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• W1974133434 abstract "The Arabidopsis thaliana AtHMA1 protein is a member of the PIB-ATPase family, which is implicated in heavy metal transport. However, sequence analysis reveals that AtHMA1 possesses a predicted stalk segment present in SERCA (sarcoplasmic/endoplasmic reticulum Ca2+ ATPase)-type pumps that is involved in inhibition by thapsigargin. To analyze the ion specificity of AtHMA1, we performed functional complementation assays using mutant yeast strains defective in Ca2+ homeostasis or heavy metal transport. The heterologous expression of AtHMA1 complemented the phenotype of both types of mutants and, interestingly, increased heavy metal tolerance of wild-type yeast. Biochemical analyses were performed to describe the activity of AtHMA1 in microsomal fractions isolated from complemented yeast. Zinc, copper, cadmium, and cobalt activate the ATPase activity of AtHMA1, which corroborates the results of metal tolerance assays. The outcome establishes the role of AtHMA1 in Cd2+ detoxification in yeast and suggests that this pump is able to transport other heavy metals ions. Further analyses were performed to typify the active Ca2+ transport mediated by AtHMA1. Ca2+ transport displayed high affinity with an apparent Km of 370 nm and a Vmax of 1.53 nmol mg–1 min–1. This activity was strongly inhibited by thapsigargin (IC50 = 16.74 nm), demonstrating the functionality of its SERCA-like stalk segment. In summary, these results demonstrate that AtHMA1 functions as a Ca2+/heavy metal pump. This protein is the first described plant P-type pump specifically inhibited by thapsigargin. The Arabidopsis thaliana AtHMA1 protein is a member of the PIB-ATPase family, which is implicated in heavy metal transport. However, sequence analysis reveals that AtHMA1 possesses a predicted stalk segment present in SERCA (sarcoplasmic/endoplasmic reticulum Ca2+ ATPase)-type pumps that is involved in inhibition by thapsigargin. To analyze the ion specificity of AtHMA1, we performed functional complementation assays using mutant yeast strains defective in Ca2+ homeostasis or heavy metal transport. The heterologous expression of AtHMA1 complemented the phenotype of both types of mutants and, interestingly, increased heavy metal tolerance of wild-type yeast. Biochemical analyses were performed to describe the activity of AtHMA1 in microsomal fractions isolated from complemented yeast. Zinc, copper, cadmium, and cobalt activate the ATPase activity of AtHMA1, which corroborates the results of metal tolerance assays. The outcome establishes the role of AtHMA1 in Cd2+ detoxification in yeast and suggests that this pump is able to transport other heavy metals ions. Further analyses were performed to typify the active Ca2+ transport mediated by AtHMA1. Ca2+ transport displayed high affinity with an apparent Km of 370 nm and a Vmax of 1.53 nmol mg–1 min–1. This activity was strongly inhibited by thapsigargin (IC50 = 16.74 nm), demonstrating the functionality of its SERCA-like stalk segment. In summary, these results demonstrate that AtHMA1 functions as a Ca2+/heavy metal pump. This protein is the first described plant P-type pump specifically inhibited by thapsigargin. Plant Ca2+ and heavy metal-ATPases belong to the superfamily of P-ATPases (1Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (735) Google Scholar, 2Axelsen K.B. Palmgren M.G. Plant Physiol. 2001; 126: 696-706Crossref PubMed Scopus (328) Google Scholar). Their common characteristic is the presence of a phosphorylated intermediary in the catalytic cycle. In Arabidopsis, the heavy metal-ATPases belong to the PIB subfamily and normally have eight predicted transmembrane domains, whereas the Ca2+-ATPases are part of the PIIA and PIIB subfamilies and have ten predicted transmembrane domains (2Axelsen K.B. Palmgren M.G. Plant Physiol. 2001; 126: 696-706Crossref PubMed Scopus (328) Google Scholar). A common feature among the PIB-ATPases is the presence of a CPX motif, which is though to play a role in metal translocation, as well as putative metal-binding domains located at the amino or carboxyl terminus (3Colangelo E.P. Guerinot M.L. Curr. Opin. Plant Biol. 2006; 9: 322-330Crossref PubMed Scopus (293) Google Scholar). On the other hand, PIIB ATPases have a calmodulin-binding domain that regulate their activity; however, the PIIA do not have this domain (4Geisler M. Axelsen K.B. Harper J.F. Palmgren M.G. Biochim. Biophys. Acta. 2000; 1465: 52-78Crossref PubMed Scopus (157) Google Scholar). The most important difference between the calcium and heavy metal-ATPases is their substrate specificity (1Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (735) Google Scholar). However, in vitro metal transport studies performed with membrane fractions isolated from seedlings suggest that one or more members of the superfamily of P-ATPases are capable of transporting calcium and heavy metals. The studies showed competitive inhibition between active transport of heavy metals (such as copper and cadmium) and calcium. 2V. R. Ordenes, L. Norambuena, and A. Orellana, unpublished results.2V. R. Ordenes, L. Norambuena, and A. Orellana, unpublished results. Interestingly, both transport activities were inhibited by the sesquiterpene lactone thapsigargin, a potent and specific inhibitor of SERCA 3The abbreviations used are:SERCAsarcoplasmic/endoplasmic reticulum Ca2+ ATPaseRTreverse transcriptionCPYcarboxypeptidase YMOPS4-morpholinepropanesulfonic acidTGthapsigarginWTwild type.3The abbreviations used are:SERCAsarcoplasmic/endoplasmic reticulum Ca2+ ATPaseRTreverse transcriptionCPYcarboxypeptidase YMOPS4-morpholinepropanesulfonic acidTGthapsigarginWTwild type.-type pumps (5Zhang Z. Sumbilla C. Lewis D. Inesi G. FEBS Lett. 1993; 335: 261-264Crossref PubMed Scopus (10) Google Scholar, 6Zhong L. Inesi G. J. Biol. Chem. 1998; 273: 12994-12998Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 7Xu Ch. Ma H. Inesi G. Al-Shawi M.K. Toyoshima C. J. Biol. Chem. 2004; 279: 17973-17979Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). To date no one has described plant ion pumps that transport calcium and heavy metals in biochemical terms, nor have scientists described genes encoding for ion pumps inhibited by thapsigargin or plant mutants with thapsigargin-sensitive/tolerant phenotypes. Based on the results of biochemical assays suggesting the existence of thapsigargin-sensitive Ca2+/heavy metal-ATPase, we searched for potential candidate proteins in the Arabidopsis genome. This was made possible by the highly conserved “stalk segment” or S3 sequence adjacent to the third transmembrane segment of the SERCA pumps (8Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1594) Google Scholar, 9Stokes D. Green N.M. Annu. Rev. Biophys. Biomol. Struct. 2003; 32: 445-468Crossref PubMed Scopus (78) Google Scholar) composed of amino acids DEFGEQLSK (5Zhang Z. Sumbilla C. Lewis D. Inesi G. FEBS Lett. 1993; 335: 261-264Crossref PubMed Scopus (10) Google Scholar, 6Zhong L. Inesi G. J. Biol. Chem. 1998; 273: 12994-12998Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 7Xu Ch. Ma H. Inesi G. Al-Shawi M.K. Toyoshima C. J. Biol. Chem. 2004; 279: 17973-17979Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This sequence was almost complete and was annotated as a stalk segment (using the topology prediction software ARAMEMNON) in the Arabidopsis heavy metal pump AtHMA1 (At4g37270). This pump belongs to the subclass of zinc/cobalt/cadmium/lead-ATPases and is the most divergent metal pump of the Arabidopsis PIB-ATPases (1Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (735) Google Scholar, 2Axelsen K.B. Palmgren M.G. Plant Physiol. 2001; 126: 696-706Crossref PubMed Scopus (328) Google Scholar, 10Cobbett C.S. Hussain D. Haydon M.J. New Phytol. 2003; 159: 315-321Crossref Scopus (54) Google Scholar, 11Williams L. Mills R. Trends Plant Sci. 2005; 10: 1360-1385Abstract Full Text Full Text PDF Scopus (286) Google Scholar) (see Fig. 1). It lacks an amino-terminal heavy metal-binding domain, such as GMXCXXC or GICC(T/S)SE, which is often found in other members of the group. It has an intramembranous SPC instead of the CP(C/H/S) motif located at the putative metal transporting site of PIB-ATPases (11Williams L. Mills R. Trends Plant Sci. 2005; 10: 1360-1385Abstract Full Text Full Text PDF Scopus (286) Google Scholar, 12Argüello J.M. J. Membr. Biol. 2003; 195: 93-108Crossref PubMed Scopus (214) Google Scholar). The pump possesses other structural characteristics related to heavy metal binding and transport, such as a poly-H motif commonly found in zinc-binding proteins, several C and CC pairs, and a HP dipeptide (11Williams L. Mills R. Trends Plant Sci. 2005; 10: 1360-1385Abstract Full Text Full Text PDF Scopus (286) Google Scholar, 12Argüello J.M. J. Membr. Biol. 2003; 195: 93-108Crossref PubMed Scopus (214) Google Scholar). Initial studies performed by Higuchi and Sonoike (13Higuchi M. Sonoike K. van der Est A. Bruce D. Photosynthesis: Fundamental Aspects to Global Perspectives. 2005: 716-718Google Scholar) show that an Arabidopsis disruption mutant of AtHMA1 is sensitive to high concentrations of zinc. Recently, it was demonstrated that AtHMA1 is localized in the chloroplast envelope, and Arabidopsis insertional mutants exhibit a lower chloroplast copper content and a diminution of the total chloroplast superoxide dismutase, activity suggesting a role for AtHMA1 in copper homeostasis (14Seigneurin-Berny D. Gravot A. Auroy P. Mazard C. Kraut A. Finazzi G. Grunwald D. Rappaport F. Vavasseur A. Joyard J. Richaud P. Rolland N. J. Biol. Chem. 2006; 281: 2882-2892Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). At this point, two key questions about the functions and ion specificities of AtHMA1 must be addressed. (i) Can AtHMA1 transport Ca2+? (ii) In which heavy metal homeostatic mechanisms is AtHMA1 involved? We expressed AtHMA1 in yeast (Saccharomyces cerevisiae) in order to perform functional complementation assays. We demonstrated that AtHMA1 complements the mutant phenotype displayed by two Ca2+ transport-deficient yeast strains. In vitro calcium transport assays showed that AtHMA1 promotes thapsigargin-sensitive Ca2+ transport. AtHMA1 also complements a Cd2+-hypersensitive yeast mutant and confers high Cd2+ tolerance onto wild-type yeast. Cadmium-stimulated ATPase activity of AtHMA1 confirms that this pump plays a role in cadmium transport. Expression of AtHMA1 also confers high zinc, copper, and cobalt tolerance onto yeast, and ATPase activity is also stimulated by the addition of these metals. Taken together, our results strongly suggest that AtHMA1 is a metal pump capable of transporting a wide range of ions including calcium. As such, AtHMA1 may represent a key element in the mechanism used to overcome ion deficiency or toxicity in Arabidopsis thaliana. sarcoplasmic/endoplasmic reticulum Ca2+ ATPase reverse transcription carboxypeptidase Y 4-morpholinepropanesulfonic acid thapsigargin wild type. sarcoplasmic/endoplasmic reticulum Ca2+ ATPase reverse transcription carboxypeptidase Y 4-morpholinepropanesulfonic acid thapsigargin wild type. Cloning of AtHMA1—Full-length AtHMA1 cDNA (2460 bp) was supplied by RIKEN (15Seki M. Narusaka M. Kamiya A. Ishida J. Satou M. Sakurai T. Nakajima M. Enju A. Akiyama K. Oono Y. Muramatsu M. Hayashizaki Y. Kawai J. Carnicini P. Itoh M. Ishii Y. Arakawa T. Shibata K. Shinagawa A. Shinozaki K. Science. 2002; 296: 141-145Crossref PubMed Scopus (541) Google Scholar, 16Seki M. Carnicini P. Nishiyama Y. Hayashizaki Y. Shinozaki K. Plant J. 1998; 15: 707-720Crossref PubMed Scopus (192) Google Scholar). It was amplified using Accu-Therm™ DNA polymerase (GeneCraft) and primers flanking the coding region designed from the genomic sequence At4g37270. Primer sequences were as follows: 5′-CGCTTGAGATCTAATTCGTCGACCATGGAA-3′ (sense, BglII restriction site underlined) and 5′-AGACAAGCGGCCGCAAGTTACCCCCTAATG-3′ (antisense, NotI restriction site underlined). After verification by sequencing, the amplification product was digested with BglII and NotI and ligated into BamHI/NotI-digested pGPD426, a uracil yeast (S. cerevisiae) expression vector (17Mumberg D. Muller R. Funk M. Gene. 1995; 156: 119-122Crossref PubMed Scopus (1553) Google Scholar) to generate pGPD426-AtHMA1. Escherichia coli strain DH5α was used to build and maintain plasmid stocks using standard molecular biology procedures (18Sambrook J. Fritsch E.F. Maniatis T. 2nd Ed. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Yeast Strains and Growth Conditions—The following S. cerevisiae strains were used in this study: YR98 (MATα, ade2 his3-Δ200 leu2-3,112 lys2-Δ201 ura3-52) and its isogenic mutant Δpmr1 (YR122) (pmr1-Δ1::Leu2); W303 (MATa, ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1) and its isogenic mutant K616 (pmr1::His3 cnb1::Leu2 pmc1::Trp1); and DTY165 (MATα, ura3-52 leu2-3,112 his3-Δ200 trp1-Δ901 lys2-801 suc2-Δ9) and its isogenic mutant Δycf1 (DTY167) (ycf1::HisG). All strains were propagated at 30 °C in rich YPD medium, except K616, which was grown in YPD medium (1% (w/v) bacto-yeast extract, 2% (w/v) bacto-peptone, and 2% (w/v) glucose) supplemented with 10 mm CaCl2 (19Liang F. Cunningham K.W. Harper J.F. Sze H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8579-8584Crossref PubMed Scopus (132) Google Scholar). Yeast transformation was performed using the LiAc method (20Gietz D. St Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2863) Google Scholar). After transformation, yeast cells were grown in a selective medium (0.67% yeast nitrogen base without amino acids, 2% glucose) and supplemented with appropriate auxotrophic requirements. Uracil-based selection was used to screen for transformants. Expression of AtHMA1 in Yeast—AtHMA1 expression in transformed yeast was verified using RT-PCR. Transformed yeast was grown on a selective medium until an A600 of 0.6. Cells were then collected by centrifugation and used to isolate total RNA following the Chomczynski phenol-chloroform extraction method (21Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). The first strand of cDNA was prepared using a RevertAid™ first strand cDNA synthesis kit (Ferrmentas) with oligo(dT) and 1 μg of total RNA. After reverse transcription of mRNA to cDNA, a 565-bp fragment of the AtHMA1 cDNA was amplified by PCR using the following primers: 5′-ATGATGTTAACTGGGGACC-3′ (sense) and 5′-CTAATGTGCAGAGCTTAAACTGTTGCTGCTGCTACT-3′ (antisense). Amplification of ACT1 cDNA was used as an internal control in all reactions (22Del Aguila E.M. Dutra M.B. Silva J.T. Paschoalin V.M.F. BMC Mol. Biol. 2005; 6: 9Crossref PubMed Scopus (16) Google Scholar). The PCR products were separated by agarose gel electrophoresis. Functional Complementation Assays—Functional complementation of Ca2+ transport-defective mutants K616 and Δpmr1 was carried out as described previously (19Liang F. Cunningham K.W. Harper J.F. Sze H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8579-8584Crossref PubMed Scopus (132) Google Scholar). Mutant and wild-type yeast strains transformed with pGPD426 or pGPD426-AtHMA1 were streaked onto growth plates lacking uracil and supplemented with 10 mm EGTA. Ca2+-depleted plates were then incubated for up to 3 days at 30 °C. For functional complementation of the cadmium-hypersensitive yeast mutant Δycf1, the complementation was carried out by streaking transformed yeast onto growth plates lacking uracil and supplemented with 70 μm CdCl2. High Cd2+ plates were incubated for up to 5 days at 30 °C. Metal Toxicity Tests—To test sensitivity to high cadmium concentrations in Δycf1 yeast transformed with AtHMA1, cells grown to log phase were diluted to an A600 of 0.1 and incubated in the presence of 70–200 μm CdCl2. Growth at 30 °C was monitored by the change in A600 every 3 h for 21 h, and the rate of exponential growth of yeast cultures was expressed as generation time in hours, i.e. the doubling time of each yeast population. Sensitivity to other transition metals was tested by growing transformed Δycf1 on solid media supplemented with 6 mm CoCl2, 4 mm CuSO4, or 28 mm ZnCl2. Plates were incubated for 5 days at 30 °C. Additionally, the capacity of AtHMA1 to confer cadmium tolerance to wild-type yeast (W303) was tested by growing transformed cells in liquid medium with 70–200 μm CdCl2, and the A600 of cultures was followed for 5 days. Carboxypeptidase Y (CPY) Detection Assay—Transformed K616 and wild-type cells were grown in 3 ml of selective medium for 2 days at 30 °C. To obtain yeast lysate samples, cells were resuspended in 200 μl of SUMEB buffer (1% SDS, 8 m urea, 10 mm MOPS, pH 6.8, 10 mm EDTA, and 0.01% bromphenol blue) plus 100 μl of 500-μm glass beads (Sigma) and then vortexed three times for 1 min. The mixture was incubated for 10 min at 65 °C and centrifuged at 2,000 × g. The supernatant was collected. Two μl of supernatant (or yeast lysate) and 600 μl of growth media were transferred simultaneously onto a nitrocellulose membrane using a dot-blot device (Bio-Rad). The membrane was then washed briefly with water and blocked with 5% milk in TBS (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Tween 20). Secreted CPY was detected by monoclonal anti-CPY antibody (1:1,000) (Molecular Probes). Anti-mouse IgG antibody-horseradish peroxidase conjugate (1: 10,000; Molecular Probes) was used as secondary antibody. Isolation of Yeast Membranes—Membranes were prepared from yeast cells as described previously (23Liang F. Sze H. Plant Physiol. 1998; 118: 817-825Crossref PubMed Scopus (66) Google Scholar). 2–5 ml of stationary phase cultures of transformed yeast was diluted into 300 ml of appropriate selective medium, grown for 24 h at 30 °C to an A600 of ∼0.8, and collected by centrifugation. After being washed with distilled water, the cells were resuspended in 1 volume of a buffer solution containing 10% sucrose, 25 mm Hepes-bis-Tris propane, pH 7.5, 2 mm MgCl2, 2 mm dithiothreitol, 1 mm EGTA, and a mixture of protease inhibitors (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, and 1 mm phenylmethylsulfonyl fluoride). One volume of 500-μm glass beads was added to the mixture, and yeast cells were disrupted by vortexing at maximum speed for 2 min. The homogenate was centrifuged at 5,000 × g for 5 min at 4 °C. The supernatant was collected and centrifuged at 110,000 × g for 1 h. The resulting pellet was resuspended in 200 μl of a buffer solution containing 10% sucrose, 20 mm Hepes-bis-Tris propane, pH 7.0, 1 mm dithiothreitol, and the mixture of protease inhibitors described above. The membrane preparations were aliquoted and stored at –80 °C until needed. Protein content was measured using the bicinchoninic acid method (Pierce Chemical) with bovine serum albumin as standard. ATPase Activity Assays—These assays were carried out as described previously (24Eren E. Argüello J.M. Plant Physiol. 2004; 136: 3712-3723Crossref PubMed Scopus (161) Google Scholar). Yeast vesicles (50 μg protein/ml) were mixed with a reaction buffer containing 50 mm Tris, pH 7.5, 3 mm MgCl2, 3 mm ATP, 20 mm Cys, 1 mm dithiothreitol, and each metal (at 1 μm) listed in Fig. 6A. The reaction mixture was incubated for 15 min at 30 °C. Released inorganic phosphate was determined colorimetrically (25Lanzetta P.A. Alvarez L.J. Reinach P.S. Candia O.A. Anal. Bichem. 1979; 100: 95-97Crossref PubMed Scopus (1794) Google Scholar). Background activity measured in the absence of metals was less than 10% of the highest metal-stimulated activity and was subtracted from the activity determined in the presence of metals. The results are the average of four independent experiments with measurements performed in triplicate. The relative activity was standardized by setting the highest value obtained from the measurements (48.15 nmol μg–1) at 100% relative activity. All subsequent values were then adjusted accordingly and the S.E. was calculated. 45Ca2+ Uptake Assays—Ca2+ uptake by membranes of transformed yeast was assayed measuring 45Ca2+ accumulation using the filtration method (26Durr G. Strayle J. Plemper R. Elbs S. Klee S.K. Catty P. Wolf D.H. Rudolph H.K. Mol. Biol. Cell. 1998; 9: 1149-1162Crossref PubMed Scopus (345) Google Scholar). Membrane samples were suspended (0.3–0.6 mg protein/ml final concentration) in a reaction buffer (3 ml) containing 250 mm sucrose, 25 mm Hepes-bis-Tris propane (pH 7.0), 10 mm KCl, and 0.4 mm NaN3. Free Ca2+ concentration in the reaction buffer was set at desired values using variable amounts of 45CaCl2 (containing 2.4 Ci of 45Ca2+ mmol–1 CaCl2, PerkinElmer Life Sciences) and EGTA. Free Ca2+ concentrations were calculated using the WinMaxC 2.05 computer program. Vesicles were incubated in the buffer at 25 °C for 1 min, and transport was initiated (time zero) by adding a mixture of ATP and MgCl2 to final concentrations of 2 and 3 mm, respectively. Samples of 200 μl were taken at different times and filtered through 0.45-μm nitrocellulose membranes (Millipore, Bedford, MA). The filters were rinsed immediately with 5 ml of ice-cold STOP buffer containing 250 mm sucrose, 2.5 mm Hepes-bis-Tris propane, pH 7.0, and 0.2 mm CaCl2. The 45Ca2+ radioactivity associated with the filters was determined by liquid scintillation counting. Ca2+ uptake was expressed as nmol Ca2+/mg protein in the presence and absence of ATP-Mg. To determine the Km for Ca2+ uptake activity, the reaction mixture contained 500 μm EGTA and various amounts of Ca2+ To provide the desired range of free Ca2+ concentration (from pCa 5.5–7.2). ATP-dependent Ca2+ uptake (nmol mg–1 min–1) was defined as the difference between the 45Ca2+ retained in the filters following incubation in the presence and absence of MgATP. The effect of thapsigargin (TG) was tested by adding the compound to a final concentration in the reaction mixture of 0.1 μm at 15 min before the uptake assay was started. 45Ca2+ uptake and ATPase activity assays were performed at least four times in triplicate. Growth Inhibition of Δpmr1 and K616 on Ca2+-deficient Media Is Overcome by the Expression of AtHMA1—Fig. 1 shows a comparison between AtHMA1 and SERCA1. Like all P-type ATPases, both enzymes are transmembrane proteins. SERCA1 possesses ten transmembrane helices, like the vast majority of characterized Ca2+-ATPases. AtHMA1, instead, has seven predicted transmembrane helices (according to the topology prediction software ARAMEMNON). This differs from the characteristic eight predicted transmembrane helices presented in the rest of plant metal-ATPases recruited in the PIB-ATPase subfamily (2Axelsen K.B. Palmgren M.G. Plant Physiol. 2001; 126: 696-706Crossref PubMed Scopus (328) Google Scholar). On the other hand, AtHMA1 and SERCA1 share the highly conserved Asp in the signature sequence DKTGT that is phosphorylated during the catalytic cycle of P-type ATPases (1Axelsen K.B. Palmgren M.G. J. Mol. Evol. 1998; 46: 84-101Crossref PubMed Scopus (735) Google Scholar, 27Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (414) Google Scholar, 28Aravind L. Galperin M.Y. Koonin E.V. Trends Biochem. Sci. 1998; 23: 127-129Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Other common features of P-type ATPases are slightly modified in AtHMA1. For example, the transduction domain TGES (29Andersen J.P. Vilsen B. FEBS Lett. 1995; 359: 101-106Crossref PubMed Scopus (114) Google Scholar) in AtHMA1 is TGEX. The GDGXNDXP and TGD motifs involved in ATP binding (27Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (414) Google Scholar, 28Aravind L. Galperin M.Y. Koonin E.V. Trends Biochem. Sci. 1998; 23: 127-129Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 30Toyoshima C. Inesi G. Annun. Rev. Biochem. 2004; 73: 269-292Crossref PubMed Scopus (291) Google Scholar, 31Jensen A.M. Sørensen T.L. Olesen C. Møller J.V. Nissen P. EMBO J. 2006; 25: 2305-2314Crossref PubMed Scopus (163) Google Scholar, 32Efremov R.G. Kosinsky Y.A. Nolde D.E. Tsivkovskii R. Arseniev A.S. Lutsenko S. Biochem. J. 2004; 382: 293-305Crossref PubMed Scopus (22) Google Scholar, 33Savinsky M.H. Mandal A.K. Argüello J.M. Rosenzweig A.C. J. Biol. Chem. 2006; 281: 11161-11166Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) in AtHMA1 are GEGINDAP and TGD, respectively. AtHMA1 also presents motifs involved in heavy metal sensing, binding, and transport. These include several C and CC pairs (34Solioz M. Vulpe C. Trends Biochem. Sci. 1996; 21: 237-241Abstract Full Text PDF PubMed Scopus (412) Google Scholar, 35Eren E. Kennedy D.C. Maroney M.J. Argüello J.M. J. Biol. Chem. 2006; 281: 33881-33891Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 36Mills R.F. Francini A. Ferreira da Rocha P.S. C. Baccarini P.J. Aylett M. Krijger G.C. Williams L.E. FEBS Lett. 2005; 579: 783-791Crossref PubMed Scopus (194) Google Scholar), a poly-H domain located at the amino terminus, an HP dipeptide (11Williams L. Mills R. Trends Plant Sci. 2005; 10: 1360-1385Abstract Full Text Full Text PDF Scopus (286) Google Scholar, 12Argüello J.M. J. Membr. Biol. 2003; 195: 93-108Crossref PubMed Scopus (214) Google Scholar, 35Eren E. Kennedy D.C. Maroney M.J. Argüello J.M. J. Biol. Chem. 2006; 281: 33881-33891Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 37Verret F. Gravot A. Auroy P. Preveral S. Forestier C. Vavasseur A. Richaud P. FEBS Lett. 2005; 579: 1515-1522Crossref PubMed Scopus (97) Google Scholar), the site SPC essential for metal transport, and a HEGG motif (11Williams L. Mills R. Trends Plant Sci. 2005; 10: 1360-1385Abstract Full Text Full Text PDF Scopus (286) Google Scholar, 12Argüello J.M. J. Membr. Biol. 2003; 195: 93-108Crossref PubMed Scopus (214) Google Scholar, 14Seigneurin-Berny D. Gravot A. Auroy P. Mazard C. Kraut A. Finazzi G. Grunwald D. Rappaport F. Vavasseur A. Joyard J. Richaud P. Rolland N. J. Biol. Chem. 2006; 281: 2882-2892Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). It is noteworthy that AtHMA1, as a heavy metal pump, possesses the signature sequence DEFGENYSK (black circles), which is very similar to the DEFGEQLSK sequence involved in the inhibitory effect of thapsigargin in SERCA-type Ca2+ pumps (6Zhong L. Inesi G. J. Biol. Chem. 1998; 273: 12994-12998Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 38Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar). To determine whether AtHMA1 plays a role in Ca2+ homeostasis, we performed functional complementation assays using mutant yeast deficient in Ca2+ transport. Heterologous expression of the complete AtHMA1 cDNA was performed under the control of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD) promoter (39Bitter G.A. Egan K.M. Gene. 1984; 32: 263-274Crossref PubMed Scopus (138) Google Scholar) and was verified using RT-PCR (Fig. 2A). Fig. 2B shows that parental wild-type yeast strains YR98 and W303 grew normally on media containing 10 mm EGTA. As demonstrated previously (26Durr G. Strayle J. Plemper R. Elbs S. Klee S.K. Catty P. Wolf D.H. Rudolph H.K. Mol. Biol. Cell. 1998; 9: 1149-1162Crossref PubMed Scopus (345) Google Scholar), Δpmr1, a strain in which the secretory pathway Ca2+-ATPase PMR1 is disrupted (40Antebi A. Fink G. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (374) Google Scholar), was unable to grow in media depleted of Ca2+ in cells transformed with the empty vector. When this mutant was transformed with pGPD426-AtHMA1, growth on Ca2+-deficient medium was restored. AtHMA1 expression also complemented the Ca2+ depletion sensitivity of the triple mutant K616, which lacks both endogenous Ca2+ pumps (PMR1 and PMC1) and calcineurin (CNB1) function (19Liang F. Cunningham K.W. Harper J.F. Sze H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8579-8584Crossref PubMed Scopus (132) Google Scholar, 41Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (354) Google Scholar, 42Harper J.F. Hong B. Hwang I. Guo H.Q. Stoddard R. Huang J.F. Palmgren M.G. Sze H.A. J. Biol. Chem. 1998; 273: 1099-1106Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Similar to Δpmr1, K616 transformed with the empty vector did not grow on a medium containing very low Ca2+ (10 mm EGTA). Nevertheless, the triple mutant transformed with pGPD426-AtHMA1 became tolerant of this condition (Fig. 2B), supporting the hypothesis that AtHMA1 encodes a functional Ca2+ pump. AtHMA1 Reverts the Missorting Phenotype Displayed by K616—Given that yeast mutants lacking the secretory pathway Ca2+-ATPase PMR1 partially secrete carboxypeptidase Y, an enzyme normally destined to the vacuole (40Antebi A. Fink G. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (374) Google Scholar), we evaluated the effect of AtHMA1 expression on this characteristic phenotype. CPY secreted due to missorting was detected using a monoclonal anti-CPY antibody. CPY was detected in a culture media sample recovered from K616 cells transformed with the empty vector as shown in Fig. 2C. However, CPY was not detected in the culture media of K616 cells transformed with pGPD426-AtHMA1. In both cases, intracellular accumulation of CPY was verified in cell lysates. The wild-type strain was used as a negative control, as CPY is not secreted under normal conditions (26Durr G. Strayle J. Plemper R. Elbs S. Klee S.K. Catty P. Wolf D.H. Rudolph H.K. Mol. Biol. Cell. 1998; 9: 1149-1162Crossref PubMed Scopus (345) Google Scholar). Active Ca2+ Transport in K616 Membrane Vesicles Is Increased upon Expres" @default.
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• W1974133434 title "AtHMA1 Is a Thapsigargin-sensitive Ca2+/Heavy Metal Pump" @default.
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• W1974133434 cites W1964205809 @default.
• W1974133434 cites W1978472424 @default.
• W1974133434 cites W1979329347 @default.
• W1974133434 cites W1983828769 @default.
• W1974133434 cites W1997301069 @default.
• W1974133434 cites W1999113498 @default.
• W1974133434 cites W2010132078 @default.
• W1974133434 cites W2019297738 @default.
• W1974133434 cites W2019780345 @default.
• W1974133434 cites W2021943964 @default.
• W1974133434 cites W2022703044 @default.
• W1974133434 cites W2023415094 @default.
• W1974133434 cites W2033738324 @default.
• W1974133434 cites W2037444850 @default.
• W1974133434 cites W2042786900 @default.
• W1974133434 cites W2043715540 @default.
• W1974133434 cites W2048826261 @default.
• W1974133434 cites W2049878158 @default.
• W1974133434 cites W2053118944 @default.
• W1974133434 cites W2066006292 @default.
• W1974133434 cites W2067999317 @default.
• W1974133434 cites W2072950818 @default.
• W1974133434 cites W2079357395 @default.
• W1974133434 cites W2081750787 @default.
• W1974133434 cites W2086660631 @default.
• W1974133434 cites W2088300489 @default.
• W1974133434 cites W2095816191 @default.
• W1974133434 cites W2097666362 @default.
• W1974133434 cites W2102544942 @default.
• W1974133434 cites W2104120677 @default.
• W1974133434 cites W2108367015 @default.
• W1974133434 cites W2109503333 @default.
• W1974133434 cites W2110949951 @default.
• W1974133434 cites W2118128739 @default.
• W1974133434 cites W2127153072 @default.
• W1974133434 cites W2129673771 @default.
• W1974133434 cites W2144905501 @default.
• W1974133434 cites W2153331570 @default.
• W1974133434 cites W2156442455 @default.
• W1974133434 cites W2158337358 @default.
• W1974133434 cites W2158448504 @default.
• W1974133434 cites W2161656526 @default.
• W1974133434 cites W2163169780 @default.
• W1974133434 cites W2167211772 @default.
• W1974133434 cites W2169297695 @default.
• W1974133434 cites W2793839956 @default.
• W1974133434 cites W367800729 @default.
• W1974133434 cites W4294216491 @default.
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