Functional expression of Acetabularia acetabulum vacuolar H(+)-pyrophosphatase in a yeast VMA3-deficient strain

The function of the translation product of cDNA for Acetabularia vacuolar H(+)-pyrophosphatase was examined using the Saccharomyces cerevisiae VMA3-deficient strain. The open reading frame of Acetabularia H(+)-pyrophosphatase was revealed to encode 751 amino acids (721 or 751 amino acids in a previous paper). The acidification of the vacuole was observed by fluorescence microscopy when the cDNA was constructed in pYES2. Immunoblot analysis also supported the localization of the translation product in the vacuolar-membrane-enriched fraction.     

A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast

V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.     

Photochemistry of Acetabularia rhodopsin II from a marine plant, Acetabularia acetabulum

Acetabularia rhodopsins are the first microbial rhodopsins discovered in a marine plant organism, Acetabularia acetabulum. Previously, we expressed Acetabularia rhodopsin II (ARII) by a cell-free system from one of two opsin genes in A. acetabulum cDNA and showed that ARII is a light-driven proton pump [Wada, T., et al. (2011) J. Mol. Biol. 411, 986-998]. In this study, the photochemistry of ARII was examined using the flash-photolysis technique, and data were analyzed using a sequential irreversible model. Five photochemically defined intermediates (P(i)) were sufficient to simulate the data. Noticeably, both P(3) and P(4) contain an equilibrium mixture of M, N, and O. Using a transparent indium tin oxide electrode, the photoinduced proton transfer was measured over a wide pH range. Analysis of the pH-dependent proton transfer allowed estimation of the pK(a) values of some amino acid residues. The estimated values were 2.6, 5.9 (or 6.3), 8.4, 9.3, 10.5, and 11.3. These values were assigned as the pK(a) of Asp81 (Asp85(BR)) in the dark, Asp92 (Asp96(BR)) at N, Glu199 (Glu204(BR)) at M, Glu199 in the dark, an undetermined proton-releasing residue at the release, and the pH to start denaturation, respectively. Following this analysis, the proton transfer of ARII is discussed.     

Effects of isoflurane on measurements of delayed lumininescence in Acetabularia acetabulum.

The volatile halogenated methyl ethyl ether, isoflurane, used as an anaesthetic, inhibits actin-based dynamics directly or indirectly in animal cells. In plant cells, most intracellular movements are related to actin pathways. We have used isoflurane in a unicellular alga, Acetabularia acetabulum, to test the dynamics of choloroplast organization. By measuring the delayed luminescence, we found that isoflurane worked efficiently in the unicellular organism and showed dose- and time-course-dependent actin-inhibition patterns. When A. acetabulum was treated with saturated solutions of isoflurane in artificial seawater (defined as 100% isoflurane) for 3 or 6 min, the delayed luminescence (DL) was decreased and was never recovered. In contrast, if treated with 75% diluted isoflurane, the DL was firstly inhibited and then recovered several hours later, and if treated with 50% diluted isoflurane, the change of DL was small. Our work proved that isoflurane can affect actin-related pathways in both animals and plants.     

Improvement of reconstitution of the Cl(-)-translocating ATPase isolated from Acetabularia acetabulum into liposomes and several anion pump characteristics.

The improved reconstitution of the Mono Q-III fraction, a Cl(-)-translocating ATPase, isolated from Acetabularia acetabulum (Ikeda et al. (1990) Biochemistry 29, 2057-2065) into liposomes rendered transport properties of this enzyme clear. The liposomes were prepared by the reversed-phase method using egg lecithin and cholesterol in a molar ratio of 2:1 and the purified ATPase was incorporated into the liposomes by a dialysis for 3 h. About 80% of the ATPase was incorporated into the liposomes. The weight ratio of the enzyme to lipid was 1:400-600. A sigmoid curve was obtained when the Cl(-)-transport activity of the enzyme was plotted against Cl- concentration. Hill's plot afforded a half-substrate concentration [S]0.5 of 45 mM and a Hill's coefficient n of 2.33. Effects of Br- and F- on the Cl(-)-transport were also examined in the reconstituted system, both halide ions decreased the 36Cl- efflux significantly. These kinetic data are in good agreement with the electrophysiological data presented by Tittor et al. ((1983) J. Membr. Biol. 75, 129-139).     

Development and organization of the central vacuole of Acetabularia acetabulum

* Here we analyzed the shape of the central vacuole of Acetabularia acetabulum by visualizing its development during diplophase (from juvenility through reproduction) and haplophase (from meiosis through mating). * Light microscopy and whole-organism applications of a pH-sensitive dye, neutral red, were used to visualize the anatomy of the central vacuole. We studied connectivity within the thallus by locally applying dye to morphologically distinct regions (rhizoid, stalk, apex, hairs) and observing dye movements. * In vegetative thalli most of the rhizoid, stalk and young hairs stained with dye. In reproductive structures (caps, gametangia) dye also stained the majority of the interiors. When applied to small areas, dye moved at different rates through each region of the thallus (e.g. within the stalk). Dye moved from younger hairs, but not from older hairs, into the stalk. Errors in incorporation of central vacuole into gametangia occurred at <10(-5). * These data indicate that the central vacuole of A. acetabulum is a ramified polar organelle with, potentially, a gel-like sap that actively remodels its morphology during development.     

ATPase activities in partially purified membranes of Acetabularia

Partly purified membranes (with plasmalemma material) of Acetabularia mediterranea were studied with respect to ATPase activity in alkali- and Ca⁺⁺-free media and its sensitivity to pH (5 – 9), oligomycin (200 ?g/mg protein), 100 ?M N-N′-dicyclohexylcarbodiimide (DCCD), and 50 ?M vanadate. Besides activities which may originate from mitochondrial H⁺ ATPase (oligomycin-sensitive, alkaline pH optimum) and tonoplast H⁺ ATPase (DCCD-sensitive, pH optimum 7.5), there is ATPase activity with a pH optimum around pH 6.5, sensitive to vanadate and insensitive to DCCD. These results strongly suggest that the electrogenic Cl^? pump in the plasmalemma of Acetabularia is an ATPase. Effects of Mg⁺⁺, Mg-ATP, ADP, GTP, UTP, CTP and HCO₃ ^? versus Cl^? on this ATPase activity are described.     

Oscillations in ultraweak photon emission of Acetabularia acetabulum (L.)

Ultraweak photon emission of dark-incubated A. acetabulum cells were measured with the use of a sensitive electronphotomultiplier of the Hamamatsu 550 type. The photon count series were subjected to Fourier analysis for 2-1020 sec period range. The average level of photon emission in samples containing 50 cells was approximately. 40% above background. Cell cultures were prepared at least 24 hr before the photon emission measurements and kept un-disturbed ("established cultures"). This paper reports results of Fourier analysis of a number of samples of Acetabularia cells. In a single population cells periodicity of light emission was not defined directly from Fourier transformation. A large number of analyses, however, if they are combined and compared with background data, reveal a cell-culture specific frequency pattern. The results suggest the idea that established cell-cultures are characterized by higher intensities of long period (minutes) oscillations occurs, while a relative decrease was observed in the short period (few seconds) range. The long period oscillations were not detected in cell cultures that were prepared within 1 hr before the photon emission measurements. It is concluded that Fourier analysis of ultraweak photon emission, even with relatively low signals, appears to be possible. It may serve as a non-invasive tool for monitoring the physiological state of cells, or for studying the control of intercellular dynamics.     

The vacuolar ATPase ofNeurospora crassa

The filamentous fungusNeurospora crassa has many small vacuoles which, like mammalian lysosomes, contain hydrolytic enzymes. They also store large amounts of phosphate and basic amino acids. To generate an acidic interior and to drive the transport of small molecules, the vacuolar membranes are densely studded with a proton-pumping ATPase. The vacuolar ATPase is a large enzyme, composed of 8–10 subunits. These subunits are arranged into two sectors, a complex of peripheral subunits called V₁ and an integral membrane complex called V₀. Genes encoding three of the subunits have been isolated.vma-1 andvma-2 encode polypeptides homologous to the and subunits of F-type ATPases. These subunits appear to contain the sites of ATP binding and hydrolysis.vma-3 encodes a highly hydrophobic polypeptide homologous to the proteolipid subunit of vacuolar ATPases from other organisms. This subunit may form part of the proton-containing pathway through the membrane. We have examined the structures of the genes and attempted to inactivate them.     

Heterogeneity of the vacuolar pyrophosphatase protein fromChenopodium rubrum

Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography, liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE) to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells of Chenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot.     

Asymmetric subcellular mRNA distribution correlates with carbonic anhydrase activity in Acetabularia acetabulum

The unicellular green macroalga Acetabularia acetabulum L. Silva is an excellent system for studying regional differentiation within a single cell. In late adults, physiologically mediated extracellular alkalinity varies along the long axis of the alga with extracellular pH more alkaline along the apical and middle regions of the stalk than at and near the rhizoid. Respiration also varies with greater respiration at and near the rhizoid than along the stalk. We hypothesized that the apical and middle regions of the stalk require greater carbonic anhydrase (CA) activity to facilitate inorganic carbon uptake for photosynthesis. Treatment of algae with the CA inhibitors acetazolamide and ethoxyzolamide decreased photosynthetic oxygen evolution along the stalk but not at the rhizoid, indicating that CA facilitates inorganic carbon uptake in the apical portions of the alga. To examine the distribution of enzymatic activity within the alga, individuals were dissected into apical, middle, and basal tissue pools and assayed for both total and external CA activity. CA activity was greatest in the apical portions. We cloned two CA genes (AaCA1 and AaCA2). Northern analysis demonstrated that both genes are expressed throughout much of the life cycle of A. acetabulum. AaCA1 mRNA first appears in early adults. AaCA2 mRNA appears in juveniles. The AaCA1 and AaCA2 mRNAs are distributed asymmetrically in late adults with highest levels of each in the apical portion of the alga. mRNA localization and enzyme activity patterns correlate for AaCA1 and AaCA2, indicating that mRNA localization is one mechanism underlying regional differentiation in A. acetabulum.     

Improved mathematical model for estimating H+ influx and H+ efflux in plant vacuolar vesicles acidified by ATPase or pyrophosphatase

To adapt to environmental changes, plant cells very likely possess a biochemical system, using vacuoles, for maintaining cytoplasmic pH homeostasis. A simple approach is to estimate the active H(+) influx and H(+) efflux of isolated vacuolar vesicles, although there is no good mathematical model to describe H(+) flux. To establish a new quantitative model, vacuolar vesicles were isolated from hypocotyls of mung bean (Vigna radiata L.), and pyrophosphate (PPi)- or ATP-dependent acidification was monitored using acridine orange. The change of pH inside the vesicles (pH(in)) was calculated using a pH calibration curve relating fluorescence quenching with DeltapH. After formation of a steady state DeltapH, passive H(+) efflux was monitored after terminating pumping with ethylenediaminetetraacetate, and the relative H(+) permeability coefficient (p(H+)) was calculated. The H(+) efflux simulated using the p(H+) corresponded to the H(+) efflux determined experimentally. H(+) influx was then calculated by subtracting the predicted H(+) efflux from the experimental net H(+) influx. H(+) influx into vesicles driven by H(+)-PPase or H(+)-ATPase decreased exponentially as the intravesicular pH(in) decreased, suggesting modulation of pumping by DeltapH, pH(in), or both. Finally, the PPi- or ATP-dependent H(+) accumulation determined experimentally was closely simulated by the predicted H(+) influx and H(+) efflux. The ability to predict H(+) flux under different conditions provides a powerful tool for studying pH homeostasis.     

Reconstitution of transport function of vacuolar H(+)-translocating inorganic pyrophosphatase.

A procedure for reconstitution of the transport function of the vacuolar H(+)-translocating inorganic pyrophosphatase (H(+)-PPase; EC 3.6.1.1) prepared from etiolated hypocotyls of Vigna radiata (mung bean) is described. The method entails sequential extraction of isolated vacuolar membrane (tonoplast) vesicles with deoxycholate and CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), combination of CHAPS-solubilized protein with phospholipid-cholesterol mixtures, dialysis, and glycerol density gradient centrifugation. The final proteoliposome preparation is 9-fold enriched for PPase activity and active in pyrophosphate (PPi)-energized electrogenic H(+)-translocation. Since both PPi hydrolysis and PPi-dependent H(+)-translocation by the proteoliposomes are indistinguishable from the corresponding activities of native tonoplast vesicles, the functional integrity of the H(+)-PPase appears to be conserved during solubilization and reconstitution. The high transport capacity and amenability of the reconstituted enzyme to both radiometric membrane filtration and fluorimetric H(+)-translocation assays, on the other hand, demonstrate its applicability to a broad range of transport studies. SDS-polyacrylamide gel electrophoresis of the proteoliposomes reveals selective enrichment of the M(r) 66,000, substrate-binding subunit of the H(+)-PPase and two additional polypeptides of M(r) 21,000 and 20,000. Although the M(r) 21,000 and 20,000 polypeptides have not been described previously, all attempts to reconstitute H(+)-PPase lacking these components were unsuccessful. It is therefore tentatively proposed that the M(r) 21,000 and 20,000 polypeptides, as well as the M(r) 66,000 subunit, are required for the productive reconstitution of PPi-dependent H(+)-translocation.     

Structure of the vacuolar ATPase by electron microscopy.

The structure of the vacuolar ATPase from bovine brain clathrin-coated vesicles has been determined by electron microscopy of negatively stained, detergent-solubilized enzyme molecules. Preparations of both lipid-containing and delipidated enzyme have been analyzed. The complex is organized in two major domains, a V(1) and V(0), with overall dimensions of 28 x 14 x 14 nm. The V(1) is a more or less spherical molecule with a central cavity. The V(0) has the shape of a flattened sphere or doughnut with a radius of about 100 A. The V(1) and V(0) are joined by a 60-A long and 40-A wide central stalk, consisting of several individual protein densities. Two kinds of smaller densities are visible at the top periphery of the V(1), and one of these seems to extend all the way down to the stalk domain in some averages. Images of both the lipid-containing and the delipidated complex show a 30-50-kDa protein density on the lumenal side of the complex, opposite the central stalk, centered in the ring of c subunits. A large trans-membrane mass, probably the C-terminal domain of the 100-kDa subunit a, is seen at the periphery of the c subunit ring in some projections. This large mass has both a lumenal and a cytosolic domain, and it is the cytosolic domain that interacts with the central stalk. Two to three additional protein densities can be seen in the V(1)-V(0) interface, all connected to the central stalk. Overall, the structure of the V-ATPase is similar to the structure of the related F(1)F(0)-ATP synthase, confirming their common origin.     

A genomic screen for yeast vacuolar membrane ATPase mutants

V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification and the basis of these growth phenotypes, approximately 4700 yeast deletion mutants were screened for growth defects at pH 7.5 in 60 mm CaCl(2). In addition to 13 of 16 mutants lacking known V-ATPase subunits or assembly factors, 50 additional mutants were identified. Sixteen of these also grew poorly in nonfermentable carbon sources, like the known V-ATPase mutants, and were analyzed further. The cwh36Delta mutant exhibited the strongest phenotype; this mutation proved to disrupt a previously uncharacterized V-ATPase subunit. A small subset of the mutations implicated in vacuolar protein sorting, vps34Delta, vps15Delta, vps45Delta, and vps16Delta, caused both Vma- growth phenotypes and lower V-ATPase activity in isolated vacuoles, as did the shp1Delta mutation, implicated in both protein sorting and regulation of the Glc7p protein phosphatase. These proteins may regulate V-ATPase targeting and/or activity. Eight mutants showed a Vma- growth phenotype but no apparent defect in vacuolar acidification. Like V-ATPase-deficient mutants, most of these mutants rely on calcineurin for growth, particularly at high pH. A requirement for constitutive calcineurin activation may be the predominant physiological basis of the Vma- growth phenotype.     

An analysis of morphogenesis of the reproductive whorl of Acetabularia acetabulum

Acetabularia acetabulum (Linn.) P.C. Silva, is a useful system for studying changes in shape because it is large, morphologically complex unicell. The middle, or gametophore lobe of the cap grows radially from the stalk axis as a disc and the fully grown cap can be one of several shapes: flat, concave, convex, and saddle. The shape of the cap normally changes during the first three and a half weeks of reproductive development: individual caps within a population change shape in a stereotypical progression, with the majority proceeding from concave to flat to saddle. Marking the existing surface of caps with carbon grains revealed that the majority of growth occurs near the center, not at the perimeter, of caps. The shape of the mature cap appeared to be independent of algal height, number of gametophores per cap, and final cap diameter. Removing the rhizoid, which contains the nucleus, suggested that the contribution of the nucleus may be important for changes in shape during early cap growth. Based on these data, we present a simple model of cap shape development that suggests both differential growth and biophysical factors may contribute to the final shape of caps of A. acetabulum. Received: 7 January 1998 / Accepted: 7 March 1998