Claudio Olivari

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.

The Potassium Channel KAT1 Is Activated by Plant and Animal 14-3-3 Proteins

14-3-3 proteins modulate the plant inward rectifier K+ channel KAT1 heterologously expressed in Xenopus oocytes. Injection of recombinant plant 14-3-3 proteins into oocytes shifted the activation curve of KAT1 by +11 mV and increased the τon. KAT1 was also modulated by 14-3-3 proteins of Xenopus oocytes. Titration of the endogenous 14-3-3 proteins by injection of the peptide Raf 621p resulted in a strong decrease in KAT1 current (∼70% at –150 mV). The mutation K56E performed on plant protein 14-3-3 in a highly conserved recognition site prevented channel activation. Because the maximal conductance of KAT1 was unaffected by 14-3-3, we can exclude that they act by increasing the number of channels, thus ruling out any effect of these proteins on channel trafficking and/or insertion into the oocyte membrane. 14-3-3 proteins also increased KAT1 current in inside-out patches, suggesting a direct interaction with the channel. Direct interaction was confirmed by overlay experiments with radioactive 14-3-3 on oocyte membranes expressing KAT1.

Chlorella virus ATCV-1 encodes a functional potassium channel of 82 amino acids.

Chlorella virus PBCV-1 (Paramecium bursaria chlorella virus-1) encodes the smallest protein (94 amino acids, named Kcv) previously known to form a functional K+ channel in heterologous systems. In this paper, we characterize another chlorella virus encoded K+ channel protein (82 amino acids, named ATCV-1 Kcv) that forms a functional channel in Xenopus oocytes and rescues Saccharomyces cerevisiae mutants that lack endogenous K+ uptake systems. Compared with the larger PBCV-1 Kcv, ATCV-1 Kcv lacks a cytoplasmic N-terminus and has a reduced number of charged amino acids in its turret domain. Despite these deficiencies, ATCV-1 Kcv accomplishes all the major features of K+ channels: it assembles into a tetramer, is K+ selective and is inhibited by the canonical K+ channel blockers, barium and caesium. Single channel analyses reveal a stochastic gating behaviour and a voltage-dependent conductance that resembles the macroscopic I/V relationship. One difference between PBCV-1 and ATCV-1 Kcv is that the latter is more permeable to K+ than Rb+. This difference is partially explained by the presence of a tyrosine residue in the selective filter of ATCV-1 Kcv, whereas PBCV-1 Kcv has a phenylalanine. Hence, ATCV-1 Kcv is the smallest protein to form a K+ channel and it will serve as a model for studying structure-function correlations inside the potassium channel pore.

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.

14‐3‐3 proteins regulate the potassium channel KAT1 by dual modes

KAT1 is a cloned plant potassium channel belonging to the superfamily of Shaker-like Kv channels. Previous studies have shown that 14-3-3 proteins significantly increase KAT1 current by modifying the channel open probability. Employing a 14-3-3 scavenger construct to lower the long-term availability of endogenous 14-3-3 proteins, we found that 14-3-3 proteins not only control the voltage dependency of the channel but also the number of channels in the plasma membrane.

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.

ACA12 is a deregulated isoform of plasma membrane Ca2+-ATPase of Arabidopsis thaliana

Abstract Plant auto-inhibited Ca2+-ATPases (ACA) are crucial in defining the shape of calcium transients and therefore in eliciting plant responses to various stimuli. Arabidopsisthaliana genome encodes ten ACA isoforms that can be divided into four clusters based on gene structure and sequence homology. While isoforms from clusters 1, 2 and 4 have been characterized, virtually nothing is known about members of cluster 3 (ACA12 and ACA13). Here we show that a GFP-tagged ACA12 localizes at the plasma membrane and that expression of ACA12 rescues the phenotype of partial male sterility of a null mutant of the plasma membrane isoform ACA9, thus providing genetic evidence that ACA12 is a functional plasma membrane-resident Ca2+-ATPase. By ACA12 expression in yeast and purification by CaM-affinity chromatography, we show that, unlike other ACAs, the activity of ACA12 is not stimulated by CaM. Moreover, full length ACA12 is able to rescue a yeast mutant deficient in calcium pumps. Analysis of single point ACA12 mutants suggests that ACA12 loss of auto-inhibition can be ascribed to the lack of two acidic residues—highly conserved in other ACA isoforms—localized at the cytoplasmic edge of the second and third transmembrane segments. Together, these results support a model in which the calcium pump activity of ACA12 is primarily regulated by increasing or decreasing mRNA expression and/or protein translation and degradation.

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.

Plant Type 2B Ca 2+ -ATPases: The Diversity of Isoforms of the Model Plant Arabidopsis thaliana

In plant cells, Ca2+ extrusion from the cytoplasm is accomplished either through Ca2+-H+ antiporters powered by a proton-motive force or through Ca2+ pumps powered by ATP hydrolysis. Plants possess two types of Ca2+-pumping ATPase, named ECAs (for ER-type Ca2+-ATPase) and ACAs (for auto-inhibited Ca2+-ATPase), which group respectively with animal sarco-endoplasmic reticulum Ca2+-ATPase in the 2A subgroup of P-type ATPases or with plasma membrane Ca2+-ATPase in the 2B subgroup. Each type comprises different isoforms, localized on different membranes. Here, we summarize available knowledge of the biochemical characteristics and the physiological role of ACAs, focusing on what we have learnt from analysis of the model plant Arabidopsis thaliana.

The Plasma Membrane Calcium Pump: Studies on Membrane Vesicles Isolated from Higher Plants

Free Ca2+ concentration in the cytoplasm of living plant cells is kept in the submicromolar range against a steep electrochemical gradient which would favour Ca2+ influx into the cytoplasm both from intracellular compartments and from the apoplast (Poovaiah and Reddy, 1987; Felle 1989; Miller, Vogg and Sanders, 1990). A variety of hormonal and environmental stimuli induce an increase in cytoplasmic free Ca2+ concentration, most likely via the opening of regulated Ca2+ channels in endomembranes and/or in the plasma membrane (PM) (Poovaiah and Reddy, 1987; Tester, 1990).