Active transport across the vacuolar components of the eukaryotic endomembrane system is energized by a specific vacuolar H+-ATPase. The amino acid sequences of the 70-and 60-kDa subunits of the vacuolar H+-ATPase are -25% identical to the .8 and a subunits, respectively, of the eubacterial-type FOFj-ATPases. We now report that the same vacuolar H+-ATPase subunits are -50% identical to the a and 13 subunits, respectively, of the sulfur-metabolizing Sulfolobus acidocaldarius, an archaebacterium (Archaeobacterium). Moreover, the homologue of an 88-amino acid stretch near the amino-terminal end of the 70-kDa subunit is absent from the FOFj-ATPase P subunit but is present in the a subunit of Sulfolobus. Since the two types of subunits (a and 13 subunits; 60-and 70-kDa subunits) are homologous to each other, they must have arisen by a gene duplication that occurred prior to the last common ancestor of the eubacteria, eukaryotes, and Sulfolobus. Thus, the phylogenetic tree of the subunits can be rooted at the site where the gene duplication occurred. The inferred evolutionary tree contains two main branches: a eubacterial branch and an eocyte branch that gave rise to Sulfolobus and the eukaryotic host cell. The implication is that the vacuolar H+-ATPase of eukaryotes arose by the internalization of the plasma membrane H+-ATPase of an archaebacterial-like ancestral cell.Recently, attention has focused on the evolutionary relationships among the H+-ATPases, particularly the F0F1-ATPases (F-type) and vacuolar (V-type) H+-ATPases. F-and VATPases exhibit a number of structural and functional similarities (1-4). Both are large, multisubunit enzymes (=500 kDa) composed of a water-soluble catalytic sector and an integral membrane proton channel complex. Each hydrophilic sector contains three copies of the catalytic subunit (F-ATPase (3 subunit or V-ATPase 70-kDa subunit), three copies of a regulatory subunit (F-ATPase a subunit or V-ATPase 60-kDa subunit), and one copy each of several minor subunits (4). Sequences obtained for several eukaryotic V-ATPase 70-and 60-kDa subunits confirmed that the Fand V-type H+-ATPases are indeed homologous (5-9). However, the low overall similarity (25%) and the presence of a large stretch of nonhomologous sequence in the 70-kDa subunit (5) suggest that they diverged early in evolution. Consistent with this view, sequences obtained for the two major subunits of the membrane H+-ATPase of Sulfolobus acidocaldarius, an archaebacterium (Archaeobacterium), indicated that the "archaebacterial-type" H+-ATPase is only distantly related to the eubacterial-type F-ATPases (10, 11). In this joint communication from four of the laboratories involved, we show that the H+-ATPase of S. acidocaldarius belongs in the V-ATPase class of proton pumps. The implications for the origin of eukaryotes are discussed. MATERIALS AND METHODSTo determine the evolutionary relationships among the different H+-ATPases, protein or DNA sequences coding for the two major subunits or parts of these subunits were aligned, ...
The pH-dependent fluorescence quenching of acridine orange was used to study the Na'-and KI-dependent H' fluxes in tonoplast vesicles isolated from storage tissue of red beet and sugar beet (Beta vulgaris L.). The Na'-dependent H' flux across the tonoplast membrane could be resolved into two components: (a) a membrane potential-mediated flux through conductive pathways; and (b) an electroneutral flux which showed Michaelis-Menten kinetics relationship to Na' concentration and was competitively inhibited by amiloride (K1 = 0.1 millimolar). The potentialdependent component of H' flux showed an approximately linear dependence on Na' concentration. In contrast, the K-dependent H' flux apparently consisted of a single component which showed an approximately linear dependence on K concentration, and was insensitive to amiloride. Based on the Na'-and Ktdependent H' fluxes, the passive permeability of the vesicle preparation to Na was about half of that to K+.The apparent K,for Na of the electroneutral Nat/H+ exchange varied by more than 3-fold (7.5-26.5 millimolar) when the internal and external pH values were changed in parallel. The results suggest a simple kinetic model for the operation of the Na+/H+ antiport which can account for the estimated in vivo accumulation ratio for Na into the vacuole.Plant cells typically maintain a high K+/Na+ ratio in the cytoplasm, and there is evidence that Na+ is actively transported out ofthe cytoplasm at both the plasmalemma and the tonoplast membranes (10,14,15,17,24,25). While some moderately sodium-tolerant species depend mainly on exclusion of sodium at the plasma membrane, halophytes such as Beta vulgaris may accumulate large amounts of sodium which are sequestered into the vacuole to serve as osmoticum. Thus, transport of sodium at the tonoplast may be particularly important in these species (9).The mechanism of sodium transport at the tonoplast has not yet been elucidated. There is evidence supporting an H+/Na+ antiport mechanism at the plasma membrane of barley roots (19). The existence of a proton transporting ATPase at the tonoplast of various species (5, 7) including B. vulgaris (2,18,27) suggests the possibility of a similar mechanism for sodium transport into the vacuole. In the present work, we present evidence for an Na+/H+ antiport in tonoplast vesicles isolated from B. vulgaris. Membrane Preparations. Tonoplast vesicles were isolated as described elsewhere (20). For the identification of the membrane preparation as tonoplast, see "Discussion." The vesicles were preloaded with a buffer of desired ionic composition by suspension and sedimentation at 100,OOOg for 30 min. Final membrane pellets were resuspended in the same media to 6 mg protein/ml and incubated at 4°C for 3 h.Fluorescence Assays. The fluorescence quenching of acridine orange was used to monitor the formation and dissipation of inside-acid pH gradients (3, 18) across the membranes of the tonoplast vesicles. In all experiments, tonoplast vesicles (60 yg protein) were added to 2 ml of buffer con...
The energy-dependent transport of solutes across the vacuolar membrane (tonoplast) of plant cells is driven by two H' pumps: a vacuolar ("V-type") H+-ATPase (EC 3.6.1.3) and a HW-translocating (pyrophosphate-energized) inorganic pyrophosphatase (H+-PPase; EC 3.6.1.1). The H+-PPase, like the V-type H+-ATPase, is abundant and ubiquitous in the vacuolar membranes of plant cells, and both enzymes make a substantial contribution to the transtonoplast HW-electrochemical potential difference. Here, we report the cloning and sequence of cDNAs encoding the tonoplast H+-PPase of Arabidopsis thaiana. The protein predicted from the nucleotide sequence of the cDNAs is constituted of 770 amino acids and has a molecular weight of 80,800. It is a highly hydrophobic integral membrane protein, and the structure derived from hydrophilicity plots contains at least 13 transmembrane spans. Since the tonoplast H+-PPase appears to be constituted of one polypeptide species and genomic Southern analyses indicate that the gene encoding the M, 80,800 polypeptide is present in only a single copy in the genome of Arabidopsis, it is suggested that the H+-PPase has been cloned in its entirety. The lack of sequence identities between the tonoplast H+-PPase and any other characterized H+ pump or PP-dependent enzyme implies a different evolutionary origin for this translocase.The chemiosmotic hypothesis (1) contends that membranebound H+ pumps constitute the primary transducers by means of which living cells interconvert light, chemical, and electrical energy. Through the establishment and maintenance of transmembrane electrochemical gradients, H+ pumps energize the transport of other solutes or, in the special case of the energy-coupling membranes of mitochondria, chloroplasts, and bacteria, transduce the H+ electrochemical gradient generated by membrane-linked anisotropic redox reactions to the synthesis of ATP (1). Given the multitude of biological reactions energized by ATP, primary H+ translocation and the interconversions of ATP have come to be recognized as the principal generators of usable energy in the cell. Intriguing, therefore, is the fact that the vacuolar membrane (tonoplast) of plant cells contains not only a H+-ATPase (EC 3.6.1.3) (2, 3) but also an inorganic pyrophosphate-energized H+-pyrophophatase (H+-PPase; EC 3.6.1.1) (2). Both enzymes catalyze inward electrogenic H+ translocation (from cytosol to vacuole lumen), but the H+-PPase is unusual in its exclusive use of PPj as energy source (4).The tonoplast H+-PPase appears to be important for plant cell function: it is widespread, active, and abundant. The enzyme is ubiquitous in the vacuolar membranes of plant cells (2) and capable of establishing a H+ gradient of similar, and often greater, magnitude than the H+-ATPase on the same membrane (2, 5-7). The Mr 64,500-73,000 substrate (MgPPi)-binding subunit of the H+-PPase constitutes between 1% (8) and 10%1o (6, 7) of total vacuolar membrane protein and the purified enzyme has a turnover number of between 50 and 100 s-1, ...
Cold-acclimation-specific (CAS) gene expression has been examined by screening a cDNA library prepared from poly(A)+ RNA of cold-acclimated seedlings of a freezing-tolerant variety of alfalfa (Medcago falcata cv Anik). Three CAS cDNA clones, pSM784, pSM2201, and pSM2358, representing different sequence species, have been used to investigate the relative abundance and time-course of accumulation of corresponding transcripts. Results obtained show that the expression of these CAS genes is regulated in a coordinated manner most likely at the level of transcription. The expression of genes, as measured by mRNA abundance corresponding to the three CAS cDNA clones, is not stimulated or induced by heat shock, water stress, abscisic acid, or wounding. A positive correlation is observed between the expression of these cloned sequences and the degree of freezing-tolerance in four alfalfa cultivars.Freezing temperatures constitute one ofthe most important environmental constraints limiting the productivity and distribution of plants. Although plants are known to differ in their ability to withstand freezing temperatures, the molecular/genetic basis of this differential freezing-tolerance is unclear. It is known, however, that a prior exposure of plants to low nonfreezing temperatures (cold-acclimation) increases their tolerance to subsequent freezing (14). Many physiological and biochemical changes are known to occur in plants during cold-acclimation (5, 10, 13, 21) and it has been suggested (26) Cold-acclimation at 4°C was carried out as described (17) for the time periods mentioned in the text or figure legends. Tests of freezing tolerance were also carried out as described previously (17) except that several temperatures, namely, -50C, -8C, -12C, and -15C were used and LT50 values4 were determined. Administration of StressSeedlings were subjected to water stress, heat shock, wounding, and ABA treatment. ABA was used because it has been implicated in plant responses to environmental stresses (14), particularly to water stress ( 14) and low temperature stress ( 1,3). Water stress was imposed by placing the seedlings in polyethylene glycol-6000 (water potential of -15 bars), heat 4Abbreviations: LT5o, temperature at which 50% seedlings fail to
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