Controlled Proteolysis Activates the Plasma Membrane Ca2+ Pump of Higher Plants (A Comparison with the Effect of Calmodulin in Plasma Membrane from Radish Seedlings)

Abstract

The effects of calmodulin and of controlled trypsin treatments on the activity of the Caz+ pump were investigated in plasma membrane purified from radish (Raphanus sativus 1.) seedlings. Treatment of the plasma membrane with ethylenediaminetetraacetate (EDTA), which removed about two-thirds of the plasma membrane-associated calmodulin, markedly increased the stimulation of the Caz+ pump by calmodulin. In EDTA-treated plasma membrane, stimulation by calmodulin of the Caz+ pump activity was maximal at low free Caz+ (2-5 p~) and decreased with the increase of free CaZ+ concentration. l h e CaZ+ pump activity was stimulated also by a controlled treatment of the plasma membrane with trypsin: the effect of trypsin treatment depended on the concentration of both trypsin and plasma membrane proteins and on the duration of incubation. Stimulation of the CaZ+ pump activity by trypsin treatment of the plasma membrane was similar to that induced by calmodulin both in extent and in dependence on the free CaZ+ concentration in the assay medium. Moreover, the Caz+ pump of trypsin-treated plasma membrane was insensitive to further stimulation by calmodulin, suggesting that limited proteolysis preferentially cleaves a regulatory domain of the enzyme that is involved in its activation by calmodulin.In plant cells, the extrusion of Ca2+ from the cytoplasm to the apoplast is catalyzed by a Mg-ATP-dependent Ca2+ pump. During the last few years, the transport and hydrolytic activities of the plant PM Ca2+ pump have been characterized in some detail both in native PM vesicles and in proteoliposomes reconstituted with the solubilized and partially purified enzyme (reviewed in Briskin, 1990; Evans et al., 1991;De Michelis et al., 1992a). The most striking characteristics of the plant PM Ca2+ pump are its ability to use ITP or GTP besides ATP as a substrate (Williams et al., 1990; Camelli et al., 1992) and its high sensitivity to inhibition by fluorescein derivatives such as erythrosin B (Rasi-Caldogno et al., 1987, This work was supported by a grant of the Italian Ministry for University and Scientific and Technologic Research (40% quote) and by the Italian Ministry of Agriculture in the framework of the project 'Resistenze genetiche delle piante agrarie agli stress biotici ed abiotici."

Modulation of an Intracellular Calmodulin-Stimulated Ca2+-Pumping ATPase in Cauliflower by Trypsin (The Use of Calcium Green-5N to Measure Ca2+ Transport in Membrane Vesicles)

“…Tryptic digestion of the cauliflower Ca2+-ATPase resulted in a Kn(Ca2+) of 0.5 p~, whereas a value of about 2 p~ was obtained with untreated membranes in the presence of CaM. In agreement with these results, it was reported that activation of the Ca2+-ATPase in radish plasma membranes by trypsin was maximal at low Ca2+ concentrations (Rasi-Caldogno et al, 1993). CaM had no effect on the V,,, or the Ca2+ affinity with trypsin-treated cauliflower membranes, as expected from the 1251-CaM-binding studies, which showed that the CaM-binding region was rapidly lost during trypsin treatment (Fig.…”

“…For this purpose, an antiserum was raised against the CaM-stimulated CaZf-ATPase from the low-density membranes, and a fluorometric method for continuous measurement of Ca2+ transport in membrane vesicles was developed. The results are discussed in relation to what is known about the Ca2+-ATPase in animal plasma membranes and to recent reports of the effect of trypsin treatment of the CaM-stimulated CaZf-ATPase in plasma membranes from radish seedlings (Rasi-Caldogno et al, 1993, 1995.…”

“…In animals, only the plasma-membrane Ca2+-ATPase is directly stimulated by CaM (Carafoli, 1994). In contrast, CaM-stimulated Ca2+-ATPases in plants have been reported to be present in the ER (Hsieh et al, 1991;Askerlund and Evans, 1992;Gilroy and Jones, 1993;Liss and Weiler, 1994;Logan and Venis, 1995), the vacuolar membrane (Gavin et al, 1993), and the chloroplast envelope (Nguyen and Siegenthaler, 1985), as well as the plasma membrane (Robinson et al, 1988;Rasi-Caldogno et al, 1993, 1995 Olbe and M. Sommarin, unpublished data; for a review, see Askerlund and Sommarin, 1996). CaM-stimulated Ca2+-ATPase in cauliflower (Brassica olevacea L.) was earlier found to be located mainly in a low-density intracellular membrane fraction that was suggested to be the ER, but also in the plasma membrane (Askerlund and Evans, 1992).…”

Single Point Mutations in the Small Cytoplasmic Loop of ACA8, a Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana, Generate Partially Deregulated Pumps

“…Fig. 3 also shows the response of WT and D291A ACA8 to controlled proteolysis with trypsin, which selectively cleaves the N terminus of plant PM Ca 2ϩ -ATPase (12,28,29). Under the applied conditions tryptic treatment of the membranes effectively, albeit not completely, cleaved the N terminus of WT and D291A ACA8 (Fig.…”

Auto-inhibition of Drs2p, a Yeast Phospholipid Flippase, by Its Carboxyl-terminal Tail

“…Various regulatory activities can relieve the auto-inhibition caused by this R domain. For example, the P2 B -ATPase subgroup, comprising the plasma membrane Ca 2ϩ pumps, contains an R domain in the C-terminal tail (in animals) (8,9,18) or in the N-terminal tail (in plants) (13)(14)(15)(16)19). When calmodulin interacts with this R domain, it displaces the autoinhibitory tail and activates the protein for Ca 2ϩ transport.…”