S406ThePlant CellH+/Ca2+-antiport activity conducted with oat root vacuolesCalciumExchangers(Schumaker and Sze, 1986).The ionic specificity of CAX1 isH+/Ca2+ antiporters can in principle drive "uphill" transportpartially determined by a 9-amino-acid sequencefollowing theof Ca2+,in which a proton motive force is maintained, al-first predicted transmembrane domain (Shigaki et al., 2001).though in plants this usually requires an H+/Ca2+ stoichiom-In Arabidopsis, there are 12 genes predicted to encodeetry of at least three (Blackford et al., 1990). The first plantantiporters closely related to CAX1 (Maser et al., 2001; http://H+/Ca2+antiportertobeclonedandfunctionallyexpressedplantst.sdsc.edu/plantst/html/2.A.19.shtml).Whetherallofthese related antiporters can transportcalciumhas notbeenwasCAX1(calciumexchanger 1;Hirschietal.,1996;Hirschi,2001)and its projected membrane topology is shown in Fig-determined.CAX2appearstotransportMninadditiontoCaure 4A The gene was identified by its ability to restore(Hirschi, et al., 2000). Although CAX1 is expected to be lo-growth on high-Ca2+ media to a yeast mutant defective incalized to the plant vacuole, there is evidence for H+/Ca2+vacuolarCa2+transport.CAX1appearstotransportCa2+withantiporters in other locations, such as the plasma mem-brane (Kasai and Muto,1990).The subcellular locations ofa low affinity (Km is ~13 μM), consistent with kinetic studies onAArabidopsis Calcium-Proton Exchanger (CAX1)H+Ca2+Auto-COOHInhibitorCytosolNE,B Arabidopsis Autoinhibited Calcium ATPase (ACAtype ATPase)410COOHCa2tCytosolAuto-VInhibitorAsp-PATP-bindingCaM-晨BindingTNH,Figure 4. Topology Models of Systems Catalyzing Ca2+ Efflux from the Cytosol.(A) Ca2+/H+ antiporters. The topology of CAX1 is based on speculation from hydropathy analyses. The number of transmembrane domains pre-dicted varies from eight to eleven. CAX1 has recently been shown to have an N-terminal autoinhibitor. The blue highlight indicates the position ofa 9-amino-acid sequence implicated in providing transport specificity for cations.(B) Ca2+-ATPase (ACA type). The topology of calcium pumps is well-supported by homology modeling based on a crystal structure of a mam-malian sacro(endo)plasmic reticulum-type Ca2+ ATPase pump. ECA-type calcium pumps are predicted to have similar topologies, but lack thedistinguishing feature of an N-terminal auotoinhibitor and calmodulin binding site
S406 The Plant Cell Calcium Exchangers H/Ca2 antiporters can in principle drive “uphill” transport of Ca2, in which a proton motive force is maintained, although in plants this usually requires an H/Ca2 stoichiometry of at least three (Blackford et al., 1990). The first plant H/Ca2 antiporter to be cloned and functionally expressed was CAX1 (calcium exchanger 1; Hirschi et al., 1996; Hirschi, 2001) and its projected membrane topology is shown in Figure 4A. The gene was identified by its ability to restore growth on high-Ca2 media to a yeast mutant defective in vacuolar Ca2 transport. CAX1 appears to transport Ca2 with a low affinity (Km is 13 M), consistent with kinetic studies on H/Ca2-antiport activity conducted with oat root vacuoles (Schumaker and Sze, 1986). The ionic specificity of CAX1 is partially determined by a 9-amino-acid sequence following the first predicted transmembrane domain (Shigaki et al., 2001). In Arabidopsis, there are 12 genes predicted to encode antiporters closely related to CAX1 (Maser et al., 2001; http:// plantst.sdsc.edu/plantst/html/2.A.19.shtml). Whether all of these related antiporters can transport calcium has not been determined. CAX2 appears to transport Mn in addition to Ca (Hirschi, et al., 2000). Although CAX1 is expected to be localized to the plant vacuole, there is evidence for H/Ca2 antiporters in other locations, such as the plasma membrane (Kasai and Muto, 1990). The subcellular locations of Figure 4. Topology Models of Systems Catalyzing Ca2 Efflux from the Cytosol. (A) Ca2/ H antiporters. The topology of CAX1 is based on speculation from hydropathy analyses. The number of transmembrane domains predicted varies from eight to eleven. CAX1 has recently been shown to have an N-terminal autoinhibitor. The blue highlight indicates the position of a 9-amino-acid sequence implicated in providing transport specificity for cations. (B) Ca2-ATPase (ACA type). The topology of calcium pumps is well-supported by homology modeling based on a crystal structure of a mammalian sacro(endo)plasmic reticulum-type Ca2 ATPase pump. ECA-type calcium pumps are predicted to have similar topologies, but lack the distinguishing feature of an N-terminal auotoinhibitor and calmodulin binding site.
CalciumattheCrossroadsofSignalingS407all twelve Arabidopsis CAX1-related isoforms still need toACA2 (ER; Liang et al., 1997; Hong, et al., 1999) and ACA4bedetermined(small vacuoles; Geisler et al., 2000b). In addition, evidenceRecently,the activityofCAX1wasshown tobe regulatedbysuggests that ACA1 is located in the plastid inner envelopeanN-terminalautoinhibitor(PittmanandHirschi,2001).Theex-membrane (Huang et al., 1993).Thesubcellularlocationspression of a "deregulated" CAX1 in tobacco resulted in plantshave not been determined for ACA7, 9, 10, 11, 12, and 13.with increased accumulation of Ca (Hirschi, 1999), consistentwiththeincreased activity ofanantiporterthatfunctionstosequester calcium into an endomembrane compartment. In-Functional Overlap?terestingly,the plants displayed growth phenotypes that mim-icked calcium deficiency symptoms. In addition, the plantsGiven the probability of at least 26 calcium pumps and anti-displayed hypersensitivity to K and Mgand increased sensitiv-porters in Arabidopsis, it is likely that multiple efflux systemsity to various stresses, including cold. Thus, aspects of plantlocated in the same membrane system will be found.For ex-development and stress tolerance are dependenton regulationample, in vacuoles, there is evidenceforboth an H+/Ca2+an-of CAX1 activity.An important challenge is to understand howtiporter (such as CAX1p) and an autoinhibited calcium pump,theactivityof CAX1and related antiporters is controlled.suchasACA4.IntheER,thereis evidencefortwo differenttypes of calcium pumps, ECA1andACA2.An importantchal-lenge is to delineate the specific and redundant functions forPumpseach of the different efflux pathways (Harper, 2001)CalciumpumpsbelongtothesuperfamilyofP-typeATPasesthat directly use ATP to drive ion translocation. Two distinctRegulationCa2+pumpfamilieshavebeenproposedonthebasisofpro-tein sequence identities (Geisler et al., 2000a; Axelsen andIt is now clear that transport activities are regulated forPalmgren,2001;http://biobase.dk/~axe/Patbase.html).Mem-CAX1 and most members of the ACA-type calcium pumps,bers of the type lIA and lB families, respectively, include theas indicated by experimental evidence and structural anal-ER-typecalciumATPases (ECAs)andtheautoinhibited cal-ogy.However, in plants, there is no experimental evidencecium ATPases (ACAs). The ACAs are distinguished fromfortheregulationofECAactivity.lntheory;theplantECAECAsbythreebiochemicalfeatures:1)Thepresenceofanpathwaymayprovideaconstitutive"house-keeping"activ-N-terminal autoinhibitor, 2) direct activation through bindingity that simply “cleans up" at a constant rate after any cal-Ca/calmodulin,and3)insensitivitytoinhibitionbycyclopiaz-cium release. Nevertheless, there are two rationales foronic acid and thapsigargin (Figure 4B; Sze et al., 2000). Inter-expecting some kind of regulatory control. First, the mostestingly,a pump showing mixed characteristics of both ECAclosely related ER-type calcium pumps in animal systemsand ACA pumps was identified in maize (Subbaiah and Sachs,are highly regulated. In animals, the activity of the sacro-2000).However,a corresponding"chimeric gene"has not(endo)plasmic reticulum-type Ca2+ ATPase pump is tightlybeen found in the Arabidopsis genome, suggesting that thisregulated by the phosphorylation status of an inhibitory sub-unusual pump is not common to all land plants.unit, phospholamban (East, 2000). In addition, there is evi-In Arabidopsis, there are four ECA- and ten ACA-type cal-dence thattheanimal ER-typepumps canbe regulated by aciumpumps(Axelsen andPalmgren,2001).IsoformECA1feedback system that maintains an appropriate Ca2+ loadappears to be located in the ER, as determined by mem-within the ER lumen (Bhogal and Colyer, 1998; Mogami et al.,brane fractionation and immunodetection (Liang etal.,1997;1998).Second, the observation in plants of theregulation ofHong et al., 1999). However, the potential for other isoformsbothCAX1and ACApathways supportsa speculation thattargetingto non-ER locations mustbe considered.In to-all major efflux pathways in plant cells are carefully regu-mato,there is evidence from membrane fractionation andlated. Assuming that ECAs are regulated, an important chal-immunodetection suggesting that related ER-type calciumlenge is to determine for each specific efflux pathway whetherpumps (LCA1-related) are present in the vacuolar andthe transporter's regulation is as a tuning mechanism toplasma membranes (Ferrol and Bennett, 1996)control the magnitude or duration of a calcium spike (i.e.,aTheACA subgroup ofplant Ca2+ATPases ismost closelysignalingfunction),orasfeedbackcontroltoadjustthedis-related to the plasma membrane-type pumps found in ani-tribution andlevels ofcalcium at thecell surfaceor indiffer-mals. However, they form a distinct subgroup distinguishedent endomembrane compartments (ie., a nutritional function).by two features: 1)a unique structural arrangement with theautoinhibitory domain at the N terminus instead of the C terminus, and 2) representatives that target to membranesDECODINGCALCIUMSIGNALSother than the plasma membrane (i.e., endomembranes).WhileACA8 has been found at the plasma membraneThe initial perception of a calcium signal occurs through the(Bonzaetal.,2000),asexpectedonthebasisoftheanimalbinding of calcium to many different calcium sensors. Sensorsparadigm,endomembrane locationshavebeenidentifiedfor
Calcium at the Crossroads of Signaling S407 all twelve Arabidopsis CAX1–related isoforms still need to be determined. Recently, the activity of CAX1 was shown to be regulated by an N-terminal autoinhibitor (Pittman and Hirschi, 2001). The expression of a “deregulated” CAX1 in tobacco resulted in plants with increased accumulation of Ca (Hirschi, 1999), consistent with the increased activity of an antiporter that functions to sequester calcium into an endomembrane compartment. Interestingly, the plants displayed growth phenotypes that mimicked calcium deficiency symptoms. In addition, the plants displayed hypersensitivity to K and Mg and increased sensitivity to various stresses, including cold. Thus, aspects of plant development and stress tolerance are dependent on regulation of CAX1 activity. An important challenge is to understand how the activity of CAX1 and related antiporters is controlled. Pumps Calcium pumps belong to the superfamily of P-type ATPases that directly use ATP to drive ion translocation. Two distinct Ca2 pump families have been proposed on the basis of protein sequence identities (Geisler et al., 2000a; Axelsen and Palmgren, 2001; http://biobase.dk/~axe/Patbase.html). Members of the type IIA and IIB families, respectively, include the ER-type calcium ATPases (ECAs) and the autoinhibited calcium ATPases (ACAs). The ACAs are distinguished from ECAs by three biochemical features: 1) The presence of an N-terminal autoinhibitor, 2) direct activation through binding Ca/calmodulin, and 3) insensitivity to inhibition by cyclopiazonic acid and thapsigargin (Figure 4B; Sze et al., 2000). Interestingly, a pump showing mixed characteristics of both ECA and ACA pumps was identified in maize (Subbaiah and Sachs, 2000). However, a corresponding “chimeric gene” has not been found in the Arabidopsis genome, suggesting that this unusual pump is not common to all land plants. In Arabidopsis, there are four ECA- and ten ACA-type calcium pumps (Axelsen and Palmgren, 2001). Isoform ECA1 appears to be located in the ER, as determined by membrane fractionation and immunodetection (Liang et al., 1997; Hong et al., 1999). However, the potential for other isoforms targeting to non-ER locations must be considered. In tomato, there is evidence from membrane fractionation and immunodetection suggesting that related ER-type calcium pumps (LCA1-related) are present in the vacuolar and plasma membranes (Ferrol and Bennett, 1996). The ACA subgroup of plant Ca2ATPases is most closely related to the plasma membrane–type pumps found in animals. However, they form a distinct subgroup distinguished by two features: 1) a unique structural arrangement with the autoinhibitory domain at the N terminus instead of the C terminus, and 2) representatives that target to membranes other than the plasma membrane (i.e., endomembranes). While ACA8 has been found at the plasma membrane (Bonza et al., 2000), as expected on the basis of the animal paradigm, endomembrane locations have been identified for ACA2 (ER; Liang et al., 1997; Hong, et al., 1999) and ACA4 (small vacuoles; Geisler et al., 2000b). In addition, evidence suggests that ACA1 is located in the plastid inner envelope membrane (Huang et al., 1993). The subcellular locations have not been determined for ACA7, 9, 10, 11, 12, and 13. Functional Overlap? Given the probability of at least 26 calcium pumps and antiporters in Arabidopsis, it is likely that multiple efflux systems located in the same membrane system will be found. For example, in vacuoles, there is evidence for both an H/Ca2 antiporter (such as CAX1p) and an autoinhibited calcium pump, such as ACA4. In the ER, there is evidence for two different types of calcium pumps, ECA1 and ACA2. An important challenge is to delineate the specific and redundant functions for each of the different efflux pathways (Harper, 2001). Regulation It is now clear that transport activities are regulated for CAX1 and most members of the ACA-type calcium pumps, as indicated by experimental evidence and structural analogy. However, in plants, there is no experimental evidence for the regulation of ECA activity. In theory, the plant ECA pathway may provide a constitutive “house-keeping” activity that simply “cleans up” at a constant rate after any calcium release. Nevertheless, there are two rationales for expecting some kind of regulatory control. First, the most closely related ER-type calcium pumps in animal systems are highly regulated. In animals, the activity of the sacro- (endo)plasmic reticulum-type Ca2 ATPase pump is tightly regulated by the phosphorylation status of an inhibitory subunit, phospholamban (East, 2000). In addition, there is evidence that the animal ER-type pumps can be regulated by a feedback system that maintains an appropriate Ca2 load within the ER lumen (Bhogal and Colyer, 1998; Mogami et al., 1998). Second, the observation in plants of the regulation of both CAX1 and ACA pathways supports a speculation that all major efflux pathways in plant cells are carefully regulated. Assuming that ECAs are regulated, an important challenge is to determine for each specific efflux pathway whether the transporter’s regulation is as a tuning mechanism to control the magnitude or duration of a calcium spike (i.e., a signaling function), or as feedback control to adjust the distribution and levels of calcium at the cell surface or in different endomembrane compartments (i.e., a nutritional function). DECODING CALCIUM SIGNALS The initial perception of a calcium signal occurs through the binding of calcium to many different calcium sensors. Sensors