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• W2035539991 abstract "Regulation of Ca2+ transporters is a vital component of signaling. The ArabidopsisH+/Ca2+ exchanger CAX1 contains an N-terminal autoinhibitory domain that prevents Ca2+transport when CAX1 is heterologously expressed in yeast. Using a yeast screen, we have identified three different proteins that activate CAX1. One of these, CXIP1 (CAX-interactingprotein-1; 19.3 kDa) has amino acid similarity to the C terminus of PICOT (protein kinase C-interacting cousin ofthioredoxin) proteins. Although PICOT proteins are found in a variety of organisms, a function has not been previously ascribed to a plant PICOT protein. We demonstrate that CXIP1 activated the CAX1 homolog CAX4, but not CAX2 or CAX3. An Arabidopsis homolog of CXIP1 (CXIP2) weakly activated CAX4, but not CAX1. In a yeast two-hybrid assay, CXIP1 interacted with the N terminus of CAX1. In competition analysis, CXIP1 and a CAX1 N-terminal peptide appeared to bind to similar N-terminal domains of CAX1. Chimeric CAX3 constructs containing the N terminus of CAX1 were activated by CXIP1. InArabidopsis, CXIP1transcripts, likeCAX1, accumulated in response to different metal conditions. This work thus characterizes a new class of signaling molecules in plants that may regulate CAX transporters in vivo. Regulation of Ca2+ transporters is a vital component of signaling. The ArabidopsisH+/Ca2+ exchanger CAX1 contains an N-terminal autoinhibitory domain that prevents Ca2+transport when CAX1 is heterologously expressed in yeast. Using a yeast screen, we have identified three different proteins that activate CAX1. One of these, CXIP1 (CAX-interactingprotein-1; 19.3 kDa) has amino acid similarity to the C terminus of PICOT (protein kinase C-interacting cousin ofthioredoxin) proteins. Although PICOT proteins are found in a variety of organisms, a function has not been previously ascribed to a plant PICOT protein. We demonstrate that CXIP1 activated the CAX1 homolog CAX4, but not CAX2 or CAX3. An Arabidopsis homolog of CXIP1 (CXIP2) weakly activated CAX4, but not CAX1. In a yeast two-hybrid assay, CXIP1 interacted with the N terminus of CAX1. In competition analysis, CXIP1 and a CAX1 N-terminal peptide appeared to bind to similar N-terminal domains of CAX1. Chimeric CAX3 constructs containing the N terminus of CAX1 were activated by CXIP1. InArabidopsis, CXIP1transcripts, likeCAX1, accumulated in response to different metal conditions. This work thus characterizes a new class of signaling molecules in plants that may regulate CAX transporters in vivo. Ca2+ signal transduction requires the judicious control of cytosolic Ca2+ levels. Cytosolic “Ca2+ spikes” are either directly or indirectly translated into biological responses that govern all aspects of growth and development (1McAnish M.R. Heatherington A.M. Trends Plant Sci. 1998; 3: 32-36Google Scholar, 2Sanders D. Pelloux J. Brownlee C. Harper J.F. Plant Cell. 2002; 14: S401-S417Google Scholar). Endomembrane Ca2+ transporters are believed to play an important role in specifying the duration and amplitude of these cytosolic Ca2+ fluctuations (2Sanders D. Pelloux J. Brownlee C. Harper J.F. Plant Cell. 2002; 14: S401-S417Google Scholar, 3Harper J.F. Trends Plant Sci. 2001; 6: 395-397Google Scholar). Understanding the regulatory mechanisms of these Ca2+transporters is a fundamental component in dissecting signaling specificity. Expression in yeast has facilitated the cloning of numerous plant genes, including plant endomembrane Ca2+ transporters (4Hirschi 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,5Sze H. Liang F. Hwang I. Curran A.C. Harper J.F. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 433-462Google Scholar). Initially, plant H+/Ca2+ antiporter genes were cloned by their ability to suppress the Ca2+-hypersensitive phenotype of a Saccharomyces cerevisiae mutant (4Hirschi 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, 6Ueoka-Nakanishi H. Tsuchiya T. Sasaki M. Nakanishi Y. Cunningham K.W. Maeshima M. Eur. J. Biochem. 2000; 267: 3090-3098Google Scholar). These genes are termedCAX for cation exchangers, and CAX1 from Arabidopsis thaliana is a high capacity Ca2+ transporter. Arabidopsis appears to have up to 11 other putative cation/H+ antiporters (CAX2–11 and MHX) (7Mäser 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). CAX1, CAX2, CAX4, and MHX have been shown to localize to the plant vacuole (8Shaul O. Hilgemann D.W. de-Almeida-Engler J. Van Montagu M.V. Inze D. Galili G. EMBO J. 1999; 18: 3973-3980Google Scholar, 9Hirschi K.D. Korenkov V.D. Wilganowski N.L. Wagner G.J. Plant Physiol. 2000; 124: 125-134Google Scholar, 10Cheng N.-H. Pittman J.K. Shigaki T. Hirschi K.D. Plant Physiol. 2002; 128: 1245-1254Google Scholar, 11Cheng N.-H. Pittman J.K. Barkla B.J. Shigaki T. Hirschi K.D. Plant Cell. 2003; (in press)Google Scholar). The activity of CAX1 appears to be regulated by an N-terminal autoinhibitory domain that was absent in the initial clone characterized by heterologous expression in yeast (12Pittman J.K. Hirschi K.D. Plant Physiol. 2001; 127: 1020-1029Google Scholar, 13Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar). Ectopic expression of deregulated CAX1 missing the N-terminal autoinhibitor (sCAX1) in tobacco increases Ca2+ levels in the plants and causes numerous stress-sensitive phenotypes often associated with Ca2+ deficiencies (14Hirschi K.D. Plant Cell. 1999; 11: 2113-2122Google Scholar, 15Hirschi K. Trends Plant Sci. 2001; 6: 100-104Google Scholar). Thus, a wide range of environmental responses appear to require regulation of CAX1 transport activity; however, the mechanism by which CAX1 transport becomes activated is currently not understood. From recent studies, it has become clear that the N termini of particular plant Ca2+-ATPases act as points of convergence between Ca2+-signaling molecules that can both positively and negatively regulate Ca2+ transport (3Harper J.F. Trends Plant Sci. 2001; 6: 395-397Google Scholar, 5Sze H. Liang F. Hwang I. Curran A.C. Harper J.F. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2000; 51: 433-462Google Scholar). A Ca2+-dependent protein kinase competes with calmodulin for binding to the N termini of these Ca2+-ATPases to negatively regulate pump activity (16Hwang I. Sze H. Harper J.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6224-6229Google Scholar). Interestingly, the N terminus of CAX1 does not contain a calmodulin-binding site, and biochemical studies show that CAX1 cannot be activated by exogenous calmodulin (12Pittman J.K. Hirschi K.D. Plant Physiol. 2001; 127: 1020-1029Google Scholar). Moreover, expression of an activated Ca2+-dependent protein kinase in yeast strains harboring CAX1 does not alter activity. 1J. K. Pittman and K. D. Hirschi, unpublished observations. 1J. K. Pittman and K. D. Hirschi, unpublished observations. These findings strongly suggest that a unique set of signal transduction molecules physically interact with the N terminus of CAX1 to modulate Ca2+ transport. We propose that one possible mechanism of activation of CAX1 is through a protein cofactor that directly interacts with the antiporter. In this work, we have utilized autoinhibited CAX1 in a yeast screen to identify and characterize plant gene products that activate CAX1-mediated H+/Ca2+ transport. We have characterized the specificity of this activation and the regions of CAX1 involved in the physical interaction between CAX1 and one of these activators. This study details a new class of signaling molecules in plants that may be involved in activation of H+/Ca2+ transporters in plants. S. cerevisiaestrain K667 (MAT a cnb1::LEU1 pmc1::TRP1 vcx1Δ ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1) (17Cunningham K.W. Fink G.R. Mol. Cell. Biol. 1996; 16: 2226-2237Google Scholar) was used in all yeast experiments involving the expression of CAX genes with or withoutCXIP (CAX-interactingprotein) genes. Yeast strain Y190 (MAT a gal4 gal180 his3 trp1-901 ade2-101 ura3-52 leu2-3,112 + URA3::GAL→ lacZ, LYS2::GAL(UAS) →HIS3 cyh r) was used in the yeast two-hybrid assay (18Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Google Scholar). The A. thaliana Columbia ecotype was used in this study. Yeast strain K667 expressing CAX1, which is hypersensitive to high concentrations of Ca2+, was transformed with an A. thaliana (ecotype Landsberg) cDNA library constructed in the episomal yeast-Escherichia colishuttle vector pFL61 (19Minet M. Dufour M.E. Lacroute F. Plant J. 1992; 2: 417-422Google Scholar). Stable transformants were selected on synthetic complete medium lacking His and Ura (20Sherman F. Hicks J.B. Fink G.R. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar). Of these, ≈2 × 105 colonies were replicated onto yeast extract/peptone/dextrose (YPD) medium supplemented with 200 mm CaCl2 to identify Ca2+-tolerant transformants. cDNA clones of interest were isolated, and plasmid DNA was retransformed into the CAX1-expressing K667 yeast cells to confirm that the activation of CAX1 Ca2+ transport activity was attributable to the Arabidopsis cDNA inserts. All cDNA inserts were subcloned into pBluescript and completely sequenced. Yeast cells expressing aCAX gene (CAX1, CAX3, orCAX4 or chimeric CAX constructs) with the CXIP1gene were assayed on YPD medium supplemented with 200 mmCaCl2 (13Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar, 21Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). Chimeric CAX constructs sCAX3-β, sCAX3-γ, sCAX3-δ, sCAX1-α, sCAX3-α1, and sCAX1-9 were constructed in a previous study (21Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). Amino acid substitutions were introduced into the CXIP1 PICOT (protein kinase C-interacting cousin ofthioredoxin) homology domain (HD)2 using a modified phoenix mutagenesis protocol with the Type IIS restriction enzymeBsmBI (22Shigaki T. Hirschi K.D. Anal. Biochem. 2001; 298: 118-120Google Scholar). To change amino acids CGFS to AAAA, forward primer 5′-GAA TCC CGT CTC CCC ATG GCT GCG GCA GCC AAC ACT GTG GTT CAG ATT TTG-3′ (where italic letters indicate the mutated residues) and reverse primer 5′-G AAA GGA ACG AGA GAC TTC CCC ATG GAG ACG GAA TTC-3′ were used. To change amino acids SWNPT to AAAAA, forward primer 5′-GAA TCC CGT CTC GAG TAT GCT GCA GCA GCC GCG TTT CCT CAG CTT TAT ATC-3′ was used along with reverse primer 5′-TTG AGG CAA GGA CTT AAA GAG TAT GAG ACG GAA TTC-3′. All mutant constructs were completely sequenced and subcloned into the yeast expression vector piHGpd (23Nathan D.F. Vos M.H. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1409-1414Google Scholar). mRNA transcript expression in yeast cells was confirmed by reverse transcription-PCR (24Witte S. Villalba M. Bi K. Liu Y.-H. Isakov N. Altman A. J. Biol. Chem. 2000; 275: 1902-1909Google Scholar). A. thaliana (ecotype Columbia) seeds were germinated in artificial soil (Metro-Mix 200, Scotts, Marysville, OH) and grown under continuous light. Sample preparation and RNA extraction were performed as previously described (14Hirschi K.D. Plant Cell. 1999; 11: 2113-2122Google Scholar). To analyze gene expression, 20 μg of total RNA was loaded onto a formaldehyde-containing 1.0% agarose gel and blotted onto nylon membrane (Amersham Biosciences, Buckinghamshire, United Kingdom). Filters were hybridized overnight at 42 °C with a32P-labeled gene-specific probe in 2× SSC and 50% formamide, washed twice with 2× SSC and 0.1% SDS and twice with 0.1× SSC and 0.1% SDS (both at 55 °C), and exposed to x-ray film (Eastman Kodak Co.) at −80 °C for an appropriate time. Microsomal membrane-enriched vesicles were isolated from yeast as described previously (13Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar). Time-dependent H+/Ca2+ transport into endomembrane vesicles was examined using the direct filtration method as described previously (12Pittman J.K. Hirschi K.D. Plant Physiol. 2001; 127: 1020-1029Google Scholar). Measurement of Ca2+transport in the presence of a synthetic peptide was performed as previously described (13Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar). The yeast two-hybrid system used in this study was described previously (13Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar, 18Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Google Scholar). The two CAX1 N-terminal fragments (residues 1–65 and 37–73) were amplified by PCR and cloned in-frame into the NcoI and BamHI sites of pAS2. CXIP1 was amplified by PCR and cloned in-frame into theNcoI and XhoI sites of pACT2. Protein expression in yeast was confirmed as previously described (13Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar). A pmc1vcx1cnb1 yeast strain (K667) lacking the vacuolar Ca2+ transporters Pmc1 and Vcx1 and calcineurin (Cnb1) is hypersensitive to high levels of Ca2+(17Cunningham K.W. Fink G.R. Mol. Cell. Biol. 1996; 16: 2226-2237Google Scholar). CAX1-expressing K667 cells grow well in standard medium, but do not grow in medium containing high levels of Ca2+ (12Pittman J.K. Hirschi K.D. Plant Physiol. 2001; 127: 1020-1029Google Scholar). Only expression of deregulated CAX1 (containing mutations or truncations of the N terminus) can suppress the yeast vacuolar Ca2+ transport defect (4Hirschi 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, 13Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar). Using a yeast expressionArabidopsis cDNA library (19Minet M. Dufour M.E. Lacroute F. Plant J. 1992; 2: 417-422Google Scholar), we have isolatedArabidopsis cDNAs that allow full-length CAX1-expressing K667 strains to grow in medium containing high levels of Ca2+. The cDNAs were isolated from the yeast and then retransformed into K667 strains with or without CAX1 to verify that they do not suppress the Ca2+ sensitivity of K667 by themselves and to confirm that the clones are indeed required for CAX1 to suppress the Ca2+ sensitivity of K667. We have used the term CXIP (CAX-interactingprotein) for the protein products of these cDNAs. Six different CXIP cDNA clones have been identified using this yeast activation approach (TableI). One of the cDNA clones,CXIP1, which is the focus of this study, encodes a novelArabidopsis protein with sequence homology at the C terminus to PICOT proteins (24Witte S. Villalba M. Bi K. Liu Y.-H. Isakov N. Altman A. J. Biol. Chem. 2000; 275: 1902-1909Google Scholar, 25Rahlfs S. Fischer M. Becker K. J. Biol. Chem. 2001; 276: 37133-37140Google Scholar, 26Rodriguez-Manzaneque M.T. Tamarit J. Bellı́ G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Google Scholar). We cloned an Arabidopsis homolog of CXIP1 that we have termed CXIP2, which encodes a protein of 293 amino acids (32 kDa). The yeast activation approach also identified CXIP3, which encodes an FKBP protein (A. thaliana FKBP15-2) (27Luan S. Kudla J. Gruissem W. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6964-6969Google Scholar). Four of the six cDNA clones identified in the yeast activation screen are identical and encode a protein whose function has not been previously characterized. We have termed this gene CXIP4.Table ISummary of CXIPsCXIP1CXIP2aA homolog of CXIP1 that was cloned from an Arabidopsis gene expression library using PCR amplification.CXIP3CXIP4GeneBank™/EBI accession no.AY157988AY157989U52047AY163162Location in Arabidopsis genomeChromosome 3Chromosome 2Chromosome 5Chromosome 2mRNA (bp)522882662999Amino acids173293163332Molecular mass (kDa)bDeduced using the protein analysis program ProtParam Tool (available at www.expasy.ch/cgi_bin/protparam).19.3321537.8BLAST search resultscDetermined using the BLAST program.A. thaliana PICOT proteinA. thaliana PICOT proteinA. thaliana FKBP15–2A. thalianaunknown proteinMotif or functionPICOT domainPICOT domainFK506- and rapamycin-sensitive rotamase activitya A homolog of CXIP1 that was cloned from an Arabidopsis gene expression library using PCR amplification.b Deduced using the protein analysis program ProtParam Tool (available at www.expasy.ch/cgi_bin/protparam).c Determined using the BLAST program. Open table in a new tab K667 yeast cells expressing CXIP1 or CAX1 alone were unable to grow on 200 mm CaCl2, whereas coexpression of CXIP1 with CAX1 significantly enhanced the growth of K667 (Fig.1 A). However, growth of the (CAX1 + CXIP1)-expressing strain was not as strong as growth of K667 cells expressing N-terminally truncated sCAX1. The ability to suppress the Ca2+ sensitivity of the yeast mutants inferred activation of autoinhibited CAX1; however, it was important to demonstrate directly through biochemical studies that the combination of full-length CAX1 and CXIP1 mediated H+/Ca2+antiport activity in yeast. Therefore, we measured ΔpH-dependent 10 μm45CaCl2 uptake into microsomal vesicles isolated from K667 yeast strains expressing both ArabidopsiscDNAs. H+/Ca2+ transport activity was observed in membrane vesicles from a (CAX1 + CXIP1)-expressing strain and an sCAX1-expressing strain (Fig. 1 B). However, no H+/Ca2+ antiport activity was detectable in vesicles from full-length CAX1-expressing or CXIP1-expressing yeast strains (Fig. 1 B and data not shown). The H+/Ca2+ antiport activity measured in CAX1 + CXIP1 vesicles was consistently ∼10% of the activity measured in sCAX1 vesicles. The CXIP1 cDNA consists of 522 base pairs (GenBankTM/EBI accession number AY157988). The amino acid sequence consists of 173 amino acids (with a predicted molecular mass of 19.3 kDa) and has extensive homology to PICOT proteins found in a broad taxonomic distribution, including mammals, yeast, and bacteria (28Isakov N. Witte S. Altman A. Trends Biochem. Sci. 2000; 25: 537-539Google Scholar). In other organisms, these proteins appear to play a negative regulatory role in cellular stress responses (24Witte S. Villalba M. Bi K. Liu Y.-H. Isakov N. Altman A. J. Biol. Chem. 2000; 275: 1902-1909Google Scholar, 29Isakov N. Altman A. Annu. Rev. Immunol. 2002; 20: 761-794Google Scholar). The primary amino acid sequence of CXIP1 (Fig. 2) is 26% identical (38% similar) to the 171-amino acid GLP-1 PICOT protein from Plasmodium falciparum (PfGLP-1) and 33% identical (45% similar) to the 335-amino acid human PICOT (HsPICOT) protein. The PICOT proteins contain a modular unit of 84 amino acid located in the C terminus that is highly conserved (PICOT-HD) (Fig. 2). The human PICOT protein has two tandem PICOT-HD repeats, whereas both CXIP1, CXIP2, and P. falciparum GLP-1 have a single PICOT-HD in their C termini. Within the PICOT-HD, several stretches of amino acids appear to be highly conserved, for example, -CGFS- and -SNWPT- (amino acids 97–100 and 133–137, respectively) (Fig. 2). Among the various PICOT proteins, several include a single thioredoxin (Trx) HD in their N termini. The human PICOT protein is involved in the interaction with protein kinase C through this Trx-HD and negatively regulates the c-Jun N-terminal kinase/AP-1 and NF-κB pathway (24Witte S. Villalba M. Bi K. Liu Y.-H. Isakov N. Altman A. J. Biol. Chem. 2000; 275: 1902-1909Google Scholar). CXIP2 is 30% identical (43% similar) to CXIP1 overall and is >54% identical (71% similar) to CXIP1 within the PICOT-HD. CXIP2 contains a Trx-HD in the N terminus like that of the human PICOT protein (Fig.2). Using the yeast Ca2+suppression assay, we were interested in determining whether CXIP1 and CXIP2 can activate various CAX transporters. Three close homologs of CAX1 (CAX2–4) also appear to be N-terminally regulated, and neither full-length CAX3 nor CAX4 is able to suppress the Ca2+sensitivity of K667 (10Cheng N.-H. Pittman J.K. Shigaki T. Hirschi K.D. Plant Physiol. 2002; 128: 1245-1254Google Scholar, 30Pittman J.K. Sreevidya C.S. Shigaki T. Ueoka-Nakanishi H. Hirschi K.D. Plant Physiol. 2002; 130: 1054-1062Google Scholar). Unlike CXIP1, CXIP2 could not activate CAX1 (Fig. 3 A). CXIP1 and CXIP2 could both weakly activate full-length CAX4 (Fig. 3 B); however, both CXIP1 and CXIP2 failed to activate CAX2 2The abbreviations used are: HD, homology domain; Trx, thioredoxin and CAX3 (data not shown). As an initial step toward deducing the mechanism of CAX1 activation by CXIP1, we tested the ability of CXIP1 to activate various chimeric CAX constructs. CAX3 could not suppress the K667 Ca2+-sensitive phenotype. In fact, an N-terminal truncation of CAX3 (sCAX3) (21Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar) was also unable to suppress the Ca2+ toxicity despite lacking its autoinhibitory domain (Fig. 4 A). Just as CXIP1 was unable to activate full-length CAX3, CXIP1 coexpressed with sCAX3 was unable to suppress the yeast Ca2+ sensitivity (Fig.4 A). Numerous sCAX3-CAX1 constructs + CXIP1 were unable to suppress the Ca2+ sensitivity of K667 yeast cells. For example, K667 strains in which CXIP1 was coexpressed with chimeric sCAX3-CAX1 constructs containing the central region or C terminus of CAX1 were unable to suppress the Ca2+-sensitive phenotype (Fig. 4, B and C). However, when a chimeric construct (called sCAX3-α1) was used that contained 37 amino acids of the N-terminal region of CAX1 (Met37–Leu73) fused to CAX3, CXIP1 could activate this chimeric construct (Fig. 4 C). We have previously shown that a 9-amino acid domain within CAX1, which we termed the “Ca2+ domain,” is required for Ca2+ transport (21Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). Furthermore, when this 9-amino acid domain from CAX1 is swapped into an N-terminally truncated CAX3 construct (to give sCAX3-9), this “activated” CAX3 mutant can transport Ca2+ (21Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). The activation of CAX3 (sCAX3-α1) in the presence of CXIP1 demonstrated in Fig. 4 C did not require the CAX1 9-amino acid Ca2+ domain. The chimera sCAX3-α1, when coexpressed with CXIP1 in K667 yeast strains, demonstrated ΔpH-dependent 10 μm45CaCl2 uptake into microsomal vesicles (Fig.4 D). This H+/Ca2+ antiport activity was comparable to that measured for sCAX1 (data not shown). The sCAX1-CAX3 chimera sCAX1-9 (21Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar), with the 9-amino acid Ca2+domain removed and replaced with the equivalent 9-amino acid region of CAX3, was not able to strongly suppress the Ca2+sensitivity of K667; however, expression of CXIP1 + sCAX1-9 strongly suppressed the Ca2+ toxicity (Fig. 4 C). To determine whether this activation of CAX1 by CXIP1 is caused by a physical interaction between CXIP1 and the CAX1 N terminus, a yeast two-hybrid experiment was performed. CXIP1 coexpressed with the N terminus of CAX1 (Met37–Leu73 or Met1–Asn65) caused the lacZ gene in the Y190 yeast strain to be activated, indicating that a direct interaction between CXIP1 and the CAX1 N terminus had occurred (Fig.5 A). No color reaction occurred in the absence of CXIP1 or in the absence of the CAX1 N-terminal fragments. We have previously demonstrated that a synthetic peptide corresponding to the first 36 amino acids of the CAX1 N terminus is able to inhibit H+/Ca2+ transport mediated by N-terminally truncated CAX1 (sCAX1), but does not inhibit Ca2+ transport by other truncated H+/Ca2+ antiporters (13Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar). The interaction of this N-terminal peptide with CAX1 was mapped to include residues 56–62 within CAX1 (13Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar). We were interested in coexpressing CXIP1 and sCAX1 to determine whether the presence of CXIP1 would alter the amount of synthetic peptide needed to inhibit N-terminally truncated CAX1. A peptide concentration of 5 ± 1.5 μm was sufficient to inhibit 50% of 10 μmCa2+ transport activity mediated by sCAX1 (Fig.5 B) (13Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar), whereas 12 ± 2 μm peptide was required to inhibit 50% of sCAX1 transport in the presence of CXIP1 (Fig. 5 B). To determine whether the CXIP1 PICOT-HD is required for activation of full-length CAX1, we mutated two highly conserved regions within the PICOT-HD (Fig. 2). The conserved CGFS domain (amino acids 97–100) was changed to AAAA, and the SNWPT domain (amino acids 133–137) was changed to AAAAA. Growth of CAX1-expressing K667 cells on 200 mm CaCl2 was virtually absent in the presence of either of the CXIP1 mutants (data not shown). Northern blot analysis was used to assess the expression of CXIP1 mRNA in variousArabidopsis tissues. Hybridization with the CXIP1probe revealed expression of CXIP1 in all tissues, with abundant expression in leaves and low level expression in roots and flowers, compared with almost equal expression of CAX1 in leaf, stem, and flower tissue and very low expression in roots (Fig.6). When seedlings were exposed to different metals, there were alterations in the expression ofCXIP1. CXIP1 levels were modestly increased when Ca2+ was added to the medium and decreased when Na+, Mn2+, and Ni2+ were added to the medium (Fig. 6). Using a yeast-based approach to activate full-length CAX1, we have identified an Arabidopsis cDNA termedCXIP1 gene, encoding a predicted protein containing a PICOT domain (Figs. 1 and 2). This domain is highly conserved throughout evolution, and comparative sequence analysis indicates that the PICOT-HD is distinct from all protein domains (28Isakov N. Witte S. Altman A. Trends Biochem. Sci. 2000; 25: 537-539Google Scholar). The physiological function of the PICOT-HD has not been identified; and until this study, no plant gene with this motif has been functionally characterized. In this work, we have demonstrated that, in a yeast expression system, CXIP1 interacted with the N terminus of CAX1 to modify vacuolar H+/Ca2+ antiport activity. This study thus ascribes, for the first time, a function to a plant gene containing a PICOT-HD. Like all PICOT-HD-containing proteins, CXIP1 appears to be a soluble protein. Two other non-transmembranous proteins that activated CAX1 Ca2+ transport in yeast were also identified in this study (Table I). CXIP3 is identical to a previously characterized protein, A. thaliana FKBP15-2, a member of the FKBP-type immunophilin family (27Luan S. Kudla J. Gruissem W. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6964-6969Google Scholar), whereas CXIP4 is a novel protein of unknown function that appears to be unique to plants. We chose to confine this study to further analysis of CXIP1. Several proteins that contain a PICOT-HD also contain a Trx-HD, including Arabidopsis CXIP2, which weakly activated CAX4 Ca2+ transport (Fig. 3 B). Proteins containing the Trx-HD are important in a range of cellular process, including controlling the redox state of the cell (31Balmer Y. Buchanan B.B. Trends Plant Sci. 2002; 7: 191-193Google Scholar). In plant Ca2+ signaling, there have been some reports linking the plasma membrane redox state with release of Ca2+ from intracellular stores (32Long J.C. Jenkins G.I. Plant Cell. 1998; 10: 2077-2086Google Scholar); however, the mechanisms for transducing these signals have not been elucidated. The inability of CXIP2 to activate CAX1 suggests specificity in the CXIP1-CAX1 interaction. Three different experimental approaches suggest this specificity depends on the N terminus of CAX1. Two-hybrid analysis demonstrated an interaction between CXIP1 and the N terminus of CAX1 (Fig. 5 A). This interaction did not require the first 36 amino acids of CAX1, as a construct containing Met37–Leu73 interacted with CXIP1 in a manner indistinguishable from the CAX1 Met1–Asn65construct. The second approach utilized chimeric CAX3 constructs to demonstrate the importance of the N terminus of CAX1. Only the chimeric CAX3 construct containing Met37–Leu73 from CAX1, but lacking the first 36 amino acids of CAX1 (sCAX3-α1), was capable of being activated by CXIP1 (Fig. 4, C andD). The third approach utilized a competition experiment to suggest that CXIP1 binds to the N terminus of CAX1. Our previous studies have demonstrated that a synthetic peptide corresponding to amino acids 1–36 of CAX1 specifically inhibits Ca2+transport mediated by sCAX1 lacking these 36 N-terminal amino acids (13Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar). Furthermore, we showed that these 36 amino acids physically interact with amino acids 56–62 at the N terminus of CAX1 to facilitate autoinhibition of CAX1 (13Pittman J.K. Shigaki T. Cheng N.-H. Hirschi K.D. J. Biol. Chem. 2002; 277: 26452-26459Google Scholar). We hypothesized that if CXIP1 binds to this same region (amino acids 56–62) of CAX1, more peptide should be required to cause Ca2+ transport inhibition. Indeed, approximately double the peptide concentration was needed to inhibit 50% of sCAX1 transport activity in the presence of CXIP1 (Fig. 5 B). The requirement for the CXIP1 PICOT-HD in this interaction was inferred by the inability of mutant forms of CXIP1 to activate CAX1 (data not shown). However, at this time, we cannot rule out the possibility that these mutants perturb protein expression or stability. Computer analysis suggests that the PICOT-HD may form a globular topology, which includes three α-helices and intervening β-strands (26Rodriguez-Manzaneque M.T. Tamarit J. Bellı́ G. Ros J. Herrero E. Mol. Biol. Cell. 2002; 13: 1109-1121Google Scholar). It has been proposed that this structure can (i) form a transient intermolecular interaction; (ii) bind to substrates, regulators, or cofactors; or (iii) tether proteins to specific subcellular compartments. In the future, it will be interesting to further delineate the mechanism of CXIP1-mediated CAX1 regulation. We suggest that CXIP1 may activate CAX1 through a direct interaction that disrupts autoinhibition and thus alters the confirmation of autoinhibited CAX1 to allow Ca2+ transport. However, the Ca2+ transport mediated by CXIP1-activated CAX1 is much less than that of deregulated sCAX1 (Fig. 1). Evidence suggests that activation of CAX1 by N-terminal truncation (as with sCAX1) is artificial and does not occur in planta (11Cheng N.-H. Pittman J.K. Barkla B.J. Shigaki T. Hirschi K.D. Plant Cell. 2003; (in press)Google Scholar), and we believe that the strong Ca2+ transport activity mediated by sCAX1 may not be realized by fully activated CAX1. For example, Ca2+ transport mediated by a version of full-length CAX1 activated by point mutations within the N terminus is less than that meditated by sCAX1.1 Similarly, Ca2+ transport mediated by the calmodulin-activated ArabidopsisCa2+-ATPase ACA2 is significantly less than that mediated by an N-terminally truncated version of ACA2 lacking its calmodulin-binding autoinhibitory domain (33Hwang I. Harper J.F. Liang F. Sze H. Plant Physiol. 2000; 122: 157-168Google Scholar). However, the level of H+/Ca2+ antiport activity measured for CXIP1-activated CAX1 is still much less than the activity measured inArabidopsis tissue. It is possible that additional protein interactions, such as with a multiple protein complex, are required to fully activate CAX1. Other proteins may be required to interact directly with CAX1 or cooperatively with CXIP1, or CXIP1 itself may require additional regulation. Interestingly, it appears that CXIP1 is able to strongly activate some chimeric CAX constructs. The level of Ca2+ transport measured in yeast vesicles expressing CXIP1-activated sCAX3-α1 was equivalent to the level of activity in vesicles expressing sCAX1 (Fig.4 D). Our previous findings show that the sCAX1-CAX3 chimera sCAX1-9 is unable to transport Ca2+ (21Shigaki T. Cheng N.-H. Pittman J.K. Hirschi K. J. Biol. Chem. 2001; 276: 43152-43159Google Scholar). This construct contains an N-terminal truncation, but also contains the 9-amino acid region from CAX3 (residues 87–95) that replaced the equivalent 9-amino acid Ca2+ domain of CAX1 and was thought to abolish function due to the inability of this CAX3 domain to allow Ca2+ transport. However, we show here that sCAX1-9 could also be activated by coexpression of CXIP1 and that this activity was equivalent to that of sCAX1 (Fig. 4 C). Previously, we demonstrated that truncations or additions to the N terminus of CAX1 or CAX4 can activate these Ca2+transporters in yeast (10Cheng N.-H. Pittman J.K. Shigaki T. Hirschi K.D. Plant Physiol. 2002; 128: 1245-1254Google Scholar). Similarly, CAX3 can also be activated by the addition of amino acids to the N terminus (10Cheng N.-H. Pittman J.K. Shigaki T. Hirschi K.D. Plant Physiol. 2002; 128: 1245-1254Google Scholar). These findings imply that any protein binding to the N-terminal region could activate CAX1 transport in yeast by altering the autoinhibitory region or the conformation of the transporter. Thus, although CXIP1 clearly interacts with CAX1 at the N terminus in our various yeast assays, we cannot discriminate between gene products that activate or repress CAX1 transport in plants. The expression pattern of CXIP1RNA mirrors the expression pattern of CAX1in plants (Fig. 6). That is, they were both expressed in all tissues; both were modestly induced by exogenous Ca2+; and both were repressed by certain ions. Interestingly, aside from the low level expression of CXIP1in flowers, the gene appears to be expressed at higher levels thanCAX1. The expression patterns of CXIP3(A. thaliana FKBP15-2) and CXIP4were both also induced by Ca2+ and regulated by different ions (data not shown); however, the expression levels and tissue distribution of these genes differed from those of CXIP1, suggesting that thoseCXIP genes might play different roles in the regulation of CAX1 activity in planta. The use of yeast to reconstitute a plant response pathway has been achieved with genes involved in Na+ homeostasis (34Quintero F.J. Ohta M. Shi H. Zhu J.-K. Pardo J.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9061-9066Google Scholar). However, these genes were first identified using standard genetic approaches (35Zhu J.-K. Curr. Opin. Plant Biol. 2001; 4: 401-406Google Scholar). Here we have demonstrated that CXIP1, but not its homolog CXIP2, can regulate the Ca2+ transport activity of CAX1 in yeast. Furthermore, CXIP1 and CXIP2 may be involved in the regulation of another putative H+/Ca2+antiporter, CAX4. Indeed, we have previously proposed that each CAX transporter may be regulated by different pathways (30Pittman J.K. Sreevidya C.S. Shigaki T. Ueoka-Nakanishi H. Hirschi K.D. Plant Physiol. 2002; 130: 1054-1062Google Scholar). We speculate that multiple components (or CXIPs) are required for regulation of CAX1 and other plant CAX transporters and that each CXIP may differ in its interaction with CAX1. Future work using reverse genetics inArabidopsis will help clarify the contribution of CXIPs to CAX-mediated H+/Ca2+ transport. We thank Sherry LeClere and Jon Pittman for comments." @default.
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• W2035539991 title "Cloning and Characterization of CXIP1, a Novel PICOT Domain-containing Arabidopsis Protein That Associates with CAX1" @default.
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