TcSCA Complements Yeast Mutants Defective in Ca2+ Pumps and Encodes a Ca2+-ATPase That Localizes to the Endoplasmic Reticulum of Trypanosoma cruzi

Abstract

Intracellular Ca2+in Trypanosoma cruzi is mainly located in an acidic compartment named the acidocalcisome, which among other pumps and exchangers possesses a plasma membrane-type Ca2+-ATPase. Evidence for an endoplasmic reticulum-located Ca2+ uptake has been more elusive and based on indirect results. Here we report the cloning and sequencing of a gene encoding a sarcoplasmic-endoplasmic reticulum-type Ca2+-ATPase from T. cruzi. The protein (TcSCA) predicted from the nucleotide sequence of the gene has 1006 amino acids and a molecular mass of 109.7 kDa. Several sequence motifs found in sarcoplasmic-endoplasmic reticulum-type Ca2+-ATPases were present in TcSCA. Expression of TcSCA in yeast mutants deficient in the Golgi and vacuolar Ca2+pumps (pmr1 pmc1 cnb 1) restored growth on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with TcSCA, and it was found that the TcSCA polypeptide formed a Ca2+-dependent and hydroxylamine-sensitive32P-labeled phosphoprotein of 110 kDa in the presence of [γ-32P]ATP. Cyclopiazonic acid, but not thapsigargin, blocked this phosphoprotein formation. Transgenic parasites expressing constructs of TcSCA with green fluorescent protein exhibited co-localization of TcSCA with the endoplasmic reticulum proteins BiP and calreticulin. An endoplasmic reticulum location was also found in amastigotes and trypomastigotes using a polyclonal antibody against a COOH-terminal region of the protein. The ability of TcSCA to restore growth of mutant pmr1 pmc1 cnb 1 on medium containing Mn2+ suggests that TcSCA may also regulate Mn2+homeostasis by pumping Mn2+ into the endoplasmic reticulum of T. cruzi. Intracellular Ca2+in Trypanosoma cruzi is mainly located in an acidic compartment named the acidocalcisome, which among other pumps and exchangers possesses a plasma membrane-type Ca2+-ATPase. Evidence for an endoplasmic reticulum-located Ca2+ uptake has been more elusive and based on indirect results. Here we report the cloning and sequencing of a gene encoding a sarcoplasmic-endoplasmic reticulum-type Ca2+-ATPase from T. cruzi. The protein (TcSCA) predicted from the nucleotide sequence of the gene has 1006 amino acids and a molecular mass of 109.7 kDa. Several sequence motifs found in sarcoplasmic-endoplasmic reticulum-type Ca2+-ATPases were present in TcSCA. Expression of TcSCA in yeast mutants deficient in the Golgi and vacuolar Ca2+pumps (pmr1 pmc1 cnb 1) restored growth on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with TcSCA, and it was found that the TcSCA polypeptide formed a Ca2+-dependent and hydroxylamine-sensitive32P-labeled phosphoprotein of 110 kDa in the presence of [γ-32P]ATP. Cyclopiazonic acid, but not thapsigargin, blocked this phosphoprotein formation. Transgenic parasites expressing constructs of TcSCA with green fluorescent protein exhibited co-localization of TcSCA with the endoplasmic reticulum proteins BiP and calreticulin. An endoplasmic reticulum location was also found in amastigotes and trypomastigotes using a polyclonal antibody against a COOH-terminal region of the protein. The ability of TcSCA to restore growth of mutant pmr1 pmc1 cnb 1 on medium containing Mn2+ suggests that TcSCA may also regulate Mn2+homeostasis by pumping Mn2+ into the endoplasmic reticulum of T. cruzi. sarcoplasmic-endoplasmic reticulum-type Ca2+-ATPase T. cruzi SERCA-type Ca2+-ATPase polymerase chain reaction base pair(s) open reading frame green fluorescent protein 4-morpholinepropanesulfonic acid synthetic complete medium minus uracil endoplasmic reticulum Trypanosoma cruzi, the etiologic agent of Chagas' disease, is a parasitic protozoan that invades mammalian cells and develops intracellularly as amastigotes. Invasion of cells by T. cruzi is dependent upon an elevation in the concentration of cytosolic free calcium in the invading trypomastigote (1Moreno S.N.J. Silva J. Vercesi A.E. Docampo R. J. Exp. Med. 1994; 180: 1535-1540Crossref PubMed Scopus (181) Google Scholar, 2Docampo R. Moreno S.N.J. Parasitol. Today. 1996; 12: 61-65Abstract Full Text PDF PubMed Scopus (57) Google Scholar). Unlike mammalian cells, T. cruzi possesses most of its intracellular Ca2+ in an acidic compartment named the acidocalcisome (3Docampo R. Scott D.A. Vercesi A.E. Moreno S.N.J. Biochem. J. 1995; 310: 1005-1012Crossref PubMed Scopus (180) Google Scholar, 4Scott D.A. Docampo R. Dvorak J.A. Shi S. Leapman R.D. J. Biol. Chem. 1997; 272: 28020-28029Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 5Scott D.A. de Souza W. Benchimol M. Zhong L. Lu H.-G. Moreno S.N.J. Docampo R. J. Biol. Chem. 1998; 273: 22151-22158Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 6Lu H.-G. Zhong L. de Souza W. Benchimol M. Moreno S.N.J. Docampo R. Mol. Cell. Biol. 1998; 18: 2309-2323Crossref PubMed Google Scholar, 7Scott D.A. Docampo R. J. Biol. Chem. 2000; 275: 24215-24221Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 8Docampo R. Moreno S.N.J. Parasitol. Today. 1999; 15: 443-448Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The molecular and biochemical characterization of this organelle has provided evidence that it has an orthovanadate-sensitive plasma membrane-type Ca2+ ATPase for Ca2+ uptake (3Docampo R. Scott D.A. Vercesi A.E. Moreno S.N.J. Biochem. J. 1995; 310: 1005-1012Crossref PubMed Scopus (180) Google Scholar, 5Scott D.A. de Souza W. Benchimol M. Zhong L. Lu H.-G. Moreno S.N.J. Docampo R. J. Biol. Chem. 1998; 273: 22151-22158Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 6Lu H.-G. Zhong L. de Souza W. Benchimol M. Moreno S.N.J. Docampo R. Mol. Cell. Biol. 1998; 18: 2309-2323Crossref PubMed Google Scholar, 7Scott D.A. Docampo R. J. Biol. Chem. 2000; 275: 24215-24221Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Evidence for non-mitochondrial and endoplasmic reticulum-located Ca2+ uptake has been more elusive and based on the presence of a low capacity and high affinity orthovanadate-sensitive Ca2+ uptake in permeabilized cells and the ability of these cells to buffer [Ca2+] in the range 0.05–0.1 µm (9Vercesi A.E. Hoffmann M.E. Bernardes C.F. Docampo R. Cell Calcium. 1991; 12: 361-369Crossref PubMed Scopus (44) Google Scholar), features in common with the sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA)1 of animal cells (10Carafoli E. Brini M. Curr. Opin. Chem. Biol. 2000; 4: 152-161Crossref PubMed Scopus (132) Google Scholar). In addition, like mammalian cells (11Parodi A. Annu. Rev. Biochem. 2000; 69: 69-93Crossref PubMed Scopus (532) Google Scholar), calcium is needed for the correct folding and assembly of proteins in the endoplasmic reticulum of T. cruzi, which depends on chaperones such as the Ca2+-binding protein, calreticulin (12Labriola C. Cazzulo J.J. Parodi A. Mol. Biol. Cell. 1999; 10: 1381-1394Crossref PubMed Scopus (78) Google Scholar). Biochemical distinction of the different Ca2+ pumps present in T. cruzi has been hampered by the lack of distinguishing features such as specific inhibitor sensitivity. Orthovanadate inhibits all types of Ca2+ ATPases (10Carafoli E. Brini M. Curr. Opin. Chem. Biol. 2000; 4: 152-161Crossref PubMed Scopus (132) Google Scholar), whereas thapsigargin, a specific inhibitor of animal SERCA-type Ca2+-ATPases (13Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2988) Google Scholar), is ineffective in inhibiting Ca2+ uptake in permeabilizedT. cruzi (14Moreno S.N.J. Vercesi A.E Pignataro O.P. Docampo R. Mol. Biochem. Parasitol. 1992; 52: 251-262Crossref PubMed Scopus (59) Google Scholar, 15Docampo R. Moreno S.N.J. Vercesi A.E. Mol. Biochem. Parasitol. 1993; 59: 305-314Crossref PubMed Scopus (44) Google Scholar). Thus, a molecular approach to studying individual pumps is necessary. Expression of genes identified as encoding Ca2+-ATPases and localization of the corresponding proteins is necessary, as the plasma membrane-type Ca2+ATPase/SERCA paradigm does not necessarily apply in non-animal cells; a SERCA-type gene in tomato is expressed in different parts of the cell (16Ferrol N. Bennett A.B. Plant Cell. 1996; 8: 1159-1169Crossref PubMed Google Scholar). Also, a Tca1 gene has been identified in T. cruzi (6Lu H.-G. Zhong L. de Souza W. Benchimol M. Moreno S.N.J. Docampo R. Mol. Cell. Biol. 1998; 18: 2309-2323Crossref PubMed Google Scholar), which encodes a protein with homology to mammalian plasma membrane Ca2+-ATPases but with characteristics that place it in a novel category of Ca2+-ATPases along with the vacuolar Ca2+-ATPases described in Saccharomyces cerevisiae (17Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (362) Google Scholar), Dictyostelium discoideum (18Moniakis J. Coukell M.B. Forer A. J. Biol. Chem. 1995; 270: 28276-28281Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar),Entamoeba histolytica (19Ghosh S.K. Rosenthal B. Rogers R. Samuelson J. Mol. Biochem. Parasitol. 2000; 108: 125-130Crossref PubMed Scopus (12) Google Scholar), and Toxoplasma gondii(20Luo S. Vieira M. Graves J. Zhong L. Moreno S.N.J. EMBO J. 2001; 20: 55-64Crossref PubMed Scopus (79) Google Scholar). The gene is expressed at a high level in the amastigote stage and is localized to acidocalcisomes and the plasma membrane of the parasite (6Lu H.-G. Zhong L. de Souza W. Benchimol M. Moreno S.N.J. Docampo R. Mol. Cell. Biol. 1998; 18: 2309-2323Crossref PubMed Google Scholar). Here we demonstrate that a gene from T. cruzi(TcSCA) complemented yeast mutants defective in Ca2+ pumps by restoring their growth in EGTA. The protein by the TcSCA gene to the endoplasmic reticulum of different of the parasite in to the Ca2+-ATPase (6Lu H.-G. Zhong L. de Souza W. Benchimol M. Moreno S.N.J. Docampo R. Mol. Cell. Biol. 1998; 18: 2309-2323Crossref PubMed Google Scholar), it is expressed at in the different of the T. cruzi amastigotes and trypomastigotes were from the medium of by a of the of and PubMed Scopus Google Scholar), as we described (14Moreno S.N.J. Vercesi A.E Pignataro O.P. Docampo R. Mol. Biochem. Parasitol. 1992; 52: 251-262Crossref PubMed Scopus (59) Google Scholar, 15Docampo R. Moreno S.N.J. Vercesi A.E. Mol. Biochem. Parasitol. 1993; 59: 305-314Crossref PubMed Scopus (44) Google Scholar). The of trypomastigotes with amastigotes and of amastigotes with trypomastigotes was T. were at in medium M. PubMed Scopus (132) Google with cerevisiae a K.W. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus (133) Google was provided by W. of The and was in yeast and and inhibitor were from were from polymerase were from The in and were from the and were from and were from The was from and the protein were from The were from was a from T. cruzi was a from against T. BiP and T. were provided by and The antibody against the Ca2+-ATPase was described (6Lu H.-G. Zhong L. de Souza W. Benchimol M. Moreno S.N.J. Docampo R. Mol. Cell. Biol. 1998; 18: 2309-2323Crossref PubMed Google Scholar). The antibody was from described J. T. the the polymerase chain reaction was with of for 1 for 1 and for 1 using of polymerase with of T. cruzi 1 µm and in a The were in an and into The of the for the that to in the region acids and which to in the region acids a T. cruzi the was by and of were from the and into The was by with the as a in a described J. T. Scholar). sequencing S. Proc. Natl. Acad. U. S. A. PubMed Scopus Google was with the sequencing and a at the of at and amino acid were with the was by using the and J. J. Mol. Biol. PubMed Scopus Google Scholar). The was by using and as a and by using corresponding to the T. cruzi sequence and a TcSCA sequence were the as described that the was the for TcSCA in the a that of the region and of the of the TcSCA open reading frame was into S. cerevisiae at the of was with by the and to the yeast sequence M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: PubMed Scopus Google and a to the of the The of the of the TcSCA were to the at the by by the and The was named and to yeast the for green protein protein a that the TcSCA was into of was to a in the to into the region of TcSCA. this a of and that to a of the region acids containing a and to other were to the with the by using The was by to the and to The was by with a and and the Mol. Biochem. Parasitol. 1996; PubMed Scopus Google was by and into the of the TcSCA in The containing the TcSCA with the was into the of T. 1992; 20: PubMed Scopus Google Scholar). The was named and was to T. from was with and on with acid, 1 and to The was with was isolated by J. T. Scholar). the was isolated from and amastigotes of T. cruzi with to the was in with and 1 and to were using and a of polymerase and were from with of the sequence using polymerase in and The cruzi protein M. J. Exp. Med. 1992; PubMed Scopus Google as a in the was by T. cruzi by with corresponding to and of the sequence of the M. J. Exp. Med. 1992; PubMed Scopus Google Scholar). of were with an and using the of and a level of of this gene in all T. C. Docampo R. Moreno S.N.J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). were the were by into the of to in different encoding the acid, COOH-terminal of TcSCA protein was by polymerase chain reaction using T. cruzi as a 1) containing an and a encoding an were The was into the and of the in a that the protein to a that its on was by sequencing to that the correct been The was into the of E. and cells were in was by at a concentration of 1 the cell an of The cells were a at with at in and and at at for for into and The which was to TcSCA for antibody the for in the The protein was by and to of protein with complete were at using of protein in was the the and 1 the specific antibody been the a and was by 1 The was and at of of different of T. cruzi containing of protein were with an of buffer and and for of were using and using a by were in containing were blocked at with and with antibody for 1 at were for 1 with antibody and for the of the of to 1 were with parasites were with in for 1 at and to to for with for and with in for 1 the parasites were with a of the antibody against the expressed protein by of a at were with the were with an (6Lu H.-G. Zhong L. de Souza W. Benchimol M. Moreno S.N.J. Docampo R. Mol. Cell. Biol. 1998; 18: 2309-2323Crossref PubMed Google Scholar, Zhong L. Docampo R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). with TcSCA, the were that the against T. cruzi Tca1 Ca2+-ATPase was at a and an antibody was at was transformed with the by the Curr. 1992; PubMed Scopus Google Scholar). were by on synthetic complete medium minus uracil K.W. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus (133) Google Scholar). from a were in medium with at The cell was to the medium to an of was to the medium at to at was by the of Mn2+ yeast in was into medium at an of and the at for of T. cruzi were transformed with by at the of growth were with medium µm and in the medium at a concentration of 1 The cell was with the with a gene at with a and a and in of medium with an at was at for of the were isolated by a of a K.W. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus (133) Google Scholar). were to of buffer 1 inhibitor and of in the of a and for which in cell The were by with and the were at for The from this were on of a of in and in a at for The was in 1 and at for The was in was with the protein phosphoprotein was to and N. Acad. Sci. PubMed Scopus Google with The reaction 1 and of protein. was to of the was to a concentration of in a free Ca2+ concentration of The reaction was by to a concentration of µm and at by of and acid by in was to the and for at with acid the was in of buffer and to acidic B. A. J. Biol. Chem. Full Text PDF PubMed Google and the SERCA-type Ca2+-ATPase gene of T. cruzi, a region containing a Ca2+-ATPase sequence was from T. cruzi amino acid of SERCA-type Ca2+-ATPases of different were from and with the were and were to these and the was with T. cruzi as a of the with a from the SERCA-type Ca2+-ATPase Zhong L. Docampo R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google by and it was into The amino acid sequence of this was and to the of the SERCA-type Ca2+-ATPases of L. Zhong L. Docampo R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google and Trypanosoma E. Mol. Biochem. Parasitol. 1991; PubMed Scopus Google Scholar, E. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), T. cruzi was and using this as a was found to the sequence of the region of the SERCA-type Ca2+-ATPase gene of T. cruzi. the sequence of the region of the was and the gene was by using corresponding to the sequence and a specific sequence present in the a that the region of the gene a of with the sequence of the with the the sequence the of the encoding 1006 amino acid which was named TcSCA for T. cruzi SERCA-type Ca2+-ATPase The TcSCA amino acid sequence is to the T. SERCA-type Ca2+-ATPase sequence E. Mol. Biochem. Parasitol. 1991; PubMed Scopus Google Scholar, E. J. Biol. Chem. 1994; Full Text PDF PubMed Google and to the L. SERCA-type Ca2+-ATPase sequence Zhong L. Docampo R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google has to SERCA-type Ca2+-ATPase of and to plasma membrane-type Ca2+-ATPases from different of the TcSCA amino acid sequence that this gene all the and found in other ATPases such as the and PubMed Scopus Google U. S. PubMed Scopus Google Scholar). of the amino acid sequence a to of other calcium pumps containing is in with from of a mammalian SERCA-type Ca2+-ATPase at of C. 1998; PubMed Scopus Google Scholar). TcSCA also all the and by the in 1) that were identified as the high affinity Ca2+-binding in the of the and PubMed Scopus Google Scholar). with other SERCA-type TcSCA the amino acid sequence with found the of mammalian plasma membrane-type Ca2+ ATPase T. M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The amino acid sequence which was found to for the of the Ca2+-ATPase of reticulum with T. M. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), is in TcSCA. the located in for of Ca2+-ATPases A. B. J. Biol. Chem. 1994; Full Text PDF PubMed Google different in TcSCA of in different as with In with these we were to in in cells in the presence of low of (14Moreno S.N.J. Vercesi A.E Pignataro O.P. Docampo R. Mol. Biochem. Parasitol. 1992; 52: 251-262Crossref PubMed Scopus (59) Google Scholar, 15Docampo R. Moreno S.N.J. Vercesi A.E. Mol. Biochem. Parasitol. 1993; 59: 305-314Crossref PubMed Scopus (44) Google Scholar). were also in with the expressed in yeast TcSCA has protein which common to all SERCA-type Ca2+-ATPases of parasites but not present in of other by TcSCA a in for not that TcSCA is a The by to the presence of an in the the presence of a of in of the of T. cruzi of the by that the TcSCA gene is expressed at in all of T. cruzi. is in with the gene for the L. SERCA-type Ca2+ that is and more expressed in intracellular amastigotes Zhong L. Docampo R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). the localization of TcSCA, the sequence of the of was to the TcSCA and into the T. cruzi Mol. Biochem. Parasitol. 1996; PubMed Scopus Google Scholar, 1992; 20: PubMed Scopus Google Scholar). The region in TcSCA was for the of of to with a T. cruzi were with this and the were by Transgenic parasites expressing the exhibited as a the and in a from its an endoplasmic reticulum these cells were also with against BiP and chaperones in the of protein folding A. Biol. 1995; PubMed Scopus Google Scholar). BiP and calreticulin with TcSCA and the localization of TcSCA. In of cells with against the Ca2+-ATPase of T. cruzi (6Lu H.-G. Zhong L. de Souza W. Benchimol M. Moreno S.N.J. Docampo R. Mol. Cell. Biol. 1998; 18: 2309-2323Crossref PubMed Google in a different from the and localization of TcSCA also the localization of TcSCA in other of T. cruzi. from different cruzi were to with against a acid, COOH-terminal of TcSCA. a of kDa to the predicted molecular mass of TcSCA, in trypomastigote and amastigote was using not In indirect using the TcSCA was in trypomastigotes and amastigotes as a the and in a from its to the protein was the was the of TcSCA, of the TcSCA were into a yeast the of a and the the was to yeast mutant The yeast mutant is defective in the Golgi 1) and the vacuolar 1) Ca2+ pumps and also 1) mutant an for the of individual Ca2+ pumps from other K.W. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus (133) Google Scholar, Plant 1998; PubMed Scopus Google Scholar). The mutant transformed with on a medium containing low Ca2+ was at low of the of growth and the mutant transformed with TcSCA of the encodes a The of Ca2+ was by on of Mn2+ was by of the Mn2+ of the mutant The mutant with TcSCA in the gene is not not In was growth of the in the presence of whereas growth of yeast to S. was transformed as in and in containing the of growth in was at the TcSCA was to a like other Ca2+-ATPases L. PubMed Scopus Google Scholar, Annu. Rev. PubMed Scopus Google Scholar), we isolated from and with phosphoprotein of 110 kDa was formed in isolated from but was in yeast transformed with was dependent on the presence of the level of the phosphoprotein The phosphoprotein was to the of an J.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google formed by these evidence that TcSCA is a Ca2+-dependent Cyclopiazonic acid, an inhibitor of animal the of TcSCA whereas at low and The of TcSCA was also by and a of that to with high affinity and A. Fink G.R. Mol. Biol. Cell. 1992; PubMed Scopus Google Scholar). yeast as a K.W. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus (133) Google Scholar), we the evidence that a SERCA-type T. cruzi gene encodes a Ca2+-ATPase. Expression of the T. cruzi TcSCA gene restored the growth in a medium containing of Ca2+ of a yeast mutant defective in Ca2+ pumps Several of evidence that TcSCA encodes an Ca2+ 1) its amino acid sequence with pumps and with plasma membrane-type TcSCA motifs M. 2000; PubMed Scopus Google Scholar), at the and in the TcSCA is mainly localized to the membrane and to a in different of T. cruzi and TcSCA with calreticulin and endoplasmic reticulum chaperones with SERCA-type Ca2+ ATPases J.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), TcSCA to to (14Moreno S.N.J. Vercesi A.E Pignataro O.P. Docampo R. Mol. Biochem. Parasitol. 1992; 52: 251-262Crossref PubMed Scopus (59) Google Scholar, 15Docampo R. Moreno S.N.J. Vercesi A.E. Mol. Biochem. 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Chem. 1994; Full Text PDF PubMed Google Scholar). the of TcSCA with and the and and amino acid that different in TcSCA as with and not for the in to thapsigargin, for a that an in and is in the a a in and is that these in and for the in to of these In mammalian cells, in calcium in Ca2+ is from the by acid upon J. 1997; Scopus Google Scholar, EMBO J. 2000; PubMed Scopus Google Scholar). the to T. cruzi is The isolated has not been in but the most of calcium for the acidocalcisome R. Moreno S.N.J. Parasitol. Today. 1999; 15: 443-448Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), is not to from T. D.A. Docampo R. J. Biol. Chem. 2000; 275: 24215-24221Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). A. to in by L. Zhong L. Docampo R. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google and by T. cruzi the of (1Moreno S.N.J. Silva J. Vercesi A.E. Docampo R. J. Exp. 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Cell. 1998; PubMed Scopus Google Scholar). were found in with mammalian cells M. 1994; PubMed Scopus Google Scholar, N. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar), in of these it was that the were to Ca2+ to In cells, of a is upon of the cells with that with protein folding the Golgi C. M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Evidence from also a for the in the and of plasma membrane cell proteins M.E. EMBO J. 1999; 18: PubMed Scopus Google Scholar). in T. cruzi, the TcSCA may in the of Ca2+ Mn2+ for and of proteins the of of cell proteins in with for the yeast for for for antibody against Parodi for antibody against and for