The pH of intracellular compartments in eukaryotic cells is a carefully controlled parameter that affects many cellular processes, including intracellular membrane transport, prohormone processing and transport of neurotransmitters, as well as the entry of many viruses into cells. The transporters responsible for controlling this crucial parameter in many intracellular compartments are the vacuolar (H+)-ATPases (V-ATPases). Recent advances in our understanding of the structure and regulation of the V-ATPases, together with the mapping of human genetic defects to genes that encode V-ATPase subunits, have led to tremendous excitement in this field.

Sphingosine-1-phosphate (S1P) is a secreted lipid mediator that functions in vascular development; however, it remains unclear how S1P secretion is regulated during embryogenesis. We identified a zebrafish mutant, ko157, that displays cardia bifida (two hearts) resembling that in the S1P receptor-2 mutant. A migration defect of myocardial precursors in the ko157 mutant is due to a mutation in a multipass transmembrane protein, Spns2, and can be rescued by S1P injection. We show that the export of S1P from cells requires Spns2. spns2 is expressed in the extraembryonic tissue yolk syncytial layer (YSL), and the introduction of spns2 mRNA in the YSL restored the cardiac defect in the ko157 mutant. Thus, Spns2 in the YSL functions as a S1P transporter in S1P secretion, thereby regulating myocardial precursor migration.

Sphingosine-1-phosphate (S1P), a sphingolipid metabolite that is produced inside the cells, regulates a variety of physiological and pathological responses via S1P receptors (S1P1–5). Signal transduction between cells consists of three steps; the synthesis of signaling molecules, their export to the extracellular space and their recognition by receptors. An S1P concentration gradient is essential for the migration of various cell types that express S1P receptors, such as lymphocytes, pre-osteoclasts, cancer cells and endothelial cells. To maintain this concentration gradient, plasma S1P concentration must be at a higher level. However, little is known about the molecular mechanism by which S1P is supplied to extracellular environments such as blood plasma. Here, we show that SPNS2 functions as an S1P transporter in vascular endothelial cells but not in erythrocytes and platelets. Moreover, the plasma S1P concentration of SPNS2-deficient mice was reduced to approximately 60% of wild-type, and SPNS2-deficient mice were lymphopenic. Our results demonstrate that SPNS2 is the first physiological S1P transporter in mammals and is a key determinant of lymphocyte egress from the thymus.

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, ...

Sphingosine 1-phosphate (S1P) is accumulated in platelets and released on stimulation by thrombin or Ca 21 . Thrombin-stimulated S1P release was inhibited by staurosporin, whereas Ca 21 -stimulated release was not. When the platelet plasma membrane was permeabilized with streptolysin O (SLO), S1P leaked out with cytosol markers, whereas granular markers remained in the platelets. The SLOinduced S1P leakage required BSA, probably for solubilization of S1P in the medium. These results indicate that S1P is localized in the inner leaflet of the plasma membrane and that its release is a carrier-mediated process. We also used alpha-toxin (ATX), which makes smaller pores in the plasma membrane than SLO and depletes cytosolic ATP without BSA-dependent S1P leakage. The addition of ATP drove S1P release from ATX platelets. The ATP-driven S1P release from ATX platelets was greatly enhanced by thrombin. An ATP binding cassette transporter inhibitor, glyburide, prevents ATP-and thrombin-induced S1P release from platelets. Ca 21 also stimulated S1P release from ATX platelets without ATP, whereas the Ca 21 -induced release was not inhibited by glyburide. Our results indicate that two independent S1P release systems might exist in the platelet plasma membrane, an ATP-dependent system stimulated by thrombin and an ATP-independent system stimulated by Ca

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.

We have identified cDNAs encoding three isoforms (a1, a2, and a3) of the 100-kDa a subunit of the mouse vacuolar proton-translocating ATPase (V-ATPase). The predicted protein sequences of the three isoforms are 838, 856, and 834 amino acids, respectively, and they display approximately 50% identity between isoforms. Northern blot analysis demonstrated that all three isoforms are expressed in most tissues examined. However, the a1 isoform is expressed most heavily in brain and heart, a2 in liver and kidney, and a3 in liver, lung, heart, brain, spleen, and kidney. We also identified multiple alternatively spliced variants for each isoform. Reverse transcriptase-mediated polymerase chain reaction revealed that one splicing variant of the a1 isoform (a1-I) was expressed only in brain, whereas two other variants (a1-II and a1-III) were expressed in tissues other than brain. These alternatively spliced forms differ in the presence or absence of 6 -7 amino acid residues near the amino and carboxyl termini of the proteins encoded. The a3 isoform is also encoded by three alternatively spliced variants, two of which are predicted to encode a protein that is truncated near the border of the aminoand carboxyl-terminal domains of the a subunit and therefore lacks the integral transmembrane-spanning helices thought to participate in proton translocation. Expression of each isoform (with the exception of a1-I) was detectable at all developmental stages investigated, with a1-I absent only in day 7 embryos. The results obtained suggest that isoforms of the 100-kDa a subunit may contribute to tissue-specific functions of the V-ATPase.The vacuolar (H ϩ )-ATPases (or V-ATPases) 1 function as ATPdependent proton pumps to acidify intracellular compartments in eukaryotic cells. V-ATPases are present in a variety of intracellular compartments, including clathrin-coated vesicles, endosomes, lysosomes, Golgi-derived vesicles, chromaffin granules, synaptic vesicles, and the central vacuoles of yeast, Neurospora, and plants (1-9). Vacuolar acidification is essential for such processes as receptor-mediated endocytosis, intracellular targeting, protein processing and degradation, and coupled transport. V-ATPases have also been identified in the plasma membrane of certain specialized cells, where they function in processes such as renal acidification (7), cytoplasmic neutralization (10), bone resorption (11), tumor metastasis (12), and coupled transport of K ϩ (13). These plasma membrane functions of the V-ATPase require that cells be able to target VATPases to the cell surface, but the mechanisms involved in this selective targeting are not understood.The V-ATPases of animals, plants, and fungi are structurally very similar and are composed of two functional domains (1-9). The V 1 domain is a peripheral complex (molecular mass, 600 -650 kDa) composed of eight different subunits ranging in molecular mass from 70 to 14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V 0 domain is a 260-kDa integral complex composed of five subun...