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• W1978951191 abstract "CadA, the Cd2+-ATPase from Listeria monocytogenes, belongs to the Zn2+/Cd2+/Pb2+-ATPase bacterial subfamily of P1B-ATPases that ensure detoxification of the bacteria. Whereas it is the major determinant of Listeria resistance to Cd2+, CadA expressed in Saccharomyces cerevisiae severely decreases yeast tolerance to Cd2+ (Wu, C. C., Bal, N., Pérard, J., Lowe, J., Boscheron, C., Mintz, E., and Catty, P. (2004) Biochem. Biophys. Res. Commun. 324, 1034–1040). This phenotype, which reflects in vivo Cd2+-transport activity, was used to select from 33 point mutations, shared out among the eight transmembrane (TM) segments of CadA, those that affect the activity of the protein. Six mutations affecting CadA were found: M149A in TM3; E164A in TM4; C354A, P355A, and C356A in TM6; and D692A in TM8. Functional studies of the six mutants produced in Sf9 cells revealed that Cys354 and Cys356 in TM6 as well as Asp692 in TM8 and Met149 in TM3 could participate at the Cd2+-binding site(s). In the canonical Cys-Pro-Cys motif of P1B-ATPases, the two cysteines act at distinct steps in the transport mechanism, Cys354 being directly involved in Cd2+ binding, while Cys356 seems to be required for Cd2+ occlusion. This confirms an earlier observation that the two equivalent Cys of Ccc2, the yeast Cu+-ATPase, also act at different steps. In TM4, Glu164, which is conserved among P1B-ATPases, may be required for Cd2+ release. Finally, analysis of the role of Cd2+ in the phosphorylation from ATP and from Pi of the mutants suggests that two Cd2+ ions are involved in the reaction cycle of CadA. CadA, the Cd2+-ATPase from Listeria monocytogenes, belongs to the Zn2+/Cd2+/Pb2+-ATPase bacterial subfamily of P1B-ATPases that ensure detoxification of the bacteria. Whereas it is the major determinant of Listeria resistance to Cd2+, CadA expressed in Saccharomyces cerevisiae severely decreases yeast tolerance to Cd2+ (Wu, C. C., Bal, N., Pérard, J., Lowe, J., Boscheron, C., Mintz, E., and Catty, P. (2004) Biochem. Biophys. Res. Commun. 324, 1034–1040). This phenotype, which reflects in vivo Cd2+-transport activity, was used to select from 33 point mutations, shared out among the eight transmembrane (TM) segments of CadA, those that affect the activity of the protein. Six mutations affecting CadA were found: M149A in TM3; E164A in TM4; C354A, P355A, and C356A in TM6; and D692A in TM8. Functional studies of the six mutants produced in Sf9 cells revealed that Cys354 and Cys356 in TM6 as well as Asp692 in TM8 and Met149 in TM3 could participate at the Cd2+-binding site(s). In the canonical Cys-Pro-Cys motif of P1B-ATPases, the two cysteines act at distinct steps in the transport mechanism, Cys354 being directly involved in Cd2+ binding, while Cys356 seems to be required for Cd2+ occlusion. This confirms an earlier observation that the two equivalent Cys of Ccc2, the yeast Cu+-ATPase, also act at different steps. In TM4, Glu164, which is conserved among P1B-ATPases, may be required for Cd2+ release. Finally, analysis of the role of Cd2+ in the phosphorylation from ATP and from Pi of the mutants suggests that two Cd2+ ions are involved in the reaction cycle of CadA. P-type ATPase is the generic name for a class of membrane proteins that pump cations against their electrochemical gradient at the expense of ATP. Whatever their origin, location, and ionic selectivity, P-type ATPases are thought to share the same overall fold and reaction mechanism. The P-type ATPase fold, determined to a few angstroms resolution in the Ca2+-ATPase SERCA1a (1Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1613) Google Scholar), is made of two domains, one cytoplasmic, bearing the catalytic site, the other membranous, comprising the cation transport site(s). The P-type ATPase reaction mechanism can be reduced to a four-step process (Scheme 1) where vectorial events (cation binding (step 1) and cation release (step 3)) and chemical events (phosphorylation (step 2) and dephosphorylation (step 4)) alternate (for recent reviews, see Refs. 2Kuhlbrandt W. Nat. Rev. Mol. Cell. Biol. 2004; 5: 282-295Crossref PubMed Scopus (447) Google Scholar, 3Toyoshima C. Inesi G. Annu. Rev. Biochem. 2004; 73: 269-292Crossref PubMed Scopus (297) Google Scholar, 4Møller J.V. Nissen P. Sørensen T.L. le Maire M. Curr. Opin. Struct. Biol. 2005; 15: 387-393Crossref PubMed Scopus (110) Google Scholar). The P1B-ATPases (nomenclature from data base maintained by K. B. Axelsen) constitute a subfamily that assures the transport of heavy metals like Cu+, Cu2+, Ag+, Cd2+, Zn2+, Pb2+, or Co2+ (5Nies D.H. FEMS Microbiol. Rev. 2003; 27: 313-339Crossref PubMed Scopus (1092) Google Scholar). They differ from most of the P-type ATPases by a smaller membranous domain made of 8 transmembrane helices instead of 10 and the presence at their NH2 terminus of 1–6 metal-binding domains (6Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (418) Google Scholar, 7Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (417) Google Scholar). Most of these NH2-terminal metal-binding domains are made of 70 amino acids comprising a Cys-X-X-Cys motif whose cysteines are directly involved in metal binding. In some cases, represented by the subgroup P1B-3, the NH2 terminus consists of a histidine-rich sequence (8Argüello J.M. J. Membr. Biol. 2003; 195: 93-108Crossref PubMed Scopus (219) Google Scholar). P-type ATPases possess in their membranous domain one or more sites which bind the cation(s) to be transported. These so-called transport sites have been mainly studied on Ca2+-, Na+/K+-, and H+/K+-ATPases (P2 subfamily) and H+-ATPases (P3 subfamily). It emerged from these studies that whatever the ionic selectivity of the pump, the transport site(s) involved at least three transmembrane helices, including those two that are directly linked to the catalytic loop. For instance, in SERCA1a, the Ca2+-transport sites involve amino acids from four trans-membrane helices (1Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1613) Google Scholar). In the transport sites of P2- and P3-ATPases, Ca2+, Na+, K+, or H+, which are all hard Lewis acids, are bound to carbonyl, carboxyl, or hydroxyl groups, all hard Lewis bases, thereby verifying the principle of hard and soft acids and bases (9Pearson R.G. J. Am. Chem. Soc. 1963; 85: 3533-3539Crossref Scopus (8203) Google Scholar). For instance, in SERCA1a, the Ca2+-transport sites consist of Glu, Asp, Asn, and Thr, which bind Ca2+ through their side chain (10Andersen J. Vilsen B. Acta Physiol. Scand. Suppl. 1998; 643: 45-54PubMed Google Scholar), plus some hydrophobic amino acids, which participate through their backbone carbonyl group, and also a water molecule. Much less is known about the transport site of P1B-ATPases. Nevertheless, various early studies on Cu+/Ag+-ATPases suggested a key role of the cysteines of the Cys-Pro-Cys motif located in TM6, 6The abbreviations used are: TM, transmembrane helix; HA, hemagglutinin; MOPS, 4-morpholinepropanesulfonic acid; MES, 4-morpholineethanesulfonic acid. the upstream transmembrane helix linked to the catalytic loop (11Forbes J.R. Cox D.W. Am. J. Hum. Genet. 1998; 63: 1663-1674Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 12Yoshimizu T. Omote H. Wakabayashi T. Sambongi Y. Futai M. Biosci. Biotechnol. Biochem. 1998; 62: 1258-1260Crossref PubMed Scopus (31) Google Scholar, 13Haas R. Gutierrez-Rivero B. Knoche J. Boker K. Manns M.P. Schmidt H.H. Hum. Mutat. 1999; 14: 88Crossref PubMed Scopus (17) Google Scholar, 14Tümer Z. Møller L.B. Horn N. Ad. Exp. Med. Biol. 1999; 448: 83-95Crossref PubMed Scopus (58) Google Scholar, 15Bissig K.D. Wunderli-Ye H. Duda P.W. Solioz M. Biochem. J. 2001; 357: 217-223Crossref PubMed Scopus (50) Google Scholar, 16Fan B. Rosen B.P. J. Biol. Chem. 2002; 277: 46987-46992Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 17Mandal A.K. Argüello J.M. Biochemistry. 2003; 42: 11040-11047Crossref PubMed Scopus (89) Google Scholar, 18Bal N. Wu C.C. Catty P. Guillain F. Mintz E. Biochem. J. 2003; 369: 681-685Crossref PubMed Scopus (26) Google Scholar, 19Lowe J. Vieyra A. Catty P. Guillain F. Mintz E. Cuillel M. J. Bio. Chem. 2004; 279: 25986-25994Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This exemplifies once again the hard and soft acids and bases principle, soft Lewis acids (Cu+, Ag+) interacting with a soft Lewis base (S–) (9Pearson R.G. J. Am. Chem. Soc. 1963; 85: 3533-3539Crossref Scopus (8203) Google Scholar). However, as described for the yeast Cu+-ATPase Ccc2p (19Lowe J. Vieyra A. Catty P. Guillain F. Mintz E. Cuillel M. J. Bio. Chem. 2004; 279: 25986-25994Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and for model peptides of the human Cu+-ATPase ATP7B (20Myari A. Hadjiliadis N. Fatemi N. Sarkar B. J. Inorg. Biochem. 2004; 98: 1483-1494Crossref PubMed Scopus (15) Google Scholar), the two cysteines of the Cys-Pro-Cys motif are likely to play distinct roles in ion transport. In ATP7B, two mutations flanking the Cys-Pro-Cys motif were found to be responsible for Wilson disease, an autosomal recessive defect of copper transport. Expressed in Saccharomyces cerevisiae, the ATP7B mutants T977M and P922L were unable to complement a ccc2 mutant and had a reduced Fet3p activity, suggesting a defect in copper transport (11Forbes J.R. Cox D.W. Am. J. Hum. Genet. 1998; 63: 1663-1674Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Not only TM6 but also TM8 is likely to be involved in the transport site of P1B-ATPases. Indeed, the toxic milk mouse considered as an animal model for Wilson disease was shown to bear a mutation in the murine ATP7B homologue. Located in TM8, this mutation, the replacement by valine of a methionine highly conserved in the P1B-1 subgroup, was shown to abolish Cu+-transport (21La Fontaine S.L. Firth S.D. Camakaris J. Englezou A. Theophilos M.B. Petris M.J. Howie M. Lockhart P.J. Greenough M. Brooks H. Reddel R.R. Mercer J.F. J. Biol. Chem. 1998; 273: 31375-31380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 22Voskoboinik I. Greenough M. La Fontaine S. Mercer J.F. Camakaris J. Biochem. Biophys. Res. Commun. 2001; 281: 966-970Crossref PubMed Scopus (70) Google Scholar). A recent study used sequence alignments to predict which amino acids from TM6, TM7, and TM8 could constitute the transport site of P1B-ATPases, assuming that these transmembrane helices can play the same role as those forming the transport sites in P2- and P3-ATPases. Alignment of over 200 sequences revealed some amino acids in TM7 and TM8 that are conserved and were therefore used to define five P1B-ATPase subgroups. As these subgroups display different ionic selectivity, these amino acids were predicted as putative components of the transport site of P1B-ATPases (8Argüello J.M. J. Membr. Biol. 2003; 195: 93-108Crossref PubMed Scopus (219) Google Scholar). The prediction has been verified for the Cu+/Ag+-ATPase CopA from Archeoglobus fulgidus, a member of subgroup IB-1, whose mutations at Tyr682 (TM7), Asn683 (TM7), Met711 (TM8), and Ser715 (TM8) were shown to severely affect ATPase activity and phosphoenzyme formation from ATP (23Mandal A.K. Yang Y. Kertesz T.M. Argüello J.M. J. Biol. Chem. 2004; 279: 54802-54807Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The inventory of the ATP7B mutations causing Wilson disease suggests that transmembrane segments other than TM6, TM7, or TM8 can participate in the transport site of the P1B-type ATPases. Indeed, among the 33 mutations of the transmembrane domain of ATP7B found in patients with Wilson disease, only 11 are located in TM6, TM7, or TM8 (see the Wilson disease data base). The experimental studies carried out so far on the transport site of P1B-ATPases have all focused on Cu+/Ag+-ATPases, which constitute the largest subgroup of heavy metal pumps known today. In the present study, we have searched for the amino acids involved in the transport site of CadA, the Cd2+-ATPase from L. monocytogenes, a member of subgroup P1B-2. To do so, we first used a phenotypic test in yeast to identify among 33 point mutations of the transmembrane domain of CadA those that affect Cd2+-transport. We then produced the selected mutants in Sf9 cells to assess how Cd2+ transport was affected. We found that (i) the two cysteines of the Cys-Pro-Cys motif (TM6) act at distinct steps of the transport process, Cys354 being directly involved in Cd2+ binding, whereas Cys356 is required for Cd2+ occlusion; (ii) Asp692 in TM8 would be directly involved in Cd2+ binding; (iii) Glu164 in TM4 would be required for Cd2+ release. In addition, we propose that two Cd2+ ions are involved in the reaction cycle of CadA. Molecular Biology Procedures—JM109 Escherichia coli strain was used for DNA subcloning and amplification. Restriction and modification enzymes were from Fermentas or Invitrogen. DNA fragments were isolated using the Gene Clean Spin kit (Q-Biogene). Plasmids were purified by anion exchange chromatography (Qiagen). Site-directed mutagenesis was done using the QuikChange method (Stratagene). DNA sequencing was performed by Genome Express Inc. (Meylan, France). Yeast Manipulations—Yeast procedures are detailed in Ref. 24Wu C.C. Bal N. Pérard J. Lowe J. Boscheron C. Mintz E. Catty P. Biochem. Biophys. Res. Commun. 2004; 324: 1034-1040Crossref PubMed Scopus (11) Google Scholar. The yeast strain used in the present study, W303-1A (25Wallis J.W. Chrebet G. Brodsky G. Rolfe M. Rothstein R. Cell. 1989; 58: 409-419Abstract Full Text PDF PubMed Scopus (453) Google Scholar), was transformed following the method of Kuo and Campbell (26Kuo C.L. Campbell J.L. Mol. Cell. Biol. 1983; 3: 1730-1737Crossref PubMed Scopus (87) Google Scholar) and grown at 30 °C, with continuous stirring at 200 rpm for liquid cultures. Yeast transformants were grown in synthetic minimal DOB medium (2% glucose, 0.17% yeast nitrogen base without amino acids, 0.5% ammonium sulfate, Q-Biogene) supplemented with dropout powder without uracil (CSM-URA, Q-Biogene). HA-tagged CadA mutants were expressed from a centromeric vector derived from pRS316 (27Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), under the control of the strong constitutive PMA1 promoter. The sensitivity to Cd2+ of the different mutants was determined by drop tests, and their expression level was assessed by immunodetection of the HA epitope on a membrane preparation. Protein Preparation and Detection—The wild-type and mutant CadA proteins were produced using the Bac-to-Bac™ Baculovirus Expression System (Invitrogen). After 3 days of culture at 27 °C, 100 ml of cells (2 × 106 cells/ml) were harvested by centrifugation (10 min, 500 × g, 4 °C). The pellet was suspended in 18 ml of 10 mm MOPS/Tris (pH 7), 10 μg/ml RNase, 20 μg/ml DNase I, half a protease inhibitor mixture tablet (Roche Applied Science). Cells were lysed by potterization and the homogenate was complemented so as to reach 10 mm MOPS/Tris (pH 7), 80 mm KCl, 0.2 mm MgCl2, 300 mm sucrose. The sample was centrifuged (1 h, 100,000 × g, 4 °C), and the P100 pellet corresponding to a crude membrane fraction was suspended in 3 ml of 50 mm MOPS/Tris (pH 7), 100 mm KCl, 1 mm MgCl2, 300 mm sucrose. Aliquots were homogenized by potterization, rapidly frozen in liquid nitrogen and stored at –80 °C. Protein concentration was determined using the DC Protein Assay (Bio-Rad), with bovine serum albumin as standard. Immunodetection was carried out using the monoclonal antibody anti-HA-peroxidase (3F10) and the BM Chemiluminescence Western blotting kit from Roche Applied Science. ATPase Activity Measurements—ATPase activities were measured at 28 °C with continuous stirring, in a medium containing 50 mm MOPS/Tris (pH 7), 100 mm KCl, 300 mm sucrose, 5 mm MgCl2, 4 mm ATP, and 0.5 mg/ml of Sf9 membrane preparations. ATP hydrolysis was measured following NADH absorbance changes at 340 nm, using a coupled enzyme assay as described in Ref. 28Miras R. Cuillel M. Catty P. Guillain F. Mintz E. Protein Expression Purif. 2001; 22: 299-306Crossref PubMed Scopus (17) Google Scholar. Phosphorylations—The phosphoenzyme intermediates formed in the presence of 32Pi or [γ-32P]ATP were quantitated as a function of Cd2+ concentration. In the two types of reactions, the acid-quenched sample was submitted to an acidic SDS-PAGE as described by Weber and Osborn (29Weber K. Osborn M. J. Biol. Chem. 1969; 244: 4406-4412Abstract Full Text PDF PubMed Google Scholar). The phosphorylation signal was revealed by autoradiography and analyzed using the Optiquant Software (Cyclone, PerkinElmer Life Sciences). Later, polyacrylamide gels were stained with Coomassie Blue to check the amounts of loaded protein. Phosphorylations from Pi were performed at 25 °C in a medium containing 0.5 mg/ml of Sf9 membrane preparations, 50 mm MES pH 6, 10 mm MgCl2, 20% Me2SO, in the presence of CdCl2 or EGTA as indicated in the figure legend to Fig. 5. After 10-min incubation at 25 °C, the reaction was started by addition of 100 μm 32Pi at 10–100 μCi/nmol and stopped 10 min later by addition of 1 ml of ice-cold 1 mm KH2PO4 in 7% trichloroacetic acid. Phosphorylations from ATP were performed at 0 °C in a medium containing 0.5 mg/ml of Sf9 membrane preparations, 50 mm MOPS/Tris (pH 7), 100 mm KCl, 5 mm MgCl2, 300 mm sucrose, and CdCl2 as indicated. The reaction was started by addition of 1 μm [γ-32P]ATP at 50–500 μCi/nmol and stopped 15 s later by addition of 1 ml of ice-cold 1 mm KH2PO4 in 7% trichloroacetic acid. The present work aims to identify the amino acids of the CadA transmembrane domain that are involved in Cd2+ binding and release during the transport cycle. CadA Topology—The initial step of this study consisted of the proper delimitation of the transmembrane segments of CadA, using the results of 10 prediction programs and a comparison with the experimentally determined topology of a P1B-ATPase from Helicobacter pylori (30Melchers K. Weitzenegger T. Buhmann A. Steinhilber W. Sachs G. Schafer K.P. J. Biol. Chem. 1996; 271: 446-457Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Eight transmembrane segments were defined, hereafter named TM1 to TM8, and we must emphasize the uncertainty concerning the delimitation of TM3 and TM4 as well as TM7 and TM8 (Fig. 1). Point mutations were performed on negatively charged (Asp, Glu), polar (Ser, Thr, and Asn), and sulfur-containing amino acids (Cys, Met) liable to coordinate Cd2+ or to participate in its interaction with CadA (Fig. 1). Phenotypic Sorting of CadA Mutants—Expression of CadA in S. cerevisiae was shown to strongly increase yeast sensitivity to Cd2+. As CadA was found inserted in the membrane of the endoplasmic reticulum, this phenotype was explained by the toxicity of Cd2+ accumulated in this compartment (24Wu C.C. Bal N. Pérard J. Lowe J. Boscheron C. Mintz E. Catty P. Biochem. Biophys. Res. Commun. 2004; 324: 1034-1040Crossref PubMed Scopus (11) Google Scholar). In the following, we used this phenotype as an assessment of in vivo Cd2+-transport activity to sort CadA mutants produced by site-directed mutagenesis. Three classes of mutants were defined (Fig. 1). Class 1 comprises all the mutants (27 of the 33 tested) that induce growth arrest at 1 μm Cd2+ as does the wild-type protein (24Wu C.C. Bal N. Pérard J. Lowe J. Boscheron C. Mintz E. Catty P. Biochem. Biophys. Res. Commun. 2004; 324: 1034-1040Crossref PubMed Scopus (11) Google Scholar). Class 3 comprises mutants that do not alter yeast response to Cd2+. The reference in this class is D398A, a mutant of the phosphorylation site (DKTGT) that does not transport Cd2+ (24Wu C.C. Bal N. Pérard J. Lowe J. Boscheron C. Mintz E. Catty P. Biochem. Biophys. Res. Commun. 2004; 324: 1034-1040Crossref PubMed Scopus (11) Google Scholar). To this class belong the mutants C354A and C356A of the Cys-Pro-Cys motif cysteines in TM6 and the mutant D692A in TM8. Class 2 was defined as intermediate between class 1 and class 3, as some mutants induce yeast growth arrest at 10 μm Cd2+. In this class are found the mutants M149A, E164A, and P355A, which belong to TM3, TM4, and TM6, respectively. The expression level of the mutants was assessed by immunodetection of the HA-epitope added at the COOH-terminal end of the protein. The mutants were all equally produced in yeast, whatever the class they belong to (Fig. 2). Functional Study of the Selected CadA Mutants—Yeast was a very efficient system for sorting 6 mutants from 33 and, according to the phenotype, C354A, C356A, and D692A (class 3) are expected to be unable to transport Cd2+, whereas in M149A, E164A, and P355A (class 2) lower transport efficiency is expected. To assess the role of each of these 6 amino acids in the Cd2+-transport cycle of CadA, in vitro biochemical studies of the mutants were necessary, and yeast was not well suited anymore. The main limitations were the low expression level of the proteins and the high endogenous ATPase activity of yeast membranes. This is the reason why the six mutants selected from the phenotypic test were produced in Sf9 cells for functional studies. In this system, all the mutants exhibited the same expression level and they were all studied in a crude membrane preparation. Four methods were used to assess the functionality of the mutants:limited proteolysis, activity measurements, phosphorylation from ATP, and phosphorylation from Pi. In all these experiments, the non-functional D398A mutant was used to measure the endogenous activity of membranes from transfected Sf9 cells. Limited Proteolysis—Limited proteolysis by trypsin was used to compare the conformational states of the mutants with that of CadA. As shown in Fig. 3 (lane D), the digestion pattern resulting from CadA proteolysis in the absence of ATP displays two major bands of about 46 and 10 kDa, as well as a doublet around 27 kDa. Considering the location of the HA tag at the COOH-terminal end of CadA, these bands could result from cleavages at the end of the A domain, at the end of the large cytoplasmic loop and in the N domain, respectively. The presence of 5 mm ATP clearly modifies the proteolysis profile of CadA (lane A) that displays only one proteolytic band of about 55 kDa resulting from a cleavage in the middle of the A domain. As shown in supplemental Fig. S1, the six selected mutants display the same digestion patterns as CadA, indicating that mutations did not affect the overall conformation of the protein and that all the mutants are able to bind ATP. ATPase Activity Measurements—A membrane fraction from Sf9 cells expressing CadA displays a Cd2+-dependent ATPase activity of about 50 nmol of ATP hydrolyzed per min and mg of protein. This value results from two effects of Cd2+, which on the one hand inhibits the endogenous ATPase activity, as illustrated in the membrane fraction containing the non-functional D398A mutant, and on the other hand activates CadA (Ref. 31Bal N. Mintz E. Guillain F. Catty P. FEBS Lett. 2001; 506: 249-252Crossref PubMed Scopus (35) Google Scholar and Fig. 4). ATPase activity measurements on membrane fractions from Sf9 cells expressing the various mutants showed that two of the mutants, C354A and D692A, were inactive. The other four mutants M149A, E164A, P355A, and C356A all displayed an ATPase activity in the same Cd2+ concentration range as CadA. The maximal activities were lower than that of CadA and ranged from 10 nmol of ATP hydrolyzed per min and per mg of protein for M149A and P355A to 20 for C356A and 40 for E164A. ATPase activity measurements (in vitro) were in complete agreement with the phenotype-based classification (in vivo), for five of the six mutants. As expected, the class 3 mutants C354A and D692A are inactive, and the class 2 mutants M149A, E164A, and P355A partially active. The fact that C356A, a class 3 mutant, displayed a Cd2+-dependent ATPase activity is more puzzling and suggests that mutation of Cys356 uncouples ATP hydrolysis and Cd2+ transport. In addition, these results show that the two cysteines of the Cys-Pro-Cys motif have a distinct role in Cd2+ transport. The Phosphorylated Intermediates—CadA can be phosphorylated from ATP in the forward direction (Scheme 1, step 2) and from Pi in the backward direction (Scheme 1, step 4) (31Bal N. Mintz E. Guillain F. Catty P. FEBS Lett. 2001; 506: 249-252Crossref PubMed Scopus (35) Google Scholar, 32Wu C.C. Gardarin A. Catty P. Guillain F. Mintz E. Biochimie (Paris). 2006; doi: 10.1016/j.biochi.2006.06.013Google Scholar). These two reactions depend on Cd2+. The presence of Cd2+ at the transport site of CadA (Scheme 1, CdnE) is required for the phosphorylation from ATP (Scheme 1, step 2), whereas Cd2+ binding at that site (Scheme 1, step 1) competes with the phosphorylation from Pi (Scheme 1, reverse of step 4). Hence, both phosphorylation reactions give access to an apparent affinity of the Cd2+-transport site. Phosphorylation from Pi—Membrane fractions from Sf9 cells expressing CadA are phosphorylated from Pi at equilibrium (Fig. 5A). In the absence of Cd2+, full phosphorylation is reached, hence the 100% value. Cd2+ inhibits CadA phosphorylation from Pi with an apparent affinity of 0.5 μm (Fig. 5A). It is commonly admitted for P-type ATPases that such an inhibition reflects the binding of the transported ion to the transport site (see Ref. 33Guimaraes-Motta H. de Meis L. Arch. Biochem. Biophys. 1980; 203: 395-403Crossref PubMed Scopus (15) Google Scholar for SERCA1a). However, like most of the P1B-ATPases, CadA possesses an additional metal-binding domain in its cytoplasmic NH2-terminal end (34Banci L. Bertini I. Ciofi-Baffoni S. Su X.C. Miras R. Bal N. Mintz E. Catty P. Shokes J.E. Scott R.A. J. Mol. Biol. 2006; 356: 638-650Crossref PubMed Scopus (51) Google Scholar) that was shown to regulate CadA activity (31Bal N. Mintz E. Guillain F. Catty P. FEBS Lett. 2001; 506: 249-252Crossref PubMed Scopus (35) Google Scholar). To verify whether such a domain could participate in the inhibition of the phosphorylation from Pi, we performed similar experiments on two CadA mutants, one truncated from its NH2 terminus (ΔMBD), the other mutated at the two cysteines of the Cys-X-X-Cys motif (CadA-AA). Cd2+ inhibits CadA, ΔMBD, and CadA-AA phosphorylation from Pi at the same concentration, reflecting its binding to the transmembrane domain (Fig. 5B). When the same experiments were performed on the class 2 and class 3 mutants, the same phosphorylation level was measured in the absence of Cd2+ (Fig. 5A). The apparent affinity for Cd2+ is similar to that of CadA for M149A (0.5 μm) and P355A (0.7 μm), suggesting that Cd2+ binding is not altered in these two mutants. On the other hand, D692A (5.1 μm) and C354A (7.5 μm) displayed more than 10-fold decrease in their apparent affinity for Cd2+, suggesting an essential role of Cys354 and Asp692 in Cd2+ coordination, in agreement with the finding that these two mutants do not have any ATPase activity (Fig. 4). To a lesser extent, C356A (2 μm) and E164A (2.3 μm) also exhibited a reduced apparent affinity for Cd2+. Phosphorylation from ATP—In the presence of Cd2+, addition of ATP to membrane fractions from Sf9 cells expressing CadA triggers the enzymatic cycle (Scheme 1). The phosphoenzyme level, measured 15 s later, reflects the ratio between the phosphorylation and dephosphorylation rates of the cycle (Fig. 6A). The effect of Cd2+ on CadA phosphorylation from ATP displayed two phases. First, the phosphoenzyme increased from 0to20 μm Cd2+ and stabilized at a level corresponding to 25% of the maximum level reached by CadA, until 100 μm Cd2+ was reached. It should be emphasized that the apparent affinity for Cd2+ in this first phase (0.75 μm) is similar to that previously estimated from Pi phosphorylation (Fig. 5A). The second phase of the phosphorylation peaked at 1 mm Cd2+ (the phosphorylation level decreased at higher Cd2+ concentrations). Referring to Scheme 1, the second phase could reflect an inhibitory effect of Cd2+ on Cd2+ release (step 3), leading to the accumulation of CdnE∼P. Hence, the first phase results from Cd2+ binding to the cytoplasmic-facing site before ATP phosphorylation and the second from Cd2+ binding from the extracellular-facing site after ATP phosphorylation. As illustrated by “isotopic dilution” experiments where CadA labeling decreased upon [γ-32P]ATP dilution, CadA phosphoenzymes formed at low and high Cd2+ were transient (Fig. 6C). It should be mentioned here that the maximum level of phosphoenzyme reached by CadA in the presence of ATP (1 mm Cd2+) represented only 10% of the level reached in the presence of Pi (EGTA), the latter corresponding to the maximum amount of phosphorylatable enzyme. The low phosphorylation level with ATP primarily results from the low concentration of ATP used in these experiments (about 10-fold lower than the apparent affinity for ATP, data not shown) but also from the relative rate constants of the reaction cycle. Such a low phosphorylation level for the wild-type protein authorizes “over”-phosphorylation levels such as those shown below for the mutants C356A and E164A (Fig. 6B). As shown in Fig. 6A, C354A and D692A, two class 3 mutants characterized by a low apparent affinity for Cd2+ and no ATPase activity, do not produce any phosphorylated intermediates from ATP, a result that seems to contradict the inhibition of the phosphorylation from Pi by Cd2+ shown in Fig. 5A. Unlike C354A and D692A, C356A, the third class 3 mutant, is phosphorylatable from ATP (Fig. 6, A and B). However, the phosphorylation profile of C356A has two main differences from that of CadA, a shift of the first phase toward higher Cd2+ concentrations an" @default.
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