Global Co-ordination of Protein Translocation by the SecA IRA1 Switch
2004; Elsevier BV; Volume: 279; Issue: 21 Linguagem: Inglês
10.1074/jbc.m401008200
ISSN1083-351X
AutoresEleftheria Vrontou, Spyridoula Karamanou, Catherine Baud, Giorgos Sianidis, Anastassios Economou,
Tópico(s)DNA Repair Mechanisms
ResumoSecA, the dimeric ATPase subunit of protein translocase, contains a DEAD helicase catalytic core that binds to a regulatory C-terminal domain. We now demonstrate that IRA1, a conserved helix-loop-helix structure in the C-domain, controls C-domain conformation through direct interdomain contacts. C-domain conformational changes are transmitted to the DEAD motor and alter its conformation. These interactions establish DEAD motor/C-domain conformational cross-talk that requires a functional IRA1. IRA1-controlled binding/release cycles of the C-domain to the DEAD motor couple this cross-talk to protein translocation chemistries, i.e. DEAD motor affinities for ligands (nucleotides, preprotein signal peptides, and SecYEG, the integral membrane component of translocase) and ATP turnover. IRA1-mediated global co-ordination of SecA catalysis is essential for protein translocation. SecA, the dimeric ATPase subunit of protein translocase, contains a DEAD helicase catalytic core that binds to a regulatory C-terminal domain. We now demonstrate that IRA1, a conserved helix-loop-helix structure in the C-domain, controls C-domain conformation through direct interdomain contacts. C-domain conformational changes are transmitted to the DEAD motor and alter its conformation. These interactions establish DEAD motor/C-domain conformational cross-talk that requires a functional IRA1. IRA1-controlled binding/release cycles of the C-domain to the DEAD motor couple this cross-talk to protein translocation chemistries, i.e. DEAD motor affinities for ligands (nucleotides, preprotein signal peptides, and SecYEG, the integral membrane component of translocase) and ATP turnover. IRA1-mediated global co-ordination of SecA catalysis is essential for protein translocation. Bacterial protein translocase comprises the membrane proteins SecYEG, the dimeric peripheral ATPase SecA (1Driessen A.J. Manting E.H. van der Does C. Nat. Struct. Biol. 2001; 8: 492-498Crossref PubMed Scopus (178) Google Scholar, 2Economou A. Mol. Membr. Biol. 2002; 19: 159-169Crossref PubMed Scopus (40) Google Scholar, 3Manting E.H. van Der Does C. Remigy H. Engel A. Driessen A.J. EMBO J. 2000; 19: 852-861Crossref PubMed Scopus (169) Google Scholar, 4Breyton C. Haase W. Rapoport T.A. Kuhlbrandt W. Collinson I. Nature. 2002; 418: 625-662Crossref Scopus (213) Google Scholar, 5Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (449) Google Scholar, 6Brundage L. Hendrick J.P. Schiebel E. Driessen A.J. Wickner W. Cell. 1990; 62: 649-657Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 7Douville K. Price A. Eichler J. Economou A. Wickner W. J. Biol. 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FEBS Lett. 1992; 298: 6-8Crossref PubMed Scopus (48) Google Scholar) and intramolecular regulator of ATPase2 (IRA2) (16Sianidis G. Karamanou S. Vrontou E. Boulias K. Rapanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 20. 2001; 5: 961-970Crossref Scopus (95) Google Scholar). Specificity is provided by two appendages unique to SecA. (a) The substrate specificity domain (SSD; Figs. 1 (A and B) and 5) (17Baud C. Karamanou S. Sianidis G. Vrontou E. Politou A.S. Economou A. J. Biol. Chem. 2002; 277: 13724-13731Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) that contains a globular "bulb" domain and a "stem" that is formed by two anti-parallel β strands (stem "in" and "out") and "sprouts out" of NBD (18Hunt J.F. Weinkauf S. Henry L. Fak J.J. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Crossref PubMed Scopus (241) Google Scholar, 19Sharma V. Arockiasamy A. Ronning D.R. Savva C.G. Holzenburg A. Braunstein M. Jacobs Jr., W.R. Sacchettini J.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2243-2248Crossref PubMed Scopus (155) Google Scholar). SSD has been implicated in preprotein binding (17Baud C. Karamanou S. Sianidis G. Vrontou E. Politou A.S. Economou A. J. Biol. Chem. 2002; 277: 13724-13731Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 23Kimura E. Akita M. Matsuyama S.I. Mizushima S. J. Biol. Chem. 1991; 206: 6600-6606Abstract Full Text PDF Google Scholar, 24Kourtz L. Oliver D. Mol. Microbiol. 2000; 37: 1342-1356Crossref PubMed Scopus (38) Google Scholar). (b) The C-domain, fused C-terminally to IRA2, participates in SecA dimerization (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar, 25Hirano M. Matsuyama S. Tokuda H. Biochem. Biophys. Res. Commun. 1996; 229: 90-95Crossref PubMed Scopus (36) Google Scholar) and contains four substructures (Fig. 1A) (18Hunt J.F. Weinkauf S. Henry L. Fak J.J. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Crossref PubMed Scopus (241) Google Scholar, 19Sharma V. Arockiasamy A. Ronning D.R. Savva C.G. Holzenburg A. Braunstein M. Jacobs Jr., W.R. Sacchettini J.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2243-2248Crossref PubMed Scopus (155) Google Scholar): the scaffold domain (SD), a 46-aa-long bent α-helix, which docks the C-domain to the DEAD motor by acting as a molecular staple binding both NBD and IRA2; the flexible wing domain (WD); IRA1 (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar), a conserved helix-loop-helix (H1-L-H2) that fits between SD and SSD (Fig. 5); and the extreme C-terminal region (CTD), which is largely crystallographically unresolved and binds lipid and SecB (26Breukink E. Nouwen N. van Raalte A. Mizushima S. Tommassen J. de Kruijff B. J. Biol. Chem. 1995; 270: 7902-7907Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 27Fekkes P. de Wit J.G. van der Wolk J.P. Kimsey H.H. Kumamoto C.A. Driessen A.J. Mol. Microbiol. 1998; 29: 1179-1190Crossref PubMed Scopus (102) Google Scholar).Fig. 5Mutated IRA1 residues (red) in the SecA structure (see text for details). Figure shows data from B. subtilis SecA (Protein Data Bank code 1M6N (see Ref. 18Hunt J.F. Weinkauf S. Henry L. Fak J.J. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Crossref PubMed Scopus (241) Google Scholar)). A, top-side and B, front view. White arrow, SD α-helix bent. Blue, residues connecting to NBD.View Large Image Figure ViewerDownload (PPT) Energy conversion to mechanical work remains a central unresolved issue in several DEAD helicases (20Caruthers J.M. McKay D.B. Curr. Opin. Struct. Biol. 2002; 12: 123-133Crossref PubMed Scopus (455) Google Scholar, 21Delagoutte E. von Hippel P.H. Q. Rev. Biophys. 2002; 35: 431-478Crossref PubMed Scopus (147) Google Scholar, 28Singleton M.R. Wigley D.B. EMBO J. 2003; 22: 4579-4583Crossref PubMed Scopus (14) Google Scholar) as well as in protein translocation. The mechanism is expected to involve cross-talk between the ATP motor and specificity domains (13Economou A. Mol. Microbiol. 1998; 27: 511-518Crossref PubMed Scopus (65) Google Scholar, 16Sianidis G. Karamanou S. Vrontou E. Boulias K. Rapanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 20. 2001; 5: 961-970Crossref Scopus (95) Google Scholar, 17Baud C. Karamanou S. Sianidis G. Vrontou E. Politou A.S. Economou A. J. Biol. Chem. 2002; 277: 13724-13731Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In SecA, evidence for this is provided by the finding that, in the absence of tight C-domain association, the DEAD motor becomes a hyperactivated ATPase (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar, 18Hunt J.F. Weinkauf S. Henry L. Fak J.J. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Crossref PubMed Scopus (241) Google Scholar, 19Sharma V. Arockiasamy A. Ronning D.R. Savva C.G. Holzenburg A. Braunstein M. Jacobs Jr., W.R. Sacchettini J.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2243-2248Crossref PubMed Scopus (155) Google Scholar). Importantly, SecA with a short IRA1 deletion also becomes an unregulated, hyperactivated ATPase that is nevertheless incompetent for translocation (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar). This observation led us to propose that IRA1 is a molecular switch essential for coupling ATP hydrolysis to translocation work (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar). We now show that IRA1 contacts other SecA subdomains and through these it controls association and conformational cross-talk between the DEAD motor and the C-domain. Modulation of these physical contacts allows IRA1 to regulate DEAD motor subactivities. We propose that SecA ATP binding and hydrolysis become coupled to protein translocation through IRA1 acting as a global co-ordinator of translocase catalysis and conformation. Bacterial Strains and Recombinant DNA Experiments—Escherichia coli strains were grown and manipulated as described (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar, 16Sianidis G. Karamanou S. Vrontou E. Boulias K. Rapanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 20. 2001; 5: 961-970Crossref Scopus (95) Google Scholar, 29Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar). IRA1 mutations were constructed on pIMBB38 (secA in BamHI-EcoRI sites of pALTER-EX1) using Altered sites (Promega) using primers: X61 (CAGGTGCTCTTTCGCCAGGGAGTCAAG; W775A), X62 (GATACCCTGACGCCGATAGTCCATCGC; L785R), X63 (GCCACGCAGGTGGCGACCCTGACGCAG; I789R), X64 (CTGTGCGTAGCCAGCCAGGTGGATACC; R792A), X65 (GTATTCCTGCTTCGCATCTTTCTGTGC; P799A), X66 (TTCACGTTTGTATGCCTGCTTCGGATC; E802A), X67 (CGATTCACGTTTGGCTTCCTGCTTCGG; Y803A), X68 (CATGGAGAACGATGCACGTTTGTATTC; E806A), and X69 (CAGCATCGCTGCAGCCATGGAGAACGA; F811A). Next, the 2.9-kb NcoI fragment of pIMBB7 (HisSecA) was replaced by that of mutant genes, giving rise to pIMBB105, pIMBB106, pIMBB202, pIMBB107, pIMBB200, pIMBB108, pIMBB109, pIMBB110, and pIMBB201, respectively. The 0.83-kb EcoRI-MfeI fragment of secA IRA1 mutants was also cloned into the corresponding sites of pIMBB70 (HisC34) giving rise to pIMBB165, pIMBB166, pIMBB167, pIMBB168, pIMBB169, pIMBB181, pIMBB182, pIMBB183, and pIMBB184, respectively. C34 truncations were constructed by PCR, using pIMBB70 as template. For C609-834 and C669-834, we used forward primers X110 (GGCCCGTACATATGAAACTGGGTATGAAGCCAGGC) and X109 (GGCCCGTACATATGGTCAGCGATGTGAGCGAAACC), respectively, and the reverse primer X107 (CGCGGATCCTTAAGGCATACGTACCTGAACTTTG). For C609-757, we used forward primer X110 (GGCCCGTACATATGAAACTGGGTATGAAGCCAGGC) and reverse primer X111 (CGCGGATCCTTACTCAGCACCAACCACTTCTTC). PCR fragments were digested with NdeI/BamHI and inserted into pET16b (Invitrogen), resulting in pIMBB203, pIMBB186, and pIMBB185, respectively. ATP Hydrolysis Measurements—All assays were in buffer B (50 mm Tris-Cl, pH 8, 50 mm KCl, 5 mm MgCl2, 1 mm dithiothreitol), 1 mg/ml BSA, 1 mm ATP. SecA or N68 derivatives were added at 0.1 mg/ml. For membrane ATPase SecYEG-proteoliposomes (200 μg/ml) were added. For translocation ATPase proOmpA (0.2 mg/ml) was further added. For N68 suppression assays C34 derivatives were added as indicated in figure legends. Released phosphate was measured using malachite green detection (10Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (466) Google Scholar) or using thin layer chromatography (TLC). For TLC [γ-32P]ATP was mixed with ATP to a final concentration of 20-1000 μm (16Sianidis G. Karamanou S. Vrontou E. Boulias K. Rapanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 20. 2001; 5: 961-970Crossref Scopus (95) Google Scholar). Kcat values were determined as described (16Sianidis G. Karamanou S. Vrontou E. Boulias K. Rapanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 20. 2001; 5: 961-970Crossref Scopus (95) Google Scholar) using Prism 4.0 (GraphPad). Fluorescence Measurements—Measurements were carried out in quartz cuvettes (1 ml; Hellma) with a Cary Eclipse fluorimeter supplemented with a four-position cuvette holder and a Peltier temperature controller (Varian). For determination of equilibrium dissociation constants, SecA was added at increasing concentrations (0.025-5 μm) to MANT-ADP (0.1 μm; Molecular Probes) in buffer B. Emission spectra of MANT-ADP were recorded at each step of the binding curve (380-550 nm; excitation 356 nm; slits at 2.5 and 20 nm). KD was determined by plotting the change of MANT-ADP emission spectra upon SecA addition against SecA concentration using the equation (F1 - F0)/F0 integral values (F0 = no SecA added, F1 = SecA added). To determine apparent Tm, we monitored changes in intrinsic tryptophan fluorescence emission of SecA or C34 derivatives (0.25 μm in buffer B) as a function of increasing temperature (4 - 82 °C; ramping rate 0.8 °C/min; excitation 297 nm/emission 345 nm; slits at 2.5 and 20 nm; data acquisition interval = 0.5 min), in the presence or absence of ADP (2 mm). All data were collected using Cary Eclipse software (Bio Package; Varian) and analyzed by nonlinear regression using Origin 5.0 (Microcal). Surface Plasmon Resonance Assays—Optical biosensor measurements were on an IBIS II instrument (Echochemie). 3K7L (3 μgin50 μl from a 60 μg/ml stock in 10 mm Hepes, pH 8.5) was added onto carboxymethylated dextran-coated gold sensor disks (CMD20; Xantec) and was cross-linked via NH2-specific N-ethyl-N′-(dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide. The surface was regenerated with 100 mm HCl. Data were collected (750 s) and analyzed using IBIS Kinetic Analysis software (17Baud C. Karamanou S. Sianidis G. Vrontou E. Politou A.S. Economou A. J. Biol. Chem. 2002; 277: 13724-13731Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). SecA Binding to SecYEG—Binding of 35S-labeled SecA proteins to inner membrane vesicles (IMVs) was performed as described elsewhere (5Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (449) Google Scholar). Briefly, urea-treated IMVs (64 μg/ml) were mixed with a range of 35S-labeled SecA concentrations (0.5-2000 nm; buffer B; 1 mg/ml BSA; 15 min; 4 °C). Reactions were overlaid on equal volume of buffer B, 0.2 m sucrose, 1 mg/ml BSA, in centrifuge tubes preblocked with BSA and sedimented (320,000 × g; 30 min; 4 °C; Beckman TLX120 ultracentrifuge). Pellets, rinsed (two times; 100 μl of buffer B) and resuspended by sonication, were spotted on nylon membranes in a vacuum manifold (Bio-Rad). Bound radioactivity was quantitated by phosphorimaging. Data were fitted to hyperbolae using nonlinear regression in Prism (GraphPad). Chemicals and Biochemicals—Proteases, inhibitors and nucleotides were from Roche; E. coli phospholipids were from Avanti Polar Lipids; DNA enzymes from Minotech; oligonucleotides from MWG; dNTPs from Promega; [γ-32P]ATP (5000 Ci/mmol), [35S]methionine (1000 Ci/mmol), and chromatography materials (except Ni2+ affinity; Qiagen) from Amersham Biosciences; all other chemicals from Sigma. Biochemicals were purified as described elsewhere (6Brundage L. Hendrick J.P. Schiebel E. Driessen A.J. Wickner W. Cell. 1990; 62: 649-657Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 10Lill R. Dowhan W. Wickner W. Cell. 1990; 60: 271-280Abstract Full Text PDF PubMed Scopus (466) Google Scholar, 15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar, 16Sianidis G. Karamanou S. Vrontou E. Boulias K. Rapanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 20. 2001; 5: 961-970Crossref Scopus (95) Google Scholar). Miscellaneous—Protein concentration was determined using the Bradford reagent (Bio-Rad) with BSA as a standard or by UV absorbance or by amino acid analysis. Biochemical assays, trypsinolysis experiments, preparation of SecYEG inner membrane vesicles and proteoliposomes, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 35S-labeled SecA binding to IMVs, and SecA reconstitution from N68 and C34 were as described (5Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (449) Google Scholar, 15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar, 16Sianidis G. Karamanou S. Vrontou E. Boulias K. Rapanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 20. 2001; 5: 961-970Crossref Scopus (95) Google Scholar, 17Baud C. Karamanou S. Sianidis G. Vrontou E. Politou A.S. Economou A. J. Biol. Chem. 2002; 277: 13724-13731Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). CD spectroscopy assays were performed as described previously (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar). Native PAGE was carried out using a Bio-Rad Mini-PROTEAN II system. α-NBD and α-IRA2 rabbit polyclonal antibodies were raised against purified N1-263 and N462-610, respectively. α-SSD antibodies were purified as described (17Baud C. Karamanou S. Sianidis G. Vrontou E. Politou A.S. Economou A. J. Biol. Chem. 2002; 277: 13724-13731Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Radioactivity was quantitated by phosphorimaging (Storm 840; Amersham Biosciences). N-terminal sequencing and amino acid analysis were done at AltaBioscience (United Kingdom). Structures were analyzed with SwissPDBViewer. IRA1 Mutations Compromise SecA-mediated Protein Translocation—To understand IRA1 function, we mutated its nine most highly conserved residues (Figs. 1 (A and B) and 5) (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar, 16Sianidis G. Karamanou S. Vrontou E. Boulias K. Rapanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 20. 2001; 5: 961-970Crossref Scopus (95) Google Scholar). The ability of IRA1 mutants to complement the chromosomal thermosensitive secA gene of strain BL21.19 (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar, 29Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar) was examined (Fig. 1C). Six of the mutants (secAL785R, secAI789R, secAP799A, secAE802A, secAY803A, and secAE806A) were barely viable, as was partial deletion of IRA1 (secAΔ783-795) (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar, 30Jarosik G.P. Oliver D.B. J. Bacteriol. 1991; 173: 860-868Crossref PubMed Google Scholar). Three mutants (secAW775A, secAF811A, and secAR792A) complemented the thermosensitive strain, albeit not as efficiently as secA. Oligohistidinyl-tagged SecA IRA1 mutants were purified and shown, like SecAΔ783-795 (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar, 30Jarosik G.P. Oliver D.B. J. Bacteriol. 1991; 173: 860-868Crossref PubMed Google Scholar), to be stable, folded, and α-helical (far UV CD; data not shown) and dimeric (size exclusion chromatography, blue native PAGE, sedimentation equilibrium; data not shown). We concluded that IRA1 mutant proteins do not have significantly altered structures and were characterized biochemically. To verify that the in vivo phenotypes (Fig. 1C) are the result of defective protein export, HisSecA IRA1 mutants were used in an in vitro translocation assay with SecYEG-proteoliposomes and the secretory protein proOmpA (Fig. 1D) (6Brundage L. Hendrick J.P. Schiebel E. Driessen A.J. Wickner W. Cell. 1990; 62: 649-657Abstract Full Text PDF PubMed Scopus (392) Google Scholar). Of all IRA1 mutants, only SecAW775A (lane 5) supports protein translocation (lanes 6-13). SecAR792A (lane 8) and SecAF811A (lane 13) do not translocate in vitro, although they partially complement in vivo (Fig. 1C). Clearly, the more stringent and suboptimal in vitro proteoliposome assay exacerbates their defects. IRA1 Mutations Alter SecA ATPase Activities—To test whether IRA1 mutants are defective in ATP catalysis, we determined their basal, membrane, and translocation ATPase activities (Table I). Basal ATP catalysis is enhanced, either significantly (>13-fold; SecAW775A; >5-fold SecAF811A) or slightly (up to 2-fold; all other mutants except SecAI789R). Stimulation of basal ATPase upon addition of SecYEG-proteoliposomes (membrane ATPase) or proteoliposomes plus proOmpA (translocation ATPase) is seen only with SecAW775A, SecAR792A, and SecAF811A in agreement with the in vivo complementation test (Fig. 1C). All other IRA1 mutant proteins fail to further stimulate their basal ATPase.Table ISteady state ATPase kinetic constants of SecA IRA1 mutantsSecA derivativeKcatBasalMembraneTranslocationmin-1WT3.7 (±0.4)6.2 (±0.6)25 (±6)Δ783-79545 (±6)43 (±7)43.2 (±7)W775A50.4 (±7)54 (±10)76 (±13)L785R5.7 (±0.6)4.6 (±0.4)6.8 (±0.7)I789R3 (±0.3)3 (±0.3)2.6 (±0.2)R792A4.3 (±0.4)16 (±2.5)24.7 (±5)P799A5.2 (±0.5)3.2 (±0.3)9 (±1)E802A5.5 (±0.5)5.7 (±0.4)7.4 (±0.6)Y803A8.4 (±0.6)7.3 (±0.6)7 (±0.5)E806A4.1 (±0.3)1.3 (±0.2)5.4 (±0.4)F811A18.7 (±4)32 (±7)54 (±11) Open table in a new tab Based on the above results, IRA1 mutants fall into three classes: (i) the functional/hyperactivated W775A, (ii) the less-functional R792A and F811A, and (iii) the severely compromised L785R, I789R, P799A, E802A, Y803A, and E806A. To simplify presentation we will focus hereafter on three representative mutants: SecAW775A, SecAR792A, and SecAI789R. IRA1 Mutations Alter SecA Affinity for Nucleotide—To understand how IRA1 influences SecA ATP catalysis, we examined the effect of IRA1 single point mutations or partial deletion on nucleotide binding to SecA. To this end we developed a fluorescent MANT-ADP binding assay (Table II).Table IIEquillibrium dissociation constants of SecA and derivatives for MANT-ADP, at the indicated temperature Triplicates of 40-60 data points were used for each binding curve.SecA derivativeKDi4 °C37 °CμmSecA0.14 (±0.02)0.28 (±0.03)N680.28 (±0.03)>100Δ783-7950.75 (±0.1)8.18 (±2.5)W775A0.17 (±0.04)4.58 (±1.1)R792A0.4 (±0.08)0.38 (±0.02)I789R1.02 (±0.19)0.7 (±0.07) Open table in a new tab At 4 °C, MANT-ADP binds to SecA with high affinity (KD = 0.14 μm). This is in agreement with values obtained with other methods (29Mitchell C. Oliver D. Mol. Microbiol. 1993; 10: 483-497Crossref PubMed Scopus (188) Google Scholar, 31den Blaauwen T. van der Wolk J.P. van der Does C. van Wely K.H. Driessen A.J. FEBS Lett. 1999; 458: 145-150Crossref PubMed Scopus (23) Google Scholar). N68, the polypeptide carrying the complete DEAD motor bereft of the C-domain (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar), exhibits similarly high affinity (KD = 0.28 μm). C34, the polypeptide carrying the C-domain alone (15Karamanou S. Vrontou E. Sianidis G. Baud C. Roos T. Kuhn A. Politou A.S. Economou A. Mol. Microbiol. 1999; 34: 1133-1145Crossref PubMed Scopus (118) Google Scholar), has no measurable nucleotide binding (data not shown). In agreement with biochemical (16Sianidis G. Karamanou S. Vrontou E. Boulias K. Rapanas K. Kyrpides N. Politou A.S. Economou A. EMBO J. 20. 2001; 5: 961-970Crossref Scopus (95) Google Scholar) and structural analysis (18Hunt J.F. Weinkauf S. Henry L. Fak J.J. McNicholas P. Oliver D.B. Deisenhofer J. Science. 2002; 297: 2018-2026Crossref PubMed Scopus (241) Google Scholar, 19Sharma V. Arockiasamy A. Ronning D.R. Savva C.G. Holzenburg A. Braunstein M. Jacobs Jr., W.R. Sacchettini J.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2243-2248Crossref PubMed Scopus (155) Google Scholar), these data demonstrate that the DEAD motor domain of SecA is necessary and sufficient for nucleotide binding. Temperature does not affect nucleotide binding to the DEAD motor in SecA (37 °C; Table II). In contrast, it drastically reduces binding to the isolated DEAD motor (>350-fold). This suggests that the C-domain acts in trans to determine DEAD motor nucleotide affinity at physiological temperature. Partial deletion or single point mutations in IRA1 reduce SecA nucleotide affinity, suggesting that IRA1 may be part of this mechanism. IRA1 Mutations Alter SecA Affinity for SecYEG—Because membranes do not stimulate the ATPase of most IRA1 mutants (Table I), their binding to SecYEG might be defective. To investigate this we determined the equilibrium dissociation constants of SecA IRA1 derivatives for SecYEG (Fig. 1E). SecA binds to SecYEG in IMVs with high affinity (KD = 30 nm; lane 1) (5Hartl F.U. Lecker S. Schiebel E. Hendrick J.P. Wickner W. Cell. 1990; 63: 269-279Abstract Full Text PDF PubMed Scopus (449) Google Scholar). N68 binding to SecYEG is 4-fold reduced (KD = 110 nm; lane 2), whereas no C34 binding was measurable (data not shown) (32Dapic V. Oliver D. J. Biol. Chem. 2000; 275: 25000-25007Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). These data indicate that SecA binds to SecYEG through the DEAD motor, but the presence of the C-domain optimiz
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