Michael Forgac

The vacuolar (H+)-ATPases — nature's most versatile proton pumps

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.

Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology

The acidity of intracellular compartments and the extracellular environment is crucial to various cellular processes, including membrane trafficking, protein degradation, bone resorption and sperm maturation. At the heart of regulating acidity are the vacuolar (V-)ATPases — large, multisubunit complexes that function as ATP-driven proton pumps. Their activity is controlled by regulating the assembly of the V-ATPase complex or by the dynamic regulation of V-ATPase expression on membrane surfaces. The V-ATPases have been implicated in a number of diseases and, coupled with their complex isoform composition, represent attractive and potentially highly specific drug targets.

Structure and Properties of the Vacuolar (H+)-ATPases

Function of V-ATPases The vacuolar (H)-ATPases (or V-ATPases) are a family of ATPdependent proton pumps that are responsible for acidification of intracellular compartments in eukaryotic cells (1–3). Acidification of vacuolar compartments plays an important role in a variety of cellular processes, particularly membrane traffic processes. In receptor-mediated endocytosis, acidification of endosomes serves as a signal that activates release of internalized ligands (such as low density lipoprotein and insulin) from their receptors. This uncoupling allows unoccupied receptors to recycle to the cell surface where they can be reutilized, whereas the ligands are targeted to lysosomes for degradation (1). Acidification of endosomes is also required for the formation of endosomal carrier vesicles (which are involved in moving ligands along the endocytic pathway (4)) and in activating fusion between the endosomal membrane and internalized envelope viruses (such as influenza virus) that is necessary for viral infection (5). In intracellular targeting of newly synthesized lysosomal enzymes, vacuolar acidification plays a very similar role to that observed for endocytosis. Thus, lysosomal enzymes bound to the mannose 6-phosphate receptor are delivered to a late recycling compartment where the low pH causes dissociation, allowing receptors to recycle to the trans-Golgi and the enzymes to be targeted to lysosomes (6). Disruption of vacuolar acidification in yeast also causes a perturbation in vacuolar targeting (7), although the molecular basis of this effect is not yet clear. In lysosomes and other digestive organelles, like the central vacuole of yeast, the low pH is required to support the activity of acid hydrolases involved in degradation of macromolecules. In both central vacuoles and secretory vesicles, such as synaptic vesicles and chromaffin granules, the proton gradient or membrane potential generated by the VATPase is utilized to drive the coupled transport of small molecules into the vesicle lumen (8, 9). In addition to their role in intracellular compartments, V-ATPases have also been shown to play an important role in the plasma membrane of various specialized cells. Thus V-ATPases in the apical membrane of renal intercalated cells function in renal acidification (10), whereas plasma membrane V-ATPases in macrophages and neutrophils assist in pH homeostasis (11). Osteoclasts (12) and tumor cells (13) are able to target V-ATPases to the plasma membrane where they create an acidic extracellular environment that is necessary for bone resorption or tumor metastasis, respectively. V-ATPases in the midgut of insects have been shown to establish a membrane potential across the apical membrane that drives K transport via an electrogenic H/K antiporter (14). Finally, using the specific V-ATPase inhibitor bafilomycin (15), V-ATPases have been implicated in apoptosis (16). It is thus clear that V-ATPases serve a wide variety of roles in eukaryotic cells.

Regulation and Isoform Function of the V-ATPases

The vacuolar (H(+))-ATPases are ATP-dependent proton pumps that acidify intracellular compartments and, in some cases, transport protons across the plasma membrane of eukaryotic cells. Intracellular V-ATPases play an important role in normal physiological processes such as receptor-mediated endocytosis, intracellular membrane trafficking, pro-hormone processing, protein degradation, and the coupled uptake of small molecules, such as neurotransmitters. They also function in the entry of various pathogenic agents, including many envelope viruses, like influenza virus, and toxins, like anthrax toxin. Plasma membrane V-ATPases function in renal pH homeostasis, bone resorption and sperm maturation, and various disease processes, including renal tubular acidosis, osteopetrosis, and tumor metastasis. V-ATPases are composed of a peripheral V(1) domain containing eight different subunits that is responsible for ATP hydrolysis and an integral V(0) domain containing six different subunits that translocates protons. In mammalian cells, most of the V-ATPase subunits exist in multiple isoforms which are often expressed in a tissue specific manner. Isoforms of one of the V(0) subunits (subunit a) have been shown to possess information that targets the V-ATPase to distinct cellular destinations. Mutations in isoforms of subunit a lead to the human diseases osteopetrosis and renal tubular acidosis. A number of mechanisms are employed to regulate V-ATPase activity in vivo, including reversible dissociation of the V(1) and V(0) domains, control of the tightness of coupling of proton transport and ATP hydrolysis, and selective targeting of V-ATPases to distinct cellular membranes. Isoforms of subunit a are involved in regulation both via the control of coupling and via selective targeting. This review will begin with a brief introduction to the function, structure, and mechanism of the V-ATPases followed by a discussion of the role of V-ATPase subunit isoforms and the mechanisms involved in regulation of V-ATPase activity.

Function, structure and regulation of the vacuolar (H+)-ATPases

The vacuolar ATPases (or V-ATPases) are ATP-driven proton pumps that function to both acidify intracellular compartments and to transport protons across the plasma membrane. Intracellular V-ATPases function in such normal cellular processes as receptor-mediated endocytosis, intracellular membrane traffic, prohormone processing, protein degradation and neurotransmitter uptake, as well as in disease processes, including infection by influenza and other viruses and killing of cells by anthrax and diphtheria toxin. Plasma membrane V-ATPases are important in such physiological processes as urinary acidification, bone resorption and sperm maturation as well as in human diseases, including osteopetrosis, renal tubular acidosis and tumor metastasis. V-ATPases are large multi-subunit complexes composed of a peripheral domain (V(1)) responsible for hydrolysis of ATP and an integral domain (V(0)) that carries out proton transport. Proton transport is coupled to ATP hydrolysis by a rotary mechanism. V-ATPase activity is regulated in vivo using a number of mechanisms, including reversible dissociation of the V(1) and V(0) domains, changes in coupling efficiency of proton transport and ATP hydrolysis and changes in pump density through reversible fusion of V-ATPase containing vesicles. V-ATPases are emerging as potential drug targets in treating a number of human diseases including osteoporosis and cancer.

V-ATPase functions in normal and disease processes

Eukaryotic cells have evolved a family of ATP-dependent proton pumps known as the vacuolar (H+)-ATPases (or V-ATPases) to regulate the pH of intracellular compartments, the extracellular space, and the cytoplasm. V-ATPases present within intracellular compartments are important for such normal cellular processes as receptor-mediated endocytosis and intracellular membrane traffic, protein processing and degradation and coupled transport of small molecules and ions. They also facilitate the entry of a number of envelope viruses and bacterial toxins, including influenza virus and anthrax toxin. V-ATPases present in the plasma membranes of cells are also important in normal physiology. They facilitate bone resorption by osteoclasts, acid secretion by intercalated cells of the kidney, pH homeostasis in macrophages and neutrophils, angiogenesis by endothelial cells, and sperm maturation and storage in the male reproductive tract. In the insect midgut, they establish a membrane potential used to drive K+ secretion. Plasma membrane V-ATPases are especially important in human disease, with genetic defects in V-ATPases expressed in osteoclasts and intercalated cells leading to the diseases osteopetrosis and renal tubule acidosis, respectively. Plasma membrane V-ATPases have also been implicated in tumor cell invasion. V-ATPases are thus emerging as potential targets in the treatment of diseases such as osteoporosis and cancer.

Structure, function and regulation of the vacuolar (H+)-ATPases

The vacuolar (H+)‐ATPases (or V‐ATPases) function to acidify intracellular compartments in eukaryotic cells, playing an important role in such processes as receptor‐mediated endocytosis, intracellular membrane traffic, protein degradation and coupled transport. V‐ATPases in the plasma membrane of specialized cells also function in renal acidification, bone resorption and cytosolic pH maintenance. The V‐ATPases are composed of two domains. The V1 domain is a 570‐kDa peripheral complex composed of 8 subunits (subunits A–H) of molecular weight 70–13 kDa which is responsible for ATP hydrolysis. The V0 domain is a 260‐kDa integral complex composed of 5 subunits (subunits a–d) which is responsible for proton translocation. The V‐ATPases are structurally related to the F‐ATPases which function in ATP synthesis. Biochemical and mutational studies have begun to reveal the function of individual subunits and residues in V‐ATPase activity. A central question in this field is the mechanism of regulation of vacuolar acidification in vivo. Evidence has been obtained suggesting a number of possible mechanisms of regulating V‐ATPase activity, including reversible dissociation of V1 and V0 domains, disulfide bond formation at the catalytic site and differential targeting of V‐ATPases. Control of anion conductance may also function to regulate vacuolar pH. Because of the diversity of functions of V‐ATPases, cells most likely employ multiple mechanisms for controlling their activity.

Function of a subunit isoforms of the V-ATPase in pH homeostasis and in vitro invasion of MDA-MB231 human breast cancer cells.

It has previously been shown that highly invasive MDA-MB231 human breast cancer cells express vacuolar proton-translocating ATPase (V-ATPases) at the cell surface, whereas the poorly invasive MCF7 cell line does not. Bafilomycin, a specific V-ATPase inhibitor, reduces the in vitro invasion of MB231 cells but not MCF7 cells. Targeting of V-ATPases to different cellular membranes is controlled by isoforms of subunit a. mRNA levels for a subunit isoforms were measured in MB231 and MCF7 cells using quantitative reverse transcription-PCR. The results show that although all four isoforms are detectable in both cell types, levels of a3 and a4 are much higher in MB231 than in MCF7 cells. Isoform-specific small interfering RNAs (siRNA) were employed to selectively reduce mRNA levels for each isoform in MB231 cells. V-ATPase function was assessed using the fluorescent indicators SNARF-1 and pyranine to monitor the pH of the cytosol and endosomal/lysosomal compartments, respectively. Cytosolic pH was decreased only on knockdown of a3, whereas endosome/lysosome pH was increased on knockdown of a1, a2, and a3. Treatment of cells with siRNA to a4 did not affect either cytosolic or endosome/lysosome pH. Measurement of invasion using an in vitro transwell assay revealed that siRNAs to both a3 and a4 significantly inhibited invasion of MB231 cells. Immunofluorescence staining of MB231 cells for V-ATPase distribution revealed extensive intracellular staining, with plasma membrane staining observed in ∼18% of cells. Knockdown of a4 had the greatest effect on plasma membrane staining, leading to a 32% reduction. These results suggest that the a4 isoform may be responsible for targeting V-ATPases to the plasma membrane of MB231 cells and that cell surface V-ATPases play a significant role in invasion. However, other V-ATPases affecting the pH of the cytosol and intracellular compartments, particularly those containing a3, are also involved in invasion.

Structure of the Vacuolar ATPase by Electron Microscopy

The structure of the vacuolar ATPase from bovine brain clathrin-coated vesicles has been determined by electron microscopy of negatively stained, detergent-solubilized enzyme molecules. Preparations of both lipid-containing and delipidated enzyme have been analyzed. The complex is organized in two major domains, a V1 and V0, with overall dimensions of 28 × 14 × 14 nm. The V1 is a more or less spherical molecule with a central cavity. The V0 has the shape of a flattened sphere or doughnut with a radius of about 100 Å. The V1 and V0 are joined by a 60-Å long and 40-Å wide central stalk, consisting of several individual protein densities. Two kinds of smaller densities are visible at the top periphery of the V1, and one of these seems to extend all the way down to the stalk domain in some averages. Images of both the lipid-containing and the delipidated complex show a 30–50-kDa protein density on the lumenal side of the complex, opposite the central stalk, centered in the ring of c subunits. A large trans-membrane mass, probably the C-terminal domain of the 100-kDa subunita, is seen at the periphery of the c subunit ring in some projections. This large mass has both a lumenal and a cytosolic domain, and it is the cytosolic domain that interacts with the central stalk. Two to three additional protein densities can be seen in the V1-V0 interface, all connected to the central stalk. Overall, the structure of the V-ATPase is similar to the structure of the related F1F0-ATP synthase, confirming their common origin.

Molecular Cloning and Expression of Three Isoforms of the 100-kDa a Subunit of the Mouse Vacuolar Proton-translocating ATPase

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 amino- and 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.