Maria Chiara Pugliarello

Controlled Proteolysis Mimics the Effect of Fusicoccin on the Plasma Membrane H+-ATPase.

We analyzed the effects of controlled treatments with trypsin of plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings on the activity of the PM H+-ATPase, and we compared them with those of fusicoccin (FC). Mild treatments of the PM with trypsin, which led to a decrease of the molecular mass of the peptide of about 10 kD, markedly increased the H+-ATPase activity. The effect strongly increased with the increase of pH of the assay medium from 6.1 to 7.5, so the pH optimum of the enzyme activity shifted from 6.8 in untreated PM to 7.1 in trypsin-treated PM. The proteolytic treatment activated only the portion of PM H+-ATPase activity that is stable to preincubation in assay medium in the absence of ATP and determined a strong increase of Vmax and a less marked decrease of the apparent Km for Mg-ATP. All of these effects were very similar to those determined by FC, which activated the PM H+-ATPase without promoting its proteolytic cleavage. FC did not further activate the H+-ATPase activity of trypsin-treated PM under conditions in which the FC receptor was protected from the attack of trypsin. Conversely, trypsin treatment had little effect on the PM H+-ATPase preactivated with FC. Moreover, the activity of the PM H+-ATPase preactivated with FC was not further activated by Iysolecithin. These results indicate that the modification of the PM H+-ATPase of higher plants triggered by the FC-receptor complex hinders the inhibitory interaction of the regulatory C-terminal domain with the active site.

Fusicoccin stimulates the H+-ATPase of plasmalemma in isolated membrane vesicles from radish.

The effect of fusicoccin on the plasmalemma H+-ATPase has been investigated in a membrane fraction from 24 h old radish seedlings, in which Mg:ATP-dependent H+-transport is mediated only by the plasmalemma H+-ATPase. Fusicoccin stimulated the plasmalemma H+-ATPase - i.e. Mg:ATP-dependent intravesicular acidification, hyperpolaryzation of delta psi and ATPase activity -, when these activities were measured at the physiologically relevant pHs of 7.3 to 7.6. No effect of FC on the plasmalemma H+-ATPase was evident at pH 6.6.

Two distinct proton translocating ATPases are present in membrane vesicles from radish seedlings

Two distinct systems of Mg‐ATP‐dependent transport of protons have been put in evidence in microsomal vesicles from radish seedlings by investigating the effects of vanadate and of NO− 3 on the initial rate of acridine orange accumulation. One system is vanadate‐resistant and is inhibited by NO− 3 (I 50 1.3 mM), the other is inhibited by vanadate (I 50 43 μM) and unaffected by NO− 3. The two systems differently distribute along a continuous sucrose density gradient: the NO− 3‐sensitive system of Mg‐ATP‐dependent transport of protons peaks at d = 1.13–1.14, while the vanadate‐sensitive one peaks at d = 1.16–1.17.

Active transport of Ca2+ in membrane vesicles from pea: Evidence for a H+/Ca2+ antiport

Two non mitochondrial systems involved in ATP-dependent Ca2+ accumulation have been described and characterized in two membrane fractions from pea internodes purified on a metrizamide-sucrose discontinuous gradient. In the lighter membrane fraction an ATP-dependent Ca2+ accumulation system, which shows the characteristics of an ATP-dependent H+/Ca2+ antiport, predominates. This system is inhibited by FCCP and nigericin and stimulated by 50 mM KCl. It is saturated by 0.8–1.0 mM MgSO4-ATP, strictly requires ATP and is severely inhibited by an excess of free Mg2+ or Mn2+. A second system of ATP-dependent Ca2+ accumulation, recovered mainly in the heavier membrane fraction, is insensitive to FCCP, is saturated by 8–10 mM MgSO4-ATP, can utilize also ITP or other nucleoside triphosphates although at lower rate than ATP and is only scarcely affected by an excess of free Mg2+ or Mn2+. This system is interpreted as corresponding to the (Ca2+ + Mg2+)-ATPase described by Dieter, P. and Marme, D. ((1980) Planta 150, 1–8).

Evidence that H+ extrusion in Elodea densa leaves is mediated by an ATP-driven H+ pump

Abstract Data are presented on the ATP-driven proton extrusion mechanism in Elodea densa leaves, in the absebce of photosynthetic activity. In this material, in the dark, or in the light with photosynthesis inhibited by DCMU, H + extrusion at pH 6 was very low in the absence of permeating cations and became significant (approx. 1.8 μmol/g fresh wt.) in the presence of K + . Rb + and, much less so, Na + , could partially substitute for K + while Li + , Cs + , Ca 2+ , Co 2+ , Ni 2+ were ineffective. Fusicoccin (FC), a toxin which stimulates the plasmalemma H + ATPase activity in several plant tissues, strongly increased H + extrusion only in the presence of K + , Rb + , and, to a much lesser extent, Na + . The capacity of cations to stimulate FC-induced H + extrusion decreased in the same order as their E m -depolarizing activity (K + > Rb + ⪢ Na + ). Increasing concentrations of FC, between 10 −7 and 5 × 10 +4 M, showed a saturating effect for H + extrusion at 10 −4 M FC. The stimulating effect of FC on H + extrusion was accompanied by a hyperpolarization of the transmembrane electrical potential (by approx. 30 mV), larger in the absence than in the presence of K + , and by a marked increase of K + uptake. FC-induced H + extrusion was strongly inhibited by orthovanadate and erythrosin B, two inhibitors of the plasmalemma H + transporting ATPase. The activity of an ATPase and an ATP-dependent H + transport, both sensitive to vanadate and erythrosin B, were also demonstrated in membrane vesicle preparations. These results are interpreted as indicating the presence in E. densa leaves of an electrogenic H + extrusion mechanism which appears identical to the K + -dependent, FC-sensitive, ATP-driven proton pump described in several terrestrial plant cells.

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase (III. Is There a Direct Interaction between the Fusicoccin Receptor and the Plasma Membrane H+-ATPase?)

A radioimmunoassay using antibodies raised against bovine serum albumin-conjugated fusicoccin (FC) was applied to measure FC bound to the plasma membrane (PM) isolated from seedlings of radish (Raphanus sativus L.) and of Arabidopsis thaliana treated in vivo plus or minus the toxin. FC bound to the PM from seedlings treated with 5 [mu]M FC was 2-fold (radish) to 7-fold (A. thaliana) higher than the binding capacity of control PM. FC binding depended on the duration of the in vivo treatment but was unaffected by cycloheximide. When FC binding and the PM H+-ATPase activity were compared under different conditions (in vivo or in vitro treatment of different lengths or with different concentrations of FC), a strict linear relation between FC binding and the activation of the PM H+-ATPase was observed in both plant materials under all the conditions tested. Comparison between the maximum binding capacity and the amount of H+-ATPase observed in PM from the two plant materials suggest a one-to-one stoichiometry between the FC receptor and the PM H+-ATPase.

Reconstitution of proton pumping activity of a plasma membrane ATPase purified from radish

Abstract Plasma membrane ATPase partially purified from radish seedlings ( Raphanus sativum L.) (2.4–3.5 μmol P i min −1 mg −1 protein) has been reconstituted in proteoliposomes by the cholate-dialysis technique. Proteoliposomes are able to acidify their internal volume in the presence of Mg:ATP. Mg:ATP-dependent proton pumping is prevented by N , N ′-dicyclohexylcarbodiimide (DCCD) and by vanadate at the same concentrations which are effective on the phosphohydrolyzing activity of the plasma membrane ATPase.

Effect of Mg2+, tritorn X-100 and temperature on basal and FC- stimulated plasma membrane ATPase activity

Abstract H + -pumping driven by the plasma membrane H + -ATPase in membrane vesicles from 24-hour-old radish seedlings is stimulated by pretreatment of the membranes with fusicoccin (FC) (Rasi-Caldogno et al., Plant Physiol., 82 (1986) 121). FC-pretreatment stimulates also the ATPase activity, but to a lesser extentthan H + -pumping. More than 80% of the ATPase activity is inhibited by 100 μM vanadate or by 3 mM Ca 2+ . Preincubation of diluted membranes in the presence of 5 mM MgSO 4 without ATP lowers both ATPase and H + -pumping activity by 20—30% without affecting FC-stimulated activities (i.e. the differences between FC-treated samples and the controls). After preincubation with MgSO 4 , ATPase activity of membranes pretreatedwith or without FC is delivery affected by Triton X-100 and by temperature: Triton X-100 activates FC-stimulated ATPase more than that of the controls and an increase of temperature (between 13 and 33°C) enhances ATPase activity of the controls more than the FC-stimulated one. These results have been interpreted as suggesting that, while H + -pumping in this membrane fraction is driven only by the plasma membrane H + -ATPase, ATP-hydrolysis is catalyzed by two different enzymes (or forms of the same enzxxyme) diversely sensitive to FC, Triton X-100 and temperature and possibly diversely involved in H + -pumping.

The plasma membrane Ca2+ pump: Potential role in Ca2+ homeostasis

Plant cells, like those of other organisms, maintain the concentration of free Ca2+ in the cytoplasm in the submicromolar range, against a steep electrochemical gradient which would favour Ca2+influx into the cytoplasm from both the apoplast and endocellular compartments (Poovaiah and Reddy, 1987; Felle, 1989; Miller et al. 1990). An increasing amount of evidence indicates that also in plant cells transient increases of cytoplasmic free Ca2+ concentration can be brought about by a variety of environmental or endogenous stimuli and play a crucial role in the regulation of metabolic events. Such increases appear to be mediated by the opening of Ca2+ channels localized in the plasma membrane and/or in endomembranes (Poovaiah and Reddy, 1987; Tester, 1990). Regulation of Ca2+ channel opening and the role of Calf in signal transduction is discussed in other papers in this book.

Effects of penconazole on plasma membranes isolated from radish seedlings

Abstract The triazole derivative penconazole at concentrations from 10−4 to 3 × 10−4 M inhibited the plasma membrane H+-ATPase when its activity was assayed both on native membranes and on the partially purified enzyme. The same concentrations of penconazole also inhibited the plasma membrane Ca2+-ATPase and increased the membrane permeability to ions. A possible dependence of these three effects on the interaction between penconazole and plasma membrane phospholipids is proposed.