Dissecting the Role of a Conserved Motif (the Second Region of Homology) in the AAA Family of ATPases

Escherichia coli FtsH is an ATP-dependent protease that belongs to the AAA protein family. The second region of homology (SRH) is a highly conserved motif among AAA family members and distinguishes these proteins in part from the wider family of Walker-type ATPases. Despite its conservation across the AAA family of proteins, very little is known concerning the function of the SRH. To address this question, we introduced point mutations systematically into the SRH of FtsH and studied the activities of the mutant proteins. Highly conserved amino acid residues within the SRH were found to be critical for the function of FtsH, with mutations at these positions leading to decreased or abolished ATPase activity. The effects of the mutations on the protease activity of FtsH correlated strikingly with their effects on the ATPase activity. The ATPase-deficient SRH mutants underwent an ATP-induced conformational change similar to wild type FtsH, suggesting an important role for the SRH in ATP hydrolysis but not ATP binding. Analysis of the data in the light of the crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes an intermolecular catalytic role for the SRH. Escherichia coli FtsH is an ATP-dependent protease that belongs to the AAA protein family. The second region of homology (SRH) is a highly conserved motif among AAA family members and distinguishes these proteins in part from the wider family of Walker-type ATPases. Despite its conservation across the AAA family of proteins, very little is known concerning the function of the SRH. To address this question, we introduced point mutations systematically into the SRH of FtsH and studied the activities of the mutant proteins. Highly conserved amino acid residues within the SRH were found to be critical for the function of FtsH, with mutations at these positions leading to decreased or abolished ATPase activity. The effects of the mutations on the protease activity of FtsH correlated strikingly with their effects on the ATPase activity. The ATPase-deficient SRH mutants underwent an ATP-induced conformational change similar to wild type FtsH, suggesting an important role for the SRH in ATP hydrolysis but not ATP binding. Analysis of the data in the light of the crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes an intermolecular catalytic role for the SRH. second region of homology alkaline phosphatase polyacrylamide gel electrophoresis isopropyl-1-thio-β-d-galactopyranoside 5′-adenylyl-β, γ-imidodiphosphate The AAA protein family (for ATPasesassociated with diverse cellular activities) is a distinct subfamily of the Walker-type ATPases that has been defined on the basis of amino acid sequence homology (for reviews see Refs.1Confalonieri F. Duguet M. Bioessays. 1995; 17: 639-665Crossref PubMed Scopus (314) Google Scholar, 2Beyer A. Protein Sci. 1997; 6: 2043-2058Crossref PubMed Scopus (211) Google Scholar, 3Patel S. Latterich M. Trends Cell Biol. 1998; 8: 65-71Crossref PubMed Google Scholar). Walker-type ATPases have two consensus motifs, Walker A and B. In addition to these consensus motifs, AAA proteins have another highly conserved amino acid sequence within their ATPase domain that has been termed the second region of homology (SRH)1 (4Swaffield J.C. Bromberg J.F. Johnston S.A. Nature. 1992; 357: 698-700Crossref PubMed Scopus (165) Google Scholar). A 200–250-amino acid residue sequence that encompasses the Walker A and B motifs and the SRH is named the AAA module, and proteins that contain one or two copies of this module are classified into the AAA protein family. Thus, the SRH is the defining feature that distinguishes the AAA family from other Walker-type ATPases. FtsH of Escherichia coli is the first identified prokaryotic member of the AAA family (5Ogura T. Tomoyasu T. Yuki T. Morimura S. Begg K.J. Donachie W.D. Mori H. Niki H. Hiraga S. Res. Microbiol. 1991; 142: 279-282Crossref PubMed Scopus (83) Google Scholar, 6Tomoyasu T. Yuki T. Morimura S. Mori H. Yamanaka K. Niki H. Hiraga S. Ogura T. J. Bacteriol. 1993; 175: 1344-1351Crossref PubMed Scopus (204) Google Scholar). FtsH is a membrane-bound ATP-dependent protease with two N-terminal transmembrane segments (Fig. 1) (7Tomoyasu T. Yamanaka K. Murata K. Suzaki T. Bouloc P. Kato A. Niki H. Hiraga S. Ogura T. J. Bacteriol. 1993; 175: 1352-1357Crossref PubMed Scopus (143) Google Scholar, 8Tomoyasu T. Gamer J. Bukau B. Kanemori M. Mori H. Rutman A.J. Oppenheim A.B. Yura T. Yamanaka K. Niki H. Hiraga S. Ogura T. EMBO J. 1995; 14: 2551-2560Crossref PubMed Scopus (365) Google Scholar). It possesses one copy of the AAA module and at its C terminus possesses a Zn2+-binding motif, which is thought to be the catalytic center for proteolysis. Several substrates of FtsH have been identified, including cytoplasmic proteins, such as the heat shock transcription factor ς32 (8Tomoyasu T. Gamer J. Bukau B. Kanemori M. Mori H. Rutman A.J. Oppenheim A.B. Yura T. Yamanaka K. Niki H. Hiraga S. Ogura T. EMBO J. 1995; 14: 2551-2560Crossref PubMed Scopus (365) Google Scholar, 9Herman C. Thévenet D. D'Ari R. Bouloc P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3516-3520Crossref PubMed Scopus (274) Google Scholar), LpxC (10Ogura T. Inoue K. Tatsuta T. Suzaki T. Karata K. Young K. Su L.-H. Fierke C.A. Jackman J.E. Raetz C.R.H. Coleman J. Tomoyasu T. Matsuzawa H. Mol. Microbiol. 1999; 31: 833-844Crossref PubMed Scopus (193) Google Scholar), SsrA-tagged proteins (11Herman C. Thévenet D. Bouloc P. Walker G.C. D'Ari R. Genes Dev. 1998; 12: 1348-1355Crossref PubMed Scopus (242) Google Scholar), λ CII (12Shotland Y. Koby S. Teff D. Mansur N. Oren D.A. Tatematsu K. Tomoyasu T. Kessel M. Bukau B. Ogura T. Oppenheim A.B. Mol. Microbiol. 1997; 24: 1303-1310Crossref PubMed Scopus (126) Google Scholar), λ CIII (13Herman C. Thévenet D. D'Ari R. Bouloc P. J. Bacteriol. 1997; 179: 358-363Crossref PubMed Scopus (78) Google Scholar), and λ Xis (14Leffers G.G. Gottesman S. J. Bacteriol. 1998; 180: 1573-1577Crossref PubMed Google Scholar), as well as integral membrane proteins such as SecY (15Kihara A. Akiyama Y. Ito K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4532-4536Crossref PubMed Scopus (212) Google Scholar, 16Akiyama Y. Kihara A. Tokuda H. Ito K. J. Biol. Chem. 1996; 271: 31196-31201Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), subunit a of the proton ATPase FO sector (17Akiyama Y. Kihara A. Ito K. FEBS Lett. 1996; 399: 26-28Crossref PubMed Scopus (108) Google Scholar), and YccA (18Kihara A. Akiyama Y. Ito K. J. Mol. Biol. 1998; 279: 175-188Crossref PubMed Scopus (99) Google Scholar). Although FtsH is essential for cell growth (5Ogura T. Tomoyasu T. Yuki T. Morimura S. Begg K.J. Donachie W.D. Mori H. Niki H. Hiraga S. Res. Microbiol. 1991; 142: 279-282Crossref PubMed Scopus (83) Google Scholar, 19Akiyama Y. Ogura T. Ito K. J. Biol. Chem. 1994; 269: 5218-5224Abstract Full Text PDF PubMed Google Scholar), cells lacking ftsH can grow if the sfhC mutation coexists (10Ogura T. Inoue K. Tatsuta T. Suzaki T. Karata K. Young K. Su L.-H. Fierke C.A. Jackman J.E. Raetz C.R.H. Coleman J. Tomoyasu T. Matsuzawa H. Mol. Microbiol. 1999; 31: 833-844Crossref PubMed Scopus (193) Google Scholar, 20Tatsuta T. Tomoyasu T. Bukau B. Kitagawa M. Mori H. Karata K. Ogura T. Mol. Microbiol. 1998; 30: 583-594Crossref PubMed Scopus (97) Google Scholar). Interestingly, several findings have also raised the possibility that FtsH has a chaperone-like activity. Some ftsH mutations cause abnormal protein translocation and defects in protein export. In particular, it has been shown that the PhoA moiety of SecY-PhoA fusions, in which PhoA is attached to the C-terminal cytoplasmic region of SecY, is abnormally translocated (Std phenotype) in ftsH mutant strains (19Akiyama Y. Ogura T. Ito K. J. Biol. Chem. 1994; 269: 5218-5224Abstract Full Text PDF PubMed Google Scholar, 21Akiyama Y. Shirai Y. Ito K. J. Biol. Chem. 1994; 269: 5225-5229Abstract Full Text PDF PubMed Google Scholar). These phenotypes are differentially suppressed by overproduction of other chaperones (22Shirai Y. Akiyama Y. Ito K. J. Bacteriol. 1996; 178: 1141-1145Crossref PubMed Scopus (50) Google Scholar). The data also indicated that FtsH binds to denatured but not intact PhoA polypeptide and that this binding does not result in proteolysis of PhoA (23Akiyama Y. Ehrmann M. Kihara A. Ito K. Mol. Microbiol. 1998; 28: 803-812Crossref PubMed Scopus (38) Google Scholar). The AAA family has been growing rapidly for the past several years with more than 200 family members identified to date. Members of this family are spread among various species in all kingdoms, archaea, bacteria, and eukaryotes and participate in various cellular activities. The AAA family can be divided into at least six subfamilies: metalloproteases including FtsH, subunits of the 26 S proteasome, proteins involved in vesicle-mediated secretion, homotypic fusion, peroxisome biogenesis, and meiosis/mitochondrial functions. No clue to the common function of AAA proteins, besides ATP binding and/or hydrolysis, has yet emerged. There appears to be a confusing contrast between the extreme sequence conservation within the module and the diversity of function of the AAA proteins. There must be a very important reason for this high conservation of sequence both among proteins with a common function in evolutionarily distant species, as well as among AAA proteins with a variety of functions within the cell of a given species. Elucidation of the basic function of the module represents an exciting challenge, and it must provide a clue to understanding the common function of the AAA proteins. Because the SRH is a part of the ATPase domain, it is assumed to have some relation to the ATPase activity. However, there have as yet been no reports of an experimental investigation into the function of the SRH. To elucidate SRH function, we introduced a series of mutations into the SRH sequence of E. coli FtsH and studied their consequences both in vivo and in vitro. The results indicate that the SRH is important for the ATPase activity. AR3289 (sfhC21 zad220::Tn 10) (10Ogura T. Inoue K. Tatsuta T. Suzaki T. Karata K. Young K. Su L.-H. Fierke C.A. Jackman J.E. Raetz C.R.H. Coleman J. Tomoyasu T. Matsuzawa H. Mol. Microbiol. 1999; 31: 833-844Crossref PubMed Scopus (193) Google Scholar, 20Tatsuta T. Tomoyasu T. Bukau B. Kitagawa M. Mori H. Karata K. Ogura T. Mol. Microbiol. 1998; 30: 583-594Crossref PubMed Scopus (97) Google Scholar), AR423 (met gal supE hsdR sfiCΔ(srl-recA)306::Tn 10ΔftsH3::kan [pAR171, ftsH + rep ts cam R]) (19Akiyama Y. Ogura T. Ito K. J. Biol. Chem. 1994; 269: 5218-5224Abstract Full Text PDF PubMed Google Scholar), AR754 (thr-1 leu-6 thi-1 supE44 lacY1 tonA21 zha-6::Tn 10 ftsH1) (24Begg K.J. Tomoyasu T. Donachie W.D. Khattar M. Niki H. Yamanaka K. Hiraga S. Ogura T. J. Bacteriol. 1992; 174: 2416-2417Crossref PubMed Google Scholar), and BL21(DE3) (F− ompT hsdS gal dcm [DE3]) (25Studier F.W. Moffatt B.A. J. Mol. Biol. 1986; 189: 113-130Crossref PubMed Scopus (4842) Google Scholar) have been described. AR5074 (BL21[DE3] sfhC21 zad220::Tn 10) was constructed by P1 transduction of sfhC21 zad220::Tn 10 from AR3289 into BL21(DE3). AR5088 (AR5074 ΔftsH3::kan) was constructed by P1 transduction of ΔftsH3::kan from AR423 into AR5074. XL1-Blue (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F'::Tn 10, proA + B + lacI q Z ΔM15]) was used as a host strain to construct plasmids in this study. L medium (10 g of tryptone, 5 g of yeast extract, 5 g NaCl/liter, pH 7.4) was used. Ampicillin (100 μg/ml) was added for growing strains carrying plasmids. pUC118 carries the gene for ampicillin resistance (amp R). pET29b is a plasmid vector of the pET system carrying the T7 promoter (Novagen). pKY297 carries a truncated ftsH gene that lacks the sequence encoding the two transmembrane segments and the Walker motifs (21Akiyama Y. Shirai Y. Ito K. J. Biol. Chem. 1994; 269: 5225-5229Abstract Full Text PDF PubMed Google Scholar). pSTD401 carries the wild type ftsH gene with its own Shine-Dalgarno sequence under the control of the lac promoter (19Akiyama Y. Ogura T. Ito K. J. Biol. Chem. 1994; 269: 5218-5224Abstract Full Text PDF PubMed Google Scholar). pSTD41 carries the K201N mutant ftsH gene (21Akiyama Y. Shirai Y. Ito K. J. Biol. Chem. 1994; 269: 5225-5229Abstract Full Text PDF PubMed Google Scholar). pQM21 carries the H421Y mutant ftsH gene (26Qu J.N. Makino S. Adachi H. Koyama Y. Akiyama Y. Ito K. Tomoyasu T. Ogura T. Matsuzawa H. J. Bacteriol. 1996; 178: 3457-3461Crossref PubMed Scopus (41) Google Scholar). pIFH100 is the plasmid vector used in this study and was constructed as follows. The Nde I-Bam HI fragment of pET29b was deleted to eliminate the S·Tag sequence, and the Nsi I short fragment of the resulting plasmid was replaced with the Bsp HI amp R-containing fragment of pUC118 by blunt end ligation after treatment with Klenow fragment. pIFH108 is the wild type FtsH expressing plasmid constructed by inserting the Eco RI-Hin dIII ftsH fragment of pSTD401 into pIFH100. We checked the whole sequence of the cloned ftsH gene on pIFH108 and found a silent mutation of Ala163 (GCA to GCG) in the ftsH coding region. All mutant ftsH plasmids were constructed by replacing a segment of ftsH on pIFH108 with a mutated fragment. The numbering of amino acid residues of FtsH was according to Wang et al. (27Wang R.-F. O'Hara E.B. Aldea M. Bargmann C.I. Gromley H. Kushner S.R. J. Bacteriol. 1998; 180: 1929-1939Crossref PubMed Google Scholar). The mutants are as follows: 1) K201N (AAA to AAC): the Kpn I-Pst I segment of pIFH108 was replaced with the corresponding segment of pSTD41. 2) V296A (GTT to GCA), T300A (ACT to GCT), N301A (AAC to GCA), P303A (CCG to GCG), D304A (GAC to GCC), D307A (GAC to GCG), D307N (GAC to AAC), D307E (GAC to GAG), L310A (CTG to GCG), R312A (CGT to GCT), R312L (CGT to CTT), R312K (CGT to AAA), G314A (GGC to GCA), R315A (CGT to GCA), R315L (CGT to CTT), and R315K (CGT to AAA): mutagenized ftsH fragments were produced by sequential polymerase chain reaction steps using synthetic primers (28Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Albright L.M. Coen D.M. Varki A. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1991Google Scholar). The Kpn I-Nco I segment of pIFH108 was replaced with each mutated fragment. 3) E418Q (GAA to CAG): the mutated fragment was produced by sequential polymerase chain reaction steps. The fragment digested with Mlu I and Bsu 36I was cloned into pKY297. The Kpn I-Pst I fragment was then subcloned into pIFH108. 4) H421Y (CAT to TAT): the Kpn I-Pst I segment of pQM21 was subcloned into pIFH108. We checked the whole sequence of ftsH to confirm that no other mutations had been introduced adventitiously. AR5088 cells carrying pIFH108 were grown in 2 liters of L medium containing ampicillin (100 μg/ml) at 37 °C to a cell density corresponding to 50 Klett units. At this point, expression of FtsH was induced by the addition of IPTG (1 mm) followed by growth for a further 3 h. Crude membranes were prepared and homogenized as described (8Tomoyasu T. Gamer J. Bukau B. Kanemori M. Mori H. Rutman A.J. Oppenheim A.B. Yura T. Yamanaka K. Niki H. Hiraga S. Ogura T. EMBO J. 1995; 14: 2551-2560Crossref PubMed Scopus (365) Google Scholar) and solubilized by 0.2% sodium lauryl sarcosinate (Sarkosyl). The solubilized preparation was dialyzed against buffer A (20 mm monoethanolamine HCl, pH 9.0, 1 mm EDTA, 1 mm dithiothreitol, 10% glycerol, 0.5% Nonidet P-40). MonoQ fractionation was carried out with buffer A and buffer B (buffer A containing 1 m NaCl). Pooled MonoQ fractions were applied to a Superose 6 size fractionation column in buffer A. Pooled Superose 6 fractions were applied to a MonoP fractionation column with buffers A and B. The peak fraction of FtsH was dialyzed against dialysis buffer (20 mm monoethanolamine HCl, pH 9.0, 5 mmmagnesium acetate, 10% glycerol, 0.5% Nonidet P-40) and used for in vitro assays. The proteolytic activity of purified wild type and mutant FtsH proteins was assayed as described (8Tomoyasu T. Gamer J. Bukau B. Kanemori M. Mori H. Rutman A.J. Oppenheim A.B. Yura T. Yamanaka K. Niki H. Hiraga S. Ogura T. EMBO J. 1995; 14: 2551-2560Crossref PubMed Scopus (365) Google Scholar). NTPase activities were assayed using ATP, GTP, CTP, or UTP as described (16Akiyama Y. Kihara A. Tokuda H. Ito K. J. Biol. Chem. 1996; 271: 31196-31201Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Conformational changes of FtsH induced by ATP were analyzed according to the procedures described by Akiyama et al. (23Akiyama Y. Ehrmann M. Kihara A. Ito K. Mol. Microbiol. 1998; 28: 803-812Crossref PubMed Scopus (38) Google Scholar). The reaction was started by the addition of 2.5 μg/ml of trypsin. Samples were removed at intervals, and analyzed by 10% SDS-polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting using the anti-FtsH serum and detected by ECL (Amersham Pharmacia Biotech). 1 ml of culture (Klett unit = 50) was harvested by centrifugation. Pelleted cells were suspended in 100 μl of the SDS loading buffer. 20 μl of the samples were analyzed by SDS-PAGE followed by immunoblotting using anti-FtsH or anti-ς32 serum (29Gamer J. Bujard H. Bukau B. Cell. 1992; 69: 833-842Abstract Full Text PDF PubMed Scopus (255) Google Scholar) and detected by ECL. To examine the activity of mutant FtsH proteins in vivo and to purify them without contamination from wild type FtsH, it is necessary to use a strain lacking the ftsH gene as a host. Recently, we have identified a suppressor mutation, sfhC, that allows cells lacking ftsH to survive (10Ogura T. Inoue K. Tatsuta T. Suzaki T. Karata K. Young K. Su L.-H. Fierke C.A. Jackman J.E. Raetz C.R.H. Coleman J. Tomoyasu T. Matsuzawa H. Mol. Microbiol. 1999; 31: 833-844Crossref PubMed Scopus (193) Google Scholar, 20Tatsuta T. Tomoyasu T. Bukau B. Kitagawa M. Mori H. Karata K. Ogura T. Mol. Microbiol. 1998; 30: 583-594Crossref PubMed Scopus (97) Google Scholar). To take advantage of the efficient pET expression vectors that utilize the T7 promoter, we constructed a derivative of BL21(DE3) carrying ΔftsH and sfhC, AR5088, and cloned the ftsH gene on a derivative of pET29b, pIFH108. In this system, intact FtsH protein is expressed from pIFH108, and its expression is inducible by IPTG. We found that this system has an additional merit. In the absence of IPTG, expression of FtsH from pIFH108 in AR5088 was leaky such that levels of FtsH comparable with that derived from the wild type chromosomal ftsH gene are produced (see Fig. 2, A and B, lanes 1, 2, and 5). This property allows the in vivo activity of mutant FtsH proteins in AR5088 to be assayed. In principle, FtsH cannot be induced from pIFH108 in host strains other than λDE3 lysogens owing to the absence of T7 RNA polymerase. Indeed as expected, we observed negligible expression of FtsH from pIFH108 in such strains in the absence of IPTG. However, somewhat surprisingly, in the presence of IPTG, the level of expression of FtsH from pIFH108 was comparable with that from the chromosomal ftsH gene (data not shown). Although the mechanism underlying this observation is not understood, this property was exploited in the complementation experiments with the temperature-sensitive mutant strain (see Fig.3). Several amino acid residues such as Thr300, Asn301, Asp307, Ala309, Arg312, and Arg315 in the SRH of FtsH are very highly conserved among the AAA family proteins (Fig. 1 B), whereas others are less well conserved. Changing each residue to Ala, in what is termed “alanine scanning,” is a useful strategy for identifying functionally important residues in proteins (30Cunningham B.C. Wells J.A. Science. 1989; 244: 1081-1085Crossref PubMed Scopus (1097) Google Scholar). To determine whether or not the SRH is important for FtsH function and, if so, which amino acid residues in the SRH are important, we introduced Ala substitutions into some conserved and less conserved residues in the SRH. Plasmids expressing wild type or mutant FtsH were introduced into AR5088, and the levels of ς32, which is one of the known specific substrates of the FtsH protease (8Tomoyasu T. Gamer J. Bukau B. Kanemori M. Mori H. Rutman A.J. Oppenheim A.B. Yura T. Yamanaka K. Niki H. Hiraga S. Ogura T. EMBO J. 1995; 14: 2551-2560Crossref PubMed Scopus (365) Google Scholar, 9Herman C. Thévenet D. D'Ari R. Bouloc P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3516-3520Crossref PubMed Scopus (274) Google Scholar), were analyzed by Western blotting (Fig. 2 A). In untransformed AR5088 (ΔftsH) or AR5088 harboring the vector pIFH100, ς32 accumulated (Fig. 2 A, lanes 3 and 4). When wild type FtsH was expressed from pIFH108, the accumulation of ς32 was not observed (Fig. 2 A, lane 5). In the case of mutants of less conserved residues such as V296A, P303A, D304A, and G314A, the accumulation of ς32 was not observed (Fig. 2 A, lanes 6, 9, 10, and 14), indicating that these mutants have almost the same activity as wild type FtsH and that these less conserved residues are not important for the in vivo protease activity of FtsH. On the other hand, with the mutants of the highly conserved residues N301A, D307A, R312A, and R315A, ς32 accumulated to the same levels as those in AR5088 harboring the vector (Fig. 2 A, lanes 8, 11, 13, and 15), indicating that these highly conserved residues are essential for the in vivo protease activity of FtsH. In cells expressing the T300A or L310A mutants, the accumulation of ς32 was partially suppressed (Fig. 2 A, lanes 7 and 12), suggesting that these mutants have a low protease activity. This implies that Thr300 and Leu310 are less important. Overall, these results indicate that the SRH is important for FtsH function and that there is good correlation between the functional importance and the extent of conservation of the amino acid residues in the SRH. Because highly conserved residues such as Asp307, Arg312, and Arg315 were found to be important for FtsH function by alanine scanning mutagenesis, we constructed further mutants that introduce less drastic substitutions at these positions. Specifically, we changed Asp307 (negatively charged) to Asn (polar but uncharged) or Glu (similarly negatively charged) or Arg312 and Arg315 (positively charged) to Leu (bulky and uncharged) or Lys (similarly positively charged). The mutants D307N, R312L, R312K, R315L, and R315K showed no detectable protease activity (Fig. 2 B, lanes 7–12). The mutant D307E exhibited a low but nevertheless significant protease activity similar to T300A and L310A. Thus, the negative charge of the Asp307 side chain seems to be important for FtsH activity. On the other hand, the two conserved Arg residues, Arg312and Arg315, could not be functionally substituted by Lys even though the positive charge is retained with this substitution. For comparison, we constructed additional mutants that have mutations outside the SRH. The K201N substitution resides in the Walker A motif, which is believed to be essential for ATPase activity, whereas the E418Q and H421Y mutations are in the Zn2+-binding motif believed to form the active site of the protease domain. As expected, the mutants E418Q and H421Y had no protease activity (Fig.2 B, lanes 13 and 14). The mutant K201N also showed no detectable protease activity (Fig. 2 B, lane 6), confirming the notion that the protease activity is dependent on the ATPase activity. To determine whether or not SRH mutants retain the essential functions of FtsH for cell growth, the ability of the mutants to complement the temperature-sensitive ftsH1 mutation was examined. Wild type and mutant pIFH108 plasmids were introduced into the ftsH1 strain AR754, and the growth of the transformants was examined at 42 °C in the presence of IPTG (Fig.3). Under these conditions, wild type and mutant plasmid encoded FtsH proteins are expressed at levels comparable with that from the chromosomal ftsH gene (data not shown). Wild type FtsH and the mutants T300A and D307E, which exhibit a low in vivo protease activity, complemented ftsH1, whereas the other SRH mutants lacking the protease activity did not. Because the mutants K201N and H421Y also failed to complement ftsH1, it is clear that the ability of FtsH to complement ftsH1 is related to the in vivo protease activity. To examine the activities of these mutants in vitro, we purified wild type FtsH and the mutants K201N, T300A, D307N, D307E, R312L, R315L, and H421Y to near homogeneity. All of the mutant proteins purified in this study behaved as high molecular mass complexes upon Superose 6 size fractionation (see “Experimental Procedures”) similar to the wild type protein (data not shown), indicating that their oligomeric states are not significantly affected by the mutations. Using these purified FtsH proteins, we tested the in vitro protease activity toward the ς32 substrate (Fig. 4). ATP-dependent degradation of C-terminally histidine-tagged ς32 was observed only with wild type FtsH. Significant proteolysis was not observed with any of the mutant FtsH proteins. The low proteolytic activities of the mutants T300A and D307E observed in vivo were not detected in the less sensitive in vitro assay used. Overall, these in vitro results seem to be consistent with in vivo results described above. We measured the NTPase activities of the purified wild type and mutant FtsH proteins (TableI). Among the four nucleotides, FtsH hydrolyzed ATP and CTP efficiently. This is consistent with the observation that CTP, but not GTP or UTP, can substitute for ATP in the degradation of ς32 and SecY (8Tomoyasu T. Gamer J. Bukau B. Kanemori M. Mori H. Rutman A.J. Oppenheim A.B. Yura T. Yamanaka K. Niki H. Hiraga S. Ogura T. EMBO J. 1995; 14: 2551-2560Crossref PubMed Scopus (365) Google Scholar, 16Akiyama Y. Kihara A. Tokuda H. Ito K. J. Biol. Chem. 1996; 271: 31196-31201Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Interestingly, however, FtsH also hydrolyzed GTP and UTP at appreciable rates. The reason that GTP and UTP do not support the proteolysis reaction is unknown.Table INTPase activity of wild type and mutant FtsH proteinsFtsHSpecific activityKm for ATPATPGTPCTPUTPnmol/mg/minμmWild type45921249312383K201N<10<10<10<10ND1-aND, not determined.T300A70<1054<10467D307N<10<10<10<10NDD307E40<1054<10817R312L<10<10<10<10NDR315L<10<10<10<10NDH421Y1073959136621-a ND, not determined. Open table in a new tab The specific activity of wild type FtsH in ATP hydrolysis is ∼459 nmol/min/mg. As expected, the mutant K201N showed no detectable ATPase activity. This also excludes the possibility of significant contamination of any other ATPases in FtsH preparations purified by the procedures used. Among the SRH mutants, T300A and D307E, which both have low protease activity, have low specific ATPase activities of ∼70 and ∼40 nmol/min/mg, respectively. The other SRH mutants D307N, R312L, and R315L, which have lost the protease activity completely, showed no detectable ATPase activity. Thus, it seems that the SRH is important for both the ATPase and the protease activity of FtsH, presumably because these two activities are tightly coupled. Interestingly, the ATPase activity of the mutant H421Y decreased to about a quarter of that of wild type FtsH (∼107 nmol/min/mg), even though this mutation is in the Zn2+-binding motif essential for the protease activity. The H421Y mutant did not rescue the lethality of the ftsH1 mutation at the nonpermissive temperature (Fig. 3), indicating that the protease activity of FtsH is essential for cell growth regardless of its ATPase activity. As some SRH mutants showed defects in nucleotide hydrolysis, it is important to establish whether or not they are defective in nucleotide binding. We have tried unsuccessfully to demonstrate ATP binding by FtsH in gel filtration assays, ATP agarose chromatography assays, and UV cross-linking assays employing radiolabeled ATP. 2K. Karata, and T. Ogura, unpublished results.We therefore followed the procedures developed by Akiyama et al. (23Akiyama Y. Ehrmann M. Kihara A. Ito K. Mol. Microbiol. 1998; 28: 803-812Crossref PubMed Scopus (38) Google Scholar), which allowed us to detect conformational changes in FtsH induced by ATP binding. As shown in Fig. 5 A, wild type FtsH undergoes a conformational change in the presence of ATP, which is characterized by partial protection of an ∼33-kDa ATPase domain from trypsin digestion. This conformational change is not detected for the Walker motif mutant K201N, which does not bind ATP (Fig.5 B). We examined three representative SRH mutants, D307N, R312L, and R315L, which showed no detectable ATPase activity as described above, for the ATP-induced conformational change. As shown in Fig. 5 (C–E), the ∼33-kDa fragment of all three mutants was protected from proteolytic degradation by the presence of ATP to a similar extent to that seen for wild type FtsH. Thus, it is most likely that mutations in the SRH do not significantly affect the ability of FtsH to bind ATP, indicating that the SRH plays a catalytic rather than simply a binding role in ATP hydrolysis. There is no obvious difference in the pattern of fragments generated by trypsin digestion of wild type and mutant FtsH proteins, suggesting that the amino acid alterations in these mutants do not significantly affect the overall structure of FtsH. In this paper, we have investigated the function of the SRH in FtsH. The results indicate that the highly conserved residues in the SRH are important for its ATPase activity. Among the