The 100-kDa "a" subunit of the vacuolar proton-translocating ATPase (V-ATPase) is encoded by two genes in yeast, VPH1 and STV1. The Vph1p-containing complex localizes to the vacuole, whereas the Stv1p-containing complex resides in some other intracellular compartment, suggesting that the a subunit contains information necessary for the correct targeting of the V-ATPase. We show that Stv1p localizes to a late Golgi compartment at steady state and cycles continuously via a prevacuolar endosome back to the Golgi. V-ATPase complexes containing Vph1p and Stv1p also differ in their assembly properties, coupling of proton transport to ATP hydrolysis, and dissociation in response to glucose depletion. To identify the regions of the a subunit that specify these different properties, chimeras were constructed containing the cytosolic amino-terminal domain of one isoform and the integral membrane, carboxyl-terminal domain from the other isoform. Like the Stv1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Stv1p localized to the Golgi and the complex did not dissociate in response to glucose depletion. Like the Vph1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Vph1p localized to the vacuole and the complex exhibited normal dissociation upon glucose withdrawal. Interestingly, the V-ATPase complex containing the chimera with the carboxyl-terminal domain of Vph1p exhibited a higher coupling of proton transport to ATP hydrolysis than the chimera containing the carboxylterminal domain of Stv1p. Our results suggest that whereas targeting and in vivo dissociation are controlled by sequences located in the amino-terminal domains of the subunit a isoforms, coupling efficiency is controlled by the carboxyl-terminal region.The V-ATPases 1 are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells (1-8). Acidification of these compartments is crucial for such processes as receptor-mediated endocytosis, intracellular trafficking, the processing and degradation of macromolecules, and the coupled transport of small molecules. In addition, V-ATPases in the plasma membrane of specialized cells function in such processes as pH homeostasis (9), bone resorption (10), renal acidification (11), potassium transport (12), and tumor metastasis (13). In yeast, the VATPase functions to create the driving force for uptake of small molecules and ions into the vacuole (14) and is important for post-Golgi protein trafficking (15-17).The V-ATPase complex is composed of the following two domains: a soluble V 1 domain responsible for ATP hydrolysis and an integral V 0 domain responsible for proton translocation (1-8). The V 1 domain is a 500-kDa complex composed of eight different subunits (subunits A-H) of molecular masses 70 to 14 kDa, whereas the V 0 domain is a 250-kDa complex containing five different subunits (subunits a, d, c, cЈ, and cЉ) of molecular masses 100 to 16 kDa (1-8). The ...
The 100 kDa a-subunit of the yeast vacuolar (H ؉ )-ATPase (V-ATPase) is encoded by two genes, VPH1 and STV1. These genes encode unique isoforms of the a-subunit that have previously been shown to reside in different intracellular compartments in yeast. Vph1p localizes to the central vacuole, whereas Stv1p is present in some other compartment, possibly the Golgi or endosomes. To compare the properties of V-ATPases containing Vph1p or Stv1p, Stv1p was expressed at higher than normal levels in a strain disrupted in both genes, under which conditions V-ATPase complexes containing Stv1p appear in the vacuole. Complexes containing Stv1p showed lower assembly with the peripheral V 1 domain than did complexes containing Vph1p. When corrected for this lower degree of assembly, however, V-ATPase complexes containing Vph1p and Stv1p had similar kinetic properties. Both exhibited a K m for ATP of about 250 M, and both showed resistance to sodium azide and vanadate and sensitivity to nanomolar concentrations of concanamycin A. Stv1p-containing complexes, however, showed a 4 -5-fold lower ratio of proton transport to ATP hydrolysis than Vph1p-containing complexes. We also compared the ability of V-ATPase complexes containing Vph1p or Stv1p to undergo in vivo dissociation in response to glucose depletion. Vph1p-containing complexes present in the vacuole showed dissociation in response to glucose depletion, whereas Stv1p-containing complexes present in their normal intracellular location (Golgi/endosomes) did not. Upon overexpression of Stv1p, Stv1p-containing complexes present in the vacuole showed glucose-dependent dissociation. Blocking delivery of Vph1p-containing complexes to the vacuole in vps21⌬ and vps27⌬ strains caused partial inhibition of glucose-dependent dissociation. These results suggest that dissociation of the V-ATPase complex in vivo is controlled both by the cellular environment and by the 100-kDa a-subunit isoform present in the complex.The vacuolar (H ϩ )-ATPases (or V-ATPases) 1 are a family of ATP-dependent proton pumps found in a variety of intracellular compartments that function in both endocytic and secretory pathways (1-8). Acidification of these compartments is essential for many cellular processes, including receptor-mediated endocytosis, intracellular targeting, protein processing and degradation, and coupled transport. V-ATPases are also present in the plasma membrane of certain specialized cells, including osteoclasts (9), renal intercalated cells (10), and neutrophils (11), where they function in such processes as bone resorption, renal acidification, and pH homeostasis, respectively.The V-ATPases from fungi, plants, and animals are structurally very similar and are composed of two domains (1-8).The V 1 domain is a peripheral complex of molecular mass 570 kDa composed of eight different subunits of molecular mass 70 -14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V 0 domain is a 260-kDa integral complex composed of five subunits of molecular mass 100 -17 kDa (subunits a, d, ...
The vacuolar (H ؉ )-ATPases (V-ATPases) are ATP-dependent proton pumps that acidify intracellular compartments and pump protons across specialized plasma membranes. Proton translocation occurs through the integral V0 domain, which contains five different subunits (a, d, c, c, and c؆). Proton transport is critically dependent on buried acidic residues present in three different proteolipid subunits (c, c, and c؆). Mutations in the 100-kDa subunit a have also influenced activity, but none of these residues has proven to be required absolutely for proton transport. On the basis of previous observations on the F-ATPases, we have investigated the role of two highly conserved arginine residues present in the last two putative transmembrane segments of the yeast V-ATPase a subunit (Vph1p). Substitution of Asn, Glu, or Gln for Arg-735 in TM8 gives a V-ATPase that is fully assembled but is totally devoid of proton transport and ATPase activity. Replacement of Arg-735 by Lys gives a V-ATPase that, although completely inactive for proton transport, retains 24% of wild-type ATPase activity, suggesting a partial uncoupling of proton transport and ATP hydrolysis in this mutant. By contrast, nonconservative mutations of Arg-799 in TM9 lead to both defective assembly of the V-ATPase complex and decreases in activity of the assembled V-ATPase. These results suggest that Arg-735 is absolutely required for proton transport by the VATPases and is discussed in the context of a revised model of the topology of the 100-kDa subunit a.
The vacuolar H + -ATPases (or V-ATPases) are a family of ATP-dependent proton pumps responsible for acidi¢-cation of intracellular compartments and, in certain cases, proton transport across the plasma membrane of eukaryotic cells. They are multisubunit complexes composed of a peripheral domain (V 1 ) responsible for ATP hydrolysis and an integral domain (V 0 ) responsible for proton translocation. Based upon their structural similarity to the F 1 F 0 ATP synthases, the V-ATPases are thought to operate by a rotary mechanism in which ATP hydrolysis in V 1 drives rotation of a ring of proteolipid subunits in V 0 . This review is focused on the current structural knowledge of the V-ATPases as it relates to the mechanism of ATPdriven proton translocation.
We have identified a cDNA encoding a novel isoform of the mouse V-ATPase d subunit (d2). The protein encoded is 350 amino acids in length and shows 42 and 67% identity to the yeast d subunit (Vma6p) and the mouse d1 isoform, respectively. Reverse transcriptase-PCR analysis using isoform-specific primers demonstrate that d2 is expressed mainly in kidney and at lower levels in heart, spleen, skeletal muscle, and testis. Although d1 and d2 show similar levels of sequence homology to Vma6p, only the d1 isoform can complement the phenotype of a yeast strain in which VMA6 has been disrupted when cells are grown at 30°C. The d2 isoform, however, can complement the vma6⌬ phenotype when cells are grown at 25°C. Moreover, partial assembly of the VATPase complex on the vacuolar membrane can be detected under these conditions, although assembly is significantly lower than that observed for the strain expressing Vma6p. This reduced assembly is also reflected in a reduced level of concanamycin-sensitive ATPase activity and proton transport in isolated vacuoles. Comparison of the kinetic properties of V-ATPase complexes containing Vma6p and d1 demonstrate that although the K m for ATP hydrolysis is similar (0.26 and 0.31 mM, respectively), the coupling ratio (proton transport/ATP hydrolysis) is ϳ3-6-fold higher for d1-containing complexes than for Vma6p-containing complexes. These results suggest that subunit d may play a role in coupling of proton transport and ATP hydrolysis.The vacuolar (H ϩ )-ATPase (or V-ATPase) 1 functions as an ATP-dependent proton pump to acidify intracellular compartments in eukaryotic cells. The V-ATPases are present in a variety of intracellular compartments, including clathrincoated vesicles, endosomes, lysosomes, Golgi-derived vesicles, chromaffin granules, synaptic vesicles, and the central vacuoles of yeast, Neurospora, and plants (1-8). Vacuolar acidification plays an important role in many cellular processes, including receptor-mediated endocytosis, intracellular targeting, protein processing and degradation, and coupled transport. In certain mammalian cells, V-ATPases also function in the plasma membrane to transport protons from the cytoplasm to the extracellular environment (9 -13). In osteoclasts, plasma membrane V-ATPases play a role in bone resorption (11), whereas in intercalated cells in the kidney they function in renal acidification (9). V-ATPases in the plasma membrane of tumor cells have also been implicated in metastasis (13).The V-ATPases from fungi, plants, and animals are structurally very similar and are composed of two functional domains, V 1 and V 0 (1-8). The V 1 domain is a peripheral complex with molecular mass of 640 kDa composed of eight different subunits of molecular mass 70 -14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V 0 domain is a 260-kDa integral complex composed of five subunits of molecular masses 100 -17 kDa (subunits a, d, c, cЈ, and cЉ) that is responsible for proton translocation. In yeast cells, all subunits are encoded by single gene...
Sphingosine 1-phosphate (S1P) is an intercellular signaling molecule present in blood. Erythrocytes have a central role in maintaining the S1P concentration in the blood stream. We previously demonstrated that S1P is exported from erythrocytes by a glyburide-sensitive S1P transporter. However, the gene encoding the S1P transporter in erythrocytes is unknown. In this study, we found that the mouse erythroid cell line, MEDEP-E14, has S1P export activity and exhibits properties that are consistent with those of erythrocytes. Using microarray analysis of MEDEP-E14 cells and its parental cell line, E14TG2a, we identified several candidate genes for S1P export activity. Of those genes, only one gene, Mfsd2b, showed S1P transport activity. The properties of S1P release by MFSD2B were similar to those in erythrocytes. Moreover, knockout of MFSD2B in MEDEP-E14 cells decreased S1P export from the cells. These results strongly suggest that MFSD2B is a novel S1P transporter in erythroid cells.
Subunit A is the catalytic nucleotide binding subunit of the vacuolar proton-translocating ATPase (or V-ATPase) and is homologous to subunit  of the F 1 F 0 ATP synthase (or F-ATPase). Amino acid sequence alignment of these subunits reveals a 90-amino acid insert in subunit A (termed the non-homologous region) that is absent from subunit . To investigate the functional role of this region, site-directed mutagenesis has been performed on the VMA1 gene that encodes subunit A in yeast. Substitutions were performed on 13 amino acid residues within this region that are conserved in all available A subunit sequences. Most of the 18 mutations introduced showed normal assembly of the V-ATPase. Of these, one (R219K) greatly reduced both proton transport and ATPase activity. By contrast, the P217V mutant showed significantly reduced ATPase activity but higher than normal levels of proton transport, suggesting an increase in coupling efficiency. Two other mutations in the same region (P223V and P233V) showed decreased coupling efficiency, suggesting that changes in the non-homologous region can alter coupling of proton transport and ATP hydrolysis. It was previously shown that the V-ATPase must possess at least 5-10% activity relative to wild type to undergo in vivo dissociation in response to glucose withdrawal. However, four of the mutations studied (G150A, D157E, P177V, and P223V) were partially or completely blocked in dissociation despite having greater than 30% of wild type levels of activity. These results suggest that changes in the non-homologous region can also alter in vivo dissociation of the V-ATPase independent of effects on activity.The vacuolar proton-translocating ATPases (or V-ATPases) 1 are ATP-driven proton pumps present in both intracellular compartments and the plasma membrane of eukaryotic cells (1-8). They couple the energy released upon ATP hydrolysis to the active transport of protons from the cytoplasm to either the lumen of various intracellular compartments or to the extracellular environment. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking of lysosomal enzymes, degradation of macromolecules, uptake of neurotransmitters, and the entry of various envelope viruses and toxins (1-8). Plasma membrane V-ATPases have also been implicated in many normal and disease processes, including bone resorption, renal acidification, pH homeostasis, and tumor metastasis (9 -13). Defects in specific V-ATPase subunits have been shown to be responsible for a number of human genetic diseases, including autosomal recessive osteopetrosis and renal tubular acidosis (9, 14 -16).The V-ATPases are multi-subunit complexes composed of two functional domains (1-8). The 640-kDa peripheral V 1 domain is responsible of ATP hydrolysis and consists of 8 different subunits (subunits A-H) with molecular masses of 70 -14 kDa. The V 0 domain is a 260-kDa integral complex composed of five different subunits (subunits a, d, cЈЈ, cЈ, and c with mole...
Proton translocation by the vacuolar (H ؉ )-ATPase (or V-ATPase) has been shown by mutagenesis to be dependent upon charged residues present within transmembrane segments of subunit a as well as the three proteolipid subunits (c, c, and c؆). Interaction between R735 in TM7 of subunit a and the glutamic acid residue in the middle of TM4 of subunits c and c or TM2 of subunit c؆ has been proposed to be essential for proton release to the luminal compartment. In order to determine whether the helical face of TM7 of subunit a containing R735 is capable of interacting with the helical face of TM4 of subunit c containing the essential glutamic acid residue (Glu-145), cysteine-mediated crosslinking between these subunits in yeast has been performed. Cys-less forms of subunits a and c as well as forms containing unique cysteine residues were constructed, introduced together into a strain disrupted in both endogenous subunits, and tested for growth at neutral pH, for assembly competence and for cross-linking in the presence of cupric-phenanthroline by SDS-PAGE and Western blot analysis. Four different cysteine mutants of subunit a were each tested pairwise with ten different unique cysteine mutants of subunit c. Strong cross-linking was observed for the pairs aS728C/cI142C, aA731C/cE145C, aA738C/cF143C, aA738C/cL147C, and aL739C/cL147C. Partial cross-linking was observed for an additional 13 of 40 pairs analyzed. When arrayed on a helical wheel diagram, the results suggest that the helical face of TM7 of subunit a containing Arg-735 interacts with the helical face of TM4 of subunit c centered on Val-146 and bounded by Glu-145 and Leu-147. The results are consistent with a possible rotational flexibility of one or both of these transmembrane segments as well as some flexibility of movement perpendicular to the membrane.The vacuolar (H ϩ )-ATPases (or V-ATPases) 1 are ATP-driven proton pumps that acidify a variety of intracellular compartments in eukaryotic cells, including endosomes, lysosomes, clathrin-coated vesicles, Golgi-derived vesicles, secretory vesicles, and the central vacuoles of plants and fungi (1-8). Acidification of these intracellular compartments is important for many cellular processes, including receptor-mediated endocytosis, intracellular trafficking, protein processing, and degradation, coupled transport of small molecules and entry of viruses and toxins (1). V-ATPases also exist in the plasma membrane of certain cells, where they function in such processes as renal acidification, bone resorption, pH homeostasis, coupled potassium transport, and tumor invasion (9 -13).V-ATPases are composed of a peripheral V 1 domain responsible for ATP hydrolysis and an integral V 0 domain that carries out proton transport (1-8). V 1 is composed of eight different subunits (A-H) of molecular mass 70 -13 kDa that are present in a stoichiometry of A 3 B 3 C 1 D 1 E 1 F 1 G 2 H 1-2 (14, 15) and that form a complex of ϳ640 kDa. Both the A and B subunits participate in nucleotide binding, with the catalytic sites located o...
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