Ancient origin of the vacuolar H+-ATPase 69-kilodalton catalytic subunit superfamily

Recently, two distinct cDNA clones encoding the catalytic subunit of the vacuolar H⁺-ATPase (V-ATPase) were isolated from the allotetraploid cotton species Gossypium hirsutum L. cv Acala SJ-2 (Wilkins 1992, 1993). Differences in the nucleotide sequence of these clones were used as molecular markers to explore the organization and structure of the V-ATPase catalytic subunit genes in the A and D genomes of diploid and allotetraploid cotton species. Nucleotide sequencing of polymerase chain reaction (PCR) products amplified from G. arboreum (A₂, 2n=26), G. raimondii (D₅, 2n=26), and G. hirsutum cv Acala SJ-2 [(AD)₁, 2n=4x=52] revealed a V-ATPase catalytic subunit organization more complex than indicated hitherto in any species, including higher plants. In the genus Gossypium, the V-ATPase catalytic subunit genes are organized as a superfamily comprising two diverse but closely related multigene families, designated as vat69A and vat69B, present in both diploid and allotetraploid species. As expected, each vat69 subfamily is correspondingly more complex in the allotetraploid species due to the presence of both A and D alloalleles. Because of this, about one-half of the complex organization of V-ATPase catalytic subunit genes predates polyploidization and speciation of New World tetraploid species. Comparison of plant and fungal V-ATPase catalytic subunit gene structure indicates that introns accrued in the plant homologs following the bifurcation of plant and fungi but prior to the gene duplication event that gave rise to the vat69A and vat69B genes approximately 45 million years ago. The structural complexity of plant V-ATPase catalytic subunit genes is highly conserved, indicating the presence of at least ten introns dispersed throughout the coding region.     

Subunit C of the Vacuolar-Type ATPase from the Vanadium-Rich Ascidian Ascidia sydneiensis samea Rescued the pH Sensitivity of Yeast vma5 Mutants

A vanadium-accumulating ascidian, Ascidia sydneiensis samea, expresses vacuolar-type H⁺-ATPases (V-ATPases) on the vacuole membrane of the vanadium-containing blood cells known as vanadocytes. Previously, we showed that the contents of their vacuoles are extremely acidic and that a V-ATPase-specific inhibitor, bafilomycin A₁, neutralized the contents of the vacuoles. To understand the function of V-ATPase in vanadocytes, we isolated complementary DNA encoding subunit C of V-ATPase from vanadocytes because this subunit has been known to be responsible for the assembly of V-ATPases and to regulate the ATPase activity of V-ATPases. The cloned cDNA was 1443 nucleotides in length, and encoded a putative 384 amino acid protein. By expressing the ascidian cDNA for subunit C under the control of a galactose-inducible promoter, the pH-sensitive phenotype of the corresponding vma5 mutant of a budding yeast was rescued. This result showed that the ascidian cDNA for subunit C functioned in yeast cells. Received August 11, 2000; accepted March 5, 2001.     

Retrieval of the Vacuolar H+-ATPase from Phagosomes Revealed by Live Cell Imaging

Background

The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized.

Methodology

To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins.

Principal Findings

We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved.

Conclusions/Signficance

Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway.     

A Cytotoxic Type III Secretion Effector of Vibrio parahaemolyticus Targets Vacuolar H+-ATPase Subunit c and Ruptures Host Cell Lysosomes

Vibrio parahaemolyticus is one of the human pathogenic vibrios. During the infection of mammalian cells, this pathogen exhibits cytotoxicity that is dependent on its type III secretion system (T3SS1). VepA, an effector protein secreted via the T3SS1, plays a major role in the T3SS1-dependent cytotoxicity of V. parahaemolyticus. However, the mechanism by which VepA is involved in T3SS1-dependent cytotoxicity is unknown. Here, we found that protein transfection of VepA into HeLa cells resulted in cell death, indicating that VepA alone is cytotoxic. The ectopic expression of VepA in yeast Saccharomyces cerevisiae interferes with yeast growth, indicating that VepA is also toxic in yeast. A yeast genome-wide screen identified the yeast gene VMA3 as essential for the growth inhibition of yeast by VepA. Although VMA3 encodes subunit c of the vacuolar H⁺-ATPase (V-ATPase), the toxicity of VepA was independent of the function of V-ATPases. In HeLa cells, knockdown of V-ATPase subunit c decreased VepA-mediated cytotoxicity. We also demonstrated that VepA interacted with V-ATPase subunit c, whereas a carboxyl-terminally truncated mutant of VepA (VepAΔC), which does not show toxicity, did not. During infection, lysosomal contents leaked into the cytosol, revealing that lysosomal membrane permeabilization occurred prior to cell lysis. In a cell-free system, VepA was sufficient to induce the release of cathepsin D from isolated lysosomes. Therefore, our data suggest that the bacterial effector VepA targets subunit c of V-ATPase and induces the rupture of host cell lysosomes and subsequent cell death.     

Biochemical characterization of glyceraldehyde-3-phosphate dehydrogenase from <Emphasis Type="Italic">Thermococcus kodakarensis</Emphasis> KOD1

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an essential role in glycolysis by catalyzing the conversion of d-glyceraldehyde 3-phosphate (d-G3P) to 1,3-diphosphoglycerate using NAD⁺ as a cofactor. In this report, the GAPDH gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (GAPDH-tk) was cloned and the protein was purified to homogeneity. GAPDH-tk exists as a homotetramer with a native molecular mass of 145 kDa; the subunit molecular mass was 37 kDa. GAPDH-tk is a thermostable protein with a half-life of 5 h at 80–90°C. The apparent K _m values for NAD⁺ and d-G3P were 77.8 ± 7.5 μM and 49.3 ± 3.0 μM, respectively, with V _max values of 45.1 ± 0.8 U/mg and 59.6 ± 1.3 U/mg, respectively. Transmission electron microscopy (TEM) and image processing confirmed that GAPDH-tk has a tetrameric structure. Interestingly, GAPDH-tk migrates as high molecular mass forms (~232 kDa and ~669 kDa) in response to oxidative stress.     

Probing Subunit-Subunit Interactions in the Yeast Vacuolar ATPase by Peptide Arrays

Background

Vacuolar (H⁺)-ATPase (V-ATPase; V₁V_o-ATPase) is a large multisubunit enzyme complex found in the endomembrane system of all eukaryotic cells where its proton pumping action serves to acidify subcellular organelles. In the plasma membrane of certain specialized tissues, V-ATPase functions to pump protons from the cytoplasm into the extracellular space. The activity of the V-ATPase is regulated by a reversible dissociation mechanism that involves breaking and re-forming of protein-protein interactions in the V₁-ATPase - V_o-proton channel interface. The mechanism responsible for regulated V-ATPase dissociation is poorly understood, largely due to a lack of detailed knowledge of the molecular interactions that are responsible for the structural and functional link between the soluble ATPase and membrane bound proton channel domains.

Methodology/Principal Findings

To gain insight into where some of the stator subunits of the V-ATPase associate with each other, we have developed peptide arrays from the primary sequences of V-ATPase subunits. By probing the peptide arrays with individually expressed V-ATPase subunits, we have identified several key interactions involving stator subunits E, G, C, H and the N-terminal domain of the membrane bound a subunit.

Conclusions

The subunit-peptide interactions identified from the peptide arrays complement low resolution structural models of the eukaryotic vacuolar ATPase obtained from transmission electron microscopy. The subunit-subunit interaction data are discussed in context of our current model of reversible enzyme dissociation.     

Molecular Evidence for the Occurrence of H+-Transporting V-ATPase Subunit D and Two Different Forms of Subunit E in Leaves of the Obligate CAM Species Kalanchoë daigremontiana

Membrane proteins of purified tonoplast vesicles from leaves of Kalanchoë daigremontiana Hamet et Perrier were solubilized by the non-ionic detergent Triton X-114 and subsequently separated by MonoQ® anion-exchange chromatography. Special attention was given to the range of molecular masses around 30 kDa comprising the central stalk subunit peptides of the H⁺-transporting V-ATPase. Three polypeptides of apparent molecular masses of 32, 33 and 34 kDa were separated. Proteolytic fragments were obtained by trypsin digestion. Analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry of tryptic fragments of the 32 and 33 kDa peptides and protein data- bank comparisons showed that they are two different forms of subunit E. N-terminal amino acid sequencing of tryptic fragments of the 34 kDa peptide showed that it is subunit D. This work provides for the first time unequivocal molecular evidence that the central stalk of the V-ATPase of the obligate CAM plant K. daigremontiana includes subunit D and different forms of subunit E.     

Organ-Specific Responses of Vacuolar H~+-ATPase in the Shoots and Roots of C_3 Halophyte Suaeda salsa to NaCl

Suaeda salsa L. is a halophytic species that is well adapted to high salinity. In order to understand its salt tolerance mechanism, we examined the growth and vacuolar H⁺-ATPase (V-ATPase) response to NaCl within the shoots and roots. The growth of shoots, but not roots, was dramatically stimulated by NaCl. Cl⁻ and Na⁺ were mainly accumulated in shoots. V-ATPase activity was significantly increased by NaCl in roots and especially in shoots. Interestingly, antisera ATP95 and ATP88b detected three V₁ subunits (66, 55 and 36 KDa) of V-ATPase only in shoots, while an 18 kDa V₀ subunit of V-ATPase was detected by both antisera in shoots and roots. It suggested that the tissue-specific characteristics of V-ATPase were related to the different patterns of growth and ion accumulation in shoots and roots of S. salsa.     

Characterization of<Emphasis Type="Italic"> Schizosaccharomyces pombe</Emphasis> mutants defective in vacuolar acidification and protein sorting

The vacuolar H⁺-ATPases (V-ATPases) are ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. To investigate the functional roles of the V-ATPase in Schizosaccharomyces pombe, the gene vma1 encoding subunit A or vma3 encoding subunit c was disrupted. Both deletion mutants lost the capacity for vacuolar acidification in vivo, and showed sensitivity to neutral pH or high concentrations of divalent cations including Ca²⁺. The delivery of FM4-64 to the vacuolar membrane and accumulation of Lucifer Yellow CH were strongly inhibited in the vma1 and vma3 mutants. Moreover, deletion of the S. pombe vma1 ⁺ or vma3 ⁺ gene resulted in pleiotropic phenotypes consistent with lack of vacuolar acidification, including the missorting of vacuolar carboxypeptidase Y, abnormal vacuole morphology, and mating defects. These findings suggest that V-ATPase is essential for endocytosis, ion and pH homeostasis, and for intracellular targeting of vacuolar proteins and vacuolar biogenesis in S. pombe.Communicated by M. Johnston     

New Insight into the Structure and Regulation of the Plant Vacuolar H+-ATPase

Plant cells are characterized by a highly active secretory system that includes the large central vacuole found in most differentiated tissues. The plant vacuolar H⁺-ATPase plays an essential role in maintaining the ionic and metabolic gradients across endomembranes, in activating transport processes and vesicle dynamics, and, hence, is indispensable for plant growth, development, and adaptation to changing environmental conditions. The review summarizes recent advances in elucidating the structure, subunit composition, localization, and regulation of plant V-ATPase. Emerging knowledge on subunit isogenes from Arabidopsis and rice genomic sequences as well as from Mesembryanthemum illustrates another level of complexity, the regulation of isogene expression and function of subunit isoforms. To this end, the review attempts to define directions of future research on plant V-ATPase.     

Vacuolar-type H<Superscript>+</Superscript>-ATPase with the <Emphasis Type="Italic">a</Emphasis>3 isoform is the proton pump on premature melanosomes

The melanosome, an organelle specialized for melanin synthesis, is one of the lysosome-related organelles. Its lumen is reported to be acidified by vacuolar-type H⁺-ATPase (V-ATPase). Mammalian V-ATPase exhibits structural diversity in its subunit isoforms; with regard to membrane intrinsic subunit a, four isoforms (a1–a4) have been found to be localized to distinct subcellular compartments. In this study, we have shown that the a3 isoform is co-localized with a melanosome marker protein, Pmel17, in mouse melanocytes. Acidotropic probes (LysoSensor and DAMP) accumulate in non-pigmented Pmel17-positive melanosomes, and DAMP accumulation is sensitive to bafilomycin A1, a specific inhibitor of V-ATPase. However, none of the subunit a isoforms is associated with highly pigmented mature melanosomes, in which the acidotropic probes are also not accumulated. oc/oc mice, which have a null mutation at the a3 locus, show no obvious defects in melanogenesis. In the mutant melanocytes, the expression of the a2 isoform is modestly elevated, and a considerable fraction of this isoform is localized to premature melanosomes. These observations suggest that the V-ATPase keeps the lumen of premature melanosomes acidic, whereas melanosomal acidification is less significant in mature melanosomes. Ge-Hong Sun-Wada and Yoh Wada contributed equally to this study. This study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by the Hayashi and Noda Foundations.     

Molecular cloning and chromosomal assignment of the porcine 54 and 56 kDa vacuolar H(+)-ATPase subunit gene (V-ATPase)

Vacuolar proton-translocating ATPases (V-ATPase) are multisubunit enzyme complexes located in the membranes of eukaryotic cells regulating cytoplasmic pH. So far, nothing is known about the genomic organization and chromosomal location of the various subunit genes in higher eukaryotes. Here we describe the isolation and analysis of a cDNA coding for the 54- and 56-kDa porcine V-ATPase subunit alpha and beta isoforms. We have determined the genomic structure of the V-ATPase subunit gene spanning at least 62 kb on Chromosome (Chr) 4q14-q16. It consists of 14 exons with sizes ranging from 54 bp to 346 bp, with a non-coding first exon and an alternatively spliced seventh exon leading to two isoforms. The 5′ end of the V-ATPase cDNA was isolated by RACE-PCR. The V-ATPase alpha isoform mRNA, lacking the seventh exon, has an open reading frame of 1395 nucleotides encoding a hydrophilic protein of 465 amino acids with a calculated molecular mass of 54.2 kDa and a pI of 7.8, whereas the beta isoform has a length of 1449 nucleotides encoding a protein of 483 amino acids with a calculated molecular mass of 55.8 kDa. Amino acid and DNA sequence comparison revealed that the porcine V-ATPase subunit exhibits a significant homology to the VMA13 subunit of Saccharomyces cerevisiae V-ATPase complex and V-ATPase subunit of Caenorhabditis elegans. Received: 14 May 1998 / Accepted: 20 October 1998     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H-V-ATPase

Vacuolar (H⁺)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an α-helical conformation for peptide MTM7 and in DMSO three α-helical regions are identified by 2D ¹H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an α-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Functional complementation of yeast vma1 delta cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H(+)-ATPase activity

The ability of a vacuolar H(+)-ATPase (V-ATPase) subunit homolog (subunit A) from plants to rescue the vma mutant phenotype of yeast was investigated as a first step towards investigating the structure and function of plant subunits in molecular detail. Heterologous expression of cotton cDNAs encoding near-identical isoforms of subunit A in mutant vma1 delta yeast cells successfully rescued the mutant vma phenotype, indicating that subunit A of plants and yeast have retained elements essential to V-ATPases during the course of evolution. Although vacuoles become acidified, the plant-yeast hybrid holoenzyme only partially restored V-ATPase activity (approximately 60%) in mutant yeast cells. Domain substitution of divergent N- or C-termini only slightly enhanced V-ATPase activity, whereas swapping both domains acted synergistically, increasing coupled ATP hydrolysis and proton translocation by approximately 22% relative to the native plant subunit. Immunoblot analysis indicated that similar amounts of yeast, plant or plant-yeast chimeric subunits are membrane-bound. These results suggest that subunit A terminal domains contain structural information that impact V-ATPase structure and function.     

The role of iron uptake in pathogenicity and symbiosis in <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis> TT01

Background  

Photorhabdus are Gram negative bacteria that are pathogenic to insect larvae whilst also having a mutualistic interaction with nematodes from the family Heterorhabditis. Iron is an essential nutrient and bacteria have different mechanisms for obtaining both the ferrous (Fe²⁺) and ferric (Fe³⁺) forms of this metal from their environments. In this study we were interested in analyzing the role of Fe³⁺ and Fe²⁺ iron uptake systems in the ability of Photorhabdus to interact with its invertebrate hosts.     

Dimer formation of subunit G of the yeast V-ATPase

The G subunit of the vacuolar ATPase (V-ATPase) is a component of the stalk connecting the V(1) and V(O) sectors of the enzyme and is essential for normal assembly and function. Subunit G (Vma10p) of the yeast V-ATPase was expressed in Escherichia coli as a soluble protein and was purified to homogeneity. The molecular mass of subunit G, determined by Native-polyacrylamide gel electrophoresis, gel filtration analysis and small-angle X-ray scattering, was approximately 28+/-2 kDa, indicating that this protein is dimeric. With a radius of gyration (R(g)) and a maximum size (D(max)) of 2.7+/-0.2 nm and 8.0+/-0.3 nm, respectively, the G-dimer is rather elongated. To understand which region of subunit G is required to mediate dimerization, a G(38-144) form (the carboxyl-terminus) was expressed and purified. G(38-144) is homogeneous, with a molecular mass of approximately 12+/-3 kDa, indicating a monomeric form in solution.     

Optic Nerve Compression and Retinal Degeneration in Tcirg1 Mutant Mice Lacking the Vacuolar-Type H+-ATPase a3 Subunit

Background

Vacuolar-type proton transporting ATPase (V-ATPase) is involved in the proper development of visual function. Mutations in the Tcirg1 (also known as Atp6V0a3) locus, which encodes the a3 subunit of V-ATPase, cause severe autosomal recessive osteopetrosis (ARO) in humans. ARO is often associated with impaired vision most likely because of nerve compression at the optic canal. We examined the ocular phenotype of mice deficient in Tcirg1 function.

Methodology/Principal Findings

X-ray microtomography showed narrowed foramina in the skull, suggesting that optic nerve compression occurred in the a3-deficient (Tcirg1 ^−/−) mice. The retina of the mutant mice had normal architecture, but the number of apoptotic cells was increased at 2–3 wks after birth. In the ocular system, the a3 subunit accumulated in the choriocapillary meshwork in uveal tissues. Two other subunit isoforms a1 and a2 accumulated in the retinal photoreceptor layer. We found that the a4 subunit, whose expression has previously been shown to be restricted to several transporting epithelia, was enriched in pigmented epithelial cells of the retina and ciliary bodies. The expression of a4 in the uveal tissue was below the level of detection in wild-type mice, but it was increased in the mutant choriocapillary meshwork, suggesting that compensation may have occurred among the a subunit isoforms in the mutant tissues.

Conclusions

Our findings suggest that a similar etiology of visual impairment is involved in both humans and mice; thus, a3-deficient mice may provide a suitable model for clinical and diagnostic purposes in cases of ARO.     

Assembly and Regulation of the Yeast Vacuolar H+-ATPase

The yeast vacuolar proton-translocating ATPase (V-ATPase) is an excellent model for V-ATPases in all eukaryotic cells. Activity of the yeast V-ATPase is reversibly down-regulated by disassembly of the peripheral (V₁) sector, which contains the ATP-binding sites, from the membrane (V₀) sector, which contains the proton pore. A similar regulatory mechanism has been found in Manduca sexta and is believed to operate in other eukaryotes. We are interested in the mechanism of reversible disassembly and its implications for V-ATPase structure. In this review, we focus on (1) characterization of the yeast V-ATPase stalk subunits, which form the interface between V₁ and V₀, (2) potential mechanisms of silencing ATP hydrolytic activity in disassembled V₁ sectors, and (3) the structure and function of RAVE, a recently discovered complex that regulates V-ATPase assembly.