A Ca 2ϩ -ATPase was purified from plasma membranes (PM) isolated from Arabidopsis cultured cells by calmodulin (CaM)-affinity chromatography. Three tryptic fragments from the protein were microsequenced and the corresponding cDNA was amplified by polymerase chain reaction using primers designed from the microsequences of the tryptic fragments. At-ACA8 (Arabidopsis-autoinhibited Ca 2ϩ -ATPase, isoform 8, accession no. AJ249352) encodes a 1,074 amino acid protein with 10 putative transmembrane domains, which contains all of the characteristic motifs of Ca 2ϩ -transporting P-type Ca 2ϩ -ATPases. The identity of At-ACA8p as the PM Ca 2ϩ -ATPase was confirmed by immunodetection with an antiserum raised against a sequence (valine-17 through threonine-31) that is not found in other plant CaM-stimulated Ca 2ϩ -ATPases. Confocal fluorescence microscopy of protoplasts immunodecorated with the same antiserum confirmed the PM localization of At-ACA8. At-ACA8 is the first plant PM localized Ca 2ϩ -ATPase to be cloned and is clearly distinct from animal PM Ca 2ϩ -ATPases due to the localization of its CaM-binding domain. CaM overlay assays localized the CaM-binding domain of At-ACA8p to a region of the N terminus of the enzyme around tryptophan-47, in contrast to a C-terminal localization for its animal counterparts. Comparison between the sequence of At-ACA8p and those of endomembranelocalized type IIB Ca 2ϩ -ATPases of plants suggests that At-ACA8 is a representative of a new subfamily of plant type IIB Ca 2ϩ -ATPases.

Ca 2+ play a key role in cell signaling across organisms. The question of how a simple ion can mediate specific outcomes has spurred research into the role of Ca 2+ signatures and their encoding and decoding machinery. Such studies have frequently focused on Ca 2+ alone and our understanding of how Ca 2+ signaling is integrated with other responses is poor. Using in vivo imaging with different genetically encoded fluorescent sensors in Arabidopsis (Arabidopsis thaliana) cells, we show that Ca 2+ transients do not occur in isolation but are accompanied by pH changes in the cytosol. We estimate the degree of cytosolic acidification at up to 0.25 pH units in response to external ATP in seedling root tips. We validated this pH-Ca 2+ link for distinct stimuli. Our data suggest that the association with pH may be a general feature of Ca 2+ transients that depends on the transient characteristics and the intracellular compartment. These findings suggest a fundamental link between Ca 2+ and pH dynamics in plant cells, generalizing previous observations of their association in growing pollen tubes and root hairs. Ca 2+ signatures act in concert with pH signatures, possibly providing an additional layer of cellular signal transduction to tailor signal specificity.

The initial rate of quenching of quinacrine fluorescence was used to monitor Mg:ATP-dependent Hf-pumping in membrane vesicles from corn (Zea mays L. cv WF9 x M017) roots and obtain a preparation in which vanadate-sensitive H"-pumping could be observed. Separation of membranes on a linear sucrose density gradient resulted in two distinct peaks of Hf-pumping activity: a major one, at density 1.11 grams per cubic centimeter, was sensitive to N03-and resistant to vanadate, while a minor one, at density 1.17 grams per cubic centimeter, was substantially resistant to NO3-and sensitive to vanadate. A membrane fraction enriched in the vanadate-sensitive Hf-pump could be obtained by washing microsomes prepared in the presence of 10% glycerol with 0.25 molar KI. The kinetics of inhibition of H -pumping by vanadate in this membrane preparation indicated that most of the Hf-pumping activity in this fraction is sensitive to inhibition by vanadate, 50% inhibition being reached at about 60 micromolar vanadate. This value is fairly close to that observed for inhibition by vanadate of the ATPase activity in similar experimental conditions (40 micromolar). The inhibitor sensitivity, divalent cation dependence, pH optimum (6.5), and K,,, for ATP (0.7 millimolar) of the Hf-pumping activity match quite closely those reported for the plasma membrane ATPase of corn roots and other plant materials.somal vesicles, probably due to leakiness of plasma membrane vesicles and/or to a predominantly right-side-out orientation of sealed plasma membrane vesicles (16,25). In particular, no vanadate-sensitive H+-pumping could be demonstrated in most membrane preparations from corn roots or coleoptiles (12,15,20). Only very recently has a peak of NO3-resistant H+-pumping, coincident with that of vanadate-sensitive ATPase, been found in isopycnic density gradients of corn coleoptile membranes (14). However, such NO3-resistant H+-pumping activity was only slightly sensitive to vanadate (25% inhibition at 200 ,uM vanadate). Thus, the involvement of the plasma membrane ATPase of corn in proton transport has been inferred mainly from in vivo work (10) and by analogy with other plant materials.In this paper we show that it is possible to obtain a membrane fraction from maize roots endowed with vanadate-sensitive H+-pumping activity and very low in NO3-sensitive and/or vanadate-resistant activity. The characteristics of this H+-pump are shown to correlate well with those of the plasma membrane ATPase of the same material (13, 24). MATERIALS AND METHODSAt least two types of H+-ATPases have been identified in membrane vesicles from different plant materials on the basis of their biochemical characteristics, sensitivity to inhibitors, and distribution on density gradients. One is N03 -sensitive, vanadate-resistant, and localized at the tonoplast (3); the other, sensitive to vanadate, is associated with the plasma membrane (29). A third kind of H+-ATPase, insensitive to vanadate and perhaps also to NQ3r, has been demonstrated recently in Golgi apparat...

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Microsomal vesicles from 24-hour-old radish (Raphanas sativus L.) seedlings accumulate Ca2+ upon addition of MgATP. MgATP-dependent Ca"2 uptake co-migrates with the plasma membrane H'-ATPase on a sucrose gradient. Ca2" uptake is insensitive to oligomycin, inhibited by vanadate (ICs. 40 micromolar) and erythrosin B (ICo 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca2+ uptake is insensitive to protonophores. These results indicate that Ca2 transport in these microsomal vesicles is catalyzed by a Mg8-dependent ATPase localized on the plasma membrane. Ca2o strongly reduces ApH generation by the plasma membrane H'-ATPase and increases MgATP-dependent membrane potential difference (A4') generation. These effects of Ca2" on ApH and A4, generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca2" uptake into plasma membrane vesicles. The Ca2"-induced increase of A4, is collapsed by permeant anions, which do not affect Ca2"-induced decrease of ApH generation by the plasma membrane H'-ATPase. The rate of decay of MgATP-dependent ApH, upon inhibition of the plasma membrane H'-ATPase, is accelerated by MgATPdependent Ca2" uptake, indicating that the decrease of ApH generation induced by Ca2 reflects the efflux of H' coupled to Ca2" uptake into plasma membrane vesicles. It is therefore proposed that Ca2" transport at the plasma membrane is mediated by a Mg2"-dependent ATPase which catalyzes a nH'/Ca2" exchange.activated Ca2'-transport ATPase, whose proposed localization at the plasma membrane has not been supported with convincing experimental evidence (8,9,30). Secondary active systems, nH+/ Ca2`antiporters which utilize the AAH+ built up by a H+-pumping ATPase to drive Ca2+ transport (1,3,10,16,(20)(21)(22)(23)(27)(28)(29)(30), have been described in tonoplast vesicles isolated from higher plants and yeast (1,3,16,20,21,27) and in plasma membrane vesicles isolated from Neurospora (28).The starting point of this work was to ascertain whether the plasma membrane of higher plant cells is endowed with a nH+/ Ca2+ antiporter analogous to that described in Neurospora. We approached this problem using microsomal vesicles isolated from radish seedlings germinated for 24 h. We have previously demonstrated that microsomes isolated at this stage of development exhibit MgATP-dependent H+-pumping catalyzed only by the vanadate-sensitive plasma membrane H+-ATPase with no activity of the tonoplast H+-ATPase detectable (25). Thus, any contribution ofthe nH+/Ca2+ antiporter ofthe tonoplast to MgATPdependent Ca2' uptake in this membrane fraction is minimal.Consequently, this membrane fraction is well suited for investigating whether ApH generated by the plasma membrane H+-ATPase of higher plants drives active Ca2+ transport through a nH+/Ca2' antiporter.The data reported in this paper lead us to conclude that a nH+/Ca2+ antiport driven by ApH does not occur on the plasma membrane of radish seedlings. Our results indicate that Ca...