, 1983Randall L.L. Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation.Cell. 1983; 33: 231-240Abstract Full Text PDF PubMed Scopus (122) Google Scholar). In addition, the defect in translocation in the secA and rplW mutants resulted in a small but reproducible accumulation of MBP and β-lactamase in their cytoplasms (Figures 5C and 5D). Although the fact that MBP and β-lactamase accumulate in the mature form in the cytoplasms of strains expressing L23(FEVEVE/E89A) seems unusual, this phenomenon could be detected in different background strains (data not shown) and has been observed before in other strains with translocation defects (Schierle et al., 2003Schierle C.F. Berkmen M. Huber D. Kumamoto C. Boyd D. Beckwith J. The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway.J. Bacteriol. 2003; 185: 5706-5713Crossref PubMed Scopus (156) Google Scholar). Finally, double mutants expressing both mutant L23 and mutant SecA accumulated more precursor MBP at early time points compared to strains expressing only mutant L23 (Figure 5E). Taken together, the additive nature of the translocation defects caused by the secA and rplW mutants in combination with our finding that expression of the mutant L23 proteins causes th" @default.
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• W2005518105 date "2011-02-01" @default.
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• W2005518105 title "SecA Interacts with Ribosomes in Order to Facilitate Posttranslational Translocation in Bacteria" @default.
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