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• W2072066095 abstract "Limited proteolysis by proteinase K of rabbit SERCA1 Ca2+-ATPase generates a number of fragments which have been identified recently. Here, we have focused on two proteolytic C-terminal fragments, p20C and p19C, starting at Gly-808 and Asp-818, respectively. The longer peptide p20C binds Ca2+, as deduced from changes in migration rate by SDS-polyacrylamide gel electrophoresis performed in the presence of Ca2+ as well as from labeling with45Ca2+ in overlay experiments. In contrast, the shorter peptide p19C, a proteolysis fragment identical to p20C but for 10 amino acids missing at the N-terminal side, did not bind Ca2+ when submitted to the same experiments. Two cluster mutants of Ca2+-ATPase, D813A/D818A and D813A/D815A/D818A, expressed in the yeast Saccharomyces cerevisiae, were found to have a very low Ca2+-ATPase activity. Region 808–818 is thus essential for both Ca2+ binding and enzyme activity, in agreement with similar results recently reported for the homologous gastric H+, K+-ATPase (Swarts, H. G. P., Klaassen, C. H. W., de Boer, M., Fransen, J. A. M., and De Pont, J. J. H. H. M. (1996) J. Biol. Chem. 271, 29764–29772). However, the accessibility of proteinase K to the peptidyl link between Leu-807 and Gly-808 clearly shows that the transmembrane segment M6 ends before region 808–818. It is remarkable that critical residues for enzyme activity are located in a cytoplasmic loop starting at Gly-808. Limited proteolysis by proteinase K of rabbit SERCA1 Ca2+-ATPase generates a number of fragments which have been identified recently. Here, we have focused on two proteolytic C-terminal fragments, p20C and p19C, starting at Gly-808 and Asp-818, respectively. The longer peptide p20C binds Ca2+, as deduced from changes in migration rate by SDS-polyacrylamide gel electrophoresis performed in the presence of Ca2+ as well as from labeling with45Ca2+ in overlay experiments. In contrast, the shorter peptide p19C, a proteolysis fragment identical to p20C but for 10 amino acids missing at the N-terminal side, did not bind Ca2+ when submitted to the same experiments. Two cluster mutants of Ca2+-ATPase, D813A/D818A and D813A/D815A/D818A, expressed in the yeast Saccharomyces cerevisiae, were found to have a very low Ca2+-ATPase activity. Region 808–818 is thus essential for both Ca2+ binding and enzyme activity, in agreement with similar results recently reported for the homologous gastric H+, K+-ATPase (Swarts, H. G. P., Klaassen, C. H. W., de Boer, M., Fransen, J. A. M., and De Pont, J. J. H. H. M. (1996) J. Biol. Chem. 271, 29764–29772). However, the accessibility of proteinase K to the peptidyl link between Leu-807 and Gly-808 clearly shows that the transmembrane segment M6 ends before region 808–818. It is remarkable that critical residues for enzyme activity are located in a cytoplasmic loop starting at Gly-808. P-type transport ATPases are members of a large family of procaryotic and eucaryotic proteins, specialized in active transport of various cations such as Na+, K+, H+, Ca2+, and Cu+ (1Glynn J.M. Karlish S.J.D. Annu. Rev. Biochem. 1990; 59: 171-205Crossref PubMed Scopus (177) Google Scholar, 2Lingrel J.B. Orlowski M.M. Shull M.M. Price E.M. Prog. Nucleic Acid Res. Mol. Biol. 1990; 38: 37-89Crossref PubMed Scopus (339) Google Scholar, 3MacLennan D.H. Biophys. J. 1990; 58: 1355-1365Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 4Bamberg, E., and Schoner, W. eds. (1994) The Sodium Pump, Springer Verlag, New YorkGoogle Scholar, 5Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (417) Google Scholar, 6Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (659) Google Scholar) and perhaps also aminophospholipids (7Tang X. Halleck M.S. Schlegel R.A. Williamson P. Science. 1996; 272: 1495-1497Crossref PubMed Scopus (419) Google Scholar). Based on sequence homologies and transport specificities, these proteins can be divided into the following main groups: types I ATPases (heavy metal transporters), IIA ATPases (e.g. Na+,K+-ATPase or SERCA 1The abbreviations used are: SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; C12E8, octaethylene glycol monododecyl ether; Tes, 2-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] aminoethanesulfonic acid; pBS, plasmid Bluescript (Stratagene); PAGE, polyacrylamide gel electrophoresis; bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane. 1The abbreviations used are: SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; SR, sarcoplasmic reticulum; C12E8, octaethylene glycol monododecyl ether; Tes, 2-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] aminoethanesulfonic acid; pBS, plasmid Bluescript (Stratagene); PAGE, polyacrylamide gel electrophoresis; bis-Tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane. Ca2+-ATPase), and IIB ATPases (e.g. H+-ATPase or plasma membrane Ca2+-ATPase) (6Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (659) Google Scholar). Ordered two-dimensional or three-dimensional membrane crystals have provided information on the overall shape of SR Ca2+-ATPase (8Dux L. Taylor K.A. Ting-Beal H.P. Martonosi A. J. Biol. Chem. 1985; 260: 11730-11743Abstract Full Text PDF PubMed Google Scholar, 9Castellani L. Hardwicke P.M. Vibert P. J. Mol. Biol. 1985; 185: 579-594Crossref PubMed Scopus (63) Google Scholar, 10Toyoshima C. Sasabe H. Stokes D.L. Nature. 1993; 362: 467-471Crossref PubMed Scopus (196) Google Scholar), the H+-ATPase of Neurospora crassa (11Cyrklaff M. Auer M. Kühlbrandt W. Scarborough G.A. EMBO J. 1995; 14: 1854-1857Crossref PubMed Scopus (54) Google Scholar), Na+,K+-ATPase (12Mohraz M. Simpson M.V. Smith P.R. J. Cell Biol. 1987; 105: 1-8Crossref PubMed Scopus (33) Google Scholar, 13Skriver E. Kaveus U. Hebert H. Maunsbach A.B. J. Struct. Biol. 1992; 108: 176-185Crossref PubMed Scopus (22) Google Scholar), and H+,K+-ATPase (14Hebert H. Xian Y. Hacksell I. Mårdh S. FEBS Lett. 1992; 299: 159-162Crossref PubMed Scopus (17) Google Scholar), but the level of resolution obtained at present (down to 10 Å) does not allow a precise description of the topology. Thus the detailed organization of cytoplasmic regions and transmembrane segments in domains with defined functional properties still remains a matter of speculation. Based on amino acid similarities, it is reasonable to postulate that, within each group, the topology and the fundamental reaction mechanism exhibit common features (6Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (659) Google Scholar, 15le Maire M. Champeil P. Møller J.V. Nature. 1990; 346: 167Crossref Scopus (1) Google Scholar). Directed mutagenesis and various biochemical techniques have been successfully used to pinpoint individual amino acid residues or group of residues important for transport activities. In the case of SR Ca2+-ATPase it was initially demonstrated by Clarkeet al. (16Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (470) Google Scholar) and later considerably refined (17Chen L. Sumbilla C. Lewis D. Zhong L. Strock C. Kirtley M.E. Inesi G. J. Biol. Chem. 1996; 271: 10745-10752Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 18Rice W.J. MacLennan D.H. J. Biol. Chem. 1996; 271: 31412-31419Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 19Vilsen, B. (1995) Acta Physiol. Scand., 154, Suppl. 624, 1–146.Google Scholar, 20Andersen JP. Biosc. Rep. 1995; 15: 243-261Crossref PubMed Scopus (117) Google Scholar) that 6 amino acid residues, presumably located in the transmembrane segments, are of primary importance for Ca2+ binding to the ATPase and/or Ca2+-dependent phosphorylation from ATP. These residues are Glu-309, Glu-771, Asn-796, Thr-799, Asp-800, and Glu-908, located in the putative transmembrane segments M4, M5/M6, and M8. A model was built in which these transmembrane segments are clustered and form a channel that can admit two Ca2+ in a single row (since these bound ions appear to be stacked inside the ATPase structure (21Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar, 22Inesi G. Kirtley M.E. J. Membr. Biol. 1990; 116: 1-8Crossref PubMed Scopus (23) Google Scholar)). All of these residues, with the exception of Glu-908 (18Rice W.J. MacLennan D.H. J. Biol. Chem. 1996; 271: 31412-31419Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 23Vilsen B. Andersen J.P. J. Biol. Chem. 1992; 267: 25739-25743Abstract Full Text PDF PubMed Google Scholar), have been found to be essential for occlusion of Ca2+. In Na+,K+-ATPase and H+,K+-ATPase the homologous residues located in the M5/M6 segments have recently been demonstrated to be of crucial importance for cation binding and transport (19Vilsen, B. (1995) Acta Physiol. Scand., 154, Suppl. 624, 1–146.Google Scholar, 24Kuntzweiler T, A. Argüello J.M. Lingrel J.B. J. Biol. Chem. 1996; 271: 29682-29687Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 25Koster J.C. Blanco G. Mills P.B. Mercer R.W. J. Biol. Chem. 1996; 271: 2413-2421Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 26Pedersen P.A. Rasmunssen J.H. Jørgensen P.L. J. Biol. Chem. 1996; 271: 2514-2522Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 27Swarts H.G.P. Klaassen C.H.W. de Boer M. Fransen J.A.M. De Pont J.J.H.H.M. J. Biol. Chem. 1996; 271: 29764-29772Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 28Andersen J.P. Vilsen B. FEBS Lett. 1992; 306: 101-106Google Scholar, 29Pedersen P.A. Rasmunssen J.H. Nielsen J.M. Jørgensen P.L. FEBS Lett. 1997; 400: 206-210Crossref PubMed Scopus (56) Google Scholar). Thanks to these and other efforts (e.g. see Refs. 30Asano S. Tega Y. Konishi K. Fujioka M. Takeguchi N. J. Biol. Chem. 1996; 271: 2740-2745Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar and 31Adebayo A.O. Enyedi A. Verma A.K. Filoteo A.G. Penniston J.T. J. Biol. Chem. 1995; 270: 27812-27816Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) a consensus picture of what could be the ion pathway in the membrane is gradually emerging. An unresolved question is whether the membranous amino acid residues considered so far are the only ones that are directly involved in translocation of cations. Recently, in H+,K+-ATPase Swarts et al. (27Swarts H.G.P. Klaassen C.H.W. de Boer M. Fransen J.A.M. De Pont J.J.H.H.M. J. Biol. Chem. 1996; 271: 29764-29772Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) have identified 3 new functionally important amino acid residues (Glu-834, Glu-837, and Glu-839) in the C-terminal part of the α subunit. In most P-type ATPase alignments, these residues have been proposed to reside in a cytosolic loop (L6–7), between the 6th and 7th transmembrane segment (Ref. 6Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (659) Google Scholar and Fig. 1 A). However in H+,K+-ATPase, De Pont and co-workers (27Swarts H.G.P. Klaassen C.H.W. de Boer M. Fransen J.A.M. De Pont J.J.H.H.M. J. Biol. Chem. 1996; 271: 29764-29772Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 32Klaassen C.H.W. De Pont J.J.H.H.M. Cell. Physiol. Biochem. 1994; 4: 115-134Crossref Scopus (21) Google Scholar) have proposed a slightly different location according to which the new critical residues would be part of M6 (see Fig.1 B). This raises the question whether, due to some special elaboration of structure or mechanism for cation transport, these residues are essential in H+,K+-ATPase only. In the present communication we report data which suggest that this is not the case, since by a combined protein-chemical and site-directed mutagenesis approach we have obtained evidence that the homologous residues in SR Ca2+-ATPase are also intimately involved in Ca2+ binding and ATPase activity. Furthermore, since in Ca2+-ATPase this region is accessible to proteolytic attack, its predicted organization as a cytoplasmic loop seems to be warranted. These results therefore emphasize the possible contribution of non-membranous regions to the control of cation binding to the ATPases.Figure 1Amino acid alignments of the M6-L6/7 region of P-type ATPases. A and B, the sequences were obtained for sarcoplasmic reticulum SERCA1 Ca2+-ATPase from Brandl et al. (33Brandl C.J. Green N.M. Korczak B. MacLennan D.H. Cell. 1986; 44: 597-607Abstract Full Text PDF PubMed Scopus (592) Google Scholar), for Na+, K+,-ATPase from Shull et al. (34Shull G.E. Schwartz A. Lingrel J.B. Nature. 1985; 316: 691-695Crossref PubMed Scopus (518) Google Scholar), and for gastric H+,K+-ATPase from Shull and Lingrel (35Shull G.E. Lingrel J.B. J. Biol. Chem. 1986; 261: 16788-16791Abstract Full Text PDF PubMed Google Scholar). The alignment is made according to Refs. 6Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (659) Google Scholar and 36Green N.M. Biochem. Soc. Trans. 1989; 17: 970-972Google Scholar. The position of M6 for the Ca2+-ATPase (6Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (659) Google Scholar) is based on Ref. 33Brandl C.J. Green N.M. Korczak B. MacLennan D.H. Cell. 1986; 44: 597-607Abstract Full Text PDF PubMed Scopus (592) Google Scholar; proteolytic cleavages by proteinase K occur after Leu-807 and after Met-817 (37, see arrowheads); a tryptic cleavage site has been demonstrated just after this region, at Lys-825 (38Shin J.M. Kajimura M. Argüello J.M. Kaplan J.H. Sachs G. J. Biol. Chem. 1994; 269: 22533-22537Abstract Full Text PDF PubMed Google Scholar). The position of M6 for Na+,K+-ATPase is also given according to Ref. 6Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (659) Google Scholar and does not differ much from that proposed in Refs. 5Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (417) Google Scholar and36Green N.M. Biochem. Soc. Trans. 1989; 17: 970-972Google Scholar. A proteolytic cleavage by trypsin occurs after Arg-830 (39, seearrowhead). The position of M6 for H+,K+,-ATPase is given according to Ref. 6Møller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (659) Google Scholar inA and according to Ref. 32Klaassen C.H.W. De Pont J.J.H.H.M. Cell. Physiol. Biochem. 1994; 4: 115-134Crossref Scopus (21) Google Scholar in B. A proteolytic cleavage by trypsin occurs after Arg-852 (40, 41, not shown). Positions of M5/M6 similar to the one shown in B have also been proposed for Na+,K+,-ATPase (42Lingrel J.B. Kuntzweiler T. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar). The position of some functionally important amino acids mutated by Swarts et al. (27Swarts H.G.P. Klaassen C.H.W. de Boer M. Fransen J.A.M. De Pont J.J.H.H.M. J. Biol. Chem. 1996; 271: 29764-29772Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) are underlined. C, amino acid alignment of part of the cytoplasmic loop 6–7 of SR Ca2+-ATPase and the Ca2+-binding region of α-lactalbumin. The sequence for human milk α-lactalbumin was obtained from Hall et al. (43Hall L. Craig R.K. Edbrooke M.R. Campbell P.N. Nucleic Acids Res. 1982; 10: 3503-3515Crossref PubMed Scopus (51) Google Scholar). Aspartate residues which could be in analogous positions in both proteins are indicated by adouble line. The position of residues of the α-lactalbumin involved in Ca2+ binding is based on its three-dimensional structure (44Acharya K.R. Stuart D.I. Walker N.P.C. Lewis M. Phillips D.C. J. Mol. Biol. 1989; 208: 99-127Crossref PubMed Scopus (292) Google Scholar); the residues interacting with Ca2+ through their carbonyl or carboxyl oxygens are underlined orboxed, respectively. Besides these five coordination sites, the last two ligands of the Ca2+ ion are water molecules (44Acharya K.R. Stuart D.I. Walker N.P.C. Lewis M. Phillips D.C. J. Mol. Biol. 1989; 208: 99-127Crossref PubMed Scopus (292) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Proteolytic digestion of SR (45Champeil P. Guillain F. Vénien C. Gingold M. Biochemistry. 1985; 24: 69-81Crossref PubMed Scopus (113) Google Scholar) or purified Ca2+-ATPase vesicles (46Meissner G. Conner G.E. Fleisher S. Biochim. Biophys. Acta. 1973; 298: 246-269Crossref PubMed Scopus (324) Google Scholar) with trypsin was performed as described in Ref. 47le Maire M. Lund S. Viel A. Champeil P. Møller J.V. J. Biol. Chem. 1990; 265: 1111-1123Abstract Full Text PDF PubMed Google Scholar. Proteinase K digestion, in 100 mmbis-Tris, pH 6.5, and 0.3 mm Ca2+, was performed essentially as described in Ref. 37Juul B. Turc H. Durand M.L. Gomez de Gracia A. Denoroy L. Møller J.V. Champeil P. le Maire M. J. Biol. Chem. 1995; 270: 20123-20134Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar except that, in some cases, proteolysis took place for 4 min at 37 °C instead of 30 min at 20 °C, a modification which generated a higher amount of peptide p20C relative to p19C. SDS-PAGE gels (48Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206999) Google Scholar) were prepared with inclusion of either 1 mm Ca2+ or 0.02–0.1 mm EGTA in the stacking and separation gels (49Garrigos M. Deschamps S. Viel A. Lund S. Champeil P. Møller J.V. le Maire M. Anal. Biochem. 1991; 194: 82-88Crossref PubMed Scopus (63) Google Scholar). EGTA gels were made with 12.5 or 13% acrylamide instead of 11.8% to allow for less efficient polymerization (as indicated from migration of standard proteins). The protein samples, prepared without the addition of Ca2+ or EGTA (49Garrigos M. Deschamps S. Viel A. Lund S. Champeil P. Møller J.V. le Maire M. Anal. Biochem. 1991; 194: 82-88Crossref PubMed Scopus (63) Google Scholar), were treated with concentrated urea to prevent aggregation (50Soulié S. Møller J.V. Falson P. le Maire M. Anal. Biochem. 1996; 236: 363-364Crossref PubMed Scopus (31) Google Scholar). Gels were stained with Coomassie Blue. Electroelution of peptides was performed as described (51le Maire M. Deschamps S. Møller J.V. Le Caer J.P. Rossier J. Anal. Biochem. 1993; 214: 50-57Crossref PubMed Scopus (96) Google Scholar). 45Ca2+ overlay (52Maruyama K. Mikawa T. Ebashi S. J. Biochem. ( Tokyo ). 1984; 95: 511-519Crossref PubMed Scopus (628) Google Scholar) was performed as described in Ref. 49Garrigos M. Deschamps S. Viel A. Lund S. Champeil P. Møller J.V. le Maire M. Anal. Biochem. 1991; 194: 82-88Crossref PubMed Scopus (63) Google Scholar. Western blotting was performed as described (37Juul B. Turc H. Durand M.L. Gomez de Gracia A. Denoroy L. Møller J.V. Champeil P. le Maire M. J. Biol. Chem. 1995; 270: 20123-20134Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) with visualization by the ECL kit (Amersham Corp.). Densitometric measurements were performed on a GS-700 imaging densitometer (Bio-Rad Laboratories). Site-directed mutagenesis was performed with the Exsite kit (Stratagene Inc.) on Ca2+-ATPase SERCA1a cDNA (53Centeno F. Deschamps S. Lompré A.M. Anger M. Moutin M.J. Dupont Y. Palmgren M. Møller J.V. Falson P. le Maire M. FEBS Lett. 1994; 354: 117-122Crossref PubMed Scopus (37) Google Scholar). One single mutation E309Q, one double mutation D813A/D818A (referred to later as ADA), and one triple mutation D813A/D815A/D818A (AAA) were introduced. The presence of these mutations and the absence of unexpected mutations due to polymerase chain reaction were verified by DNA sequencing. Wild type and mutant cDNAs were inserted into the yeast expression vector pYeDP60 (a gift of Dr. D. Pompon CNRS, Gif sur Yvette). Saccharomyces cerevisiae W303.1B (a,leu2, his3, trp1, ura3,ade2-1, can r,cyr +) was transformed (53Centeno F. Deschamps S. Lompré A.M. Anger M. Moutin M.J. Dupont Y. Palmgren M. Møller J.V. Falson P. le Maire M. FEBS Lett. 1994; 354: 117-122Crossref PubMed Scopus (37) Google Scholar, 54Groves J.D. Falson P. le Maire M. Tanner M.J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12245-12250Crossref PubMed Scopus (36) Google Scholar) and selected using ura3 complementation. Growth conditions and criteria for expression of the Ca2+-ATPase were as in Ref. 53Centeno F. Deschamps S. Lompré A.M. Anger M. Moutin M.J. Dupont Y. Palmgren M. Møller J.V. Falson P. le Maire M. FEBS Lett. 1994; 354: 117-122Crossref PubMed Scopus (37) Google Scholar for the test of individual clones and as in Ref. 55Pompon D. Louerat B. Bronine A. Urban P. Methods Enzymol. 1996; 272: 51-64Crossref PubMed Google Scholar for the large scale expression and crude extract preparation. The crude extract was first centrifuged at 900 × g av for 15 min at 4 °C and then centrifuged at 10,000 ×g av for 15 min, 4 °C. The supernatant was submitted to a second centrifugation at 120,000 ×g av for 45 min (4 °C) in 10 mmHepes (Tris), pH 7.4, 0.3 m sucrose, 0.1 mmCaCl2 to pellet a “light” membrane fraction which was homogenized and adjusted to a protein concentration of about 10 mg/ml. Protein concentrations (56Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner M.D. Provenzano M.D. Fugimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18581) Google Scholar) were measured in the presence of 0.5% SDS. The amount of Ca2+-ATPase was estimated by quantitative Western blotting as in Ref. 53Centeno F. Deschamps S. Lompré A.M. Anger M. Moutin M.J. Dupont Y. Palmgren M. Møller J.V. Falson P. le Maire M. FEBS Lett. 1994; 354: 117-122Crossref PubMed Scopus (37) Google Scholar using the polyclonal antibody 577–588 (37Juul B. Turc H. Durand M.L. Gomez de Gracia A. Denoroy L. Møller J.V. Champeil P. le Maire M. J. Biol. Chem. 1995; 270: 20123-20134Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Typically, the light membranes expressed wild type or mutant Ca2+-ATPase at about 1 mg of Ca2+-ATPase per 100 mg of membrane proteins. ATP hydrolysis was assayed at 30 °C using an ATP-regenerating coupled enzyme system as in Ref. 53Centeno F. Deschamps S. Lompré A.M. Anger M. Moutin M.J. Dupont Y. Palmgren M. Møller J.V. Falson P. le Maire M. FEBS Lett. 1994; 354: 117-122Crossref PubMed Scopus (37) Google Scholar, with some modifications. To a buffer containing 10 mm Tes (Tris), pH 7.5, 50 mm KNO3, 1 mg/ml C12E8, 7 mm MgCl2, 0.1 mm CaCl2, 0.1 mg/ml lactate dehydrogenase (Boehringer Mannheim, catalog no. 127221), 50 mm phosphoric acid, and 0.225 mm NADH, we added 50 μg of protein/ml of yeast light membranes. This mixture was preincubated for 10 min at 30 °C in the presence of 5 mm NaN3, 0.05 μg/ml bafilomycin A, 0.1 mm ammonium molybdate as inhibitors of other ATPases and 0.1 μm N-aminocaproic acid, 0.1 μmphenylmethylsulfonyl fluoride, and 2.8 mmβ-mercaptoethanol as antiproteases. After addition of 1 mm phosphoenolpyruvate and 0.1 mg/ml pyruvate kinase (Boehringer Mannheim, catalog no. 109045), the reaction was started by the addition of 1 mm Na2ATP. Thapsigargin (1 μg/ml) was added after 100 s, and the rate of hydrolysis was followed for an additional 100 s. The Ca2+-ATPase activity was calculated as the difference between the slopes obtained in the presence and in the absence of thapsigargin and was corrected for a small background activity obtained with control yeast light membranes. It has previously been documented in a number of investigations that changes of electrophoretic mobility in Ca2+-containing SDS gels can be used for the detection of Ca2+-binding proteins or peptides (57Klee C.B. Crouch T.H. Krinks M.H. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6270-6273Crossref PubMed Scopus (610) Google Scholar, 58Burgess W.H. Jemiolo D.K. Kretsinger R.H. Biochim. Biophys. Acta. 1980; 623: 257-270Crossref PubMed Scopus (196) Google Scholar, 59Cox J.A. Stein E.A. Biochemistry. 1981; 20: 5430-5436Crossref PubMed Scopus (45) Google Scholar, 60Campbell K.P. MacLennan D.H. Jorgensen A.O. J. Biol. Chem. 1983; 258: 11267-11273Abstract Full Text PDF PubMed Google Scholar, 61Campbell K.P. Entman M.L. Van Winckle W.B. Sarcoplasmic Reticulum in Muscle Physiology. 1. CRC Press, Boca Raton, FL1986: 65-99Google Scholar). Examples of such changes in migration rate are shown in Fig. 2, A and B, where it can be seen that calmodulin in a Ca2+-containing gel (lane 4, Fig. 2 B) migrates at a much faster rate than in a gel containing 0.1 mm EGTA (lane 4, Fig. 2 A). Intact Ca2+-ATPase from sarcoplasmic reticulum (lanes 2) moves slightly faster in the presence of Ca2+ than in its absence (compare with the 95-kDa molecular mass marker). A more pronounced increase in migration rate is noted for fragment B (lanes 3), the C-terminal polypeptide (sequence 506–994) produced by tryptic cleavage of Ca2+-ATPase, which co-migrates with the N-terminal fragment A in the presence of EGTA but not in the presence of Ca2+. Fragment A is further cleaved to A1 (peptide 199–505) and A2 (peptide 1–198). Some increase in migration rate in the presence of Ca2+ is observed for A2 (compare with the 20-kDa molecular mass marker), whereas A1 is not affected by Ca2+ (compare with the 30-kDa molecular mass marker). The effect of Ca2+on the migration rate of Ca2+-binding proteins and some of their peptide fragments is due probably to retention of a more compact and native-like structure in the presence of SDS, resulting from the binding of Ca2+ (49Garrigos M. Deschamps S. Viel A. Lund S. Champeil P. Møller J.V. le Maire M. Anal. Biochem. 1991; 194: 82-88Crossref PubMed Scopus (63) Google Scholar, 61Campbell K.P. Entman M.L. Van Winckle W.B. Sarcoplasmic Reticulum in Muscle Physiology. 1. CRC Press, Boca Raton, FL1986: 65-99Google Scholar, 62Cozens B. Reithmeier R.A.F. J. Biol. Chem. 1984; 259: 6248-6252Abstract Full Text PDF PubMed Google Scholar). In Fig. 2, C and D, we show that still shorter C-terminal Ca2+-ATPase fragments produced by proteolysis with proteinase K (p28C, p27C, and p20C) also change their mobility in Ca2+-containing gels, whereas other fragments (p29/30, p19C) are unaffected by the presence of Ca2+. As illustrated in Fig. 3, p20C starts at Gly-808, a residue which was predicted to be close to the C-terminal border of the M5/M6 transmembrane region, and extends to the C-terminal Gly-994 while p19C starts at Asp-818 (therefore in the L6–7 loop) and also ends at Gly-994. Thus a marked difference in peptide behavior in SDS gels is observed after removal of only 10 amino acids from p20C (Fig.3 B). The Ca2+-dependent change in migration rate of p20C is observed not only in the mixture of peptides obtained after limited proteolysis (lanes 3, Fig. 2,C and D), but also after isolation of p20C by electroelution from the gel and renewed electrophoresis (lanes 4), indicating that Ca2+ binding is an intrinsic property of the peptide. After electroelution p19C is still unaffected by the presence of Ca2+ in the gel (lanes 5). 45Ca2+ overlay of proteins (52Maruyama K. Mikawa T. Ebashi S. J. Biochem. ( Tokyo ). 1984; 95: 511-519Crossref PubMed Scopus (628) Google Scholar, 64Tanaka Y. Maruyama K. Mikawa T. Hotta Y. J. Biochem. ( Tokyo ). 1988; 104: 489-491Crossref PubMed Scopus (10) Google Scholar, 65Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar, 66Sienaert I. De Smedt H. Parys J.B. Messiaen L. Vanlingen S. Sipma H. Casteels R. J. Biol. Chem. 1996; 271: 27005-27012Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) after transfer to Immobilon membranes is a complementary technique with which to detect Ca2+ binding to proteins or peptides with various affinities for Ca2+ (49Garrigos M. Deschamps S. Viel A. Lund S. Champeil P. Møller J.V. le Maire M. Anal. Biochem. 1991; 194: 82-88Crossref PubMed Scopus (63) Google Scholar). For instance this strategy has been successfully used to localize a Ca2+-binding region which does not contain an EF-hand motif in the cardiac Na+-Ca2+ exchanger (65Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar). In Fig.4 A we show the results of a45Ca2+ overlay experiment performed on purified p20C and p19C. In these experiments, the 20-kDa molecular mass marker, trypsin inhibitor (lane 3), also binds45Ca2+ (49Garrigos M. Deschamps S. Viel A. Lund S. Champeil P. Møller J.V. le Maire M. Anal. Biochem. 1991; 194: 82-88Crossref PubMed Scopus (63) Google Scholar, 52Maruyama K. Mikawa T. Ebashi S. J. Biochem. ( Tokyo ). 1984; 95: 511-519Crossref PubMed Scopus (628) Google Scholar), providing a convenient reference in this region of the gel. It is clear that only p20C binds45Ca2+ (Fig. 4 A, lane 1), while p19C is not labeled (lane 2), although present in comparable amounts, as seen by Coomassie Blue staining of the blot (Fig. 4 B). Note that these experiments are performed after extensive washing of the membrane with a non-ionic and non-denaturing detergent, C12E8, which should help to remove SDS. In addition, 45Ca2+ overlay is performed at low Ca2+ concentration (10–15 μm) and in the presence of 5 mm Mg2+, which suggests that the partially refolded binding site in p20C has retained some specificity for Ca2+ over Mg2+. The combined demonstration of a Ca2+-dependent change in migration rate and of 45Ca2+ overlay labeling is a good indication of Ca2+ binding by p20C and not by p19C. However, this does not necessarily imply that Ca2+ is bound at functionally important sites. By inspection of the 808–818 sequence we have noticed some resemblance with the Ca2+ binding site of α-lactalbumin, a binding site which in this protein is confined to a short segment (amino acid residues 82–88; cf. Fig. 1 C). In this sequence the Ca2+ binding site is formed by three carboxylic groups of aspartic acid residues and two carbonyl groups, located in a loop, and two adjoining helical regions. Preliminary attempts with molecular modeling of the Ca2+-ATPase 808–818 sequence by energy minimization according to the α-lactalbumin Ca2+ binding site suggested that the side chains of Asp-813 and Asp-818, together with the peptide carbonyl group between Asp-815 and Ile-816 could be involved in Ca2+ binding to the L6–7 loop. 2T. Menguy and J. Smith, unpublished observations. We then took advantage of the functional expression of Ca2+-ATPase in yeast (53Centeno F. Deschamps S. Lompré A.M. Anger M. Moutin M.J. Dupont Y. Palmgren M. Møller J.V. Falson P. le Maire M. FEBS Lett. 1994; 354: 117-122Crossref PubMed Scopus (37) Google Scholar) to test the activity of two cluster mutants in this region, i.e.D813A/D818A (ADA) and D813A/D815A/D818A (AAA). Note that residues Asp-813 and Asp-815 correspond to residues found to be important in the case of the H+,K+-ATPase (Fig. 1 A). After purification of a light membrane fraction from the ATPase-expressing yeasts, we verified by Western blotting and densitometric measurements that mutant and wild type ATPases had been expressed to the same extent (data not shown). To quantify ATP hydrolysis, we used a coupled system and followed the disappearance of NADH in the absence or presence of thapsigargin, a specific inhibitor of SERCA ATPase. In Fig. 5 we present histograms of specific Ca2+-ATPase activity based on several experiments. It is seen that activity in the double mutant activity is reduced by more than 90 %, and activity in the triple mutant drops to a negligible level, comparable to that seen after mutation to glutamine of the essential glutamate in M4 (E309Q; Ref. 16Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (470) Google Scholar). These activity measurements do not point to any particular mechanism for inactivation, but phosphorylation experiments suggest that the Ca2+binding step is indeed affected by the mutations. The insetof Fig. 5 shows that phosphorylation by [32P]Pi, while inhibited by Ca2+at 126 μm and 1000 μm for wild type ATPase and SR + control yeast membranes, is still possible for the ADA mutant despite these high Ca2+ concentrations. Based on the rationale of Clarke et al. (16Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (470) Google Scholar), for the six mutations cited in the introduction, this shows that the ADA mutant is Ca2+-insensitive. Our study demonstrates the involvement of the region corresponding to the L6–7 loop in the reaction mechanism of Ca2+-ATPase. It should be noted that this region is fairly well conserved, in particular among type IIA ATPases. Therefore the previous findings with the corresponding residues in H+,K+-ATPase (27Swarts H.G.P. Klaassen C.H.W. de Boer M. Fransen J.A.M. De Pont J.J.H.H.M. J. Biol. Chem. 1996; 271: 29764-29772Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) probably are of wider significance and indicate common features in the transport mechanism. However, in contrast with the interpretation proposed for H+,K+-ATPase in the latter work, there is convincing evidence for cytosolic exposure of this region in Ca2+-ATPase, since it is an easy target for proteolytic cleavage (37Juul B. Turc H. Durand M.L. Gomez de Gracia A. Denoroy L. Møller J.V. Champeil P. le Maire M. J. Biol. Chem. 1995; 270: 20123-20134Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 38Shin J.M. Kajimura M. Argüello J.M. Kaplan J.H. Sachs G. J. Biol. Chem. 1994; 269: 22533-22537Abstract Full Text PDF PubMed Google Scholar) and it reacts with sequence-specific antibody 809–827 (67Møller J.V. Juul B. Lee Y.-J. le Maire M. Champeil P. Bamberg E. Schoner W. The Sodium Pump. Springer Verlag, New York1994: 131-134Crossref Google Scholar). Therefore, we do not consider it likely, as suggested by Swarts et al. (27Swarts H.G.P. Klaassen C.H.W. de Boer M. Fransen J.A.M. De Pont J.J.H.H.M. J. Biol. Chem. 1996; 271: 29764-29772Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), that these residues are embedded inside the membrane structure, but we favor a probable location of the Ca2+ liganding groups at the cytosol/membrane border. We suggest that residues of the L6–7 loop could be involved in the initial processes of Ca2+ transport. Specifically, they could act as a site for ion approach (68Or E. David P. Shainskaya A. Tal D.M. Karlish S.J.D. J. Biol. Chem. 1993; 268: 16929-16937Abstract Full Text PDF PubMed Google Scholar, 69Shainskaya A. Karlish S.J.D. J. Biol. Chem. 1994; 269: 10780-10789Abstract Full Text PDF PubMed Google Scholar) toward the high affinity binding pocket inside the membrane and/or be part of the gate operating during calcium occlusion (70Forbush B. J. Biol. Chem. 1987; 262: 11116-11127Abstract Full Text PDF PubMed Google Scholar, 71Orlowski S. Champeil P. Biochemistry. 1991; 30: 352-361Crossref PubMed Scopus (77) Google Scholar), preventing release of bound Ca2+ toward the cytosolic side. Exploration of these possibilities requires further studies. We are grateful to Dr. J. P. Andersen, Danish Biomembrane Research Center, University of Aarhus, for communicating the results of selected mutations in loop 6–7 before publication, to Drs. D. Pompon and Ph. Urban, CNRS, Gif sur Yvette for the gift of the yeast strain and the vector, and to them and Dr. J. D. Groves, Dept. of Biochemistry, University of Bristol, for numerous helpful suggestions. We also thank the members of our Section, CEA, Gif sur Yvette for stimulating discussions, as well as Dr. J. Smith for his help with molecular modeling." @default.
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• W2072066095 title "The Cytoplasmic Loop between Putative Transmembrane Segments 6 and 7 in Sarcoplasmic Reticulum Ca2+-ATPase Binds Ca2+ and Is Functionally Important" @default.
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