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1.
Vacuolar H+-ATPase (V-ATPase), an electrogenic proton pump, is highly expressed in Plasmodium falciparum, the human malaria parasite. Although V-ATPase-driven proton transport is involved in various physiological processes in the parasite, the overall features of the V-ATPase of P. falciparum, including the gene organization and biogenesis, are far less known. Here, we report cDNA cloning of proteolipid subunit c of P. falciparum, the smallest and most highly hydrophobic subunit of V-ATPase. RT-PCR analysis as well as Northern blotting indicated expression of the proteolipid gene in the parasite cells. cDNA, which encodes a complete reading frame comprising 165 amino acids, was obtained, and its deduced amino acid sequence exhibits 52 and 57% similarity to the yeast and human counterparts, respectively. Southern blot analysis suggested the presence of a single copy of the proteolipid gene, with 5 exons and 4 introns. Upon transfection of the cDNA into a yeast null mutant, the cells became able to grow at neutral pH, accompanied by vesicular accumulation of quinacrine. In contrast, a mutated proteolipid with replacement of glutamate residue 138 with glutamine did not lead to recovery of the growth ability or vesicular accumulation of quinacrine. These results indicated that the cDNA actually encodes the proteolipid of P. falciparum and that the proteolipid is functional in yeast.  相似文献   

2.
Ji YJ  Choi KY  Song HO  Park BJ  Yu JR  Kagawa H  Song WK  Ahnn J 《FEBS letters》2006,580(13):3161-3166
Vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump, which transports protons across the membrane. It is a multi-protein complex which is composed of at least 13 subunits. The Caenorhabditis elegans vha-8 encodes the E subunit of V-ATPase which is expressed in the hypodermis, intestine and H-shaped excretory cells. VHA-8 is necessary for proper intestinal function likely through its role in cellular acidification of intestinal cells. The null mutants of vha-8 show a larval lethal phenotype indicating that vha-8 is an essential gene for larval development in C. elegans. Interestingly, characteristics of necrotic cell death were observed in the hypodermis and intestine of the arrested larvae suggesting that pH homeostasis via the E subunit of V-ATPase is required for the cell survival in C. elegans.  相似文献   

3.
The V-ATPases are ATP-dependent proton pumps present in both intracellular compartments and the plasma membrane. They function in such processes as membrane traffic, protein degradation, renal acidification, bone resorption and tumor metastasis. The V-ATPases are composed of a peripheral V1 domain responsible for ATP hydrolysis and an integral V0 domain that carries out proton transport. Our recent work has focused on structural analysis of the V-ATPase complex using both cysteine-mediated cross-linking and electron microscopy. For cross-linking studies, unique cysteine residues were introduced into structurally defined sites within the B and C subunits and used as points of attachment for the photoactivated cross-linking reagent MBP. Disulfide mediated cross-linking has also been used to define helical contact surfaces between subunits within the integral V0 domain. With respect to regulation of V-ATPase activity, we have investigated the role that intracellular environment, luminal pH and a unique domain of the catalytic A subunit play in controlling reversible dissociation in vivo.  相似文献   

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The vacuolar H+-ATPase (V-ATPase) is a major contributor to luminal acidification in epithelia of Wolffian duct origin. In both kidney-intercalated cells and epididymal clear cells, cAMP induces V-ATPase apical membrane accumulation, which is linked to proton secretion. We have shown previously that the A subunit in the cytoplasmic V1 sector of the V-ATPase is phosphorylated by protein kinase A (PKA). Here we have identified by mass spectrometry and mutagenesis that Ser-175 is the major PKA phosphorylation site in the A subunit. Overexpression in HEK-293T cells of either a wild-type (WT) or phosphomimic Ser-175 to Asp (S175D) A subunit mutant caused increased acidification of HCO3-containing culture medium compared with cells expressing vector alone or a PKA phosphorylation-deficient Ser-175 to Ala (S175A) mutant. Moreover, localization of the S175A A subunit mutant expressed in HEK-293T cells was more diffusely cytosolic than that of WT or S175D A subunit. Acute V-ATPase-mediated, bafilomycin-sensitive H+ secretion was up-regulated by a specific PKA activator in HEK-293T cells expressing WT A subunit in HCO3-free buffer. In cells expressing the S175D mutant, V-ATPase activity at the membrane was constitutively up-regulated and unresponsive to PKA activators, whereas cells expressing the S175A mutant had decreased V-ATPase activity that was unresponsive to PKA activation. Finally, Ser-175 was necessary for PKA-stimulated apical accumulation of the V-ATPase in a polarized rabbit cell line of collecting duct A-type intercalated cell characteristics (Clone C). In summary, these results indicate a novel mechanism for the regulation of V-ATPase localization and activity in kidney cells via direct PKA-dependent phosphorylation of the A subunit at Ser-175.  相似文献   

8.
Vacuolar H+-ATPases (V-ATPases) are large, multisubunit proton pumps that acidify the lumen of organelles in virtually every eukaryotic cell and in specialized acid-secreting animal cells, the enzyme pumps protons into the extracellular space. In higher organisms, most of the subunits are expressed as multiple isoforms, with some enriched in specific compartments or tissues and others expressed ubiquitously. In mammals, subunit a is expressed as four isoforms (a1-4) that target the enzyme to distinct biological membranes. Mutations in a isoforms are known to give rise to tissue-specific disease, and some a isoforms are upregulated and mislocalized to the plasma membrane in invasive cancers. However, isoform complexity and low abundance greatly complicate purification of active human V-ATPase, a prerequisite for developing isoform-specific therapeutics. Here, we report the purification of an active human V-ATPase in native lipid nanodiscs from a cell line stably expressing affinity-tagged a isoform 4 (a4). We find that exogenous expression of this single subunit in HEK293F cells permits assembly of a functional V-ATPase by incorporation of endogenous subunits. The ATPase activity of the preparation is >95% sensitive to concanamycin A, indicating that the lipid nanodisc-reconstituted enzyme is functionally coupled. Moreover, this strategy permits purification of the enzyme’s isolated membrane subcomplex together with biosynthetic assembly factors coiled-coil domain–containing protein 115, transmembrane protein 199, and vacuolar H+-ATPase assembly integral membrane protein 21. Our work thus lays the groundwork for biochemical characterization of active human V-ATPase in an a subunit isoform-specific manner and establishes a platform for the study of the assembly and regulation of the human holoenzyme.  相似文献   

9.
Amemiya T  Kanayama Y  Yamaki S  Yamada K  Shiratake K 《Planta》2006,223(6):1272-1280
The vacuole is a large, multifunctional organelle related to the processes of cell expansion, solute accumulation, regulation of cytoplasmic ion concentrations, pH homeostasis and osmoregulation, which are directly or indirectly achieved by vacuolar H+-pumps: vacuolar H+-ATPase (V-ATPase; EC 3.6.1.3) and vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1). In this study, we produced antisense-transgenic tomatoes (Lycopersicon esculentum L.) of the V-ATPase A subunit, which is under the control of the fruit-specific 2A11 promoter. One β-glucuronidase (GUS)-transgenic line (GUS control) and seven A subunit antisense-transgenic lines were obtained. The amount of V-ATPase A subunit mRNA in fruit decreased in all antisense-transgenic lines, but in leaves showed no difference compared with the GUS control line and the nontransformant, suggesting that suppression of the V-ATPase A subunit by a 2A11 promoter is limited to fruit. The antisense-transgenic plants had smaller fruits compared with the GUS control line and the nontransformant. Surprisingly, fruits from the antisense-transgenic plants, except the fruit that still had relatively high expression of A subunit mRNA, had few seeds. Sucrose concentration in fruits from the antisense-transgenic plants increased, but glucose and fructose concentrations did not change. These results show the importance of V-ATPase, not only in fruit growth, but also in seed formation and in sugar composition of tomato fruit.  相似文献   

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Apicularen A and the known vacuolar-type (H(+))-ATPase (V-ATPase) inhibitor bafilomycin A(1) induced apoptosis of RAW 264.7 cells, while apicularen B, an N-acetyl-glucosamine glycoside of apicularen A, was far less effective. Apicularen A inhibited vital staining with acridine orange of the intracellular organelles of RAW 264.7 cells, inhibited the ATP-dependent proton transport into inside-out microsome vesicles, and inhibited the bafilomycin A(1)-sensitive ATP hydrolysis. The IC(50) values of the proton transport were 0.58 nM for apicularen A, 13 nM for apicularen B, and 0.95 nM for bafilomycin A(1). Furthermore, apicularen A inhibited the bafilomycin A(1)-sensitive ATP hydrolysis more potently than apicularen B. F-ATPase and P-ATPase were not inhibited by apicularen A. We concluded that apicularen A inhibits V-ATPase, and thus induces apoptosis in RAW 264.7 cells.  相似文献   

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Salt modulation of the tonoplast H+-pumping V-ATPase and H+-PPase was evaluated in hypocotyls ofVigna unguiculata seedlings after 3 and 7 days of treatment. In 3-day-old seedlings, treatment with 100 mmol/L NaCl decreased the proton transport and hydrolytic activities of both the V-ATPase and the H+-PPase. After 7 days, the proton transport and hydrolysis activities of the V-ATPase were higher, while the H+-PPase activities were lower in seedlings. Western blot analysis of A- and B-subunits of V-ATPase revealed that the protein content of the two subunits varied in parallel with their activities, i.e. to a higher activity corresponded a higher protein content of the subunits and vice versa. Contrarily, Western blot analysis of H+-PPase levels failed to show any correlation with PPase activity, suggesting a partial enzyme inactivation. The results indicate that salt stress induces V-ATPase expression inV. unguiculata with concomitant enhancement of its activity as a homeostatic mechanism to cope with salt stress. Under the same conditions PPase is inhibited.  相似文献   

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Archaeal H(+)-ATPase (A-ATPase) is composed of an A(1) region that hydrolyzes ATP and an integral membrane part A(0) that conducts protons. Subunit E is a component of peripheral stator(s) that physically links A(1) and A(0) parts of the A-ATPase. Here we report the first crystal structure of subunit E of A-ATPase from Pyrococcus horikoshii OT3 at 1.85 A resolution. The protomer structure of subunit E represents a novel fold. The quaternary structure of subunit E is a homodimer, which may constitute the core part of the stator. To investigate the relationship with other stator subunit H, the complex of subunits EH was prepared and characterized using electrophoresis, mass spectrometry, N-terminal sequencing and circular dichroism spectroscopy, which revealed the polymeric and highly helical nature of the EH complex with equimolar stoichiometry of both the subunits. On the basis of the modular architecture of stator subunits, it is suggested that both cytoplasm and membrane sides of the EH complex may interact with other subunits to link A(1) and A(0) parts.  相似文献   

15.
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.  相似文献   

16.
Subunit “a” is associated with the membrane-bound (VO) complex of eukaryotic vacuolar H+-ATPase acidification machinery. It has also been shown recently to be involved in diverse membrane fusion/secretory functions independent of acidification. Here, we report the crystal structure of the N-terminal cytosolic domain from the Meiothermus ruber subunit “I” homolog of subunit a. The structure is composed of a curved long central α-helix bundle capped on both ends by two lobes with similar α/β architecture. Based on the structure, a reasonable model of its eukaryotic subunit a counterpart was obtained. The crystal structure and model fit well into reconstructions from electron microscopy of prokaryotic and eukaryotic vacuolar H+-ATPases, respectively, clarifying their orientations and interactions and revealing features that could enable subunit a to play a role in membrane fusion/secretion.  相似文献   

17.
Sun-Kyung Lee  Weixun Li  TaiYoun Rhim 《BBA》2010,1797(10):1687-1695
Vacuolar (H+)-ATPases, also called V-ATPases, are ATP-driven proton pumps that are highly phylogenetically conserved. Early biochemical and cell biological studies have revealed many details of the molecular mechanism of proton pumping and of the structure of the multi-subunit membrane complex, including the stoichiometry of subunit composition. In addition, yeast and mouse genetics have broadened our understanding of the physiological consequences of defective vacuolar acidification and its related disease etiologies. Recently, phenotypic investigation of V-ATPase mutants in Caenorhabditis elegans has revealed unexpected new roles of V-ATPases in both cellular function and early development. In this review, we discuss the functions of the V-ATPases discovered in C. elegans.  相似文献   

18.
We report the nucleotide sequence of a gene encoding the c ('16 kDa') subunit of the vacuolar-type H+-ATPase (V-ATPase) from a marine red alga, Porphyra yezoensis. A cDNA clone was isolated from a leafy gametophyte cDNA library and analyzed for the sequence. The genomic DNA sequence was directly determined by nested PCR. The structural gene contained four introns within a coding sequence of 483 base pairs which encodes a polypeptide of 161-amino acids with four hydrophobic transmembrane-spanning regions. Comparison of the deduced amino acid sequences showed higher similarity to the land plant Oryza sativa (69.1%) than to the Ulvophyceae Acetabularia acetabulum (64.1%). The mRNA was detected both in the leafy gametophytes and filamentous sporophytes.  相似文献   

19.
Li Z  Zhang X 《Planta》2004,219(6):948-954
The vacuolar H+-ATPase from mung bean (Vigna radiata L. cv. Wilczek) was purified to homogeneity. The purified complex contained all the reported subunits from mung bean, but also included a 40-kDa subunit, corresponding to the membrane-associated subunit d, which has not previously been observed. The structure of the V-ATPase from mung bean was studied by electron microscopy of negatively stained samples. An analysis of over 6,000 single-particle images obtained by electron microscopy of the purified complex revealed that the complex, similar to other V-ATPases, is organized into two major domains V1 and Vo with overall dimensions of 25 nm×13.7 nm and a stalk region connecting the V1 and Vo domains. Several individual areas of protein density were observed in the stalk region, indicating its complexity. The projections clearly showed that the complex contained one central stalk and at least two peripheral stalks. Subcomplexes containing subunits A, B and E, dissociated from the tonoplast membrane by KI, were purified. The structure of the subcomplex was also studied by electron microscopy followed by single-molecule analysis of 13,000 projections. Our preliminary results reveal an area of high protein density at the bottom of the subcomplex immediately below the cavity formed by the A and B subunits, indicating the position of subunit E.Abbreviations MSA Multivariate statistical analysis - 2D, 3D Two-, three-dimensional - V-ATPase Vacuolar H+-ATPase  相似文献   

20.
The stimulation of the activity of the H(+)-ATPase present in the vacuolar membrane (V-ATPase) of Saccharomyces cerevisiae is here described in response to a moderate stress induced by 2,4-dichlorophenoxyacetic acid (2,4-D). This in vivo activation (up to 5-fold) took place essentially during the adaptation period, preceding cell division under herbicide stress, in coordination with a marked activation of plasma membrane H(+)-ATPase (PM-ATPase) (up to 30-fold) and the decrease of intracellular and vacuolar pH values, suggesting that activation may be triggered by acidification. Single deletion of VMA1 and genes encoding other V-ATPase subunits led to a more extended period of adaptation and to slower growth under 2,4-D stress. Results suggest that a functional V-ATPase is required to counteract, more rapidly and efficiently, the dissipation of the physiological H(+)-gradient across vacuolar membrane registered during 2,4-D adaptation.  相似文献   

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