The Ca²⁺-Transport ATPase of Plant Plasma Membrane Catalyzes a nH⁺/Ca²⁺ Exchange

Abstract

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...

Identification of the Plasma Membrane Ca2+-ATPase and of Its Autoinhibitory Domain

“…As previously reported (Williams et al, 1990), formation of a phosphorylated intermediate during the catalytic cycle and low specificity for triphosphopurine nucleosides allow selective labeling of the PM Ca2+-ATPase with [Y-~~PIGTP. We have exploited the higli sensitivity of the enzyme to inhibition by fluorescein derivatives (Rasi-Caldogno et al, 1987De Michelis et al, 1993) to selectively label the catalytic site with FITC ( Dux et al, 1986). Finally, the PM Ca2+-ATPase retains the ability to bind CaM after SDS-PAGE and thus can be labeled by '251-CaM overlay (Evans et al, 1989;James et al, 1989).…”

“…In radish seeds at the early stages of germination, the endomembrane system is poorly developed. In Suc-density gradients a11 of ATP-dependen t Ca2+ transport and Ca2+-dependent ATP-hydrolyzing activity co-migrate with PM markers and no activity associated with ER membranes is detectable (Rasi-Caldogno et al, 1987. In this material, tonoplast markers co-migrate with PM on Suc gradients but can be easily separatetl from PM by the aqueous two-phase partitioning teclinique .…”

“…So, we decided to exploit the characteristic of the PM Ca2'-ATPase as being specifically inhibited by low concentrations of fluorescein derivatives (Rasi-Caldogno et al, 1987Graf and Weiler, 1989;Williams et al, 1990;Olbe and Sommarin, 1991;Carnelli et al, 1992;De Michelis et al, 1993) and to use FITC to specifically label the PM Ca2+-ATPase. Because this modified dye binds to Lys residues of the protein in an alkali-stable covalent manner, it could serve to identify the PM Ca2+-ATPase also on SDS gels run under standard (Laemmli, 1970) conditions.…”

“…Antimycin A-insensitive NADH-Cyt c reductase activity was assayed as described by Rasi-Caldogno et al (1987) but in the presence of 1 /AM antimycin A. Membrane protein was assayed according to the method of Markwell et al (1978).…”

“…In this work, to study the effect of controlled proteolysis on the PM Ca2+-ATPase at the molecular le\rel, besides following the migration of its phosphorylated intermediate in acidic gels, we have developed methods suitable also for SDS-PAGE according to Laemmli (1970), exploiting some characteristics of the enzyme. First, based on its high sensitivity to inhibition by fluorescein tlerivatives (Rasi-Caldogno et al, 1987Graf and Weiler, 1989;Williams et al, 1990;Olbe and Sommarin, 1991;Carnelli et al, 1992;De Michelis et al, 1993), we have selectively labeled the PM Ca2+-ATPase with FITC (Dux et al, 1986). Second, based on the high affinity of the PM Ca2+-ATPase for CaM, we have labeled it with lz5I-CaM (James et al, 1989).…”