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• W4310859957 abstract "•SecB co-translationally engages proteins of all cellular compartments•SecB binds to membrane-associated ribosome nascent chain complexes•SecB interaction with nascent IMPs is critical for membrane protein biogenesis•TF controls the SecB binding to cytoplasmic and translocated proteins The chaperone SecB has been implicated in de novo protein folding and translocation across the membrane, but it remains unclear which nascent polypeptides SecB binds, when during translation SecB acts, how SecB function is coordinated with other chaperones and targeting factors, and how polypeptide engagement contributes to protein biogenesis. Using selective ribosome profiling, we show that SecB binds many nascent cytoplasmic and translocated proteins generally late during translation and controlled by the chaperone trigger factor. Revealing an uncharted role in co-translational translocation, inner membrane proteins (IMPs) are the most prominent nascent SecB interactors. Unlike other substrates, IMPs are bound early during translation, following the membrane targeting by the signal recognition particle. SecB remains bound until translation is terminated, and contributes to membrane insertion. Our study establishes a role of SecB in the co-translational maturation of proteins from all cellular compartments and functionally implicates cytosolic chaperones in membrane protein biogenesis. The chaperone SecB has been implicated in de novo protein folding and translocation across the membrane, but it remains unclear which nascent polypeptides SecB binds, when during translation SecB acts, how SecB function is coordinated with other chaperones and targeting factors, and how polypeptide engagement contributes to protein biogenesis. Using selective ribosome profiling, we show that SecB binds many nascent cytoplasmic and translocated proteins generally late during translation and controlled by the chaperone trigger factor. Revealing an uncharted role in co-translational translocation, inner membrane proteins (IMPs) are the most prominent nascent SecB interactors. Unlike other substrates, IMPs are bound early during translation, following the membrane targeting by the signal recognition particle. SecB remains bound until translation is terminated, and contributes to membrane insertion. Our study establishes a role of SecB in the co-translational maturation of proteins from all cellular compartments and functionally implicates cytosolic chaperones in membrane protein biogenesis. Efficient synthesis of folded, correctly localized proteins is critically important for cellular integrity and is supported by multiple chaperones and targeting factors that often engage nascent polypeptides during translation.1Kramer G. Shiber A. Bukau B. Mechanisms of cotranslational maturation of newly synthesized proteins.Annu. Rev. Biochem. 2019; 88: 337-364https://doi.org/10.1146/annurev-biochem-013118-111717Crossref PubMed Scopus (77) Google Scholar,2Koubek J. Schmitt J. Galmozzi C.V. Kramer G. Mechanisms of cotranslational protein maturation in bacteria.Front. Mol. Biosci. 2021; 8689755https://doi.org/10.3389/fmolb.2021.689755Crossref Scopus (9) Google Scholar,3Steinberg R. Knüpffer L. Origi A. Asti R. Koch H.G. Co-translational protein targeting in bacteria.FEMS Microbiol. Lett. 2018; 365https://doi.org/10.1093/femsle/fny095Crossref Scopus (46) Google Scholar,4Rodnina M.V. Wintermeyer W. Protein elongation, Co-translational folding and targeting.Journal of molecular biology. 2016; 428: 2165-2185https://doi.org/10.1016/j.jmb.2016.03.022Crossref PubMed Scopus (41) Google Scholar Bacteria employ three major routes of protein maturation that are specific for the different cellular compartments. About 70%–80% of newly synthesized proteins fold in the cytosol, generally supported by a network of molecular chaperones. The remaining 20%–30% are either inserted into the inner membrane by the co-translational translocation pathway or translocated across the inner membrane to the periplasm or the outer membrane by post-translational translocation.5Crane J.M. Randall L.L. The sec system: protein export in Escherichia coli.EcoSal Plus. 2017; 7https://doi.org/10.1128/ecosalplus.ESP-0002-2017Crossref Scopus (56) Google Scholar Triaging and the initiation of folding or translocation begin early during protein synthesis and are mediated by a dynamic interplay of chaperones and targeting factors.1Kramer G. Shiber A. Bukau B. Mechanisms of cotranslational maturation of newly synthesized proteins.Annu. Rev. Biochem. 2019; 88: 337-364https://doi.org/10.1146/annurev-biochem-013118-111717Crossref PubMed Scopus (77) Google Scholar,6Pechmann S. Willmund F. Frydman J. The ribosome as a hub for protein quality control.Mol. Cell. 2013; 49: 411-421https://doi.org/10.1016/j.molcel.2013.01.020Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar,7Deuerling E. Gamerdinger M. Kreft S.G. Chaperone interactions at the ribosome.Cold Spring Harb. Perspect. Biol. 2019; 11: a033977https://doi.org/10.1101/cshperspect.a033977Crossref PubMed Scopus (33) Google Scholar,8Smets D. Loos M.S. Karamanou S. Economou A. Protein transport across the bacterial plasma membrane by the sec pathway.Protein J. 2019; 38: 262-273https://doi.org/10.1007/s10930-019-09841-8Crossref Scopus (19) Google Scholar,9Castanié-Cornet M.P. Bruel N. Genevaux P. Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane.Biochim. Biophys. Acta. 2014; 1843: 1442-1456https://doi.org/10.1016/j.bbamcr.2013.11.007Crossref PubMed Scopus (84) Google Scholar In Escherichia coli, protein translocation into and across the cytoplasmic membrane depends on two major secretion pathways that employ specific targeting factors but converge at the ubiquitously used SecYEG translocon.3Steinberg R. Knüpffer L. Origi A. Asti R. Koch H.G. Co-translational protein targeting in bacteria.FEMS Microbiol. Lett. 2018; 365https://doi.org/10.1093/femsle/fny095Crossref Scopus (46) Google Scholar,10Kuhn A. Koch H.G. Dalbey R.E. Targeting and insertion of membrane proteins.EcoSal Plus. 2017; 7https://doi.org/10.1128/ecosalplus.ESP-0012-2016Crossref Scopus (47) Google Scholar Co-translational translocation initiates early during protein synthesis by binding of the signal recognition particle (SRP) to ribosomes translating inner membrane proteins (IMPs).11Luirink J. High S. Wood H. Giner A. Tollervey D. Dobberstein B. Signal sequence recognition by an Escherichia coli ribonucleoprotein complex.Nature. 1992; 359: 741-743Crossref PubMed Scopus (154) Google Scholar,12Schibich D. Gloge F. Pöhner I. Björkholm P. Wade R.C. von Heijne G. Bukau B. Kramer G. Global profiling of SRP interaction with nascent polypeptides.Nature. 2016; 536: 219-223https://doi.org/10.1038/nature19070Crossref PubMed Scopus (88) Google Scholar SRP directly binds to ribosomes,13Schaffitzel 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 (109) Google Scholar and this interaction is selectively stabilized by emerging trans-membrane domains (TMDs) of IMPs.12Schibich D. Gloge F. Pöhner I. Björkholm P. Wade R.C. von Heijne G. Bukau B. Kramer G. Global profiling of SRP interaction with nascent polypeptides.Nature. 2016; 536: 219-223https://doi.org/10.1038/nature19070Crossref PubMed Scopus (88) Google Scholar By interaction with the membrane-associated SRP receptor FtsY, SRP mediates the docking of ribosomes to the SecYEG translocon.14Miller J.D. Bernstein H.D. Walter P. Interaction of E. coli Ffh/4.5S ribonucleoprotein and FtsY mimics that of mammalian signal recognition particle and its receptor.Nature. 1994; 367: 657-659https://doi.org/10.1038/367657a0Crossref PubMed Scopus (186) Google Scholar Membrane insertion of IMPs occurs co-translationally, largely energized by the translation process itself. Productive membrane integration of some IMPs furthermore requires the translocation activity of the ATPase SecA.10Kuhn A. Koch H.G. Dalbey R.E. Targeting and insertion of membrane proteins.EcoSal Plus. 2017; 7https://doi.org/10.1128/ecosalplus.ESP-0012-2016Crossref Scopus (47) Google Scholar,15Wang S. Yang C.I. Shan S.O. SecA mediates cotranslational targeting and translocation of an inner membrane protein.J. Cell Biol. 2017; 216: 3639-3653https://doi.org/10.1083/jcb.201704036Crossref PubMed Scopus (29) Google Scholar,16Collinson I. SecA-a new twist in the tale.J. Bacteriol. 2017; 199: 007366-e816https://doi.org/10.1128/JB.00736-16Crossref Scopus (7) Google Scholar,17Neumann-Haefelin C. Schäfer U. Müller M. Koch H.G. SRP-dependent co-translational targeting and SecA-dependent translocation analyzed as individual steps in the export of a bacterial protein.Embo Journal. 2000; 19: 6419-6426https://doi.org/10.1093/emboj/19.23.6419Crossref PubMed Scopus (109) Google Scholar Translocation of outer membrane proteins (OMPs) and periplasmic proteins (PPs) across the membrane involves the alternative, SRP-independent post-translational translocation pathway.8Smets D. Loos M.S. Karamanou S. Economou A. Protein transport across the bacterial plasma membrane by the sec pathway.Protein J. 2019; 38: 262-273https://doi.org/10.1007/s10930-019-09841-8Crossref Scopus (19) Google Scholar,18Driessen A.J.M. Nouwen N. Protein translocation across the bacterial cytoplasmic membrane.Annu. Rev. Biochem. 2008; 77: 643-667Crossref PubMed Scopus (463) Google Scholar,19Tsirigotaki A. De Geyter J. Šoštaric N. Economou A. Karamanou S. Protein export through the bacterial Sec pathway.Nat. Rev. Microbiol. 2017; 15: 21-36https://doi.org/10.1038/nrmicro.2016.161Crossref PubMed Scopus (227) Google Scholar Here, substrates are recognized by virtue of an N-terminal hydrophobic signal peptide that is removed during translocation. Many of these nascent substrates are initially bound by the ribosome-associated chaperone trigger factor (TF),20Oh E. Becker A.H. Sandikci A. Huber D. Chaba R. Gloge F. Nichols R.J. Typas A. Gross C.A. Kramer G. et al.Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo.Cell. 2011; 147: 1295-1308https://doi.org/10.1016/j.cell.2011.10.044Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar and translocation is facilitated by the targeting factors SecA and in most proteobacteria, including E. coli, by the chaperone SecB.21Sala A. Bordes P. Genevaux P. Multitasking SecB chaperones in bacteria.Front. Microbiol. 2014; 5: 666https://doi.org/10.3389/fmicb.2014.00666Crossref PubMed Scopus (56) Google Scholar SecA can engage nascent chains by binding to the ribosome, suggesting a co-translational initiation of substrate recognition and targeting.22Zhu Z. Wang S. Shan S.O. Ribosome profiling reveals multiple roles of SecA in cotranslational protein export.Nat. Commun. 2022; 13: 3393https://doi.org/10.1038/s41467-022-31061-5Crossref PubMed Scopus (0) Google Scholar,23Huber D. Rajagopalan N. Preissler S. Rocco M.A. Merz F. Kramer G. Bukau B. SecA interacts with ribosomes in order to facilitate posttranslational translocation in bacteria.Mol. Cell. 2011; 41 (S1097-2765(10)01013-0 [pii]): 343-353https://doi.org/10.1016/j.molcel.2010.12.028Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,24Karamyshev A.L. Johnson A.E. Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribosome targeting to the bacterial membrane.J. Biol. Chem. 2005; 280: 37930-37940Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar,25Huber D. Jamshad M. Hanmer R. Schibich D. Döring K. Marcomini I. Kramer G. Bukau B. SecA cotranslationally interacts with nascent substrate proteins in vivo.J. Bacteriol. 2017; 199: 006222-e716https://doi.org/10.1128/JB.00622-16Crossref Scopus (45) Google Scholar This agrees with early findings that many, but not all, substrates are translocated post-translationally.26Josefsson L.G. Randall L.L. Different exported proteins in E. coli show differences in the temporal mode of processing in vivo.Cell. 1981; 25: 151-157https://doi.org/10.1016/0092-8674(81)90239-7Abstract Full Text PDF PubMed Scopus (104) Google Scholar In contrast to the SRP-mediated co-translational translocation, the SecA-dependent translocation does not involve a direct docking of ribosomes to the translocon and is energized by the ATPase activity of the translocon-docked SecA.27Economou A. Wickner W. SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion.Cell. 1994; 78: 835-843Abstract Full Text PDF PubMed Scopus (482) Google Scholar Some chaperones have a bipartite function in protein folding and targeting, suggesting a partially functional integration of systems. One example is TF, which binds nascent cytosolic proteins (CPs), PPs, and OMPs.20Oh E. Becker A.H. Sandikci A. Huber D. Chaba R. Gloge F. Nichols R.J. Typas A. Gross C.A. Kramer G. et al.Selective ribosome profiling reveals the cotranslational chaperone action of trigger factor in vivo.Cell. 2011; 147: 1295-1308https://doi.org/10.1016/j.cell.2011.10.044Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar,28Crooke E. Brundage L. Rice M. Wickner W. ProOmpA spontaneously folds in a membrane assembly competent state which trigger factor stabilizes.EMBO J. 1988; 7: 1831-1835Crossref PubMed Scopus (82) Google Scholar,29Crooke E. Wickner W. Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly competent form.Proc. Natl. Acad. Sci. USA. 1987; 84: 5216-5220Crossref PubMed Scopus (167) Google Scholar,30Lill 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 (119) Google Scholar,31Hesterkamp T. Hauser S. Lütcke H. Bukau B. Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains.Proc. Natl. Acad. Sci. USA. 1996; 93: 4437-4441Crossref PubMed Scopus (203) Google Scholar TF may bind unfolded or loosely folded polypeptides to suppress premature and unproductive folding steps and coordinate the engagement of additional chaperones and targeting factors with the progress of translation.1Kramer G. Shiber A. Bukau B. Mechanisms of cotranslational maturation of newly synthesized proteins.Annu. Rev. Biochem. 2019; 88: 337-364https://doi.org/10.1146/annurev-biochem-013118-111717Crossref PubMed Scopus (77) Google Scholar,9Castanié-Cornet M.P. Bruel N. Genevaux P. Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane.Biochim. Biophys. Acta. 2014; 1843: 1442-1456https://doi.org/10.1016/j.bbamcr.2013.11.007Crossref PubMed Scopus (84) Google Scholar,32De Geyter J. Portaliou A.G. Srinivasu B. Krishnamurthy S. Economou A. Karamanou S. Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase.EMBO Rep. 2020; 21e49054https://doi.org/10.15252/embr.201949054Crossref Scopus (17) Google Scholar The second, much less studied example is SecB, a non-essential, ATP-independent homotetrameric protein of 69 kDa with strong antifolding activity.21Sala A. Bordes P. Genevaux P. Multitasking SecB chaperones in bacteria.Front. Microbiol. 2014; 5: 666https://doi.org/10.3389/fmicb.2014.00666Crossref PubMed Scopus (56) Google Scholar,33Weiss J.B. Ray P.H. Bassford Jr., P.J. Purified SecB protein of Eschericia coli retards folding and promotes membrane translocation of the maltose-binding protein.Proc. Natl. Acad. Sci. USA. 1988; 85: 8978-8982Crossref PubMed Scopus (185) Google Scholar,34Hartl 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 (447) Google Scholar,35Bechtluft P. van Leeuwen R.G.H. Tyreman M. Tomkiewicz D. Nouwen N. Tepper H.L. Driessen A.J.M. Tans S.J. Direct observation of chaperone-induced changes in a protein folding pathway.Science. 2007; 318: 1458-1461https://doi.org/10.1126/science.1144972Crossref PubMed Scopus (108) Google Scholar,36Randall L.L. Hardy S.J. High selectivity with low specificity: how SecB has solved the paradox of chaperone binding.Trends Biochem. Sci. 1995; 20: 65-69Abstract Full Text PDF PubMed Scopus (117) Google Scholar,37Zhou J. Xu Z. The structural view of bacterial translocation-specific chaperone SecB: implications for function.Mol. Microbiol. 2005; 58: 349-357https://doi.org/10.1111/j.1365-2958.2005.04842.xCrossref PubMed Scopus (27) Google Scholar Both chaperones, TF and SecB, bind to unfolded or largely loosely folded sections of proteins to prevent their premature folding and therefore contribute to protein homeostasis in the cell by functioning as a holdase.21Sala A. Bordes P. Genevaux P. Multitasking SecB chaperones in bacteria.Front. Microbiol. 2014; 5: 666https://doi.org/10.3389/fmicb.2014.00666Crossref PubMed Scopus (56) Google Scholar,32De Geyter J. Portaliou A.G. Srinivasu B. Krishnamurthy S. Economou A. Karamanou S. Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase.EMBO Rep. 2020; 21e49054https://doi.org/10.15252/embr.201949054Crossref Scopus (17) Google Scholar,35Bechtluft P. van Leeuwen R.G.H. Tyreman M. Tomkiewicz D. Nouwen N. Tepper H.L. Driessen A.J.M. Tans S.J. Direct observation of chaperone-induced changes in a protein folding pathway.Science. 2007; 318: 1458-1461https://doi.org/10.1126/science.1144972Crossref PubMed Scopus (108) Google Scholar The disc-shaped SecB exposes two major long hydrophobic grooves to bind multiple hydrophobic substrate segments and wrap the unfolded polypeptide around the SecB tetramer.38Crane J.M. Suo Y. Lilly A.A. Mao C. Hubbell W.L. Randall L.L. Sites of interaction of a precursor polypeptide on the export chaperone SecB mapped by site-directed spin labeling.J. Mol. Biol. 2006; 363: 63-74https://doi.org/10.1016/j.jmb.2006.07.021Crossref Scopus (35) Google Scholar,39Huang C. Rossi P. Saio T. Kalodimos C.G. Structural basis for the antifolding activity of a molecular chaperone.Nature. 2016; 537: 202-206https://doi.org/10.1038/nature18965Crossref PubMed Scopus (105) Google Scholar Supporting its role in post-translational translocation, SecB was originally identified in a genetic screen of mutants defective in the translocation of specific precursor proteins.40Kumamoto 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 Furthermore, SecB binds to a number of presecretory proteins and directly binds and functionally cooperates with SecA and TF.32De Geyter J. Portaliou A.G. Srinivasu B. Krishnamurthy S. Economou A. Karamanou S. Trigger factor is a bona fide secretory pathway chaperone that interacts with SecB and the translocase.EMBO Rep. 2020; 21e49054https://doi.org/10.15252/embr.201949054Crossref Scopus (17) Google Scholar,41Fekkes P. vanderDoes C. Driessen A.J. The molecular chaperone SecB is released from the carboxy-terminus of SecA during initiation of precursor protein translocation.Embo Journal. 1997; 16: 6105-6113https://doi.org/10.1093/emboj/16.20.6105Crossref PubMed Scopus (156) Google Scholar Finally, ΔsecB mutants display a strong cold-sensitive (Cs) phenotype below 23°C, a moderate temperature-sensitive (Ts) phenotype above 46°C, and increased protein aggregation.42Ullers 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 (63) Google Scholar Suggesting that TF and SecB act in the same pathway, altered levels of TF cause distinct growth consequences in absence of secB. The deletion of tig, encoding TF, reverses the Cs, Ts, and aggregation-prone phenotype of ΔsecB, while the toxicity of TF overexpression is exacerbated in SecB absence.42Ullers 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 (63) Google Scholar,43Genevaux P. Keppel F. Schwager F. Langendijk-Genevaux P.S. Hartl F.U. Georgopoulos C. In vivo analysis of the overlapping functions of DnaK and trigger factor.EMBO Rep. 2004; 5: 195-200Crossref PubMed Scopus (143) Google Scholar A direct role of SecB in protein folding of cytoplasmic proteins is suggested by the aggregation of some CPs in ΔsecB mutants, by the chemical crosslinking of SecB to CPs, and by the finding that overexpression of secB can suppress the aggregation of CPs and alleviate the heat-sensitivity of mutants lacking TF and the major Hsp70 chaperone DnaK.42Ullers 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 (63) Google Scholar,44Ullers R.S. Luirink J. Harms N. Schwager F. Georgopoulos C. Genevaux P. SecB is a bona fide generalized chaperone in Escherichia coli.Proc. Natl. Acad. Sci. USA. 2004; 101: 7583-7588Crossref PubMed Scopus (95) Google Scholar Implicating SecB in additional cellular processes, secB homologs not only exist in proteobacteria but are also found in viral genomes, on plasmids, and in gram-positive bacteria that lack outer membrane and periplasm, and a SecB-like protein controls a specific toxin-antitoxin pair in Mycobacterium tuberculosis.21Sala A. Bordes P. Genevaux P. Multitasking SecB chaperones in bacteria.Front. Microbiol. 2014; 5: 666https://doi.org/10.3389/fmicb.2014.00666Crossref PubMed Scopus (56) Google Scholar Several findings suggest that SecB exerts its role in protein targeting and translocation by binding to nascent chains: SecB comigrates with polysomes, reversibly engages nascent chains at a length of about 200 residues,45Randall L.L. Topping T.B. Hardy S.J. Pavlov M.Y. Freistroffer D.V. Ehrenberg M. Binding of SecB to ribosome-bound polypeptides has the same characteristics as binding to full-length, denatured proteins.Proc. Natl. Acad. Sci. USA. 1997; 94: 802-807Crossref PubMed Scopus (56) Google Scholar and can be crosslinked to ribosome nascent chain complexes (RNCs).44Ullers R.S. Luirink J. Harms N. Schwager F. Georgopoulos C. Genevaux P. SecB is a bona fide generalized chaperone in Escherichia coli.Proc. Natl. Acad. Sci. USA. 2004; 101: 7583-7588Crossref PubMed Scopus (95) Google Scholar Furthermore, SecB preferentially interacts with unfolded segments of polypeptides46Kumamoto C.A. Francetić O. Highly selective binding of nascent polypeptides by an Escherichia coli chaperone protein in vivo.J. Bacteriol. 1993; 175: 2184-2188Crossref PubMed Google Scholar,47Khisty V.J. Randall L.L. Demonstration in vivo that interaction of maltose-binding protein with SecB is determined by a kinetic partitioning.J. Bacteriol. 1995; 177: 3277-3282Crossref PubMed Google Scholar,48Knoblauch N.T. Rüdiger S. Schönfeld H.J. Driessen A.J. Schneider-Mergener J. Bukau B. Substrate specificity of the SecB chaperone.J. Biol. Chem. 1999; 274: 34219-34225Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar and was recently identified in a detailed proteomics analysis of the interactome of translating ribosomes.49Zhao L. Castanié-Cornet M.P. Kumar S. Genevaux P. Hayer-Hartl M. Hartl F.U. Bacterial RF3 senses chaperone function in co-translational folding.Mol. Cell. 2021; 81: 2914-2928.e7https://doi.org/10.1016/j.molcel.2021.05.016Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar In this study, we apply two ribosome profiling strategies to explore the role of SecB in co-translational protein folding and targeting, and the coordination of SecB with other maturation factors engaging translating ribosomes. Nascent chain-specific interaction profiles of SecB demonstrate that SecB constitutes an integral part of the chaperone network supporting protein folding and a general factor of protein translocation into and across the membrane. Supporting a so-far undiscovered role in membrane protein biogenesis, SecB promotes the targeting function of SRP and is critical for productive membrane insertion. Our study reveals that SecB constitutes a potent, co-translationally acting generalized chaperone and targeting factor that functionally contributes to membrane protein biogenesis and provides functional elasticity and redundancy to the proteostasis network. To identify nascent substrates of SecB in vivo by selective ribosome profiling (SeRP), we generated a strain that chromosomally encoded a C-terminally Avi-tagged SecB (SecB-Avi) under control of an isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible promoter. To reach wild-type (WT) expression levels of fully biotinylated SecB-Avi, we optimized the concentration of IPTG and biotin in the media (Figures S1A, S1B, and S1C). Growth analysis at different temperatures demonstrated that WT levels of SecB-Avi fully complemented the chromosomal secB deletion (Figure S1D). Using these conditions, we performed SeRP following previously described protocols.50Becker A.H. Oh E. Weissman J.S. Kramer G. Bukau B. Selective ribosome profiling as a tool for studying the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes.Nat. Protoc. 2013; 8: 2212-2239https://doi.org/10.1038/nprot.2013.133Crossref PubMed Scopus (73) Google Scholar Briefly, we harvested exponentially growing cells by rapid filtration and immediate freezing in liquid N2, prepared frozen lysates by mixer milling, treated the thawing lysate with micrococcal nuclease, and purified monosomes by sucrose cushion centrifugation. One part of the isolated monosomes was used to reveal the total translatome and the other part was used to isolate SecB-engaged monosomes using streptavidin beads (Figure S2A). The isolated ribosome footprints of both samples were converted to a cDNA library, sequenced, and bioinformatically analyzed. Obtained datasets provide snapshot of mRNA positions of all translating ribosomes (total translatome) and all SecB-bound ribosomes (SecB-bound translatome) in the cell. According to sucrose cushion centrifugations followed by SecB-specific western blotting, we estimate that, in growing cells, about 5% of total SecB or SecB-Avi is engaging ribosomes (Figure S2B). A treemap plot representation of the total translatome (Figure 1A , left) shows that about 80% of the total active ribosome pool translates open reading frames (orfs) encoding CPs, and the remaining 20% translate orfs encoding translocated proteins. Indicating compartment-specific nascent substrate preferences of SecB, the orf distribution among the SecB-bound RNCs (Figure 1A, right) is different from the total pool of ribosomes. Only 60% of SecB-bound RNCs synthesize CPs, suggesting that SecB, by virtue of its holdase activity, may contribute to the folding of nascent CPs, but prefers binding to translocated nascent chains. According to its established role in protein translocation across the membrane, SecB readily engages PPs and OMPs. Unexpectedly, SecB only slightly preferred nascent OMPs over all other proteins and PPs are not specifically enriched. Instead, we found an almost 3-fold enrichment of SecB on nascent IMPs (6.5% of IMPs in total translatome compared with 18% of IMPs in the SecB-bound translatome), revealing that IMPs are the preferred nascent substrate class of SecB. This strong preference for nascent IMPs points toward a so-far undetected function of SecB in co-translational targeting or membrane insertion of IMPs. Next, we explored how nascent chain engagement is coordinated with translation on a metagene level (Figure 1B), which reveals the averaged binding behavior of SecB to RNCs resolved by nascent chain length (further details are provided in the STAR Methods). Relevant SecB binding starts when ribosomes have reached codon 200 (and nascent chains reached a length of 200 residues). Considering that the C-terminal 30 residues are buried in the polypeptide exit tunnel of the ribosome, about 170 residues of the newly synthesized polypeptides are exposed on the ribosome surface. The SecB enrichment increases with ongoing translation and remains high until translation termination. This implies that SecB prefers longer nascent chains and contributes to protein maturation until translation is completed and possibly also post-translationally. By comparing metagene profiles of different subcellular localizations, we explored whether the length preferences of SecB may differ between substrate classes, indicating varying SecB binding behavior to nascent proteins of different subcellular localizations (Figure 1B). The metagene binding curves of CPs, PPs, and OMPs rise slowly, exceed an enrichment value of 1 only after 200–300 codons, and reach the highest values at the end of translation. In contrast, SecB engagement of ribosomes translating IMPs is sharper, reaches higher values, and initiates much earlier, when nascent chains have a length of only about 80–100 amino acids (50–70 residues exposed). These results support established roles of SecB in coordinating the folding of CPs and translocation of PPs and OMPs by virtue of its holdase function,42Ullers 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 (63) Google Scholar,44Ullers R.S. Luirink J. Harms N. Schwager F. Georgopoulos C. Genevaux P. SecB is a bona fide generalized chaperone in Escherichia coli.Proc. Natl. Acad." @default.
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• W4310859957 date "2022-12-01" @default.
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• W4310859957 title "Selective ribosome profiling reveals a role for SecB in the co-translational inner membrane protein biogenesis" @default.
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• W4310859957 doi "https://doi.org/10.1016/j.celrep.2022.111776" @default.
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