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EMBO J. Feb 15, 1994; 13(4): 954–963.
PMCID: PMC394897

SecD and SecF are required for the proton electrochemical gradient stimulation of preprotein translocation.

Abstract

Mutations in secD and secF show impaired protein translocation across the inner membrane of Escherichia coli. We investigated the effect of SecD and SecF (SecD/F) depletion on preprotein translocation into inverted inner membrane vesicles (IMVs). Both IMVs and cells which were depleted of SecD/F were defective in their ability to maintain a proton electrochemical gradient. The translocation of pre-maltose binding protein (preMBP), which is strongly delta microH+ dependent, showed a 5-fold decreased rate with IMVs lacking SecD/F. In contrast, proteolytic processing of preMBP to MBP by leader peptidase was similar in IMVs containing and lacking SecD/F, consistent with earlier findings that only ATP-dependent translocation is required for the initiation of translocation. In the absence of a delta microH+, with ATP as the sole energy source, preMBP translocation into IMVs which contained or were depleted of SecD/F was identical. Translocation of the precursor of outer membrane protein A (proOmpA) in the presence of subsaturating ATP also required a generated delta microH+ and, under these conditions, proOmpA translocation required SecD/F. With saturating concentrations of ATP, where delta microH+ has little effect on in vitro proOmpA translocation, SecD/F also had little effect on translocation. These results explain why SecD/F effects are precursor protein dependent in vitro.

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  • Akimaru J, Matsuyama S, Tokuda H, Mizushima S. Reconstitution of a protein translocation system containing purified SecY, SecE, and SecA from Escherichia coli. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6545–6549. [PMC free article] [PubMed]
  • Apell HJ, Bersch B. Oxonol VI as an optical indicator for membrane potentials in lipid vesicles. Biochim Biophys Acta. 1987 Oct 16;903(3):480–494. [PubMed]
  • Arkowitz RA, Joly JC, Wickner W. Translocation can drive the unfolding of a preprotein domain. EMBO J. 1993 Jan;12(1):243–253. [PMC free article] [PubMed]
  • Bakker EP, Randall LL. The requirement for energy during export of beta-lactamase in Escherichia coli is fulfilled by the total protonmotive force. EMBO J. 1984 Apr;3(4):895–900. [PMC free article] [PubMed]
  • Bassilana M, Wickner W. Purified Escherichia coli preprotein translocase catalyzes multiple cycles of precursor protein translocation. Biochemistry. 1993 Mar 16;32(10):2626–2630. [PubMed]
  • Breukink E, Kusters R, De Kruijff B. In-vitro studies on the folding characteristics of the Escherichia coli precursor protein prePhoE. Evidence that SecB prevents the precursor from aggregating by forming a functional complex. Eur J Biochem. 1992 Sep 1;208(2):419–425. [PubMed]
  • Bieker KL, Phillips GJ, Silhavy TJ. The sec and prl genes of Escherichia coli. J Bioenerg Biomembr. 1990 Jun;22(3):291–310. [PubMed]
  • Bieker-Brady K, Silhavy TJ. Suppressor analysis suggests a multistep, cyclic mechanism for protein secretion in Escherichia coli. EMBO J. 1992 Sep;11(9):3165–3174. [PMC free article] [PubMed]
  • Brundage L, Hendrick JP, Schiebel E, Driessen AJ, Wickner W. The purified E. coli integral membrane protein SecY/E is sufficient for reconstitution of SecA-dependent precursor protein translocation. Cell. 1990 Aug 24;62(4):649–657. [PubMed]
  • Brundage L, Fimmel CJ, Mizushima S, Wickner W. SecY, SecE, and band 1 form the membrane-embedded domain of Escherichia coli preprotein translocase. J Biol Chem. 1992 Feb 25;267(6):4166–4170. [PubMed]
  • Chang CN, Blobel G, Model P. Detection of prokaryotic signal peptidase in an Escherichia coli membrane fraction: endoproteolytic cleavage of nascent f1 pre-coat protein. Proc Natl Acad Sci U S A. 1978 Jan;75(1):361–365. [PMC free article] [PubMed]
  • Chen L, Tai PC. ATP is essential for protein translocation into Escherichia coli membrane vesicles. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4384–4388. [PMC free article] [PubMed]
  • Chen LL, Tai PC. Roles of H+-ATPase and proton motive force in ATP-dependent protein translocation in vitro. J Bacteriol. 1986 Jul;167(1):389–392. [PMC free article] [PubMed]
  • Chun SY, Strobel S, Bassford P, Jr, Randall LL. Folding of maltose-binding protein. Evidence for the identity of the rate-determining step in vivo and in vitro. J Biol Chem. 1993 Oct 5;268(28):20855–20862. [PubMed]
  • Collier DN, Bankaitis VA, Weiss JB, Bassford PJ., Jr The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein. Cell. 1988 Apr 22;53(2):273–283. [PubMed]
  • Crooke E, Wickner W. Trigger factor: a soluble protein that folds pro-OmpA into a membrane-assembly-competent form. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5216–5220. [PMC free article] [PubMed]
  • Crooke E, Brundage L, Rice M, Wickner W. ProOmpA spontaneously folds in a membrane assembly competent state which trigger factor stabilizes. EMBO J. 1988 Jun;7(6):1831–1835. [PMC free article] [PubMed]
  • Crooke E, Guthrie B, Lecker S, Lill R, Wickner W. ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle. Cell. 1988 Sep 23;54(7):1003–1011. [PubMed]
  • Cunningham K, Wickner W. Specific recognition of the leader region of precursor proteins is required for the activation of translocation ATPase of Escherichia coli. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8630–8634. [PMC free article] [PubMed]
  • Cunningham K, Lill R, Crooke E, Rice M, Moore K, Wickner W, Oliver D. SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA. EMBO J. 1989 Mar;8(3):955–959. [PMC free article] [PubMed]
  • Daniels CJ, Bole DG, Quay SC, Oxender DL. Role for membrane potential in the secretion of protein into the periplasm of Escherichia coli. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5396–5400. [PMC free article] [PubMed]
  • Date T, Goodman JM, Wickner WT. Procoat, the precursor of M13 coat protein, requires an electrochemical potential for membrane insertion. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4669–4673. [PMC free article] [PubMed]
  • De Vrije T, Tommassen J, De Kruijff B. Optimal posttranslational translocation of the precursor of PhoE protein across Escherichia coli membrane vesicles requires both ATP and the protonmotive force. Biochim Biophys Acta. 1987 Jun 12;900(1):63–72. [PubMed]
  • de Vrije T, de Swart RL, Dowhan W, Tommassen J, de Kruijff B. Phosphatidylglycerol is involved in protein translocation across Escherichia coli inner membranes. Nature. 1988 Jul 14;334(6178):173–175. [PubMed]
  • Dierstein R, Wickner W. The leader region of pre-maltose binding protein binds amphiphiles. A model for self-assembly in protein export. J Biol Chem. 1985 Dec 15;260(29):15919–15924. [PubMed]
  • Driessen AJ. Precursor protein translocation by the Escherichia coli translocase is directed by the protonmotive force. EMBO J. 1992 Mar;11(3):847–853. [PMC free article] [PubMed]
  • Emr SD, Hanley-Way S, Silhavy TJ. Suppressor mutations that restore export of a protein with a defective signal sequence. Cell. 1981 Jan;23(1):79–88. [PubMed]
  • Enequist HG, Hirst TR, Harayama S, Hardy SJ, Randall LL. Energy is required for maturation of exported proteins in Escherichia coli. Eur J Biochem. 1981 May 15;116(2):227–233. [PubMed]
  • Gardel C, Benson S, Hunt J, Michaelis S, Beckwith J. secD, a new gene involved in protein export in Escherichia coli. J Bacteriol. 1987 Mar;169(3):1286–1290. [PMC free article] [PubMed]
  • Gardel C, Johnson K, Jacq A, Beckwith J. The secD locus of E.coli codes for two membrane proteins required for protein export. EMBO J. 1990 Oct;9(10):3209–3216. [PMC free article] [PubMed]
  • Geller BL. Electrochemical potential releases a membrane-bound secretion intermediate of maltose-binding protein in Escherichia coli. J Bacteriol. 1990 Sep;172(9):4870–4876. [PMC free article] [PubMed]
  • Geller BL, Green HM. Translocation of pro-OmpA across inner membrane vesicles of Escherichia coli occurs in two consecutive energetically distinct steps. J Biol Chem. 1989 Oct 5;264(28):16465–16469. [PubMed]
  • Geller BL, Movva NR, Wickner W. Both ATP and the electrochemical potential are required for optimal assembly of pro-OmpA into Escherichia coli inner membrane vesicles. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4219–4222. [PMC free article] [PubMed]
  • Gierasch LM. Signal sequences. Biochemistry. 1989 Feb 7;28(3):923–930. [PubMed]
  • Hartl FU, Lecker S, Schiebel E, Hendrick JP, Wickner W. The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane. Cell. 1990 Oct 19;63(2):269–279. [PubMed]
  • Hendrick JP, Wickner W. SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane. J Biol Chem. 1991 Dec 25;266(36):24596–24600. [PubMed]
  • Ito K, Date T, Wickner W. Synthesis, assembly into the cytoplasmic membrane, and proteolytic processing of the precursor of coliphage M13 coat protein. J Biol Chem. 1980 Mar 10;255(5):2123–2130. [PubMed]
  • Ito K, Wittekind M, Nomura M, Shiba K, Yura T, Miura A, Nashimoto H. A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins. Cell. 1983 Mar;32(3):789–797. [PubMed]
  • Joly JC, Wickner W. The SecA and SecY subunits of translocase are the nearest neighbors of a translocating preprotein, shielding it from phospholipids. EMBO J. 1993 Jan;12(1):255–263. [PMC free article] [PubMed]
  • Klionsky DJ, Brusilow WS, Simoni RD. In vivo evidence for the role of the epsilon subunit as an inhibitor of the proton-translocating ATPase of Escherichia coli. J Bacteriol. 1984 Dec;160(3):1055–1060. [PMC free article] [PubMed]
  • Kumamoto CA. Escherichia coli SecB protein associates with exported protein precursors in vivo. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5320–5324. [PMC free article] [PubMed]
  • Kumamoto CA, Beckwith J. Mutations in a new gene, secB, cause defective protein localization in Escherichia coli. J Bacteriol. 1983 Apr;154(1):253–260. [PMC free article] [PubMed]
  • Kumamoto CA, Francetić O. Highly selective binding of nascent polypeptides by an Escherichia coli chaperone protein in vivo. J Bacteriol. 1993 Apr;175(8):2184–2188. [PMC free article] [PubMed]
  • Kumamoto CA, Gannon PM. Effects of Escherichia coli secB mutations on pre-maltose binding protein conformation and export kinetics. J Biol Chem. 1988 Aug 15;263(23):11554–11558. [PubMed]
  • Lecker S, Lill R, Ziegelhoffer T, Georgopoulos C, Bassford PJ, Jr, Kumamoto CA, Wickner W. Three pure chaperone proteins of Escherichia coli--SecB, trigger factor and GroEL--form soluble complexes with precursor proteins in vitro. EMBO J. 1989 Sep;8(9):2703–2709. [PMC free article] [PubMed]
  • Lecker SH, Driessen AJ, Wickner W. ProOmpA contains secondary and tertiary structure prior to translocation and is shielded from aggregation by association with SecB protein. EMBO J. 1990 Jul;9(7):2309–2314. [PMC free article] [PubMed]
  • Lill R, Cunningham K, Brundage LA, Ito K, Oliver D, Wickner W. SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli. EMBO J. 1989 Mar;8(3):961–966. [PMC free article] [PubMed]
  • Lill R, Dowhan W, Wickner W. The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins. Cell. 1990 Jan 26;60(2):271–280. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Matsuyama S, Fujita Y, Sagara K, Mizushima S. Overproduction, purification and characterization of SecD and SecF, integral membrane components of the protein translocation machinery of Escherichia coli. Biochim Biophys Acta. 1992 Jul 13;1122(1):77–84. [PubMed]
  • Matsuyama S, Fujita Y, Mizushima S. SecD is involved in the release of translocated secretory proteins from the cytoplasmic membrane of Escherichia coli. EMBO J. 1993 Jan;12(1):265–270. [PMC free article] [PubMed]
  • Mizushima S, Tokuda H. In vitro translocation of bacterial secretory proteins and energy requirements. J Bioenerg Biomembr. 1990 Jun;22(3):389–399. [PubMed]
  • Oliver DB, Beckwith J. E. coli mutant pleiotropically defective in the export of secreted proteins. Cell. 1981 Sep;25(3):765–772. [PubMed]
  • Park S, Liu G, Topping TB, Cover WH, Randall LL. Modulation of folding pathways of exported proteins by the leader sequence. Science. 1988 Feb 26;239(4843):1033–1035. [PubMed]
  • Pogliano JA, Beckwith J. SecD and SecF facilitate protein export in Escherichia coli. EMBO J. 1994 Feb 1;13(3):554–561. [PMC free article] [PubMed]
  • Pogliano KJ, Beckwith J. The Cs sec mutants of Escherichia coli reflect the cold sensitivity of protein export itself. Genetics. 1993 Apr;133(4):763–773. [PMC free article] [PubMed]
  • Ramos S, Schuldiner S, Kaback HR. The electrochemical gradient of protons and its relationship to active transport in Escherichia coli membrane vesicles. Proc Natl Acad Sci U S A. 1976 Jun;73(6):1892–1896. [PMC free article] [PubMed]
  • Randall LL. Translocation of domains of nascent periplasmic proteins across the cytoplasmic membrane is independent of elongation. Cell. 1983 May;33(1):231–240. [PubMed]
  • Randall LL, Hardy SJ. Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell. 1986 Sep 12;46(6):921–928. [PubMed]
  • Randall LL, Hardy SJ. Unity in function in the absence of consensus in sequence: role of leader peptides in export. Science. 1989 Mar 3;243(4895):1156–1159. [PubMed]
  • Randall LL, Topping TB, Hardy SJ. No specific recognition of leader peptide by SecB, a chaperone involved in protein export. Science. 1990 May 18;248(4957):860–863. [PubMed]
  • Rasmussen BA, MacGregor CH, Ray PH, Bassford PJ., Jr In vivo and in vitro synthesis of Escherichia coli maltose-binding protein under regulatory control of the lacUV5 promoter-operator. J Bacteriol. 1985 Nov;164(2):665–673. [PMC free article] [PubMed]
  • Reenstra WW, Patel L, Rottenberg H, Kaback HR. Electrochemical proton gradient in inverted membrane vesicles from Escherichia coli. Biochemistry. 1980 Jan 8;19(1):1–9. [PubMed]
  • Riggs PD, Derman AI, Beckwith J. A mutation affecting the regulation of a secA-lacZ fusion defines a new sec gene. Genetics. 1988 Apr;118(4):571–579. [PMC free article] [PubMed]
  • Schatz PJ, Beckwith J. Genetic analysis of protein export in Escherichia coli. Annu Rev Genet. 1990;24:215–248. [PubMed]
  • Schiebel E, Driessen AJ, Hartl FU, Wickner W. Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase. Cell. 1991 Mar 8;64(5):927–939. [PubMed]
  • Shiozuka K, Tani K, Mizushima S, Tokuda H. The proton motive force lowers the level of ATP required for the in vitro translocation of a secretory protein in Escherichia coli. J Biol Chem. 1990 Nov 5;265(31):18843–18847. [PubMed]
  • Tani K, Shiozuka K, Tokuda H, Mizushima S. In vitro analysis of the process of translocation of OmpA across the Escherichia coli cytoplasmic membrane. A translocation intermediate accumulates transiently in the absence of the proton motive force. J Biol Chem. 1989 Nov 5;264(31):18582–18588. [PubMed]
  • Tani K, Tokuda H, Mizushima S. Translocation of ProOmpA possessing an intramolecular disulfide bridge into membrane vesicles of Escherichia coli. Effect of membrane energization. J Biol Chem. 1990 Oct 5;265(28):17341–17347. [PubMed]
  • Thom JR, Randall LL. Role of the leader peptide of maltose-binding protein in two steps of the export process. J Bacteriol. 1988 Dec;170(12):5654–5661. [PMC free article] [PubMed]
  • Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. [PMC free article] [PubMed]
  • Watanabe M, Blobel G. Site-specific antibodies against the PrlA (secY) protein of Escherichia coli inhibit protein export by interfering with plasma membrane binding of preproteins. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1895–1899. [PMC free article] [PubMed]
  • Weiss JB, Ray PH, Bassford PJ., Jr Purified secB protein of Escherichia coli retards folding and promotes membrane translocation of the maltose-binding protein in vitro. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8978–8982. [PMC free article] [PubMed]
  • Wickner W, Driessen AJ, Hartl FU. The enzymology of protein translocation across the Escherichia coli plasma membrane. Annu Rev Biochem. 1991;60:101–124. [PubMed]
  • Yamada H, Tokuda H, Mizushima S. Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli. J Biol Chem. 1989 Jan 25;264(3):1723–1728. [PubMed]
  • Zilberstein D, Schuldiner S, Padan E. Proton electrochemical gradient in Escherichia coli cells and its relation to active transport of lactose. Biochemistry. 1979 Feb 20;18(4):669–673. [PubMed]
  • Zimmermann R, Wickner W. Energetics and intermediates of the assembly of Protein OmpA into the outer membrane of Escherichia coli. J Biol Chem. 1983 Mar 25;258(6):3920–3925. [PubMed]

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