P-Type Calcium Atpases in Higher Plants ± Biochemical, Molecular and Functional Properties

Biochimica et Biophysica Acta 1376Ž. 1998 1±25

P-type calcium ATPases in higher plants ± biochemical, molecular and functional properties

David E. Evans a,), Lorraine E. Williams b a School of Biological and Molecular Sciences, Oxford Brookes UniÕersity, Gipsy lane, Headington, Oxford OX3 0BP, UK b School of Biological Sciences, UniÕersity of Southampton, Bassett Crescent East, Southampton SO17 7PX, UK Received 28 July 1997; revised 31 October 1997; accepted 5 November 1997

Keywords: Calcium; ATPase; Sequence homology; SERCA; PMCA; Subcellular location;Ž. Plant

Contents

1. Introduction ...... 2

2. Subcellular locations of Ca2q-pumping ATPases in plant cells ...... 2 2.1. Plasma membrane ...... 2 2.2. Tonoplast ...... 4 2.3. Endoplasmic reticulum ...... 4 2.4. Plastids and other membranes ...... 5

3. Purification and reconstitution of Ca2q-pumping ATPases ...... 5

4. Inhibitor studies ...... 6

5. Molecular characterisation of putative plant Ca2q-pumping ATPases ...... 8 5.1. Homologues of mammalian SERCA Ca2q-pumping ATPases ...... 8 5.2. Ca2q-pumping ATPases containing an auto-inhibitory CaM binding domain ...... 12 5.3. Molecular characterisation of other cloned putative Ca2q-pumping ATPases ...... 14 5.4. Subcellular targeting of plant Ca2q-pumping ATPases ...... 16

X X Abbreviations: CaM, calmodulin; cAMP, cyclic 3 -5 adenosine monophosphate; CPA, cyclopiazonic acid; EB, erythrosin B; ER, endoplasmic reticulum; EST, expressed sequence tag; FITC, fluorescein isothiocyanate; GA, gibberellic acid; IM, intracellular membrane; 2q PCR, polymerase chain reaction; PIP2 , phosphatidyl inositol diphosphate; PM, plasma membrane; PMCA, plasma membrane Ca -pumping ATPase; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; SERCA, sarcoplasmic reticulumrendoplasmic reticulum Ca2q-pumping ATPase; SR, sarcoplasmic reticulum ) Corresponding author. Fax: q44 1865 483242; E-mail: [email protected]

6. Functions of plant Ca2q-pumping ATPases ...... 16 6.1. Ca2q-pumping ATPases and cell signalling ...... 16 6.2. Ca2q-pumping ATPases and secretion ...... 18 6.3. Putative role of Ca2q-pumping ATPases in plastids ...... 18

7. Regulation of plant Ca2q-pumping ATPases ...... 19 7.1. Regulation of Ca2q-pumping ATPase gene expression...... 20

8. Phylogenetic relationships of plant Ca2q-pumping ATPases ...... 21

9. Future perspectives: lessons from yeast ...... 22

Acknowledgements ...... 22

References ...... 22

1. Introduction 2. Subcellular locations of Ca2H-pumping ATPases in plant cells Plant and animal cells regulate cytosolic free calcium at sub-micromolar concentrationsŽ seewx 1,2 for Some of the apparent confusion in assigning 2q reviews. . This is achieved by a combination of active Ca -transport mechanisms to different subcellular Ca2q pumpsŽ. P-type ATPases and antiports ener- compartments is the result of the inherent residual gised by the primary ion-motive pumps of the cell, impurity of membranes from plant sources; it may together with regulated Ca2q-influx channels. In also indicate diverse locations in the range of cells plants, the primary motive force for the transport of and tissues studiedŽ. e.g. Tables 1 and 2 . most solutes is provided by trans-membrane proton pumps located at the plasma membrane and vacuolar 2.1. Plasma membrane membraneŽ. tonoplast ; this contrasts with the sodiumrpotassium systems of mammalian cellsŽ for Cytosolic calcium concentrations in plants are a review, seewx 1. . Recently, a variety of studies have maintained at or near 10y7 M against a steep trans- shown that plant cells contain several types of Ca2q- plasma membrane gradient Ž Ca2q s 45± pumping ATPase, including those possessing a 60 kJ moly1 wx 3,7. . It is not surprising, therefore, that calmodulin-binding autoinhibitory domain and those considerable effort has been expended in characteris- lacking such a domainwx 2±5 . Molecular and bio- ing calcium transport mechanisms at this membrane. chemical evidence indicates similarities, but also key Calcium transport assays using plasma membrane differencesŽ. particularly in subcellular locations be- purified by sucrose density gradients and aqueous tween plants and animals, e.g.wx 2±6 . two-phase partitioning indicated the presence of a It is the aim of this review to draw together current P-type Ca2q pump; studies were divided as to whether molecular and related data indicating the structure, it showed direct stimulation by calmodulinŽ see Table function and location of the various plant Ca2q pumps 1. . The pumps showed affinities for calcium appro- and to compare these with their homologues from priate for regulating cytosolic free calcium concentra- 2q other organisms. tions Ž K m Ca 0.4±10 M; see Table 1 and refer- D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 3

Table 1 Ca2q transporters identified in purified plasma membrane vesicles r 2q Species tissue KmmATP K Ca Calmodulin Estimated Nucleotide specificity pH Refer- stimulation relative optimum ence molecular mass Maize leaves 3.9 M 0.4 Mnrdnrdnrd 7.5wx 19 Red beet storage root 0.37M 1.24 M yesŽ. 2= 124,000 ATP Ž. 100 )GTPsITP Ž. 40 7±7.5wx 12 119,000 Wheat roots 0.1 mM 3 Mnrd ATPŽ. 100 )GTPsITP Ž. 40 7.4±7.8wx 65 Commelina communis L. 0.3 mM 4.4 Mnonrd ATPŽ. 100 )ITPsGTP 7.2wx 64 leaves Arabidopsis thaliana L. 60 M y yesŽ. 1.5= nrd ATP Ž.Ž. 100 )ITP 60 7.2±7.4wx 62 seedlingsŽ. ITP Radish seedlings 15M12M yCaM yesŽ. 1.5= nrd ATP Ž. 100 )GTP sITP Ž. 50 6.9±7.5wx 10 7M qCaM Cauliflower floret nrdnrdnrd 116,000 nrdnrd13wx Carrot suspension nrdnrd yes 127,000 ATPGGTP nrdwx 14,160 culture cells Bryonia dioica Jacq. 14 M10Mnrd ATPŽ. 100 )ITP Ž. 70 )GTP Ž. 50 7.2wx 118 tendrils Barley leaves nrdnrd yes 130,000 High specificity for ATP nrd15wx nrd ± not determined. ences therein. , similar to that shown by the mam- specificity, being energised by the hydrolysis of GTP malian plasma membrane PM Ca2q -ATPaseŽ. PMCA and ITP as well as ATPŽ in contrast to the mam- Ž1.5 M qCaM; 20 M yCaMwx 8. . In all cases malian PMCA which is specific for ATPwx 9. . This Ca2q transporters at the PM showed a low nucleotide property has been used by a number of authors

Table 2 Ca2q transporters identified in tonoplast r 2q Species tissue Mechanism KmmATP K Ca Calmodulin Estimated pH Refer- stimulation relative optimum ence molecular mass Carrot cells Ca2q proton antiport 0.1 mM 21 Mnrdnrd 7.0wx 161 Žsuspension culture. Ca2q -ATPase nrdnrd yes 120,000 nrd14wx Oat roots Ca2q proton antiport 0.24 mM 14 Mnrdnrd 6±7 and 8wx 162±164 Sugar beet Ca2q proton antiport nrd10M yes nrdnrdwx 165 Ž.taproot Maize roots Ca2q proton antiport; nranrdn no nrdnrdwx 166 Ca2q ATPase 0.41 mM nrdnrdnrdnrd24wx Maize roots Ca2q ATPase nrdnrd yesŽ. 3±5= nrdnrdwx 167 Apple fruit Ca2q ATPase nrd43M yesŽ. slight nrd 6.7wx 168 Cauliflower Ca2q ATPase nrd2M qCaM yes 111,000 7.2wx 6 florets nrd yCaM Tomato fruit Ca2q ATPase nrdnrd no 116,000 nrd26wx nrd ± not determined. 4 D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 biochemically to differentiate the PMCA from the A putative Ca2qrHq antiporter from Arabidopsis Hq pump in plantswx 10,11 . Assignment of a relative Ž.CAX1 which shows 33% identity to the yeast molecular mass to the phosphorylated intermediates Ca2qrHq antiporter, VCx1p has recently been cloned of these pumps indicates a monomer smaller than that wx25 . The biochemical properties of CAX1Ž as deter- of the mammalian PMCA, of between 115,000 and mined from transport studies on yeast vacuolar mem- wx 130,000 Mr 12±15 . Given that proteolytic cleavage branes following expression of the carrier in yeast. , of the calmodulin-binding domain is likely to result are similar to the tonoplast Ca2qrHq antiporter de- in the presence of a fully active pumpwx 16,17 and scribed in other plant species. CAX1 does not trans- that high levels of endogenous calmodulin are present port Mg2q wx 25 . in plasma membranewx 18 it is difficult to ascertain There is increasing evidence suggesting the pres- whether the plant PM Ca2q pump is normally regu- ence of a P-type Ca2q pump at the tonoplastŽ Table lated by calmodulin in vivo or whether the data 2. . Pfeiffer and Hagerwx 24 showed evidence for an presented in Table 1 represents species and or tissue ATP-dependent, vanadate-sensitive Ca2q pump in differences with both calmodulin-binding and non- isolated tonoplast vesicles and argued that this high- calmodulin-binding Ca2q pumps present at the plant affinity Ca2q-ATPase was the only functioning Ca2q PM. In addition, the presence of a calciumrproton transporter under in vivo conditions. In cauliflower antiport at the plasma membrane has been suggested florets it has been shown that a putative tonoplast in studies on maizewx 19,20 . This antiport may func- pump is calmodulin-regulated, with an affinity for tion as a low affinity efflux mechanism using the calcium in the presence of calmodulin comparable energy of the PM Hq pump in an analogous fashion with that of the plasma membrane Ca2q pumpŽ 2 M; to the mammalian NaqrCa2q exchangerwx 20 . wx6 and see Table 2. . This pump has been assigned a relative molecular mass of 111,000 and it can be 2.2. Tonoplast purified by calmodulin-affinity chromatographyŽwx 6

and see below. . A similar pump of 120,000 Mr has The vacuolar free calcium concentration is also also been indicated at the tonoplast of carrotwx 14 much higher than cytoplasmicŽ ca. 10y32 M; Ca q whereas in tomato tonoplast there is evidence for a 28±34 kJ moly1 ;wx 3,21,22. and many studies have non-calmodulin-stimulated P-type Ca2q pump of wx been carried out using isolated tonoplast vesicles to 116,000 Mr 26 . characterise the mechanism of calcium transport at this membrane. Early studies on the vacuolar mem- 2.3. Endoplasmic reticulum brane highlighted the presence of a calciumrproton antiport of lower apparent affinity for calcium than The endoplasmic reticulum in plants, as in ani- the P-type Ca2q pumps. The biochemical properties mals, is believed to act as an important signalling described for the tonoplast antiporter characterised in pool of calcium within the cellwx 3 . Lumenal calcium purified tonoplast fractions are presented in Table 2; concentrations have been shown to be elevated com- evidence for its role in the accumulation of Ca2q in pared with cytosolic, and while it is difficult to the vacuole has been presented by Blackford et al. quantify this accuratelyŽ seewx 3. , it is estimated that wx23 . Pfeiffer and Hager wx 24 have demonstrated a the lumenal concentration is at least 10= cytosolic Ca2qrHq antiporter at the tonoplast membrane of and probably greaterŽ e.g.wx 27. . In addition, the maize roots which also has an affinity for magne- lumen of the plant ER has been shown to contain sium; they argue that because of the differences in homologues of mammalian calcium binding proteins, concentration of these divalent cations in the cyto- including calnexinwx 28,29 , calsequestrin w 30±33 x and plasm under physiological conditions, the antiporter calreticulinwx 34±36 . is likely to function as a Mg2qrnHq exchanger The presence of a P-type Ca2q pump located at the rather than a calcium transporter and may be respon- plant ER has been suggested by transport studies with sible for maintenance of cytosolic magnesium home- ER vesicles. The properties assigned by these means ostasis. This interesting possibility requires further to plant ER Ca2q pumps are outlined in Table 3. investigation. Once again, the ER pumps appear to show affinities D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 5

Table 3 Ca2q transport identified in purified endoplasmic reticulum r 2q Species tissue Km Ca Calmodulin Nucleotide specificity Estimated Reference stimulation relative molecular mass Red Beet storage root 7.5 Mnonrd 96,000wx 41 Red Beet storage root 0.92 Mnrd ATPŽ. 100 ; GTP,ITP Ž. 10 119,000wx 12 Carrot suspension culture cells nrd yesŽ. 3±4= ATP Ž.Ž. 100 ; GTP 80 120,000wx 12,39 Barley aleurone layers nrd yesŽ. 2= nrdnrd43wx Maize coleoptiles nrd yes ATPŽ. 100 ; GTP, ITP Ž-10 . 102,000wx 37 Maize coleoptiles nrd yes nrd 140,000wx 42 nrd ± not determined. for calcium appropriate for regulating cytosolic free Ca2q pumpŽwx 43 ; for further details see ``Functions 2q 2q calcium Ž K m Ca 0.92±7.5 M; see Table 3. . Of of plant Ca pumps: secretion'' below. . the studies of nucleotide specificity carried out, two suggest a much higher specificity for ATP than the 2.4. Plastids and other membranes plant PM pumpwx 12,37 , similar to that for the mam- Micromolar levels of free calcium have been re- malian PM pumpwx 9 , though others w 14,38,39 x indi- ported in plastidswx 45 and transient changes in cate lower nucleotide specificity. Studies by phospho- q Ca2 may occur during light and dark transitions rylated intermediate formation and immuno-detection as a result of photosynthesiswx 3 . A putative plastid have generated a wide range of apparent M values, q r P-type Ca2 pump has been cloned using an antibody ranging from 96,000wx 40,41 to 140,000 wx 42 ; once raised to chloroplast envelope proteins and an anti- again, proteolytic cleavage, species and tissue differ- body specific to the pump recognises a protein in the ences and less than adequate membrane purification inner envelopeŽwx 46,47 ; see below for detailed dis- may explain this range. 2q cussion. . Biochemical studies have also indicated the In a study of the Ca -transport properties of red q presence of a calmodulin-binding Ca2 -ATPase in beet membranes, Thomson et al.wx 12,44 showed some 2q the plastid envelope which was suggested to be in- differences in the properties of Ca transporters in q volved in Ca2 exportwx 48,182 although the trans- highly purified ER and PM. The main difference was q ported ion and direction of transport was not deter- in the substrate specificity for Ca2 transport be- mined. Transport of calcium by membranes other tween the two membranes. Phosphorylated intermedi- q than the endoplasmic reticulum, tonoplast and plasma ate formation also revealed two putative Ca2 pumps membrane was reviewed by Evans et al.wx 2 . Since of closely similar M ; one of 119 kDa was present at r then, no further evidence has appeared indicating the both ER and PM while a second of 124 kDa was presence of P-type calcium pumps on membranes located at the PM only. While previous studies on other than those already discussed. It should be noted this tissue using less rigorously purified membranes 2q q that this does not imply that P-type Ca pumps are had suggested a calmodulin-stimulated Ca2 pump in absent from membranes such as the Golgi apparatus; the PMwx 10 , unequivocal demonstration of CaM- rather that their calcium transport properties have yet sensitivity was not obtained for either membrane. No to be adequately explored. evidence exists for a calciumrproton antiport at plant ER. Involvement of a P-type Ca2q pump, regulated by calmodulin, has been shown in germinating barley 3. Purification and reconstitution of Ca2H-pump- aleurone layers. Here, the stabilisation of the secreted ing ATPases enzyme -amylase requires the presence of large amounts of calcium within the ER lumen; this is The presence of vanadate-inhibited P-type Ca2q achieved by up-regulation of a calmodulin-stimulated pumps has been confirmed in several species and 6 D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25

Table 4 Properties of purified Ca2q pumps r 2q Species membrane KmmATP K Ca Calmodulin Relative Reference binding molecular mass Cauliflower floretŽ. vacuolar nrd7M yes 111,000wxw 6a 51 x Zea mays L.Ž. microsomes nrdnrd yes 140,000wx 42 a Corydalis semperÕirens Ž.microsomes 5.2 M 2.3 Mnonrd57wx Commelina communis Ž.PM 11.4 M 5.2 Mnonrd55wx Zea mays L.Ž. PM nrdnrdnonrd56wx Daucus carota L.Ž. microsomes nrdnrd yes Ž. weakly 120,000wx 14 Hordeum Õulgare L.Ž. PM nrdnrd yes 130,000wx 15 nrd ± not determined. a Functionally reconstituted.

from different membrane types by purification and Mr protein on native PAGE and appeared as a 65,000 functional reconstitutionŽ. Table 4 . The presence of Mr polypeptide on SDS-PAGE. Further information calmodulin-binding pumps was first suggested by on this enzyme is not available. Dieter and MarmeÂ wx 49 by calmodulin affinity chro- Plant Ca2q-pumping ATPases have also been puri- matography of solubilised microsomal membrane fied without recourse to calmodulin binding, two protein. In two instanceswx 42,51 calmodulin-binding from plasma membrane fractions and one from mi- ATPases have been purified from mixed microsomal crosomal membraneswx 55±57 . None of these three membranes and Ca2q-transport activity confirmed by pumps show calmodulin-binding or activation, al- functional reconstitution into proteoliposomes. Rela- though given the potential lability of calmodulin-bi- tive molecular mass values of 140,000Ž maizewx 50,52 . nding domains this is not conclusive evidence of its and 115,000Ž cauliflower floretswx 51. were assigned absence in vivo. In no case has a relative molecular by phosphorylated intermediate formation. Affinity mass been assigned to these activities. chromatography of a lightŽ. tonoplast membrane fraction from cauliflower has resulted in detection of a 2q wx Mr 111,000 Ca -ATPase 53Ž though this may be the 115,000 M pump previously reported;wx 6,51. . A r 4. Inhibitor studies recent study of barley cell membraneswx 15 has indicated the presence of a CaM-stimulated Ca2q-ATPase of ca. 130,000 Mr at the plasma membrane which Attempts to use inhibitors to distinguish between could be purified by CaM-affinity chromatography, various Ca2q-transporting ATPases have proved 2q together with a Ca -ATPase of 116,000 Mr in intra- largely unsuccessful since there are, as yet, no reli- cellular membranes which was stimulated by calmod- able specific inhibitors for different types of Ca2q ulin but was not detectable by calmodulin overlay. pumps in plants. Inhibitors such as cyclopiazonic acid The authors suggest that this indicates that the intra- Ž.CPA and thapsigargin are used routinely in well cellular membrane Ca2q-ATPase has a lower affinity characterised animal systems for specifically inhibit- for calmodulin; however, as indicated above, the ing the SRrER-type Ca2q pumpwx 58,59 and distin- intracellular membrane calmodulin-stimulated Ca2q- guishing it from the PM-type. However, there are ATPase of Brassica oleracea, which is of similar very few studies in plant systems where the effects of relative molecular mass, may be affinity purified. these inhibitors have been characterised in detailwx 44 . Purification of calmodulin-binding ATPase has also CPA, an indole tetramic acid mycotoxin, inhibits the r 2q been achieved using chloroplast envelope membranes mammalian SR ER Ca -ATPase with an I50 of wx48,54 ; this activity was found to behave as a 260,000 about 0.25 mmol my3 wx 58,183 . In carrot cells a 10- D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 7 fold higher concentration is required to inhibit Ca2q- higher concentrations of thapsigargin may be related transport activitywx 39 and in red beet there was no to differences in the hydrophobicity of the two types effect on Ca2q transport or on the formation of of membranes. It is possible that the stoichiometric phosphorylated intermediates unless much higher interaction of some of these inhibitors with particular concentrations were employedwx 44 and in this situa- Ca2q pumps may become apparent only upon solubil- tion proton transport by Hq-ATPases at the tonoplast isation and reconstitution of the enzyme; however, in and plasma membrane were also affectedwx 43 . In a the case of the reconstituted calmodulin-stimulated more recent study, Hwang et al.wx 14 concluded that Ca2q pump from Brassica oleracea only very low CPA inhibited the non-calmodulin-stimulated, ER-as- sensitivity to CPA and thapsigargin was observed sociated Ca2q-transport activity more specifically than wx51 . Ca2q transport by other Ca2q pump types but the Although Erythrosin BŽ. EB has been used to inhibition was fairly low. Therefore it is perhaps inhibit the PM Ca2q-ATPase in various plant sys- prudent to treat any conclusions reached from the use tems, the concentration dependence of inhibition of of this inhibitor in intact systemswx 60 with caution the ER calcium pump has only been investigated in a until further studies regarding its specificity are car- few instances. In red beet, results indicate that pumps ried out. in ER and PM fractions are both sensitive to this -3 wx The specificity of thapsigargin action in animal inhibitor Ž I50 of about 22 mol m ; 44. . In other cells is believed to be conferred by a recognition site studies comparing the effect of this inhibitor on PM for the inhibitor on all the SRrER ATPases that is and ERrvacuolar Ca2q pumps, the PM transporter absent from the PM Ca2q-ATPase and other P-type was slightly more sensitivewx 6 . The effect of fluores- ATPaseswx 61 . This hydrophobic reagent, as well as cein derivatives on the plasma membrane Ca2q pump other related compounds such as trilobolide, are of Arabidopsis thaliana have been comparedwx 62 . thought to interact with the E2 conformation of the The three fluorescein derivatives testedŽ EB, eosin Y ATPasewx 61 . At the concentrations used to inhibit and rose bengal. were much more effective in inhibit- SRrER Ca2q-ATPases in mammalian systems ing the Ca2q pumpŽ 50% inhibition by 5± Ž0.2 mmol my3. these inhibitors have no effect on 100 mol my3. than the Hq -ATPaseŽ 50% inhibition plant Ca2q -transport activity investigated to datewx 44 . by 5 to 50 mmol my3. . Therefore, EB may also be At higher concentrations, however, thapsigargin inhi- used for discriminating between calcium and proton bition has been observed. In red beet, it was consis- pumps and also for distinguishing primary calcium tently observed that at concentrations above pumps from secondary calcium transport mechanisms 15 mmol my3, it was a more potent inhibitor of the Že.g. Ca2qqrH antiport. when used at nanomolar transport activity of the Ca2q pump in ER-enriched concentrationswx 12,44 . fractions than that in PM fractions. However, at the Vanadate is a general inhibitor of E12 E -typeŽ P- higher concentrations, proton transport due to the type. ATPases and in red beet the vanadate sensitiv- tonoplast and PM Hq-ATPases was also affected. ity of the ER and PM Ca2q-ATPases was very similar Therefore, it was concludedwx 44 that in red beet the over the 0 to 250 mmol m-3 rangeŽ 50% inhibition of general non-selective action at high concentrations of ATP-driven Ca2q transport by around 100 mmol my3; some of these SRrER Ca2q-ATPase inhibitors wx44. . Vanadate is thought to inhibit P-type ATPases ŽŽthapsigargin, trilobolide, nonylphenol, BHQ 2,5- by blocking the shift in conformation states during diŽ.tert-butyl -1,4-benzohydroquinone . and AHQ the reaction cycle of these enzymeswx 63 . It is thought

ŽŽ2,5-di tert-amyl . -1,4 benzohydroquinone .. may be to bind to the enzyme and trap it in its E2 conforma- due to their highly hydrophobic nature. Such com- tion. There are marked variations in the values re- pounds may partition selectively into the phospho- ported for vanadate sensitivity of plant Ca2q pumps lipid component of membranes andror directly inter- wx24,57,64,65 and this may be due to species differ- act with the hydrophobic domains of membrane pro- ences or relate to the degree of purity of particular teins, thereby affecting lipid±protein interactions and membrane fractions or to the treatment conditions. general ATPase activity. The differences in apparent Lanthanum is another inhibitor which has been sensitivities of the ER and PM Ca2q pumps to the used in several studies of plant Ca2q-ATPases but its 8 D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 specificity and mechanism of action is not entirely SERCA homologues. Liang et al.wx 71 have recently clear. In animal cells it is thought to block the obtained data providing compelling evidence that one dephosphorylation of the phosphoenzyme in the reac- of the plant SERCA-type homologuesŽ. ECA1 is in tion cycle and result in an increase in its net equilib- fact a Ca2q-ATPase. ECA1 contains an open reading rium level, a reduction in turnover of the Ca2q- frame of 1061 amino acids with a deduced molecular ATPase and a reduction in calcium transportwx 66 . mass of 116 kD. It was also shown to complement Results obtained investigating the Ca2q-ATPase in yeast mutants which were defective in particular Ca2q carrot suspension cultured cells suggest a similar pumps and the gene product, ECA1p formed a cal- mechanism of action since lanthanum increased the cium-dependent phosphorylated intermediate as part levels of the phosphorylated intermediatewx 67 , results of the reaction cycle. Based on immunoblotting of similar to those obtained previously for a Arabidopsis membrane fractions with an antibody to calmodulin-stimulated calcium pump in maizewx 52 . ECA1p, the protein was predominantly localised on ER membranes. Interestingly, a role in manganese transport was also suggested, since the phosphorylated intermediate was also observed in the presence 5. Molecular characterisation of putative plant of this cationwx 71 . It should be stressed at this point Ca2H-pumping ATPases that although we will refer to the other plant SERCA homologues from hereon as Ca2q-ATPases, there are q no functional data available to date which verify this Several putative Ca2 -ATPases have now been and the discussion on them here is based purely on cloned including those showing homology to mam- sequence analysis. malian SERCAs and also those with a putative q For the tomato Ca2 -ATPaseŽ. LCA , both a partial CaM-binding domain. The major properties of these cDNAŽ. 1.8 kb and full-length genomic clone different pump types are discussed below. Ž.10.3 kb have been isolatedwx 68 . The deduced amino acid sequence of LCA specifies a polypeptide with a predicted molecular mass of 116 kD. According to 5.1. Homologues of mammalian SERCA Ca2q-pump- the nomenclature adopted by Lutsenko and Kaplan ing ATPases wx 74 , it appears to be a P2 -type ATPaseŽ non-heavy- metal-transporting P-type ATPase. . For a description The first nucleotide sequences published for such of structure we have based our discussion on the SERCA homologues in higher plants were for tomato structural model adopted by Mùller et al.wx 75 for a Ž.LCA and tobacco Ž pH27 . ,wx 68,69 and a homologue Type IIa ATPase since this is the category in which Ž.ECA1 has also been found in Arabidopsis wx70,71 . the SERCA-type ATPases have been placed. In this The cloning of another SERCA homologue in rice model, 10 transmembrane segments are present with Ž.OSCa-ATPase indicates that these pumps are pre- two major cytoplasmically located domains. Most of sent in both monocots and dicotswx 72 . In addition, a the hydrophilic regions are thought to protrude into P-type ATPaseŽ. DBCA1 showing homology to the the cytoplasmic side of the membrane with little of SERCAs has also been cloned from the halotolerant the polypeptide chain exposed to the other side. The alga Dunaliella bioculata wx73 . The percentage ho- exact number of transmembrane domains in each of mologies between the plant SERCA homologues and the P-type ATPases is still debateable and it is thought several P-type ATPases are shown in Table 5 and this to vary between 8±10 for different members of the clearly shows that the tomato, tobacco, Arabidopsis, family. From hydropathy analysis, eight transmem- rice and Dunaliella sequences show high homology brane domains have been suggested for LCA and to each other and also to animal SERCA-type AT- OsCa-ATPasewx 68,72 and 8±10 for the DBCA1, the Pases. Much lower homologies are observed for a SERCA homologue of Dunaliella wx73 . Hydropathy range of other P-type ATPases. analysis of the translated partial pH27 sequence from To date, functional data indicating a role in Ca2q tobacco indicated six potential membrane-spanning transport has only been obtained for one of the plant regions that coincide with the positions of the last six D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 9 ty of n in the Ž GenBank databases r Ž.Ž . . wx Please refer to 46,47,68,69,71±73,96,98,99,156,169±181 for further detail. non-shaded boxes in the lower triangle were obtained using the GAP programme. This is available from the Genetics Computer Group programmes Universi Table 5 Sequence homologies between a range of P-type ATPases. The % identities indicated in the shaded boxes in the upper triangle and the % similarities show Wisconsin, USA via the Seqnet facility at Daresbury Laboratory, Daresbury, UK. Sequences were taken from EMBL 10 D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 potential membrane-spanning regionsŽ. M5±M10 of regions are highly conserved while others show lower the mammalian SERCAswx 69 . However, it was ob- levels of homology. In LCA, the highly-conserved served that the pH27 sequence ends within the M10 motifs characteristic of all P-type ATPases are pre- region of the other SERCAswx 69 and it was sug- sentŽ seewx 75 for detailed description of these re- gested from the hydropathy plot that in pH27 this gions. . These include the PGD, PAD and TGES region may be too small to be a transmembrane motifs of the small cytosolic loop Ž-strand or trans- domain. Further evidence is required to confirm the duction domain. . The small cytosolic loop is pre- transmembrane helix predictions for the plant SERCA dicted to consist mainly of beta-sheets and contains a homologues. conformation-sensitive tryptic split in the mammalian The relative molecular masses of the animal SER- SERCAs. It has been suggested to have an important CAs are in the range 100±110 kDawx 76 . As men- role in energy transduction in P-type ATPases al- tioned previously the predicted molecular mass of the though this has not been clearly definedwx 75 . Muta- tomato Ca2q-ATPase from the sequence data is genesis studies on mammalian SERCAs have pro- 116 kD and immunoblotting of membrane fractions vided support for its proposed function in conforma- with an antibody raised to LCA shows a reaction tional transitionswx 75 . Also conserved is the PEGL with proteins of 116 and 120 kDwx 26 . The predicted motifŽ associated with transmembrane segment 4 in molecular mass of ECA1 is also 116 kDwx 71 , though non-heavy-metal ATPases. ; the proline is highly con- an antibody to the C-terminus recognised a protein of served among cation-transporting ATPaseswx 82 . The 106 kD in Arabidopsis membraneswx 71 . In a separate plant homologues also contain the conserved catalytic study, an antipeptide antibody to another region of phosphorylation siteŽ. DKTGT containing the phos- the same sequence has been shown to detect a protein phate accepting aspartate which is phosphorylated by of 116 kD in Arabidopsis membranesŽ Mills and ATP during Ca2q transport. In the SERCAs the Williams, unpublished data. . aspartateŽ Asp351. is indispensible for phosphoryla- A hypothetical model of the tertiary structure of tion but the enzyme can accommodate minor changes the animal SERCAs has been put forward based on in the immediate vicinity of the phosphorylation site structural and biochemical informationwx 77±80 . This wx81 . The KGAPŽ. S,V,F E motif in the large cytosolic structure contains three main parts: the cytoplasmic domain is also conserved in the tomato sequence. headpiece which is connected via the 5-membered This has been implicated in nucleotide binding in the stalk-sector to the transmembrane domain. The intra- SERCAs and modification of the lysine residue by membranous part consists of 8±10 hydrophobic fluorescein isothiocyanate has been shown to cause a transmembrane helicesŽ. M1±M10 that serves to an- loss of ATP-dependent Ca2q transportwx 83 . How- chor the molecule to the lipid bilayer and form the ever, studies characterising the mutated rabbit SERCA transmembrane channel for the passage of calcium. provided little support for the direct involvement of The cytoplasmic headpiece is further subdivided into this lysine residue in ATP binding and suggested the N-terminal region, the transduction or -domain, instead that it was involved in a complex way in the phosphorylation domain, the nucleotide binding energy transduction at the catalytic centre after the domain and the hinge domainŽ for reviews see enzyme is phosphorylatedwx 84,85 . Also in the large wx76,81. . cytoplasmic domain is the DPPR motif which is The homology between the tomato sequence and conserved in the tomato, rice, Arabidopsis and the mammalian SERCA is indicated in Fig. 1; certain Dunaliella sequences. The aspartate and proline

Fig. 1. Multiple alignment of a range of SERCA-type ATPases from plant, mammalian and insect origin. The alignment was performed with the Clustal WŽ. 1.7 format. Sequences were taken from EMBLrGenBank databases. Ž.) below the sequence indicates identical amino acids;Ž. : below the sequence indicates conservation of ``strong'' groups; Ž. . below the sequence indicates conservation of ``weak'' groups. Regions conserved in all P-type ATPases are indicated by light shading while the dark shading shows the six amino acids essential for Ca2q -transport in animal SERCAs. The sequences compared are those from tomatoŽ LCA1;wx 68.Ž , rice OsCA;wx 72. , Arabidopsis ŽECA1;wx 71.Ž , Dunaliella DBCA1;wx 73.Ž , Drosophila DRSERCA;wx 99.Ž , rabbit OCSERCA2;wx 173. . D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 11 12 D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 residues in the DPPR motif are thought to be critical domain. The mammalian calmodulin-stimulated for phosphorylation in the SERCAswx 84 . Finally, the Ž.`PM-type' Ca2q -pumping ATPase possesses a C- MVŽ. I TGD motif Ž part of the ATP-binding domain . terminal domain of 12 kDa which shows calmodulin in the large cytosolic loop and the MTGDGVN motif binding activitywx 90,91 . This domain may be cleaved in the putative ``hinge'' region clearly distinguish the by brief trypsinolysis of the intact protein, following tomato, tobacco, rice, Arabidopsis and Dunaliella which the enzyme is fully activated to a level equiva- sequences as P-type ATPase. The proposed hinge lent to that in the presence of calmodulinwx 90 . This region is one of the most conserved regions of the indicates that the calmodulin-binding domain is in SERCAs and the plant SERCA homologues also fact an autoinhibitory domain, causing inhibition of show high homology in this region. It has been calcium transport and ATP hydrolysis in the absence proposed that the hinge domain is sufficiently flexi- of calmodulin, with inhibition being progressively ble to allow the nucleotide binding and phosphoryla- removed in its presencewx 90,92 . It is thought that in tion domains to come togetherwx 86 . Towards the end the absence of Ca2qrCaM, the CaM-binding domain of the highly conserved hinge region is the sequence interacts with a specific region of the enzyme itself to LKK which is conserved in most of the SERCAs and prevent high-affinity Ca2q binding of the pumpwx 93 ; PMCAs; however, in the case of the tomato, tobacco, calmodulin is thought to interact with this C-terminal rice and Arabidopsis SERCA homologues this is region and prevent this inhibitory molecular interac- 2q LKL and this motif also occurs in some of the yeast tion. This results in a decreased K m for Ca and an 2q wx Ca -ATPase sequences. increased Vmax 90,92 . The high-affinity Ca2q-binding site of the mam- The first indications of the presence of a calmod- malian SERCA is thought to be located in the trans- ulin-binding domain in some higher plant Ca2q- membrane helices M4, M5, M6 and M8wx 76 since pumping ATPases were provided by the pioneering mutagenesis of Glu309, Glu771, Asn796, Thre799, studies of Dieter and MarmeÂ wx 49,94 using microso- Asp800 and Glu908 in the transmembrane segments mal membranes of Zea mays. Subsequent studies inhibited Ca2q -dependent reactionswx 85,87,88 . All six demonstrated calmodulin-stimulated calcium trans- of these amino acids essential for Ca2q transport are port in a variety of plant speciesŽ see Tables 1±3 and conserved in the plant homologues. references therein. . Briars and Evanswx 50 demon- The CaM-binding site in the carboxyterminus is a strated for the first time that the calmodulin-stimu- characteristic feature of animal PM-type Ca2q- lated Ca2q pump in maize formed a calcium-depen- ATPases and such sites are not present in the animal dent phosphorylated intermediate and was therefore a

SERCAs. The binding of CaM is not determined by a P-type ATPase of Mr 140,000. The functional recon- consensus sequence, rather it is the ability to form a stitution of calmodulin-stimulated Ca2q-transporting particular secondary structure composed of basic am- ATPase from both maize and cauliflower membranes phiphilic -heliceswx 89 . No such regions have been after purification to near homogeneity on calmodulin observed in the tomato, tobacco, rice, Arabidopsis agarose confirmed that the calmodulin-binding do- and Dunaliella SERCA homologues. main was intrinsicwx 42,51 and calmodulin activation was not indirectŽ e.g. via a phosphorylation or similar . 2q event . 5.2. Ca -pumping ATPases containing an auto-in- Two recent studies have attempted further charac- hibitory CaM binding domain terisation of the plant calmodulin-binding domain at a biochemical level. Evidence for a calmodulin-binding As indicated previously, numerous studies have autoinhibitory domain has been obtained in radish identified CaM-stimulated Ca2q transport and associ- plasma membranes. Here, partial trypsinolysis results ated ATP hydrolysis in plant membrane preparations. in a reduction in Mr from 133 to 118 kDa paralleling Evidence that calmodulin is exerting a direct effect an increase in basalŽ. non-calmodulin stimulated hy- Žrather than stimulating transport via a calmodulin- drolytic activity. The 133 kDaŽ. but not the 118 kDa stimulated protein kinase or similar mechanism. re- protein is able to bind calmodulin, suggesting the loss quires the demonstration of a calmodulin-binding of an inhibitory calmodulin-binding sitewx 17 . A paral- D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 13 lel studywx 6 used calcium green dye to measure Ca2q DKTGTL, SGES, GDGTNDAP. and shows greatest uptake into membrane vesicles of vacuolar origin similarityŽ. 61% amino acid sequence homology with from cauliflower. Trypsin treatment resulted in acti- the Arabidopsis thaliana Ca2q pump ACA1 which vation and loss of calmodulin-sensitivity of transport has been reported to be present on the plastid enve- and concomitant cleavage of the Ca2q pump from lopewx 46,47 . Both of these plant pumps show a 111 kDa to generate peptides of 102 and 99 to 84 kDa higher level of homology with mammalian PM 2q and a reduction in KCa from 2 to 0.5 M. Only the calmodulin-stimulated Ca pumps than with SERCA intactŽ. 111 kDa protein retained calmodulin binding pumps, including the tomato SERCA homologue LCA activity. This suggests that part of the 9 kDa section Žwx68 and see below. . Southern blotting suggests only is involved in CaM-binding. one gene for BCA1. The PCR-derived sequence pre- Further characterisation of the calmodulin-stimu- sented by MalmstromÈ et al.wx 98 does not have a lated Ca2q pump of radish plasma membranes has sufficiently large C-terminus to contain a calmodulin been carried out using fluorescein isothiocyanate binding domain. MalmstromÈ et al.wx 98 however, Ž.FITC bindingwx 62 . The pump binds FITC with a suggest that the extended N-terminus of the pump high affinity, in common with that of mammalian may be a novel calmodulin-binding domain. Residues calmodulin-stimulated Ca2q pumpswx 95 . Sensitivity 19±43 of this region show a segregation of hy- to inhibition by FITC is much higher than that of the drophobic amino acids on one side and polar residues plant plasma membrane proton pump, Ž.K 0.5 2 M; on the other when represented in -helical wheel FITC-labelling resulted in the detection of two poly- arrangement, properties appropriate for calmodulin peptides of Mr 118 and 133 kDa comensurate with binding; however it shows little homology to the the native and proteolysed Ca2q pump described calmodulin binding domains of mammalian Ca2q above. Labelling and inhibition with FITC was com- pumps. A synthetic peptide to this region was able to petitive with Mg-triphosphopurine nucleotideswx 62 bind calmodulin; however this binding was equal in while the inhibitory fluorescein derivative Eosin-Y the presence and absence of calcium. effectively inhibited FITC binding. FITC binds cova- In the second study, Subbaiah and SachsŽ Pers. lently to lysine residues; a highly conserved FITC-bi- comm.. have recently obtained sequence of a gene nding regionŽ. lys 591 has been identified in isoforms from maize which shows homology to those of mam- of the mammalian PM-type Ca2q pumpwx 96,97 . malian PMCA-type pumps, including amino acids Two studies have recently resulted in the cloning essential for calcium transport and a putative c-termi- of Ca2q pumps possessing auto-inhibitory calmodulin nal calmodulin binding domain. The protein encoded binding domains in plants. MalmstromÈ et al.wx 98 is estimated to be of about 140 kDa, similar to that reported the cloning of a putative vacuolar calmod- predicted previously by protein purificationwx 50 . Un- ulin-stimulated Ca2q -pumping ATPaseŽ. BCA1 from fortunately, the full sequence is as yet not available cauliflower with a predicted Mr of 111.7 kDa. Part of for analysis. the cDNA sequence was obtained initially by PCR The above studies suggest the presence of at least using one primer based on peptide sequencing a two types of calmodulin-stimulated Ca2q pumps in tryptic fragment of the purified plant protein and a higher plants, differing in relative molecular mass second based on a region conserved in P-type AT- and subcellular location and in the location of the Pases; full length sequence was obtained by PCR calmodulin binding domain within the proteinŽ Sub- amplification using primers from the original clone. baiah, pers. comm.;wx 98. . It is evident that these early Confirmation that the sequence represents the reports require confirmation before a complete under- calmodulin-stimulated ATPase purified by affinity standing of the nature, location and regulation of the chromatographywx 98 comes from the presence of six calmodulin-stimulated Ca2q pumps is obtained. Cer- sequences obtained from tryptic digests. Ten trans- tainly, the presence of a smaller CaM-stimulated membrane helices are predicted with hydrophilic do- Ca2q pump with an N-terminal calmodulin binding mains between transmembrane helices two and three domain opens up a variety of possibilities for further and also four and five. The sequence contains P-type study. It remains to be seen whether the calmodulin ATPase conserved domainsŽ PAD, KGAPE, PEGL, binding domains of the plasma membrane calmod- 14 D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 ulin-stimulated pump in radish and other tissues is C- While there is no functional data to confirm that or N-terminal. PEA1 is a Ca2q pump the 6 amino acids in mammalian PMCAs which correspond to the 6 amino 5.3. Molecular characterisation of other cloned acids suggested as probable constituents of the cal- putatiÕeCa2q-pumping ATPases cium-binding site in the SERCAsŽ. see Section 5.1 are conserved in the putative plastid P-type ATPase 5.3.1. Plastid enÕelope pump and so suggest that this is a Ca2q-transporting AT- A putative plastid envelope Ca2q -ATPaseŽ. ACA1 Pase. has been identified by Huang et al.wx 47,48 which The putative plastid Ca2q-ATPase sequence con- shows homology to the mammalian P-type Ca2q tains a 70-80 amino acid N-terminal region which pump, but lacks a calmodulin-binding autoinhibitory shows some similarity to the transit peptide of the domain at the C-terminus. A partial cDNA was iso- plastid envelope phosphate translocator; this is sug- lated initially by screening an Arabidopsis cDNA gested to function in targeting this protein to the expression library with an antiserum raised against plastidwx 46 . The difference in sizes of the protein as spinach chloroplast envelope proteins. The corre- estimated by immunoblotting and by sequence analy- sponding genomic clone was isolated and the com- sis would tend to support thiswx 46 . This N-terminal plete coding sequence deduced. The predicted region also contains a section of about 25 amino polypeptide PEA1p has 946 amino acids and a acids which has homology to the putative N-terminal molecular mass of 111 kDawx 47 . The hydrophobicity CaM-binding region in BCA1 from Brassica oler- profile suggests that it has ten transmembrane do- acea. Therefore the possibility exists that ACA1 is mains, a large extramembrane loop between hy- equivalent to the CaM-binding Ca2q-ATPase purified drophobic regions 4 and 5 and a smaller loop be- previously from the chloroplast envelopewx 47 . To tween region 2 and 3wx 46 . PEA1p possesses all the investigate whether PEA1pŽ. ACA1 was indeed found motifs characteristic of P-type ATPasesŽ PGD, PAD, at the plastid envelope, antiserum was raised against TGES, PEGL, DKTGTLT, KGASE, DPV RP, the purified peptide expressed from a fragment of the MVTGD, VAVTGDGTNDAPAL HEADIGLAM. PEA1aŽ. ACA1 cDNA which corresponds to amino wx75 . Instead of the RrKILL motif which has been acids Glu714 ±Ser950. . The affinity purified antisera postulated as an ER-targeting sequence, it contains was used to immunoblot various membrane fractions the sequence MILA. Alignments of the coding se- from pea leaves. This antiserum reacted specifically quence with P-type ATPases shows that this protein with a protein of 90±95 kD only in the inner enve- is 40±45% identical to various mammalian PM-type lope fraction. Although this suggests that the PEA1p Ca2q-ATPases, although it lacks the hydrophilic C- Ž.ACA1 is an inner envelope protein, further studies terminal CaM binding domainŽ. Fig. 2 . PEA1p also are required to test the specificity of the antiserum appears to be smaller than the PMCAs in the hy- and to definitively show that the polypeptide recog- drophilic regions found between the putative trans- nised by the antiserum in pea leaves is actually a pea membrane domains 1 and 2 and between those of 2 homologue of PEA1p from Arabidopsis. If so, then and 3. Highest homologyŽ 76% similarity, 62% iden- this antiserum could also be used in further immuno- tity. is shown to BCA1, the putative vacuolar Ca2q - logical studies to confirm the suggested inner enve- ATPase from cauliflowerwx 98 . lope localisation.

Fig. 2. Multiple alignment of eukaryotic Ca2q-ATPase sequences. The alignment was performed with pileup and displayed in the Clustal WŽ. 1.7 format. Sequences were taken from EMBLrGenBank databases. Ž.) below the sequence indicates identical amino acids; Ž. : below the sequence indicates conservation of ``strong'' groups;Ž. . below the sequence indicates conservation of ``weak'' groups. Regions conserved in all P-type ATPases are indicated by light shading while the dark shading indicates a putative CaM-binding domain in BCA1. The sequences compared are those from Arabidopsis ŽACA1;wx 46,47.Ž , cauliflower BCA1;wx 98.Ž , Dictyostelium discoideum PAT1; wx180.Ž , yeast PMC1;wx 157.Ž and human HPMCA2;wx 96. . D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 15 16 D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25

5.4. Subcellular targeting of plant Ca2q-pumping modium falciparum but not in other representatives of ATPases the SERCA family and therefore this is not a univer- sal targeting sequence amongst the SERCAs. The structural domains that are responsible for the subcellular targeting of plant Ca2q-ATPases are un- known but several possibilities exist. The RrKILL 6. Functions of plant Ca2H-pumping ATPases motif is conserved in the 1st transmembrane segment of the animal SERCAs but not in the PM Ca2q- While the maintenance of a low cytosolic free ATPase and has been suggested to be responsible for calcium concentration Ž.-0.1 M is a central func- targeting to the ERwx 99 . By using recombinant tion of both intracellular and plasma membrane plant chimeras of the SERCA and PMCA mammalian Ca2q pumpsŽ seewx 2±5,18 for reviews. , there is pumps, Foletti et al.wx 100 have shown that the first- significant evidence indicating that plant cell Ca2q transmembrane domain contains a sequence promot- pumps are involved in generating intracellular sig- ing the retention of chimeric constructs in the ER. nalling pools and in regulating secretion. Such possi- However they have also suggested that other struc- ble roles for plant Ca2q pumps will be considered tural determinants may also contribute to ER-reten- briefly here; further information may be found in tion of the SERCA pump. The RrKILL motif also recent reviews e.g.wx 3,103 . occurs in the tomato, rice, Arabidopsis and Dunal- liella SERCA homologues and the high conservation 6.1. Ca2q-pumping ATPases and cell signalling in the amino-terminal region between these sequences has been suggested as evidence that the We refer the reader to the excellent review by Dunaliella sequence may be localised on the ERwx 73 . Bushwx 3 for a comprehensive treatment of the role of Wimmers et al.wx 68 initially postulated an ER locali- calcium in signalling processes in plants and here we sation for the tomato SERCA homologue because of have attempted to highlight the possible roles of its sequence similarity to the mammalian SERCAs; Ca2q pumps in the signalling processes. It is now however, more recent immunoblotting studies sug- widely accepted that the response to many extracellu- gested that isoforms of LCA were localised in the lar signals is mediated in plants through a transient tonoplast and plasma membrane of cells in tomato increase in cytosolic calcium levels. The variety and rootwx 26 . If it is the case that the two isoforms are specificity of cellular responses triggered by calcium derived from a single gene, as suggestedwx 26 , then it may be explained by spatial changes in its localisa- will be interesting to determine the domains responsi- tion together with amplitude and frequency variations ble for subcellular targeting. It has been postulated in concentrationwx 3,104,105 . In addition, the speci- that the differences in size and localisation of the two ficity may be encoded by the simultaneous action of isoforms detected using an antibody results from several cellular mediatorswx 3 . Physiological processes either post-transcriptional or post-translational pro- that appear to be associated with a change in the cessing of the mRNA or protein, respectivelywx 26 . cytosolic calcium concentration include stomatal re- There are other motifs which have been implicated sponses, polarised growth, responses to hormones, as targetingrretention determinantswx 101,102 . These gravitropism, mitosis and phytochrome related events include a lysine-rich motif containing two lysine Žreviewed inwx 3. . residues three to four amino acids upstream of the Transient changes in cytosolic free calcium levels cytoplasmically exposed C terminus of multispanning are induced by extracellular signals such as hormones proteinsŽ. KKXX or KXKXX . The tomato, Ara- w106,107 x , light wx 108 and touch wx 109 . Rapid progress bidopsis and Dunaliella SERCA homologues do in- is now being achieved in identifying the components deed contain the sequences KLKAA, KQKEE, and and demonstrating their involvement in Ca2q-media- KLK respectively at their C-termini but it remains to ted signal transduction pathwayswx 104,110 . For ex- be determined whether the mature proteins retain ample, Chen et al.wx 67 have reported on the cloning these amino acids. The lysine rich motif sequence is of a Ca2q-ATPase gene in rice and explored its role also present in the SERCA homologue from Plas- in the GA-dependent signal transduction pathway D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 17 during germination. This will be discussed more fully gested that the presence of such pumps would be in the following section. It has already been observed necessary if the cortical ER was participating in that although the basic mechanisms of signalling may signal transduction and regulation of cytosolic cal- be similar in plants and animals, the actual compo- cium. nents of the signalling machinery and their regulation Changes in cytosolic calcium have been reported can differ e.g.wx 4,5 and in some cases be quite novel in response to a number of stress conditions, includ- wx110 . Ca2q -ATPases play a central role in signalling ing hypoxiawx 120 , salinity wx 121 and fungal elicitor since they function to regulate submicromolar Ca2q actionwx 109 . Two studies have reported increased concentrations. During signal transduction they are transcripts for Ca2q-ATPases in plant cells exposed presumed to primarily function in restoring cytosolic to high-salt conditions and have postulated that en- calcium levels to prestimulus valueswx 3 by transport- hanced Ca2q-ATPase gene expression and enhanced ing this cation out of the cell or into intracellular Ca2q levels represent an adaptive response to saline organelles such as the vacuole and endoplasmic retic- environmentswx 68,69 . In tomato, enhanced gene ex- ulumwx 2 . In signalling events, Ca2q -ATPases may pression was observed in the presence of only 50 mM also play a crucial role in the maintenance and regu- NaClwx 68 , whereas 428 mM NaCl was used in to- lation of Ca2q oscillations as occurs in mammalian bacco culture cellswx 69 . Furthermore, Perez-Prat et systemswx 111,112 . Evidence, in the latter indicates al.wx 69 reported that NaCl induction of the Ca2q - that Ca2q extrusion across the plasma membrane is ATPase mRNA in tobacco cell cultures appeared to required for Ca2q -spike recoverywx 113 and Tepikin et be developmentally controlled. Increased activity of al.wx 114 have demonstrated pulsating increases in Ca2q-efflux mechanisms at the PM in response to Ca2q extrusion that correspond to spikes in the inter- mild oxidant stressŽ. ozone treatment have also been nal calcium concentration. However, the role and observedwx 122 but the mechanism of this activation particularly the regulation of Ca2q-ATPases in sig- remains to be shown. Before a specific role of en- nalling processes in plant cells has largely been unex- hanced Ca2q-ATPases gene expression in stress adap- plored because it is difficult to measure their activity tation can be invoked to explain these observations it in vivo. Altered Ca2q-transport capacities of isolated must first be proven that such observations are not membranes have been observed in plants treated with simply an indirect consequence of the stress condi- a range of stimuli e.g. hormonesŽ see above; e.g. tions imposed. wx115,116,104. , mechanical stimulationwx 117,118 and The regulation of the Ca2q-ATPases is a very far-red lightwx 119 . Only a few studies have provided important process in the signalling event and because information regarding their in vivo regulation during of the possible major differences in the cellular ar- signal transduction, although calcium transients have rangement of these enzymes in plants, we can no been observed in numerous studies following various longer assume that the regulatory processes are the stimulationswx 108 . In a detailed study, Liû and Weiler same as those occurring in animal cells before a wx118 observed a rapid and transient increase in the rigorous examination is conducted. Now that a num- activity of vanadate sensitive Ca2q-ATPase in the ber of Ca2q-ATPase genes have been cloned, the tendrils of Bryonia dioica Jacq. associated with the possibility of genetically manipulating these trans- early stages of touch-associated tendril coiling. The porters and studying their effect on signalling pro- tissue was shown to possess both a calmodulin- cesses in transgenic plants should help us to under- stimulated ER Ca pump and a non-calmodulin stimu- stand their role in Ca2q homeostasis and signal translated PM Ca2q pump. Chen et al.wx 67 raised the duction. Determining the cellular organisation of possibility that a calmodulin-stimulated Ca2q-pump Ca2q pumps and other Ca2q transporters in plant may be associated with the cortical ER in carrot cells is vital to this understanding since their particu- suspension cells. This is an extensive network of lar structural organisation means that they may regu- flattened cisternae and tubules situated immediately late cytosolic calcium levels in a rather unique man- adjacent to the PM. The cortical ER is thought to ner. Certainly, the role of the vacuole may be much play an important role in anchoring the cytoskeleton more relevant in signalling processes in plants. The and facilitating secretion and Chen et al.wx 67 sug- existence of a tonoplast antiporter and Ca2q-ATPase 18 D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 suggests that this is an important calcium sink in portant together with Ca2q channels in triggering an resting conditions and the identification of a range of increase in the frequency and magnitude of the lo- Ca2q channelswx 123 indicates that the vacuole may calised changes in Ca2q levels in the cytoplasm of be a very important source of stimulus-releaseable aleurone cellswx 72 . Chen et al. wx 72 hypothesised that Ca2q. calmodulin binds to Ca2q and then activates a protein kinaseŽ. or phosphatase which regulates the expres- 6.2. Ca2q-pumping ATPases and secretion sion of the downstream -amylase gene. There have been numerous reports on the role of Calcium is thought to be essential for the function- Ca2q during pollen tube tip growthŽ one of the most ing of the secretory system and is required for the vigorous secretory systems known in plants. and we activity of chaperones in the ER which are responsi- refer readers towx 127 and references therein. Pollen ble for the correct folding and assembly of proteins tubes have been shown to contain an intracellular destined for secretionwx 14,124 . The secretion of hy- Ca2q gradient, which is limited to the tip and this is drolytic enzymes by the aleurone layer of cereals in thought to be maintained by both influx of Ca2q response to gibberellic acid secretion from the scutel- through channels located at the tip and sequestration lum is a regulated process involving calcium in the ER presumably by a Ca2q -ATPasewx 128 . Fur- wx125,126 . Upon germination, increasing gibberellic ther studies are required to determine the biochemical acid results in an increased de novo synthesisŽ inter and molecular properties of this pump. alia.Ž. of the enzyme -amylase EC 3.2.1.1 and its secretion into the endosperm. As -amylase is a 6.3. PutatiÕe role of Ca2q-pumping ATPases in calcium-binding metalloprotein, its successful secre- plastids tion requires the pumping of stoichiometric quantities of Ca2q into the lumen of the ER; without this, the Based on immunochemical data localising a puta- enzyme is unstable and is degradedwx 27 . Calcium tive Ca2q -ATPaseŽ. PEA1prACA1 to the chloroplast levels within the ER of the aleurone must be main- envelopeŽ. see Section 2.4 , Huang et al.wx 46 have tained above 5M for -amylase synthesiswx 27,116 . discussed the possible roles of such a pump. They Several studies have indicated that a calmodulin- have suggested that the envelope ATPase could be stimulated pump at the ER of aleurone cells may be responsible for the export of calcium from the stroma responsible for maintaining these concentrations into the cytosol. This is particularly relevant as cal- wx26,116,43 . The activity of this pump was shown to cium may play a role in signalling between the be higher in cells treated with gibberellic acid in the plastid and the rest of the cellwx 129 . In addition, presence of calcium as compared with controls; Ca2q PEA1p may play a role in the control of chloroplast transport in ER from untreated cells was increased in metabolism by adjusting free Ca2q in the stroma the presence of exogenous calmodulin to a level Žwx46 also, see wx 45. and thereby regulate processes equivalent to that in cells first treated with GA. within the chloroplast. Nguyen and Siegenthalerwx 48 Furthermore, GA was shown to increase calmodulin suggested that the Ca2q-ATPase may be involved in levels in aleurone cells up to ten-fold over controls the energy-dependent import of proteins into the and this increase paralleled the increase in Ca2q stroma. transport and -amylase secretionwx 43 . Since levels of PEA1Ž. ACA1 transcripts were Recently Chen et al.wx 72 have cloned a GA-re- 7±9-fold higher in roots than in leaves a role in sponsive Ca2q-ATPase gene from rice aleurone cells gravitropism has been suggested as a possibilitywx 46 . and shown that it has a regulatory effect in the It is not entirely clear what role a plastid enzyme activation of the GA-dependent downstream - would have in this response but it has been proposed amylase gene. Interestingly, they showed that overex- that the ER Ca2q-ATPase of statocytes is involved in pression of the Ca2q-ATPase cDNA could bypass the transduction of the gravity stimulationwx 60,130 . CPA need for GA induction to activate the -amylase has been observed to inhibit the gravitropic response gene. It was concluded that GA induction of the in cress roots and Sievers and Buschwx 60 have argued synthesis of Ca2q-ATPase mRNA and protein is im- that CPA disrupted the cytosolic Ca2q signal neces- D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 19 sary for graviperception. The mechanism by which patterns provide differentiation-specific expression of CPA interferes with the graviperception process re- at least 26 isoformswx 134,135 which may differ in mains to be shown and may not be attributed solely sensitivity to regulators. Strehlerwx 136 has suggested to an effect on the Ca2q-ATPase. that whereas highly conserved sequences in the different isoforms are likely to represent domains essential for catalytic and transport function and also may 7. Regulation of plant Ca2H-pumping ATPases reflect specific constraints imposed upon the enzyme structure, highly divergent sequences could reflect Given our increasing understanding of the struc- regulatory and functional specialisations of the en- ture and mechanisms of action of plant Ca2q pumps, zyme adapted to meet the specific physiological needs we are now in a strong position to elucidate their of a given tissue or cell type. At present, it is too regulation. Lutsenko and Kaplanwx 74 have high- early to know whether such isoforms of the plant lighted the possible importance of the structurally calmodulin-stimulated Ca2q pumps exist; evidence diverse region in P-type ATPases located in the for only one gene encoding the vacuolar calmodulin- central cytoplasmic loop just C-terminal to the phos- stimulated Ca2q pump in Brassica has been pre- phorylation site; the variation in sequence and size in sentedwx 98 , but EST analysis suggested several ho- this region in P-type ATPases which transport the mologues in Arabidopsis thaliana ŽPittman and same cations may reflect differences in the regulatory Williams, unpublished. . mechanisms acting on different pumps. In the mam- In addition to CaM regulation, mammalian PM- malian cardiac SERCA1 a section of this region CAs can also be modified by cAMP-dependent pro- interacts with phospholamban, an integral membrane tein kinasewx 137 and Protein kinase C wx 138 . The site protein in the cardiac sarcoplasmic reticulum which of cAMP-dependent phosphorylation is just on the regulates Ca2q -ATPase activitywx 131,132Ž. see below . C-terminal side of the calmodulin-binding region. It The corresponding region in the mammalian PMCAs is differentially spaced in PMCA isoforms and is also contains a part which has been shown to bind a absent altogether in some providing a means for synthetic peptide derived from the calmodulin-bind- differential regulation. Protein kinase C has been ing sequencewx 133 . Thus it is possible that there will found to phosphorylate the pump on the single threo- be an analogous region in the plant enzymes for nine present in the calmodulin binding domain and response to particular regulatory factors. on a serine carboxy-terminal to the domainwx 139 . The presence of calmodulin-stimulated Ca2q G-proteins of various types may stimulate the pump pumps has already been discussed in some detailŽ see directly and also indirectly via stimulation of phos- Sections 2.1, 2.2, 2.3 and 5.1. and will not be dealt pholipase C, adenylate cyclase and Kq-channel activ- with further here, other than to note that while evi- ity and additional control may be achieved by spatial dence suggests that the plant calmodulin-stimulated regulation of availability of ATP for the pumpwx 107 . Ca2q pumps have an autoinhibitory calmodulin bind- Proteases such as trypsin and calpain irreversibly ing domain, the possibility that this may, at least in cleave the C-terminal region to cause stimulation and some instances, be a novel N-terminal domainwx 98 it has been suggested that this may provide a drastic suggests that these may differ mechanistically, at remedy to elevated cytosolic calcium concentrations least at the protein level, from pumps having a C- in vivowx 93 . The physiological relevance of limited terminal regulatory domain. It is also worth noting proteolysis in plants is not known. Other regulators of that difficulties in removing endogenous calmodulin mammalian PMCAs include acidic phospholipids 2q from plant membrane Ca -pump preparations may Ž.e.g. PIP2 and long-chain polyunsaturated fatty acids indicate that calmodulin is very tightly associated wx140,141 ; these interact with a phospholipid binding with plant Ca2q pumps in vivoŽ e.g.wx 18. . Carafoli domain close to the phosphorylation site and in the wx93 has suggested that in some pumps calmodulin absence of calmodulin these can increase both the could be a permanently bound subunit of the enzyme; calcium affinity and rate of transport of the pump this may be the case in plants. In the case of the wx142 . Hsieh et al. wx 39 observed a stimulation of mammalian PMCA, four genes plus different splicing calcium transport in vesicles from carrot cells by the 20 D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 acidic phospholipids, phosphatidylserine and phos- that this may not be due to a direct activation or phatidylinositol but not by phosphatidylcholine or inactivation of the pump and is likely to reflect phosphatidylethanolamine. It was also found in this indirect modulation via changes in CaM or other system that KFŽ. a phosphatase inhibitor stimulated Ca2q -binding proteinswx 149,44 . The increased Ca2q - the calmodulin-sensitive calcium uptake and the pos- transport activity in PM-enriched fractions from plants sibility of regulation of plant Ca2q-ATPases by pro- treated with the auxin indole acetic acid prior to tein kinases was discussedwx 39 . homogenisation may also be due to increased amounts The Ca2q-ATPase in the sarcoplasmic reticulum of of CaMwx 115 . The synthetic cytokinin benzyladenine cardiac and slow-twitch skeletal muscle is also acti- has also been shown to increase the affinity and vated by calmodulin and cAMP, but this regulation activity of the Ca2q-ATPase in membranes extracted occurs indirectly through the phosphorylation of from wheat seedlings to Ca2q and CaMwx 150 . Pre- phospholambanŽ. see above . The mechanism by treatments with benzyladenine enhanced a range of which it regulates these SERCAs is similar to the both Ca2q and Mg2q ATP-hydrolytic activities at the regulation of mammalian PMCAs by their calmod- plasma membrane in wheat rootswx 151 , possibly in- ulin-binding domainwx 93 . In fact, cross-inhibition of cluding effects on a Ca2q-transporting ATPase. In the two pumps has been described with phospholam- many of the above instances, it should be noted that ban and the synthetic calmodulin-binding domain of pretreatment of cells or tissues with the hormone was the PMCAwx 93 . CaM-regulated and cAMP-dependent carried out in order to demonstrate the apparent protein kinases mediate the phosphorylation of phos- regulatory effect. It should be borne in mind that pholambanwx 143 . In its non-phosphorylated form, other membrane changesŽ for instance in activity of phospholamban binds to the calcium-free form of the the Hq -ATPase. may have also occurred and it is Ca2q pump to inhibit its action. Phosphorylation of important that research focusses on the mechanisms phospholamban prevents binding and allows the pump of regulation in order to differentiate primary and to exhibit its full activity. The activity of the mam- secondary effects on the Ca2q pumps and experimen- malian SERCA may also be regulated by several tal artefacts. factors including the lipid environment, temperature- wx dependent structural fluctuations 144 and oligomeri- 2q sationwx 145,146 . Radiation inactivation studies of 7.1. Regulation of Ca -pumping ATPase gene ex- plant membranes suggest that some plant Ca2q pumps pression function as dimerswx 147,148 although the effect of such intermolecular associations on the functioning Regulation of Ca2q transporters may also occur at of the pump remains to be investigated. the gene level. Developmental regulation of the mam- There have been only a limited number of studies malian SERCA genes is achieved by the existence of on plants investigating potential regulators of the different mRNAs generated by alternative splicing. In Ca2q-ATPases. The fungal toxin fusicoccin has been addition, alternatively-spliced forms also show a tis- shown to activate a Ca2q-ATPase in Corydalis sem- sue-specific distribution in certain caseswx 152±154 . perÕirens cell suspension cultures when added prior There are four mammalian PMCA genes and alterna- wx to homogenisation 57 . Fusicoccin lowered the K m tive splicing provides differentiation-specific expres- for ATP and it was concluded that the effect was not sion of at least 20±26 isoformswx 134 . Control of gene the result of a direct interaction between the toxin expression is regulated via at least two distinct path- and the enzyme but involved components which were ways; one involves protein kinase C while the other lost or inactivated during preparationwx 57 . In con- involves Ca2q or cAMP and there is evidence of trast, the same authors noted that no effect of fusicoc- cross-talk between the two pathwayswx 155 . In plants cin was observed on Ca2q transport in plasma mem- there is some preliminary evidence which may indi- brane vesicles from fusicoccin-treated maize coleop- cate the existence of tissue-specific and developmen- tileswx 57 . The plant hormones gibberellic acid and tally-controlled isoforms. In tobacco a single 4.4 kb abscissic acid regulate Ca2q transport in barley aleu- Ca2q-ATPase transcript was detected in decreasing rone in an antagonistic way but it has been suggested amounts in stems, roots and leaveswx 69 . In tomato, D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25 21 transcripts of 3.5, 4.2 and 6.5 kb were detected in in this group are the heavy metal translocating AT- roots and a less abundant 4.2 kb transcript was de- Pases of bacteria and the mammalian copper trans- tected in leaf tissuewx 68 . Higher transcript levels of porting ATPases. More advanced P-type ATPases the putative plastid Ca2q-ATPase were also found in having a larger, regulatory C-terminus are termed Arabidopsis roots relative to leaveswx 46 . Perez-Prat type II by Mùllerwx 75 , including both the SERCA et al.wx 69 observed higher levels of Ca2q -ATPase Ž.type IIa and PMCA Ž. type IIb Ca2q pumps, the mRNA in cultured tobacco cells relative to plant mammalian sodiumrpotassium ATPase and the plant tissues and postulated that this could be due to and fungal plasma membrane proton pumps. Clearly tissue-specific expression, to less active cell growth from the information already presented in this review, in plant organs, to gene expression under develop- plants have homologues of the SERCAŽ. type IIa mental control or to the existence of isogenes which Ca2q pumpsŽ. see Section 5.2 ; however, categorisa- are expressed differentially in tissues. Further studies tion of several plant Ca2q pumps ± the plastid pump are required to differentiate between these possibili- and the pumps bearing a calmodulin binding domain ties. In both tobacco and tomato, mRNA levels were is more difficult. Preliminary evidenceŽ Subbaiah, increased by growth under high NaCl concentrations pers. comm.. suggests that the calmodulin-stimulated indicating that salinityŽ. or possibly osmotic effects Ca2q pump cloned in maize is a type IIb pump, may modulate Ca2q-ATPase gene expression. Until showing homology to the mammalian PMCA and estimates of activity and ATPase protein are made, having a C-terminal calmodulin binding domain. however, it is not clear whether increased gene ex- However, comparison of the sequences of the puta- pression results in increased activity or acts to main- tive vacuolar calmodulin-binding Ca2q pump from tain pre-existing levels of the pump under stress Brassica wx98 and the putative plastid Ca2q pump conditions. from Arabidopsis wx46 with other Ca2q pumps pre- Chen et al.wx 72 recently proposed a model of the sents a more complex picture. They are homologous gibberellic acid-dependent signalling pathway in rice with the PMCA type pumpsŽ. see Section 5.1 , but aleurone cells in which gibberellic acid binding to a lack a C-terminal regulatory domain and have an receptor by some means activated phosphoprotein extended N-terminus which in the former case is phosphatases 1 and 2AŽ seewx 155. . They suggested proposed to be a novel CaM binding sitewx 98 and in that these phosphatases may initiate the stimulation the latter a plastid targeting sequencewx 46 . MalmstromÈ of the transcription of a Ca2q-ATPase gene which et al.wx 98 present a phylogenetic tree which empha- then contributes to localised changes in calcium lev- sises the close similarity between the plastid and els and ultimately to changes in -amylase gene vacuolar pumps and also emphasises their homology expression. with the yeast vacuolar PMC1 and a vacuolar Ca2q pump PAT1 from Dictyostelium discoideum as well as with the mammalian PMCA family. Yeast vacuo-

2H lar PMCA1 also lacks a C-terminal calmodulin bind- 8. Phylogenetic relationships of plant Ca -pumping domainwx 157 . If plastids are derived from ing ATPases prokaryotic endosymbionts similar to cyanobacteria, it would seem likely that the putative plastid enve- Evolutionary aspects of P-type ATPases have been lope Ca2q-ATPase would be related to cyanobacterial discussed in recent reviewswx 74,75,82,156 and there- Ca2q-ATPases. The pacL gene cloned from Syne- fore we highlight aspects of the phylogenies of plant choccus sp. strain 7942 which has been shown to Ca2q pumps which are likely to be helpful in devel- encode a P-type plasma membrane Ca2q-ATPase oping future research strategies. wx158,181 does not show any higher similarity to the The simplest P-type ATPases found in prokaryotes putative plastid gene than to other Ca2q-ATPases have a lower relative molecular massŽ. ca. 70 kDa wx158 . The possibility that other Ca2q -ATPases will than those in eukaryotes; Mùllerwx 75 categorises be found in cyanobacteria cytoplasmic membranes these as type I ATPases and points out that this is due which show higher homology to the plastid enzyme to a less well developed C-terminal domain; included cannot be excluded. 22 D.E. EÕans, L.E. WilliamsrBiochimica et Biophysica Acta 1376() 1998 1±25

9. Future perspectives: lessons from yeast preparation of the manuscript. Jon Pittman's assis- tance in preparing the sequence alignments is also We are now beginning to unravel the structure of gratefully acknowledged. Original work described Ca2q-ATPases in higher plants. It is increasingly was supported in part by a grant from the Biotechnol- apparent that there are a variety of Ca2q pumps ogy and Biological Sciences Research Council. Both present, with homologies to mammalian and yeast DEE and LEW are Royal Society 1983 University Ca2q pumps. This variety should not surprise us; Research Fellows. calcium signalling and regulation are vital in a variety of processes and in a variety of compartments within the cell. Even in yeast, an organism lacking References the complex patterns of differentiation of higher wx plants, sequences resulting from the yeast genome 1 H. Sze, Annu. Rev. Plant Physiol. 36Ž. 1985 175±208. wx2 D.E. Evans, S.A. Briars, L.E. Williams, J. Exp. 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