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• W2113786433 abstract "In plants and fungi, vacuolar transporters help remove potentially toxic cations from the cytosol. Metal/H+ antiporters are involved in metal sequestration into the vacuole. However, the specific transport properties and the ability to manipulate these transporters to alter substrate specificity are poorly understood. The Arabidopsis thaliana cation exchangers, CAX1 and CAX2, can both transport Ca2+ into the vacuole. There are 11 CAX-like transporters in Arabidopsis; however, CAX2 was the only characterized CAX transporter capable of vacuolar Mn2+ transport when expressed in yeast. To determine the domains within CAX2 that mediate Mn2+ specificity, six CAX2 mutants were constructed that contained different regions of the CAX1 transporter. One class displayed no alterations in Mn2+ or Ca2+ transport, the second class showed a reduction in Ca2+ transport and no measurable Mn2+transport, and the third mutant, which contained a 10-amino acid domain from CAX1 (CAX2-C), showed no reduction in Ca2+ transport and a complete loss of Mn2+ transport. The subdomain analysis of CAX2-C identified a 3-amino acid region that is responsible for Mn2+ specificity of CAX2. This study provides evidence for the feasibility of altering substrate specificity in a metal/H+ antiporter, an important family of transporters found in a variety of organisms. In plants and fungi, vacuolar transporters help remove potentially toxic cations from the cytosol. Metal/H+ antiporters are involved in metal sequestration into the vacuole. However, the specific transport properties and the ability to manipulate these transporters to alter substrate specificity are poorly understood. The Arabidopsis thaliana cation exchangers, CAX1 and CAX2, can both transport Ca2+ into the vacuole. There are 11 CAX-like transporters in Arabidopsis; however, CAX2 was the only characterized CAX transporter capable of vacuolar Mn2+ transport when expressed in yeast. To determine the domains within CAX2 that mediate Mn2+ specificity, six CAX2 mutants were constructed that contained different regions of the CAX1 transporter. One class displayed no alterations in Mn2+ or Ca2+ transport, the second class showed a reduction in Ca2+ transport and no measurable Mn2+transport, and the third mutant, which contained a 10-amino acid domain from CAX1 (CAX2-C), showed no reduction in Ca2+ transport and a complete loss of Mn2+ transport. The subdomain analysis of CAX2-C identified a 3-amino acid region that is responsible for Mn2+ specificity of CAX2. This study provides evidence for the feasibility of altering substrate specificity in a metal/H+ antiporter, an important family of transporters found in a variety of organisms. The differential partitioning of cations is crucial for life processes, and transporters play a critical role in maintaining the proper concentrations of these ions in various cellular compartments (1Cohen A. Nelson H. Nelson N. J. Biol. Chem. 2000; 275: 33388-33394Google Scholar). The inability of plants to actively avoid toxic concentrations of particular cations in the environment places a particular importance on cation transporters. One mechanism employed by plants and fungi to avoid cation toxicity is the sequestration of cations into large vacuoles (2Marty F. Plant Cell. 1999; 11: 587-599Google Scholar). A fundamental question that arises is whether there are separate vacuolar transporters for each cation, and if not, what is the metal specificity of a given transporter? Several types of active transport mechanisms exist in plants to drive cations out of the cytosol against a steep concentration gradient (3Williams L.E. Pittman J.K. Hall J.L. Biochim. Biophys. Acta. 2000; 1465: 104-126Google Scholar,4Hall J.L. J. Exp. Bot. 2002; 53: 1-11Google Scholar). One important class of transporters is the H+-coupled cation antiporters, which have been identified at the vacuolar (tonoplast) membrane and are driven by a proton electrochemical gradient (5Schumaker K.S. Sze H. Plant Physiol. 1985; 79: 1111-1117Google Scholar, 6Salt D.E. Wagner G.J. J. Biol. Chem. 1993; 268: 12297-12302Google Scholar, 7Amalou Z. Gibrat R. Trouslot P. Dauzac J. Plant Physiol. 1994; 106: 79-85Google Scholar, 8Maeshima M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001; 52: 469-497Google Scholar). These transporters have numerous functions including resetting cytosolic levels of Ca2+ post signaling, vacuolar sequestration of potentially toxic concentrations of Cd2+, Zn2+, Mn2+ and other metals, and vacuolar storage of essential micronutrients such as Zn2+, Mg2+, and Mn2+. Despite numerous descriptive reports in whole plants (6Salt D.E. Wagner G.J. J. Biol. Chem. 1993; 268: 12297-12302Google Scholar, 9Gonzalez A. Koren'kov V. Wagner G.J. Physiol. Plant. 1999; 106: 203-209Google Scholar, 10Koren'kov V.D. Shepherd R.W. Wagner G.J. Physiol. Plant. 2002; 116: 359-367Google Scholar) and the recent cloning of several of these transporters (11Hirschi K.D. Zhen R.-G. Cunningham K.W. Rea P.A. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8782-8786Google Scholar, 12Gaxiola R.A. Rao R.I. Sherman A. Grisafi P. Alper S.L. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1480-1485Google Scholar, 13Shaul O. Hilgemann D.W. de-Almeida-Engler J. Van Montagu M. Inzé D. Galili G. EMBO J. 1999; 18: 3973-3980Google Scholar), there is a dearth of information available regarding H+-coupled ion selectivity, and much less for the residues that define specific cation transport (14Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). Two Arabidopsis cation exchanger (CAX) 1The abbreviations used are: CAX, cation exchanger; CaD, Ca2+ domain; CDF, cation diffusion facilitator; MES, 4-morpholineethanesulfonic acid; sCAX, N-terminally truncated CAX; YPD, yeast extract-peptone-dextrose medium; TMD, transmembrane domain 1The abbreviations used are: CAX, cation exchanger; CaD, Ca2+ domain; CDF, cation diffusion facilitator; MES, 4-morpholineethanesulfonic acid; sCAX, N-terminally truncated CAX; YPD, yeast extract-peptone-dextrose medium; TMD, transmembrane domain genes,CAX1 and CAX2, were identified that could suppress mutants of Saccharomyces cerevisiaedefective in vacuolar Ca2+ transport (11Hirschi K.D. Zhen R.-G. Cunningham K.W. Rea P.A. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8782-8786Google Scholar). Experiments using vacuolar membranes from yeast cells expressing CAX1 (11Hirschi K.D. Zhen R.-G. Cunningham K.W. Rea P.A. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8782-8786Google Scholar) demonstrate that this protein has biochemical properties similar to those of native plant vacuolar H+/Ca2+exchangers (5Schumaker K.S. Sze H. Plant Physiol. 1985; 79: 1111-1117Google Scholar, 15Blumwald E. Poole R.J. Plant Physiol. 1986; 80: 727-731Google Scholar). In similar experiments in yeast, CAX2 appears to have a higher K m for Ca2+ than CAX1 and a lower capacity for Ca2+ transport (11Hirschi K.D. Zhen R.-G. Cunningham K.W. Rea P.A. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8782-8786Google Scholar). CAX2 localizes to the plant vacuole, and when expressed at high levels in transgenic plants, increases vacuolar metal transport and causes the plants to accumulate more Ca2+, Mn2+ and Cd2+(16Hirschi K.D. Korenkov V.D. Wilganowski N.L. Wagner G.J. Plant Physiol. 2000; 124: 125-133Google Scholar). Furthermore these transgenic plants were more tolerant to Mn2+ in the growth media. These transport properties of CAX2 suggest the potential for broad substrate specificity among the 11 CAX-like transporters found in the Arabidopsis genome (17Mäser P. Thomine S. Schroeder J.I. Ward J.M. Hirschi K. Sze H. Talke I.N. Amtmann A. Maathuis F.J.M. Sanders D. Harper J.F. Tchieu J. Gribskov M. Persans M.W. Salt D.E. Kim S.A. Guerinot M.-L. Plant Physiol. 2001; 126: 1646-1667Google Scholar). Two domains have been described that modulate CAX1 activity (14Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar, 18Pittman J.K. Hirschi K.D. Plant Physiol. 2001; 127: 1020-1029Google Scholar,19Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar). The first domain has been termed the Ca2+ domain (CaD), located between amino acids 87 and 95 in CAX1 (14Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). This domain appears to be necessary for Ca2+ transport by CAX1. Exchanging this 9-amino acid region of CAX1 into CAX2 (giving the construct CAX2–9) greatly increases its Ca2+ transport activity but does not appear to alter transport of other metals (14Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). The second domain that regulates CAX function has been termed the regulatory or autoinhibitory domain (18Pittman J.K. Hirschi K.D. Plant Physiol. 2001; 127: 1020-1029Google Scholar, 19Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar). Sequence analysis suggests that an N-terminal regulatory domain may be present in all plant CAX-like transporters (20Pittman J.K. Sreevidya C.S. Shigaki T. Ueoka-Nakanishi H. Hirschi K.D. Plant Physiol. 2002; 130: 1054-1062Google Scholar). The CAX1 and CAX2 open reading frames contain additional amino acids at the N terminus that were not found in the original “shorter” N-terminally truncated CAX (sCAX) clones (sCAX1 and sCAX2) identified by suppression of yeast vacuolar Ca2+ transport mutants (20Pittman J.K. Sreevidya C.S. Shigaki T. Ueoka-Nakanishi H. Hirschi K.D. Plant Physiol. 2002; 130: 1054-1062Google Scholar, 21Schaaf G. Catoni E. Fitz M. Schwacke R. Schneider A. von Wirén N. Frommer W.B. Plant Biol. 2002; 4: 612-618Google Scholar). These findings suggest structural features involved in regulation and Ca2+transport but do not identify domains that may confer metal specificity among these CAX transporters. The manipulation of CAX transporters, through alteration in expression and substrate specificity, is an essential component in developing plants with increased tolerance to metals or removing toxic levels of metals from soil (phytoremediation) (22Salt D.E. Smith R.D. Raskin I. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1998; 49: 643-668Google Scholar). For a successful phytoremediation strategy, it is important to understand what determines the specificity of broad range metal transporters such as CAX2. In this report, we have characterized the transport properties of the CAX2 transporter. We identify specific domains within CAX2 that mediate Mn2+ substrate specificity and alter these domains to abolish the Mn2+ transport capabilities of CAX2, thus increasing its metal specificity. These findings serve as a framework for engineering metal specificity among the various H+/cation exchangers found in bacteria, fungi, and plants. K667 (cnb1::LEU2 pmc1::TRP1 vcx1Δ (23Cunningham K.W. Fink G.R. Mol. Cell. Biol. 1996; 16: 2226-2237Google Scholar)) was the S. cerevisiae strain used to express wild type and mutant genes. The wild type or mutant genes were propagated in pBluescript II SK(+) (Stratagene, La Jolla, CA), and inserts were transferred to the shuttle vector piHGpd (24Nathan D.F. Vos M.H. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1409-1414Google Scholar) for the expression in the yeast. The plasmids were introduced into yeast by the lithium acetate/single-stranded DNA/polyethylene glycol transformation method (25Gietz R.D. Schiestl R.H. Willems A.R. Woods R.A. Yeast. 1995; 11: 355-360Google Scholar). Standard techniques were used to manipulate the DNA used in this study (26Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates/Wiley Interscience, New York1998Google Scholar). All of the CAX1 and CAX2 clones used in this study were identical to the N-terminally truncated clones originally identified (11Hirschi K.D. Zhen R.-G. Cunningham K.W. Rea P.A. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8782-8786Google Scholar). Thus the proteins encoded by sCAX1 andsCAX2 lacked the first 36 and 42 amino acids, respectively. Site-directed mutagenesis was performed by a Class IIS restriction enzyme-mediated method (27Shigaki T. Hirschi K.D. Anal. Biochem. 2001; 298: 118-120Google Scholar), usingsCAX2 in pBluescript as the template DNA. BsmBI was the Class IIS restriction enzyme used throughout this study.CAX2–9 was constructed previously (14Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). The primers used were as follows: for CAX2-A, 5′-GAA TTC CGT CTC CTA ATA ATC TCC AAG AAG TCA TTC TCG GGA CAA AAC TCA ATC TAC TAC TAC CTT TC-3′ (forward), 5′-GAA TTC CGT CTC CAT TAA GTA CGC TGT TCT TTG G-3′ (reverse); for CAX2-B, 5′-GAA TTC CGT CTC CTT TGA CCA ATA ACA AAG TGG CAG TGG TCA AAT ATT CGT TAC TAG GCT CAA TTC TGT C-3′ (forward), 5′-GAA TTC CGT CTC CCA AAG CGA AAA TTG ATA TGA TC-3′ (reverse); for CAX2-C, 5′-GAA TTC CGT CTC CTG GCA CTT CAC TCT TCT GTG GAG GAA TCG CGA ATT ACC AAA AAG ACC AAG TCT TTG-3′ (forward), 5′-GAA TTC CGT CTC CGC CAA GTA CAA GTA ACA TGT TAG AC-3′ (reverse); for CAX2-D, 5′-GAA TTC CGT CTC CCT ACT TGA AAA ACG GAG AGG CTT CGG CTG CTG TTT TGT CCG ACA TGC AAC TAG CCC TGT CAA GGT TCA G-3′ (forward), 5′-GAA TTC CGT CTC CGT AGT GAA GAA CAG CCG GGA AGA G-3′ (reverse); for CAX2-E, 5′-GAA TTC CGT CTC CGT TAT GGA CTC ACC GTC AAT TGT TCG ATG CAC TCG ATG AGG AAT CAA ATC AGA AC-3′ (forward), 5′-GAA TTC CGT CTC CTA ACT GGA AGA AGA GGT AAG-3′ (reverse); for CAX2-C1, 5′-GAA TTC CGT CTC CTG GCA CTT CAC TCT TTT GTG GTG GAC TAG TCT TTT ACC-3′ (forward; the reverse primer was the same as CAX2-C reverse); for CAX2-C2, 5′-GAA TTC CGT CTC CTG GCA TCG CGA ATT ACC AAA AAG ACC AAG TCT TTG-3′ (forward), 5′-GAA TTC CGT CTC CGC CAC CAC AAA AGA AGG CGC AG-3′ (reverse). The bold letters in the primer sequences indicate introduced mutations. All constructs were sequenced completely and then subcloned into the yeast expression vector piHGpd. CAX1-c-Myc was constructed in a previous study (20Pittman J.K. Sreevidya C.S. Shigaki T. Ueoka-Nakanishi H. Hirschi K.D. Plant Physiol. 2002; 130: 1054-1062Google Scholar). Five tandem copies of the c-Myc epitope (EQKLISEEDL) were used to produce in-frame fusions of c-Myc to the C-terminal end of CAX2 constructs. The c-mycsequence was amplified by PCR from plasmid pT7–5Xmyc using the primers 5′-GAA TTC GGA TCC GGT CGA CGG TAT CG-3′ (forward) and 5′-GAA TTC GAG CTC TTA TCC ACC AAC CCG GGG TAC CGA ATT C-3′ (reverse). A BamHI site (underlined) was included in the forward primer to ligate the tag to the engineered BamHI site at the 3′-end of the CAX2 constructs. Total protein was isolated from yeast expressing c-Myc-tagged constructs using the glass bead method (26Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Greene Publishing Associates/Wiley Interscience, New York1998Google Scholar). Protein concentration was determined by using a protein assay kit (Bio-Rad). The protein was separated by SDS-PAGE and detected by anti-c-Myc monoclonal antibody (Berkeley Antibody Co., Richmond, CA) as described (20Pittman J.K. Sreevidya C.S. Shigaki T. Ueoka-Nakanishi H. Hirschi K.D. Plant Physiol. 2002; 130: 1054-1062Google Scholar). The assay for Ca2+tolerance on solid agar media was performed as previously described (11Hirschi K.D. Zhen R.-G. Cunningham K.W. Rea P.A. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8782-8786Google Scholar). K667 cultures expressing various constructs were grown in a selective liquid medium for 16 h, and 4 μl were spotted onto yeast extract-peptone-dextrose (YPD) agar media containing appropriate concentrations of CaCl2 or MnCl2. The cultures were then allowed to grow for 48 h at 30 °C. Yeast membrane microsomes were prepared as described previously (28Ueoka-Nakanishi H. Tsuchiya T. Sasaki M. Nakanishi Y. Cunningham K.W. Maeshima M. Eur. J. Biochem. 2000; 267: 3090-3098Google Scholar). For the measurement of Ca2+ and Mn2+ uptake, membrane vesicles (30–40 μg/ml) were incubated in buffer containing 0.3 msorbitol, 5 mm Tris-MES (pH 7.6), 25 mm KCl, 0.1 mm sodium azide, and 0.2 mm sodium orthovanadate. Vacuolar H+-translocating ATPase-catalyzed H+ translocation was initiated by the addition of 1 mm MgSO4 and 1 mm ATP. The vesicles were allowed to reach steady state with respect to pH gradient for 5 min at 25 °C before the addition of 45Ca2+(6 mCi/ml; American Radiolabeled Chemicals, St. Louis, MO) or54Mn2+ (6.5 mCi/ml; PerkinElmer Life Sciences). The final concentration of Ca2+ and Mn2+ in the reaction mixture was 10 μm and 1 mm, respectively. At the indicated times, aliquots (70 μl) of the reaction mix were removed and filtered through premoistened 0.45-μm pore size cellulose acetate GS-type filters (Millipore, Bedford, MA). After a 1-ml wash with ice-cold wash buffer (0.3 msorbitol, 5 mm Tris-MES, pH 7.6, 25 mm KCl, and 0.1 mm CaCl2 or MnCl2 as appropriate), the filters were air-dried, and radioactivity was determined by liquid scintillation counting. For metal competition experiments, ΔpH-dependent 10 μm45Ca2+ uptake was measured at a 10-min time point in the presence of 100 μm or 1 mm nonradioactive metals. Yeast strains lacking functional calcineurin (for example cnb strains) display increased Mn2+ sensitivity due, in part, to decreased activity of the Golgi Ca2+/Mn2+-ATPase PMR1 (23Cunningham K.W. Fink G.R. Mol. Cell. Biol. 1996; 16: 2226-2237Google Scholar, 29Farcasanu I.C. Hirata D. Tsuchiya E. Nishiyama F. Miyakawa T. Eur. J. Biochem. 1995; 232: 712-717Google Scholar, 30Pozos T.C. Sekler I. Cyert M.S. Mol. Cell. Biol. 1996; 16: 3730-3741Google Scholar). Furthermore, the additional lack of the vacuolar Ca2+/H+ antiporter VCX1 (cnb vcx1strains) slightly exacerbates the Mn2+ sensitivity of calcineurin mutants (30Pozos T.C. Sekler I. Cyert M.S. Mol. Cell. Biol. 1996; 16: 3730-3741Google Scholar), indicating that VCX1 may transport Mn2+ in addition to Ca2+. Yeast mutants deleted in VCX1 and the vacuolar Ca2+-ATPase PMC1 are sensitive to high concentrations of Ca2+ in the media (23Cunningham K.W. Fink G.R. Mol. Cell. Biol. 1996; 16: 2226-2237Google Scholar). Expression of VCX1 and N-terminally truncated CAX2 can suppress both the Mn2+ and Ca2+ sensitivity of a yeast mutant (K667, pmc1 vcx1 cnb1) defective in calcineurin and the vacuolar Ca2+ transporters (16Hirschi K.D. Korenkov V.D. Wilganowski N.L. Wagner G.J. Plant Physiol. 2000; 124: 125-133Google Scholar). As shown previously, N-terminal truncations of CAX1 can strongly suppress the Ca2+ sensitivity of these strains, whereas N-terminal truncations of CAX4 only weakly suppress the Ca2+phenotype, and N-terminal truncations of CAX3 are unable to suppress the Ca2+ phenotype (11Hirschi K.D. Zhen R.-G. Cunningham K.W. Rea P.A. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8782-8786Google Scholar, 31Shigaki T. Hirschi K.D. Gene. 2000; 257: 291-298Google Scholar, 32Cheng N.-H. Pittman J.K. Shigaki T. Hirschi K.D. Plant Physiol. 2002; 128: 1245-1254Google Scholar). We were interested in testing whether these characterized CAX transporters could suppress the Mn2+ sensitivity of this yeast strain. As shown in Fig.1, these CAX transporters cannot suppress the Mn2+ sensitivity phenotype of the yeast mutant. Additionally, the mung bean Ca2+/H+ antiporter, VCAX1 (28Ueoka-Nakanishi H. Tsuchiya T. Sasaki M. Nakanishi Y. Cunningham K.W. Maeshima M. Eur. J. Biochem. 2000; 267: 3090-3098Google Scholar), could not suppress Mn2+ sensitivity (data not shown). Only N-terminally truncated CAX2-expressing yeast mutants were capable of growing on plates containing 10 mmMnCl2 (Fig. 1). Utilizing chimeric CAX constructs in which parts of the CAX1 and CAX3 transporters were exchanged, a 9-amino acid region between putative transmembrane domains (TMD) 1 and 2 of CAX1 has been identified as important in mediating Ca2+/H+ transport (14Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). We have termed this domain the CaD. Swapping of the equivalent 9 amino acids of CAX3 into CAX1 abolished CAX1-mediated Ca2+/H+ transport. Alternatively, swapping of the CAX1 CaD into CAX3 allowed this chimeric CAX transporter to transport Ca2+ (14Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). As CAX2 is the only plant CAX transporter characterized to date with the ability to suppress the Mn2+ sensitivity of calcineurin-deficient yeast mutants (Fig. 1), a similar series of chimeric CAX constructs utilizing CAX2 should delineate the Mn2+ specificity domain(s) of this transporter. We chose to make chimeric constructs between CAX1 and CAX2 because each construct should maintain the ability to transport Ca2+. Thus, the fidelity of each construct could be rapidly assessed through suppression of Ca2+ sensitivity, and we could then test for alterations in Mn2+ suppression. Initially we planned a systematic approach to identify CAX2 Mn2+ specificity domains, similar to that used to identify the CaD of CAX1 (14Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). We chose to divide CAX1 and CAX2 into four segments of approximately equal size and exchange each segment to create eight different chimeric constructs (33Shigaki T. Hirschi K.D. BioTechniques. 2002; 32: 736-740Google Scholar). Although the construction of the chimeric clones was successful, Ca2+antiport activity was abolished for some of the constructs, indicating that protein stability was affected (data not shown). Therefore, an alternative approach was used. At the amino acid level, CAX2 is 43% identical (56% similar) to the CAX1 open reading frame. A sequence comparison among CAX1, CAX2, CAX3, and CAX4 identified five short domains, each consisting of 9–15 amino acids, which had very low sequence similarity between CAX1 and CAX2 (Fig. 2). We have designated these domains A to E. The A domain (amino acids 65–73 of CAX1) is present at the start of TMD 1, the B domain (amino acids 150–160 of CAX1) is present between TMD 3 and 4, the C domain (amino acids 175–184 of CAX1) is present in TMD 4, the D domain (amino acids 219–233 of CAX1) is present between TMD 5 and 6, and the E domain (amino acids 257–265 of CAX1) is present between TMD 6 and the acidic motif. There is very little variation among CAX1, CAX3, CAX4, and the mung bean VCAX1 in domains A, B, C, and E, whereas domain D and the region corresponding to the CAX1 9-amino acid CaD is very divergent among all CAX sequences (Fig. 2). We created five mutants in CAX2 that contain the corresponding CAX1 A, B, C, D, or E domain. These mutants were designated CAX2-A, CAX2-B, CAX2-C, CAX2-D, and CAX2-E, respectively. For these studies we have also used the previously constructed CAX2–9 construct, which contains the CAX1 9-amino acid CaD (14Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). All of the mutants were N-terminally truncated, i.e. constructed without the first 42 amino acids of CAX2. Because we had previously found that some CAX1/CAX2 chimeric clones had altered protein stability, it was very important to verify that each construct was expressed at approximately equal levels in yeast, and so we tagged each chimeric construct with a C-terminal c-Myc epitope and analyzed protein expression (Fig. 3). Each chimera was expressed similarly to CAX1 and CAX2.Figure 3Expression levels of CAX1, CAX2, and CAX2 chimeric constructs. Western blot showing relative expression levels of various constructs with C-terminal c-Myc tags used in the tolerance assay. Equal amounts of total protein isolated from yeast strains expressing each c-Myc-tagged construct as indicated were separated by SDS-PAGE, blotted, and then subjected to Western blot analysis using an anti-c-Myc monoclonal antibody.View Large Image Figure ViewerDownload (PPT) As we anticipated, each of these constructs could suppress the Ca2+ sensitivity of the yeast mutants relatively equally when grown on 200 mmCaCl2 (Fig. 4). However, when the yeast strains expressing these constructs were grown on higher levels of Ca2+ (250 mm CaCl2), we observed growth differences. Although the CAX2-C-, CAX2-D-, CAX2-E-, and CAX2–9-expressing strains grew in a manner similar to CAX2 strains, the CAX2-A- and CAX2-B-expressing strains were significantly reduced in their growth under these media conditions (Fig. 4). In media containing 10 mm MnCl2, yeast strains harboring the CAX2, CAX2–9, CAX2-D, and CAX2-E constructs all grew (Fig.5). Like strains expressing CAX1, those expressing CAX2-A, CAX2-B, and CAX2-C completely failed to suppress Mn2+ sensitivity. Even at lower MnCl2concentrations (5 mm), the CAX2-A, CAX2-B, and CAX2-C strains were unable to grow (Fig. 5).Figure 5Identification of domains in CAX2 that are necessary for conferring Mn2+ tolerance in yeast. Suppression of Mn2+ sensitivity of thepmc1 vcx1 cnb yeast mutant, K667, by various CAX constructs. The same assay conditions were used as described in the legend for Fig.1, except yeast growth is shown on YPD containing 5 mm and 10 mm MnCl2. A representative experiment is shown.View Large Image Figure ViewerDownload (PPT) To examine the Ca2+ transport properties of these three Mn2+-negative mutants directly, ΔpH-dependent 10 μm45Ca2+ uptake into yeast membrane vesicles was measured (Fig.6 A). Ca2+ antiport activity mediated by CAX2-A and CAX2-B was significantly lower than for CAX2 (57.4 and 33.4% of CAX2 activity, respectively), whereas Ca2+ antiport activity mediated by CAX2-C was not significantly different from CAX2. The ability to suppress the Mn2+ sensitivity of the yeast mutants infers Mn2+ transport capability. However, it is important to demonstrate directly that these CAX2 constructs mediate Mn2+/H+ antiport activity in yeast. Therefore, we measured ΔpH-dependent 1 mm54MnCl2 uptake into microsomal vesicles isolated from K667 yeast strains expressing various CAX transporters. Mn2+/H+ transport activity was observed in membrane vesicles from CAX2- and CAX2-E-expressing strains (Fig.6 B), but no Mn2+/H+ antiport activity was detectable in vesicles from CAX1- or CAX2-C-expressing yeast (data not shown). The Mn2+/H+ antiport activity measured from CAX2-E vesicles was modestly greater than the activity of CAX2 (Fig. 6 B), confirming the slight increase in yeast growth of CAX2-E strains compared with CAX2 strains on high Mn2+ media (Fig. 5). The CAX2-C-expressing strains have a specific defect in Mn2+ tolerance, as the growth of these strains is largely indistinguishable from CAX2- on Ca2+-containing media (Fig. 4) despite the lack of growth on Mn2+-containing media (Fig. 5). The inability of the CAX2-A- and CAX2-B-expressing strains to suppress Mn2+sensitivity also implicates these domains as being involved in Mn2+ transport (Fig. 5). However, CAX2-A- and CAX2-B-expressing strains also exhibited diminished growth on Ca2+-containing media and decreased Ca2+/H+ antiport activity (Figs. 4 and6 A), suggesting a nonspecific reduction in transport capabilities. To examine which amino acids were involved in determining the Mn2+ specificity of CAX2-C, we divided the CAX2 C domain into two regions, each containing 3 different amino acids than those present in the CAX1 C domain. The CAX2-C1 chimera contains the 3 amino acids TSL from CAX1, replacing amino acids CAF of CAX2, and the CAX2-C2 construct contains the 3 amino acids IAN from CAX1, replacing the amino acids LVF of CAX2 (Fig. 2). Yeast strains expressing the CAX2-C2 construct were indistinguishable from CAX2, as these strains could suppress both the Ca2+ and Mn2+sensitivity of K667 yeast (Fig.7 A). Yeast strains expressing CAX2-C1 were able to strongly suppress the Ca2+ sensitivity phenotype, but there was no growth of these strains on Mn2+-containing media. When the transport properties of CAX2-C1 were determined by direct ΔpH-dependent45Ca2+ and 54Mn2+uptake measurements into membrane vesicles prepared from CAX2-C1-expressing yeast, CAX2-C1 was found to have significant Ca2+/H+ antiport activity, which was indistinguishable from that of CAX2, but no Mn2+/H+ antiport activity (Fig.7 B). To further analyze the altered transport properties of the CAX2 mutants, competition experiments were performed. This approach allowed us to determine the effect of the domain swapping between CAX1 and CAX2 on cation selectivity. ΔpH-dependent 10 μm45Ca2+ uptake was measured at a single 10-min time point into yeast microsomal vesicles isolated from strains expressing CAX1, CAX2, and the six CAX2 mutants. Ca2+ uptake determined in the absence of excess nonradioactive metal (control) was compared with Ca2+uptake determined in the presence of two concentrations (10× and 100×) of various nonradioactive metals (Fig.8). Inhibition of Ca2+ uptake by nonradioactive Ca2+ was used as an internal control, and as expected, Ca2+ uptake by each CAX transporter was strongly inhibited by excess Ca2+. Nonradioactive Ca2+, particularly the 10× concentrations, did not completely inhibit Ca2+ uptake, further highlighting the low Ca2+ affinity of the CAX transporters. We have previously demonstrated that tobacco plants ectopically expressing CAX2 have significantly increased vacuolar transport of Cd2+(16Hirschi K.D. Korenkov V.D. Wilganowski N.L. Wagner G.J. Plant Physiol. 2000; 124: 125-133Google Scholar). Ca2+ uptake by CAX1 and CAX2 were both strongly inhibited by Cd2+, and this Cd2+ inhibition was consistently observed in all of the six CAX2 mutants. Howe" @default.
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• W2113786433 title "Manganese Specificity Determinants in the ArabidopsisMetal/H+ Antiporter CAX2" @default.
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