Acidification Of Intracellular Compartments Research Articles

Study of ATPase and GTPase levels in Fibrotic Lung Disease with and without COVID-19 Vaccination

Background: In eukaryotic cells, the acidification of intracellular compartments is the responsibility of vacuolar H+ ATPase, a family of proton pumps, sometimes known as V-ATPases. Small GTPases are signaling molecules that regulate important cellular processes as well as subcellular activities making them essential players, particularly in a wide variety of coronavirus infection processes. Objectives: The purpose of this research was to assess the levels of ATPase and GTPase in fibrotic lung disease patients who had received or had not received the COVID-19 vaccination, and then to compare these levels with those of the control group. Methods: A total of 150 individuals participated in this study, divided into three groups. The first group was the control group (N=50). In the second group (N=50) patients with fibrotic lung disease () who did not get the COVID-19 vaccination. Fifty patients who had received the COVID-19 vaccination made up the third group ()(N=50). Enzyme-linked immunosorbent assay was the method that was used to determine the amounts of ATPase and GTPase. The P- P-value of 0.05 or less is considered statistically significant. ROC tests were examined for ATPase and GTPase. Results: The data analysis reported a significant rise in alkaline phosphatase, Alanine aminotransferase, and Aspartate-aminotransferase () among the three groups. Both ATPase and GTPase levels were shown to have significantly increased in Groups 3 and 2 as compared to Group 1 levels. Moreover, a substantial rise was discovered in Group 3 in comparison to Group 2 which was detected. Conclusion: ATPase and GTPase levels are increased in patients with fibrotic lung disease regardless of the COVID-19 vaccination state.

The medaka mutation tintachina sheds light on the evolution of V-ATPase B subunits in vertebrates

Vacuolar-type H+ ATPases (V-ATPases) are multimeric protein complexes that play a universal role in the acidification of intracellular compartments in eukaryotic cells. We have isolated the recessive medaka mutation tintachina (tch), which carries an inactivating modification of the conserved glycine residue (G75R) of the proton pump subunit atp6v1Ba/vatB1. Mutant embryos show penetrant pigmentation defects, massive brain apoptosis and lethality before hatching. Strikingly, an equivalent mutation in atp6v1B1 (G78R) has been reported in a family of patients suffering from distal renal tubular acidosis (dRTA), a hereditary disease that causes metabolic acidosis due to impaired kidney function. This poses the question as to how molecularly identical mutations result in markedly different phenotypes in two vertebrate species. Our work offers an explanation for this phenomenon. We propose that, after successive rounds of whole-genome duplication, the emergence of paralogous copies allowed the divergence of the atp6v1B cis-regulatory control in different vertebrate groups.

The R740S mutation in the V‐ATPase a3 subunit results in osteoclast apoptosis and defective early‐stage autophagy

Vacuolar-type H(+)-ATPases (V-ATPases) are located in lysosomes and at the ruffled border in osteoclasts. We showed previously that the R740S mutation is dominant negative for V-ATPase activity, uncouples proton transport from ATP hydrolysis and causes osteopetrosis in heterozygous mice (+/R740S). Here we show mice homozygous for R740S (R740S/R740S) have more severe osteopetrosis and die by postnatal day 14. Although R740S/R740S osteoclasts express wild-type levels of a3, it is mislocalized. Acridine orange staining of R740S/R740S osteoclasts grown on a Corning resorptive surface reveals no resorption and no acidification of intracellular compartments. Whereas osteoblast and osteocyte apoptosis is normal, R740S/R740S osteoclasts exhibit increased apoptosis compared with wild-type osteoclasts. Localization of the enzyme tartrate-resistant acid phosphatase (TRAP) is also aberrant. Transmission electron microscopy reveals that R740S/R740S osteoclasts do not polarize, lack ruffled borders, and contain fewer autophagosomes. Consistent with an early stage defect in autophagy, expression of LC3II is reduced and expression of p62 is increased in R740S/R740S compared to wild-type osteoclasts. These results indicate the importance of intracellular acidification for the early stages of autophagy as well as for osteoclast survival, maturation, and polarization with appropriate cytoplasmic distribution of key osteoclast enzymes such as TRAP.

A 4-gene panel as a marker at chromosome 8q in Asian gastric cancer patients

A widely held viewpoint is that the use of multiple markers, combined in some type of algorithm, will be necessary to provide high enough discrimination between diseased cases and non-diseased. We applied stepwise logistic regression analysis to identify the best combination of the 32 biomarkers at chromosome 8q on an independent public microarray test set of 80 paired gastric samples. A combination of SULF1, INTS8, ATP6V1C1, and GPR172A was identified with a prediction accuracy of 98.0% for discriminating carcinomas from adjacent noncancerous tissues in our previous 25 paired samples. Interestingly, the overexpression of SULF1 was associated with tumor invasion and metastasis. Function prediction analysis revealed that the 4-marker panel was mainly associated with acidification of intracellular compartments. Taken together, we found a 4-gene panel that accurately discriminated gastric carcinomas from adjacent noncancerous tissues and these results had potential clinical significance in the early diagnosis and targeted treatment of gastric cancer.

Influenza A virus-induced early activation of ERK and PI3K mediates V-ATPase-dependent intracellular pH change required for fusion

The vacuolar (H+)-ATPases (V-ATPases) facilitate the release of influenza A virus (IAV) genome into the cytoplasm by acidifying the endosomal interior. The regulation of V-ATPases by signalling pathways has been demonstrated in various model systems. However, little is known about signalling-regulated V-ATPase activation during IAV infection. Here we show that V-ATPase activity is elevated during infection of cell monolayers with IAV, as measured by intracellular pH change, via a mechanism mediated by extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K). Inhibition of IAV-induced early activation of these kinases reduced V-ATPase activity and the acidification of intracellular compartments in infected cells. IAV-activated ERK and PI3K appear to interact directly, and they colocalize with the E subunit of V-ATPase V1 domain. Further, siRNAs targeting the E2 subunit isoform significantly reduced virus titres. Interestingly, suppression of PI3K early activation, but not that of ERK or V-ATPase, negatively affected virus internalization, suggesting the involvement of the pathway in earlier, V-ATPase-independent infection-promoting events. Cell treatment with a V-ATPase-specific inhibitor impaired the nuclear localization of incoming viral ribonucleoproteins, inhibiting replication/transcription of viral RNAs. These findings highlight the importance of IAV-induced ERK and PI3K early activation as signalling mediators in V-ATPase-stimulated endosomal acidification required for fusion.

The Vacuolar Proton Pump, V-ATPase, Is Required for Notch Signaling and Endosomal Trafficking in Drosophila

We have identified Rabconnectin-3alpha and beta (Rbcn-3A and B) as two regulators of Notch signaling in Drosophila. We found that, in addition to disrupting Notch signaling, mutations in Rbcn-3A and B cause defects in endocytic trafficking, where Notch and other membrane proteins accumulate in late endosomal compartments. We show that Notch is transported to the surface of mutant cells and that signaling is disrupted after the S2 cleavage. Interestingly, the yeast homolog of Rbcn-3A, Rav1, regulates the V-ATPase proton pump responsible for acidifying intracellular organelles. We found that, similarly, Rbcn-3A and B appear to regulate V-ATPase function. Moreover, we identified mutants in VhaAC39, a V-ATPase subunit, and showed that they phenocopy Rbcn-3A and Rbcn-3B mutants. Our results demonstrate that Rbcn-3 affects Notch signaling and trafficking through regulating V-ATPase function, which implies that the acidification of an intracellular compartment in the receiving cells is crucial for signaling.

(Pro)renin Receptor and Vacuolar H + -ATPase

The discovery of a “(pro)renin receptor”1 has renewed the interest in prorenin, the inactive precursor of renin. Prorenin binding to this receptor allows prorenin to display enzymatic activity without the accompanying cleavage of the prosegment that normally occurs in the kidney when prorenin is converted to renin. The underlying concept is that binding to the (pro)renin receptor induces a conformational change in the prorenin molecule, involving unfolding of the propeptide from the enzymatic cleft so that the cleft is now accessible to angiotensinogen (“nonproteolytic activation” of prorenin). Similar conformational changes occur at low pH (acid-activation) and low temperature (cryoactivation), but whether these phenomena are of physiological relevance is uncertain. In contrast, the (pro)renin receptor provides a physiological role for prorenin, explaining how angiotensin production might occur at the tissue level and putting into perspective the relatively high prorenin levels (10-fold those of renin) in the human circulation. These levels are even higher in diabetes mellitus complicated by retinopathy and nephropathy.2 Unexpectedly, prorenin binding to its receptor also induces intracellular signaling,1,3 independent of angiotensin generation, suggesting that prorenin may act as an agonist of the receptor. Although similar observations were made on renin, prorenin binds with higher affinity to the receptor and, thus, appears to be its endogenous agonist.4 The (pro)renin receptor is a 350-amino acid protein with a single transmembrane domain. Although it was first identified on cultured human mesangial cells,1 the C-terminal part of the receptor had been described earlier by Ludwig et al5 as an 8.9-kDa fragment associated with a vacuolar H+-ATPase. Consequently, the second name of the protein is ATP6ap2 (ATPase, H+ transporting, lysosomal accessory protein 2). Vacuolar H+-ATPases play important roles in the acidification of intracellular compartments and cellular pH homeostasis,6 eg, in the …

A Trypanosoma cruzi Phosphatidylinositol 3-Kinase (TcVps34) Is Involved in Osmoregulation and Receptor-mediated Endocytosis

Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H+-ATPase and vacuolar H+-pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking.

Energizing an Invertebrate Embryo: Bafilomycin‐Dependent Respiration and the Metabolic Cost of Proton Pumping by the V‐ATPase

We examine herein the contribution of V-ATPase activity to the energy budget of aerobically developing embryos of Artemia franciscana and discuss the results in the context of quiescence under anoxia. (31)P-NMR analysis indicates that intracellular pH and NTP levels are unaffected by acute incubation of dechorionated embryos with the V-ATPase inhibitor, bafilomycin A(1). Bafilomycin A(1) also has no significant effect on oxygen consumption by isolated mitochondria. Taken together, these data indicate that bafilomycin does not affect energy-producing pathways in the developing embryo. However, the V-ATPase inhibitor exhibits a concentration-dependent inhibition of oxygen consumption in aerobic embryos. A conservative analysis of respirometric data indicates that proton pumping by the V-ATPase, and processes immediately dependent on this activity, constitutes approximately 31% of the aerobic energy budget of the preemergent embryo. Given the complete absence of detectable Na(+)K(+)-ATPase activity during the first hours of aerobic development, it is plausible that the V-ATPase is performing a role in both the acidification of intracellular compartments and the energization of plasma membranes. Importantly, the high metabolic cost associated with maintaining these diverse proton gradients requires that V-ATPase activity be downregulated under anoxia in order to attain the almost complete metabolic depression observed in the quiescent embryo.

Intestinal Expression of H+ V‐ATPase in the Mosquito Aedes albopictus is Tightly Associated with Gregarine Infection

Vacuolar ATPase (V-ATPase) is a family of ATP-dependent proton pumps expressed on the plasma membrane and endomembranes of eukaryotic cells. Acidification of intracellular compartments, such as lysosomes, endosomes, and parasitophorous vacuoles, mediated by V-ATPase is essential for the entry by many enveloped viruses and invasion into or escape from host cells by intracellular parasites. In mosquito larvae, V-ATPase plays a role in regulating alkalization of the anterior midgut. We extracted RNA from larval tissues of Aedes albopictus, cloned the full-length sequence of mRNA of V-ATPase subunit A, which contains a poly-A tail and 2,971 nucleotides, and expressed the protein. The fusion protein was then used to produce rabbit polyclonal antibodies, which were used as a tool to detect V-ATPase in the midgut and Malpighian tubules of mosquito larvae. A parasitophorous vacuole was formed in the midgut in response to invasion by Ascogregarina taiwanensis, confining the trophozoite(s). Acidification was demonstrated within the vacuole using acridine orange staining. It is concluded that gregarine sporozoites are released by ingested oocysts in the V-ATPase-energized high-pH environment. The released sporozoites then invade and develop in epithelial cells of the posterior midgut. Acidification of the parasitophorous vacuoles may be mediated by V-ATPase and may facilitate exocytosis of the vacuole confining the trophozoites from the infected epithelial cells for further extracellular development.

Structural features and nucleotide-binding capability of the C subunit are integral to the regulation of the eukaryotic V1Vo ATPases

V-ATPases (vacuolar ATPases) are responsible for acidification of intracellular compartments and, in certain cases, proton transport across the plasma membrane of eukaryotic cells. They are composed of a catalytic V1 sector, in which ATP hydrolysis takes place, and the Vo sector, which functions in proton conduction. The best established mechanism for regulating the V-ATPase activity in vivo involves reversible dissociation of the V1 and Vo domains, in which subunit C is intimately involved. In the last year, impressive progress has been made in elucidating the structure of the C subunit and its arrangement inside the V-ATPase. Nucleotide occupancy by subunit C, followed by conformational changes of this subunit has shed light on the mechanism of V-ATPase regulation.

Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H+-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells

Vacuolar H+-ATPases (V-ATPases) are a family of ATP-driven proton pumps. They maintain pH gradients between intracellular compartments and are required for proton secretion out of the cytoplasm. Mechanisms of extrinsic control of V-ATPase are poorly understood. Previous studies showed that glucose is an important regulator of V-ATPase assembly in Saccharomyces cerevisiae. Human V-ATPase directly interacts with aldolase, providing a coupling mechanism for glucose metabolism and V-ATPase function. Here we show that glucose is a crucial regulator of V-ATPase in renal epithelial cells and that the effect of glucose is mediated by phosphatidylinositol 3-kinase (PI3K). Glucose stimulates V-ATPase-dependent acidification of the intracellular compartments in human proximal tubular cells HK-2 and porcine renal epithelial cells LLC-PK1. Glucose induces rapid ATP-independent assembly of the V1 and Vo domains of V-ATPase and extensive translocation of the V-ATPase V1 and Vo domains between different membrane pools and between membranes and the cytoplasm. In HK-2 cells, glucose stimulates polarized translocation of V-ATPase to the apical plasma membrane. The effects of glucose on V-ATPase trafficking and assembly can be abolished by pretreatment with the PI3K inhibitor LY294002 and can be reproduced in glucose-deprived cells by adenoviral expression of the constitutively active catalytic subunit p110alpha of PI3K. Taken together these data provide evidence that, in renal epithelial cells, glucose plays an important role in the control of V-ATPase-dependent acidification of intracellular compartments and V-ATPase assembly and trafficking and that the effects of glucose are mediated by PI3K-dependent signaling.

Subunit structure, function, and arrangement in the yeast and coated vesicle V-ATPases.

The vacuolar (H+)-ATPases (or V-ATPases) are ATP-dependent proton pumps that function both to acidify intracellular compartments and to transport protons across the plasma membrane. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking, protein processing, and coupled transport. Plasma membrane V-ATPases function in renal acidification, bone resorption, pH homeostasis, and, possibly, tumor metastasis. This review will focus on work from our laboratories on the V-ATPases from mammalian clathrin-coated vesicles and from yeast. The V-ATPases are composed of two domains. The peripheral V1 domain has a molecular mass of 640 kDa and is composed of eight different subunits (subunits A-H) of molecular mass 70-13 kDa. The integral V0 domain, which has a molecular mass of 260 kDa, is composed of five different subunits (subunits a, d, c, c', and c'') of molecular mass 100-17 kDa. The V1 domain is responsible for ATP hydrolysis whereas the V0 domain is responsible for proton transport. Using a variety of techniques, including cysteine-mediated crosslinking and electron microscopy, we have defined both the overall shape of the V-ATPase and the V0 domain as well as the location of various subunits within the complex. We have employed site-directed and random mutagenesis to identify subunits and residues involved in nucleotide binding and hydrolysis, proton translocation, and the coupling of these two processes. We have also investigated the mechanism of regulation of the V-ATPase by reversible dissociation and the role of different subunits in this process.

Intracellular potassium and chloride channels: an update.

Channels selective for potassium or chloride ions are present in all intracellular membranes such as mitochondrial membranes, sarcoplasmic/endoplasmic reticulum, nuclear membrane and chromaffin granule membranes. They probably play an important role in events such as acidification of intracellular compartments and regulation of organelle volume. Additionally, intracellular ion channels are targets for pharmacologically active compounds, e.g. mitochondrial potassium channels interact with potassium channel openers such as diazoxide. This review describes current observations concerning the properties and functional roles of intracellular potassium and chloride channels.

Arrested maturing multivesicular endosomes observed in a Chinese hamster ovary cell mutant, LEX2, isolated by repeated flow-cytometric cell sorting.

Chinese hamster ovary (CHO) cell mutants defective in the disintegration of endocytosed low-density lipoprotein (LDL) were isolated from mutagenized cells by repeated flow-cytometric cell sorting. After seven rounds of cell sorting, we obtained mutant pools, from which nine mutant clones were established. These mutant strains were all recessive, and were categorized into three complementation groups A, B, and C. The previously established CHO mutant, LEX1 (Lysosome-Endosome X1), fell into the complementation group A. One of the newly isolated mutants, LEX2, fell into the complementation group B, and showed slower degradation of RET-LDL than LEX1 cells. LEX2 showed prominence of well-elaborated multivesicular bodies (MVBs), positive for lysosomal glycoprotein-B/cathepsin D and cation-independent mannose 6-phosphate receptor (CI-MPR), yet negative for transferrin receptor or rab7. Endocytosed intact LDL accumulated in these CI-MPR-positive structures starting at 10-15 minutes of internalization and the accumulation reached completion at 20 minutes. Intermixing of separately internalized fluorescent LDLs between the LEX2 MVBs was slow and saturable at a lower level than observed between late endosomes/lysosomes in wild-type or in LEX1 cells. The receptor recycling pathway to the plasma membrane and the acidification of intracellular compartments were normal in LEX2 cells. These results are consistent with the idea that LEX2 cells are defective in the segregation and sequestration of contents at compartments equivalent to the transport intermediates, previously referred to as endosomal carrier vesicles or maturing MVBs. This MVB stage is likely to be an earlier stage than rab7-positive, lysosome-interacting late endosomes observed in LEX1 cells. Thus, LEX1 and LEX2 mutations could be considered as landmarks for these distinct late endocytic stages, and use of these cells in biochemical and molecular genetic analyses would help to understand the as yet unidentified details of late endocytic pathways including the MVB dynamics.

Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases.

The vacuolar H(+)-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps that carry out acidification of intracellular compartments in eukaryotic cells. This review is focused on our work on the V-ATPases of clathrin-coated vesicles and yeast vacuoles. The coated-vesicle V-ATPase undergoes trafficking to endosomes and synaptic vesicles, where it functions in receptor recycling and neurotransmitter uptake, respectively. The yeast V-ATPase functions to acidify the central vacuole and is necessary both for protein degradation and for coupled transport processes across the vacuolar membrane. The V-ATPases are multisubunit complexes composed of two functional domains. The V(1) domain is a 570 kDa peripheral complex composed of eight subunits of molecular mass 73-14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V(o) domain is a 260 kDa integral complex composed of five subunits of molecular mass 100-17 kDa (subunits a, d, c, c' and c") that is responsible for proton translocation. To explore the function of individual subunits in the V-ATPase complex as well as to identify residues important in proton transport and ATP hydrolysis, we have employed a combination of chemical modification, site-directed mutagenesis and in vitro reassembly. A central question concerns the mechanism by which vacuolar acidification is controlled in eukaryotic cells. We have proposed that disulfide bond formation between conserved cysteine residues at the catalytic site of the V-ATPase plays an important role in regulating V-ATPase activity in vivo. Other regulatory mechanisms that are discussed include reversible dissociation and reassembly of the V-ATPase complex, changes in the tightness of coupling between proton transport and ATP hydrolysis, differential targeting of V-ATPases within the cell and control of the Cl(-) conductance that is necessary for vacuolar acidification.

Structure and properties of the clathrin-coated vesicle and yeast vacuolar V-ATPases.

The V-ATPases are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. This review focuses on the the V-ATPases from clathrin-coated vesicles and yeast vacuoles. The V-ATPase of clathrin-coated vesicles is a precursor to that found in endosomes and synaptic vesicles, which function in receptor recycling, intracellular membrane traffic, and neurotransmitter uptake. The yeast vacuolar ATPase functions to acidify the central vacuole and to drive various coupled transport processes across the vacuolar membrane. The V-ATPases are composed of two functional domains. The V1 domain is a 570-kDa peripheral complex composed of eight subunits of molecular weight 70-14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V0 domain is a 260-kDa integral complex composed of five subunits of molecular weight 100-17 kDa (subunits a, d, c, c' and c") that is responsible for proton translocation. Using chemical modification and site-directed mutagenesis, we have begun to identify residues that play a role in ATP hydrolysis and proton transport by the V-ATPases. A central question in the V-ATPase field is the mechanism by which cells regulate vacuolar acidification. Several mechanisms are described that may play a role in controlling vacuolar acidification in vivo. One mechanism involves disulfide bond formation between cysteine residues located at the catalytic nucleotide binding site on the 70-kDa A subunit, leading to reversible inhibition of V-ATPase activity. Other mechanisms include reversible assembly and dissociation of V1 and V0 domains, changes in coupling efficiency of proton transport and ATP hydrolysis, and regulation of the activity of intracellular chloride channels required for vacuolar acidification.

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

The vacuolar (H+)-ATPases (or V-ATPases) function in the acidification of intracellular compartments in eukaryotic cells. The V-ATPases are multisubunit complexes composed of two functional domains. The peripheral V1 domain, a 500-kDa complex responsible for ATP hydrolysis, contains at least eight different subunits of molecular weight 70-13 (subunits A-H). The integral V0 domain, a 250-kDa complex, functions in proton translocation and contains at least five different subunits of molecular weight 100-17 (subunits a-d). Biochemical and genetic analysis has been used to identify subunits and residues involved in nucleotide binding and hydrolysis, proton translocation, and coupling of these activities. Several mechanisms have been implicated in the regulation of vacuolar acidification in vivo, including control of pump density, regulation of assembly of V1 and V0 domains, disulfide bond formation, activator or inhibitor proteins, and regulation of counterion conductance. Recent information concerning targeting and regulation of V-ATPases has also been obtained.

Differential expression of vacuolar-type H+-ATPase between normal human pancreatic islet B-cells and insulinoma cells

Recent studies have shown that bafilomycin A(1)-sensitive vacuolar type H+-ATPase (V-ATPase) is responsible for the acidification of intracellular compartments in eukaryotic cells. B-cells of pancreatic islets also are known to include acidifying secretory vesicles which are the major cellular site of proinsulin to insulin conversion. This study was designed to examine immunohistochemically the level of V-ATPase protein expression in normal pancreas (five cases) and benign insulinoma (six cases), using mouse monoclonal antibody raised against the 116 kDa subunit of human V-ATPase. Light microscopic immunohistochemistry revealed that moderate to marked V-ATPase expression was observed in normal islet B-cells, while insulinoma cells in each case expressed V-ATPase faintly or not at all. By immunoelectron microscopy, the majority of secretory vesicles in insulinoma cells did not express V-ATPase protein at their endomembranes, although mild to marked V-ATPase expression was noted at the endomembrane of secretory vesicles in normal islet B-cells. Thus, differential expression of V-ATPase protein at the endomembrane of secretory vesicles was observed between normal islet B-cells and insulinoma cells. These findings suggest that the reduced activity of V-ATPase per insulin secretory vesicle in insulinoma cells have a profound effect on the efficiency of proteolytic cleavage of proinsulin.

Structure and function of the proton-conducting sector of the vacuolar H(+)-ATPase.

Introduction: the vacuolar H +-ATPase Acidification of intracellular compartments, which in many cases is essential for their function, is maintained in a variety of eukaryotic systems by the activity of the vacuolar (V-type) H+-ATPases. These endomembrane-bound proton pumps are responsible for generating the low internal pH of components of the endocytic and secretory pathways, such as clathrin-coated vesicles, Golgi and secretory vesicles, lysosomes and endosomes generated during receptor-mediated endocytosis [l-31. V-ATPases are also found in the vacuolar membranes of plants and fungi, in which the proton motive force generated by the hydrolysis of cytosolic ATP is coupled to movement of ions and solutes into the vacuole [4]. The enzyme therefore performs a central role in ionic homeostasis within the cell. V-type ATPases may also be associated with the plasma membrane. For example, a plasma membrane-bound V-type ATPase is an essential component in the mechanism of bone resorption in osteoclast cells [S]. In recent years it has become more apparent that members of this class of multiprotein complex are of fundamental importance to a great variety of physiological processes, suggesting that they will, in future, be of increasing pharmacological significance [ 61. Although analogous in organization to the F,-F,, ATP synthases of mitochondria and chloroplasts, the V-ATPases operate exclusively as ATPdriven proton pumps. The holocomplex may have a total mass of 650 kDa and comprises a soluble V, sector associated with an integral membrane V, sector [ 1 -3,7]. The V, sector, which is responsible for substrate binding and regulation of activity, may consist of as many as six types of subunit [7 ,8] . The V, sector, which contains the elements responsible