Acidification of endomembrane compartments by the vacuolar-type H+-translocating ATPase (V-ATPase) is vital to the growth and development of plants. The V-ATPase purified from oat roots is a large complex of 650×10Mr that contains 10 different subunits of 70, 60,44,42, 36, 32, 29, 16, 13 and 12 × 103Mr. This set of ten polypeptides is sufficient to couple ATP hydrolysis to proton pumping after reconstitution of the ATPase into liposomes. Unlike some animal V-ATPases, the purified and reconstituted V-ATPase from oat is directly stimulated by Cl−. The peripheral complex of the ATPase includes the nucleotide-binding subunits of 70 and 60 × 103Mr and polypeptides of 44, 42, 36 and 29 × 103Mr. Six copies of the 16 × 103Mr proteolipid together with three other polypeptides are thought to make up the integral sector that forms the H+-conducting pathway. Release of the peripheral complex from the native membrane completely inactivates the pump; however, the peripheral subunits can be reassembled with the membrane sector to form a functional H+ pump.
Comparison of V-ATPases from several plants indicates considerable variations in subunit composition. Hence, several forms of the V-ATPase may exist among, and probably within, plant species. At least four distinct cDNAs encode the 16 × 103Mr proteolipid subunit in oat. Multiple genes could encode different subtypes of the H+ pump that are regulated by the developmental stage and physiological function specific to the cell or tissue type.
In plants, several different electrogenic H+ pumps provide the energy required to take up and distribute essential mineral nutrients for growth and development. These primary active transporters are (i) a plasma membrane H+-ATPase, (ii) a vacuolar-type H+-ATPase and (iii) an H+-pumping pyrophosphatase (H+-PPase). The electrochemical gradient generated by these H+ pumps provides the driving force for the secondary transport of numerous ions and metabolites (Fig. 1) (Sze, 1985).
Two distinct H+ pumps, the V-ATPase and the H+-PPase, acidify the vacuolar compartment (Rea and Sanders, 1987). Meristematic plant cells contain numerous small vacuoles or provacuoles that originate from the trans Golgi network. As cells differentiate and elongate, the provacuoles fuse to form one or more large vacuoles characteristic of plant cells. In mature cells, the vacuole is the largest intracellular organelle, occupying about 90% of the cell volume, surrounded by a membrane called the tonoplast. Being dynamic organelles, vacuoles participate in diverse functions (Table 1) depending on the tissue, the stage of development and the signals received. These functions include transport and storage of ions and metabolites, osmoregulation, signal transduction, protein storage and turnover, and storage of secondary metabolites and pigments (see Sze et al. 1992; Boiler and Wiemken, 1986). Fig. 1 shows some of the ion channels and H+-coupled transporters of plant vacuoles that are dependent on the primary H+ pumps. The proton-motive force, generated by either the H+-ATPase or the H+-PPase, and the resulting ion and metabolite fluxes are essential or central to the vital cellular processes performed by vacuoles and other endomembranes.
V-ATPases may be an integral component of the endomembrane system in plants (Sze et al. 1992), as has been observed in animals (Forgac, 1989). Plant endomembranes, which include the Golgi network, clathrin-coated vesicles, secretory vesicles and plasma membrane as well as the tonoplast, play a major role in the biogenesis of organelles, in the deposition of materials within the organelle and in the biosynthesis and transport of material destined for extracellular secretion (Chrispeels, 1991). One important feature of the secretory pathway is the role of organelle acidification in transport and targeting, as in the Golgi compartments (Mellman et al. 1986). Evidence for acidification of the Golgi compartments and coated vesicles by a vacuolar-type H+-ATPase in plants is emerging (e.g. Chanson and Taiz, 1985; Depta et al. 1991).
In the last 10 years, remarkable progress has been made in understanding the structure and function of V-ATPases from plants (Sze et al. 1992), fungi (Bowman and Bowman, 1986) and animals (Forgac, 1989; Nelson and Taiz, 1989). The unique characteristics of the H+ pumping (pH gradient, Δ pH and membrane potential, Δ φ) and ATP hydrolysis activities in vacuolar membrane vesicles from plants have been reviewed by Sze (1985). The central role of the V-ATPase in the growth and development of plants has been discussed (Sze et al. 1992). This chapter will highlight recent advances concerning the complex structure of the plant V-ATPase using data mainly from oat roots. Comparisons with V-ATPases from other plants and from animal tissues are discussed briefly. For a comprehensive coverage of plant V-ATPases, several other reviews are available (Sze, 1985; Sze et al. 1992; Rea and Sanders, 1987).
A FUNCTIONAL H+-PUMPING ATPASE IS A LARGE COMPLEX OF 10 SUBUNITS
Initial purification of plant V-ATPases had revealed three major subunits (Rea and Sanders, 1987); however, recent studies show that the plant enzyme is as complex as those found in animal and yeast endomembranes. The V-ATPase from oat roots was solubilized with Triton X-100, and purified by gel filtration and ion exchange chromatography (Ward and Sze, 1992a). Nitrate-sensitive ATP hydrolysis is associated with a large complex of 650 × 103Mr which contains subunits of 70, 60,44,42, 36, 32, 29, 16, 13 and 12 ×103Mr. In general, the subunit compositions of the V-ATPases purified from red beet, mung bean, oat and barley are similar, though not identical (Table 2). Until recently, none of the purified plant V-ATPases had been tested for proton transport activity in a reconstituted system.
To determine whether the V-ATPase complex from oat was active in proton transport, the purified enzyme was incorporated into liposomes from Escherichia coli phospholipids by removing the Triton X-100 with SM-2 biobeads. Acidification of K+-loaded proteoliposomes, monitored by the quenching of Acridine Orange fluorescence, was stimulated by valinomycin (Fig. 2A). As the presence of K+ and valinomycin dissipates a transmembrane electrical potential, the results indicated that ATP-dependent H+ pumping is electrogenic. The reconstituted pump retained its native properties since bafilomycin, a specific inhibitor of V-ATPases, completely inhibited H+ pumping at concentrations less than 50 nmol l−1 (Fig. 2B). Thus, a set of ten polypeptides associated with the oat V-ATPase (Table 2) is sufficient to couple ATP hydrolysis to proton pumping.
Unlike the purified red beet or barley H+ pump, the oat V-ATPase does not contain a large subunit of 100×103–115×103Mr. In the bovine coated vesicle V-ATPase, a 100×103Mr subunit is required for H+ transport, though it is particularly sensitive to proteases (Adachi et al. 1990). Since the purified oat V-ATPase lacks such a subunit and is active in transport, we conclude that the oat V-ATPase enriched in roots of 4-day-old seedlings lacks a 100× 103Mr subunit. The variations in subunit composition (Table 2) would suggest the presence of several subtypes of V-ATPases among plant species and probably within them as well. Interestingly, the oat root V-ATPase is strikingly similar to the kidney microsomal or brush border V-ATPase in its subunit composition and in its enzymatic properties (Gluck and Caldwell, 1987; Wang and Gluck, 1990). Furthermore, like the oat V-ATPase, the Golgi-associated V-ATPase from rat liver lacks a 115 ×103Mr subunit (Moriyama and Nelson, 1989) (Table 2). Differences in subunit composition between ATPases of various plants (Table 2) suggest that the 100×103Mr, 52× 103Mr and 36×103Mr subunits are either compartment-or tissue-specific or are developmentally regulated.
ATP hydrolysis of the purified and reconstituted ATPase is tightly coupled to H+ translocation. Both ATP hydrolysis and H+ transport activities were 50% inhibited by bafilomycin at 1 nmol per mg protein. With a relative molecular mass of 650×103 for the V-ATPase, this inhibition would indicate there are 0.65 nmol of bafilomycin per nmol of ATPase. Hence, bafilomycin reacts stoichiometrically with the V-ATPase at a site that has yet to be determined.
Unlike some of the animal V-ATPases, the oat V-ATPase is directly stimulated by Cl− and this activation is coupled to H+-pumping. Proton transport by the bovine coated vesicles is dependent on permeant anions that lead to dissipation of the membrane potential; however, neither ATP hydrolysis nor H+ pumping by the purified and reconstituted coated-vesicle H+-ATPase is stimulated by Cl− (Arai et al. 1989). By contrast, Cl− stimulates the purified and reconstituted oat V-ATPase and the activation is coupled to H+ pumping (Ward and Sze, 1992b). Since cytoplasmic [Cl−] in plant cells is about 30–90 mmol l−1, direct Cl− stimulation of the V-ATPase may be important for regulating the pH gradient (ΔpH) and membrane potential (Δ φ) across the vacuolar as well as the Golgi compartments.
THE LARGE PERIPHERAL COMPLEX CAN BE DISSOCIATED FROM THE MEMBRANE INTEGRAL SECTOR AND THEN REASSEMBLED
The nucleotide-binding subunits of 70 and 60×103Mr were previously shown to be peripheral subunits, as they are released from the membrane by chaotropic ions (Rea et al. 1987a), low ionic strength solutions or 0.1 mmol l−1 EDTA (Lai et al. 1988). However, recent studies clearly show that the peripheral complex of the oat V-ATPase consists of six different polypeptides of 70, 60, 44, 42, 36 and 29×103Mr (Fig. 3A). Like V-ATPases from chromaffin granules or bovine coated vesicles, 0.1 moll−1 KI in the presence of 5 mmol l−1 MgATP is more effective in stripping off the peripheral complex than KI alone. Interestingly, near physiological concentrations of KCl (0.2 mol l−1) or NaCI (0.15 mol l−1) also released the peripheral subunits, which inactivated 60–80% of the H+-pumping and ATPase activities (Parry et al. 1989; Ward et al. 1992). In red beet, the peripheral complex consists of five polypeptides of 67, 55, 52, 44 and 32 × 103Mr.
Five of the peripheral polypeptides from oat V-ATPase are released as a complex of about 540× 103Mr from the native membrane. Surprisingly, the 42× 103Mr subunit is not associated with this complex, but appears to be released separately (Ward and Sze, 1992a). These findings are strikingly similar to the dissociated V-ATPase of bovine coated vesicles, in which a 40× 103Mr subunit is not associated with the V1 subcomplex of 500 ×103Mr. Recently, Forgac and coworkers (Puopolo et al. 1992) showed that the 40×103Mr subunit is not required for H+ pumping activity. Since V1V0 complexes are less stable when assembled in the absence than in the presence of a 40 or 34×103Mr subunit, they suggest that these subunits stabilize the ATPase complex. Functionally, it is possible that the 42×103Mr subunit from the oat V-ATPase is analogous to the 40×103Mr subunit of the coated-vesicle proton pump.
Unlike the Fo complex of F-type ATPases, the membrane integral sector of the V-ATPase, the Vo complex, does not form a leaky proton pore. Although KI and MgATP completely abolished ATP-dependent proton pumping in tonoplast vesicles, PPi-driven H+ pumping was not affected (Rea et al. 1987a; Ward and Sze, 1992a). The H+-PPase, localized on the same membrane as the V-ATPase in plants, is made up of 2–4 integral polypeptides of 81× 103Mr that contain at least 13 trans-membrane domains (Sarafian et al. 1992). The ability to maintain a pH gradient in V1-stripped vesicles suggests that the V0 complex does not conduct H+ passively.
The subunit composition of the V0 complex, which forms the proton-conducting pathway, is less understood in both plants and animals than is that of the V1 complex. The major component common to the V0 complex of all V-ATPases is the N,N’-dicyclohexylcarbodiimide (DCCD)-binding 16× 103Mr subunit (Forgac, 1989; Nelson and Taiz, 1989). This hydrophobic polypeptide is referred to as a proteolipid because of its solubility in chloroform/methanol. From DCCD-binding studies and partial purification of the proteolipid from oat vacuolar vesicles, we estimated that there are six copies of the 16×103Mr subunit per V0 complex (Kaestner et al. 1988). Based on elimination of the peripheral polypeptides, we have suggested that the 16×103Mr subunit together with possibly the 32, 13 and 12×103Mr subunits make up the V0 complex from oat roots (Fig. 3A). However, in red beet V-ATPase, the V0 sector would consist of the 16× 103Mr proteolipids plus the 100× 103Mr polypeptide (Parry et al. 1989).
Dissociation and inactivation of the oat V-ATPase induced by KI is reversible in vitro (Ward et al. 1992) and possibly in vivo. As for the bovine-coated vesicle H+-ATPase (Puopolo and Forgac, 1990), removal of KI and MgATP by dialysis restores activity to the oat V-ATPase. ATP hydrolysis activity increased to about 50% of that of the untreated control, and the ATPase activity was coupled to H+ pumping as seen from the recovery of H+ transport. Furthermore, disappearance of the solubilized 70 and 60×103Mr subunits from the supernatant confirmed that the V1V0 complex had reassembled during dialysis. It is unclear whether such reversible dissociation of the V-ATPase in animal cells holds any physiological significance. In plants, many multimeric enzymes dissociate in response to chilling. The loss of vacuolar H+-pumping ATPase activity in mung bean suspension cells incubated at 2°C (Yoshida, 1991) is probably caused by enzyme dissociation, which is enhanced at 4°C (Parry et al. 1989; Ward et al. 1992). Full activity is restored within 1 h after cells are returned to 22°C. Since recovery did not require de novo protein synthesis (Yoshida, 1991), the dissociated and inactive V-ATPase is suggested to have reassembled at 22°C. Hence, the ability to dissociate and reassociate may be one mechanism by which the V-ATPase is regulated in vivo in response to environmental stress.
ABUNDANCE AND TRANSPORT RATE OF V-ATPASE IN PLANT CELLS
Electron microscopy demonstrates that the plant V-ATPase is a major component of the vacuolar membrane in actively growing tissues. The peripheral sectors of the plant V-ATPases are visible on the surface of vacuolar vesicles as knob-like structures (Fig. 3B). Negatively stained vesicles reveal densely packed particles 10–12nm in diameter. These particles are removed from the surface after washing with KI or KNO3, supporting their identity as the V1 complex of the V-ATPase (Ward et al. 1992). Close examination of the surface view shows a patchy distribution of structures. Some of the particles appear trigonal whereas others contain six subparticles (Taiz and Taiz, 1991; Klink and Luttge, 1991).
The abundance of the V-ATPase enzyme is supported by purification studies. A purification of 6-to 12-fold is sufficient to obtain the purified enzyme from oat roots, mung bean hypocotyl or barley roots of young seedlings (Ward and Sze, 1992a; Matsuura-Endo et al. 1990; DuPont and Morrisey, 1992). This value indicates that the V-ATPase makes up about 8–16% of the vacuolar membrane protein in young, actively growing seedlings. By contrast, V-ATPase appears to be only 1–2% of the tonoplast protein in mature red beet roots (Parry et al. 1989). The relative abundance of V-ATPases in young tissues probably reflects the presence of more H+ pumps in Golgi membranes, which are active in sorting and secretion of cell wall matrix polysaccharides and glycoproteins (Table 1).
The turnover number of protons pumped by V-ATPases from plants is within the range of 102– 103 ions s−1 typical of ion pumps driven by ATP hydrolysis. With the exception of red beet, the specific activities of purified V-ATPases from plant tissues range from 2.7μ molADP mg−1 protein min−1 at 22°C (Randall and Sze, 1986; Ward and Sze, 1992a) to 4.1 μ mol ADP mg− 1 protein min−1 at 30°C (Matsuura-Endo et al. 1990). With a stoichiometry of 2H+ pumped per ATP hydrolysed (see Sze et al. 1992 and references therein) and an average relative molecular mass of 650× 103 for the V-ATPase (Ward and Sze, 1992a), the transport rates of vacuolar H+-ATPase from oat (Ward and Sze, 1992a) and mung bean (Matsuura-Endo et al. 1990) are 60 and 90 protons per second, respectively. These transport rates are comparable to the bovine kidney V-ATPase, which has specific activities as high as 3.1 μ mol mg−1 protein min−1 (Gluck and Caldwell, 1987).
MOLECULAR STRUCTURE AND FUNCTION OF THE 16 × 103MR PROTEOLIPID
The 16× 103Mr proteolipid is one of the most interesting subunits of the V-ATPase complex because it is similar to the 8 × 103Mr proteolipid of the Fo-ATPase and has a putative role in H+ translocation. DCCD inhibits H+-pumping and ATP hydrolysis activity by covalent modification of the 16× 103Mr subunit from oat or red beet V-ATPase (Kaestner et al. 1988; Rea et al. 1987b). The amount of [14C]DCCD bound to the 16× 103Mr polypeptide is directly proportional to the inhibition of ATPase activity. From purification studies, we estimate that there are approximately six copies of the 16× 103Mr proteolipid per ATPase complex. However, binding studies and the kinetics of noncooperative inhibition show that the enzyme is completely inactivated when only 1 mol of DCCD is bound per mol of ATPase.
To understand the structure and function of the proteolipid at the molecular level, we have obtained a cDNA encoding the complete 16× 103Mr subunit from oat roots. This cDNA was detected using a synthetic oligonucleotide corresponding to a region of the V-ATPase proteolipid from bovine chromaffin granule (Mandel et al. 1988). The predicted amino acid sequence (165 amino acids, relative molecular mass 16641) of the oat proteolipid revealed a molecule with four membrane-spanning domains (Fig. 4) (Lai et al. 1991) similar to other V-ATPase proteolipids. Unlike the 8× 103Mr proteolipid from F-ATPases, the amino acid sequence of the 16× 103Mr subunit of V-ATPases is conserved among eukaryotes, especially in transmembrane domain IV. Domain IV shows about 80% amino acid sequence identity between the oat and the yeast 16× 103Mr proteolipid. A glutamate residue within this hydrophobic region is thought to be modified by the inhibitor DCCD. Support for this hypothesis has been provided in yeast, where replacing glutamate-137 with several amino acids, except aspartate, abolished activity (Noumi et al. 1991). Based on analogies with the 8× 103Mr proteolipid, one can envisage a simple model in which a functional proton pore is formed from six copies of the 16× 103Mr proteolipid plus a few other polypeptides. DCCD modification of only one of these proteolipids blocks H+ conductance by modifying the cooperative interaction required among the six subunits to form the proton pathway.
BIOSYNTHESIS AND ORIENTATION OF THE 16× 103MR SUBUNIT
Although hydropathy plots predicted a molecule with four membrane-spanning domains (Fig. 4), the topology of the molecule within the membrane is not clear. As a first step towards resolving this question, we have examined the orientation of the 16× 103Mr subunit synthesized in vitro. In vitro transcribed RNA was obtained using T7 RNA polymerase and the cDNA encoding the complete sequence of the oat 16× 103Mr proteolipid as a template. The RNA was translated using rabbit reticulocyte lysate and the products labeled with [35S]methionine were analysed by SDS– PAGE and fluorography. A 16× 103Mr polypeptide translated in vitro was stably inserted into either dog pancreatic microsomes or oat microsomal vesicles (S. Lai and H. Sze, unpublished results). The product was soluble in chloroform/methanol, but resistant to treatment with 0.4moll−1 Na2CO3 at pH 11, which releases peripheral proteins from the membrane.
To test the orientation of the 16× 103Mr proteolipid, we made two assumptions: (i) the 16× 103Mr proteolipid synthesized in vitro inserts into the membrane with the same orientation as the native proteolipid, and (ii) the protein inserts only into cytoplasmic-side-out endoplasmic reticulum vesicles. If so, we can examine the orientation based on the sensitivity of the polypeptide to proteases. Our working hypothesis is that only regions exposed at the membrane surface are sensitive to proteases. If the N and C termini face the cytoplasmic (cyt) side, protease digestion should yield small peptides of about 6– 8× 103Mr. However, should the N and C termini face the lumen (exo) side, proteolysis would result in peptides of about 6– 8 × 103Mr and small fragments of 3– 4× 103Mr.
Our preliminary results would support a model with Ccyt and Ncyt. After in vitro translation with dog pancreatic microsomes, the membranes were pelleted and digested with trypsin or proteinase K in the absence or presence of Triton X-100. In the absence of Triton X-100, either trypsin or proteinase K produced polypeptides of 6– 8× 103Mr and 16× 103Mr. However, polypeptides of 4– 5× 103Mr, but not 8× 103Mr, were detectable after protease digestion in the presence of Triton X-100.
This assignment in orientation is not consistent with the model of the F-ATPase, in which the 8× 103Mr proteolipid has both Cexo and Nexo. Using peptide-specific antibodies, Hensel et al. (1990) showed that the conserved hydrophilic region of subunit c is exposed to the cytoplasmic side. Furthermore, the proposed topology of the oat 16× 103Mr Air proteolipid does not agree with the model proposed by Hartmann et al. (1989), in which the more positive portion of residues flanking the first internal signal-anchor sequence of the protein faces the cytosol. Clearly, further studies are needed to confirm the proposed orientation of the 16× 103Mr proteolipid, in order to understand the biosynthesis and assembly of the V-ATPase complex.
V-ATPASE SUBUNITS ARE ENCODED BY MULTIGENE FAMILIES
A gene family of at least four members encodes the 16× 103Mr proteolipid in oats (Lai et al. 1991). The four distinct cDNAs showed extensive divergence in their codon usage and in their 3’-untranslated regions; however, the deduced amino acid sequences were 97– 99% identical. Genomic Southern blot analysis suggests that there may be as many as 6– 7 members in this gene family from oat.
Since oat is a hexapioid, we have begun to study the physiological significance of multiple genes encoding V-ATPase subunits using Arabidopsis. Arabidopsis thaliana has a small haploid genome (70000 kb), and tends to have smaller gene families. Using the cDNA from oat as a probe, at least two similar, but distinct, cDNAs have been obtained. These two differed from each other mainly in their codon usage and in their 3’-untranslated regions. The primary amino acid sequences deduced from the two clones, AVA-P1 and AVA-P2 (Arabidopsis Vacuolar ATPase-proteolipid), were identical. Genomic Southern analyses showed that 3– 4 DNA fragments hybridized with AVA-P1 at high stringency. These results suggest that Arabidopsis also has a small gene family encoding the 16× 103Mr subunit (I. Perera and H. Sze, unpublished results).
The 70× 103Mr subunit may also be encoded by a multigene family in several plants. Partial sequences (252– 294 bp in length) obtained by amplifying genomic DNA with conserved primers indicate that at least two separate genes may encode the catalytic subunit in Psilotum and Equisetum, two early land plants (Starke et al. 1991). The two DNA fragments of Psilotum differ mainly in their codon usage. In carrots, preliminary results using a similar approach suggest there are at least three separate genes for the 70× 103Mr subunit (L. Taiz, personal communication). However, it is not clear whether these multiple genes are actually expressed.
The discovery of multiple genes encoding V-ATPase subunits in plants raises many interesting questions. Perhaps multiple genes encode isoforms of the enzyme which could be differentially localized or specifically regulated, depending on the developmental stage. The participation of V-ATPases in the diverse roles of plant endomembranes would necessitate that the activity and expression of these pumps should be under physiological and developmental regulation. One future challenge in this area will be to understand how H+ pumping by V-ATPases and solute fluxes are integrated into plant cell division, growth and development.
Supported in part by the National Science Foundation (DCB– 90– 06402), the Department of Energy (DE– FG05– 86ER13461) to H.S. and the Maryland Agricultural Experimental Station project J-151 (Contribution no. 8533, Scientific Article no. A6349).