共查询到20条相似文献,搜索用时 15 毫秒
1.
Nakamura S 《American journal of physiology. Cell physiology》2004,287(1):C97-105
The vacuolar H+-ATPase (V-ATPase) acidifies compartments of the vacuolar system of eukaryotic cells. In renal epithelial cells, it resides on the plasma membrane and is essential for bicarbonate transport and acid-base homeostasis. The factors that regulate the H+-ATPase remain largely unknown. The present study examines the effect of glucose on H+-ATPase activity in the pig kidney epithelial cell line LLC-PK1. Cellular pH was measured by performing ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. Intracellular acidification was induced with NH3/NH4+ prepulse, and rates of intracellular pH (pHi) recovery (after in situ calibration) were determined by the slopes of linear regression lines during the first 3 min of recovery. The solutions contained 1 µM ethylisopropylamiloride and were K+ free to eliminate Na+/H+ exchange and H+-K+-ATPase activity. After NH3/NH4+-induced acidification, LLC-PK1 cells had a significant pHi recovery rate that was inhibited entirely by 100 nM of the V-ATPase inhibitor concanamycin A. Acute removal of glucose from medium markedly reduced V-ATPase-dependent pHi recovery activity. Readdition of glucose induced concentration-dependent reactivation of V-ATPase pHi recovery activity within 2 min. Glucose replacement produced no significant change in cell ATP or ADP content. H+-ATPase activity was completely inhibited by the glycolytic inhibitor 2-deoxy-D-glucose (20 mM) but only partially inhibited by the mitochondrial electron transport inhibitor antimycin A (20 µM). The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin (500 nM) abolished glucose activation of V-ATPase, and activity was restored after wortmannin removal. Glucose activates V-ATPase activity in kidney epithelial cells through the glycolytic pathway by a signaling pathway that requires PI3K activity. These findings represent an entirely new physiological effect of glucose, linking it to cellular proton secretion and vacuolar acidification. proton secretion; glycolysis; intracellular pH; concanamycin A 相似文献
2.
Plasmalemmal vacuolar-type H+-ATPase in cancer biology 总被引:6,自引:0,他引:6
Vacuolar-type H+-adenosine triphosphatase (V-ATPase) is one of the most fundamental enzymes in nature. V-ATPases are responsible for the regulation
of proton concentration in the intracellular acidic compartments. It has similar structure with the mitochondrial F0F1-ATP synthase (F-ATPase).† The V-ATPases are composed of multiple subunits and have various physiological functions, including membrane and organelle
protein sorting, neurotransmitter uptake, cellular degradative processes, and cytosolic pH regulation. The V-ATPases have
been involved in multidrug resistance. Recently, plasma membrane V-ATPases have been involved in regulation of extracellular
acidity, essential for cellular invasiveness and proliferation in tumor metastasis. The current knowledge regarding the structure
and function of V-ATPase and its role in cancer biology is discussed.
F in F0F1 ATPase is the coupling energy factor. 相似文献
3.
Florian Timo Ludwig Albrecht Schwab Christian Stock 《Journal of cellular physiology》2013,228(6):1351-1358
Many tumor cells are characterized by an increased net acid production. They extrude the excess protons mainly through the Na+/H+‐exchanger NHE1. An increased NHE1 activity elevates the metastatic potential of tumor cells. Cell migration, a key step in the metastatic cascade, requires the formation and release of integrin‐mediated cell–matrix contacts (focal adhesions). As NHE1 has been localized to focal adhesion sites, the present study tests the hypothesis that NHE1 generates measurable pH nanodomains right at focal adhesions. In order to ratiometrically measure pH close to the plasma membrane, we established a novel application of the total internal reflection fluorescence microscopy (TIRFM). Human melanoma cells were transfected with DsRed2‐paxillin to identify focal adhesion sites. The pH‐sensitive dyes BCECF and WGA‐fluorescein were used to measure the submembranous cytosolic and the pericellular pH, respectively. Distinct pH nanodomains were found at focal adhesions, particularly at those located at the cell front, where NHE1 was concentrated. These sites featured a remarkably alkaline cytosolic and an acidic pericellular pH and thus a much steeper proton gradient across the plasma membrane compared to the rest of the cell. The generation of pH nanodomains could be assigned to NHE1‐mediated H+ export because such pH domains could not be detected in NHE1‐deficient cells. Given that both integrin avidity and mechanisms contributing to adhesion turnover are pH‐sensitive, we propose that pH nanodomains at focal adhesions, locally created and maintained by NHE1 activity especially at the cell front, modulate adhesion dynamics in migrating cells. J. Cell. Physiol. 228: 1351–1358, 2013. © 2012 Wiley Periodicals, Inc. 相似文献
4.
Internodal cells of Nitellopsis were made tonoplast-free byperfusion with a medium containing EGTA. Cytoplasmic concentrationsof solutes were controlled by a second perfusion with mediaof known composition. The electrogenic pump current (Ip), whichwas calculated from electrical data obtained from cells withand without ATP, was compared with the current carried by H+(IH+) across the plasma membrane. A close correlation betweenIp and IH+ was found under various internal and external conditions.(1) Ip and IH+ depended on the internal ATP and showed Michaelis-Mententype saturation curves. For Ip, Km was 120 µM and themaximum current Vmax was 15.1 mA m2, while for IH+, Kmwas 160 µM and Vmax was 16.6 mA m2. (2) Ip andIH+ showed almost the same IH2+ dependence. The Mg2+-dependentIp was 19.5 mA m2, while the Mg2+-dependent IH2+ was17.7 mA m2. (3) IH2+ was maximal at an external pH of8 and decreased both in acidic and alkaline pH ranges. Ip wasnearly equal to IH+ in the pH range between 8 and 5. (4) IH+became maximal at an internal pH of 7.3, which is nearly thesame as the pH for maximal electrogenecity found by Mimura andTazawa (1984). All these facts support the idea proposed in our previous paper(Takeshige et al. 1985) that the electrogenic ion pump locatedin the plasma membrane of Nitellopsis is the H+ pump.
1 Dedicated to Professor Dr. Erwin Bünning on the occasionof his 80th birthday. (Received June 21, 1985; Accepted December 20, 1985) 相似文献
5.
Michael Forgac 《FEBS letters》1998,440(3)
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. 相似文献
6.
Osteoclasts aremultinucleated cells that resorb bone by extrusion of protons andproteolytic enzymes. They display marked heterogeneity in cell size,shape, and resorptive activity. Because high resorptive activity invivo is associated with an increase in the average size of osteoclastsin areas of greater resorption and because of the importance of protonextrusion in resorption, we investigated whether the activity of thebafilomycin A1-sensitive vacuolar-typeH+-ATPase (V-ATPase) and amiloride-sensitiveNa+/H+ exchanger differed between large andsmall osteoclasts. Osteoclasts were obtained from newborn rabbit bones,cultured on glass coverslips, and loaded with the pH-sensitiveindicator2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF).Intracellular pH (pHi) was recorded in single osteoclasts by monitoring fluorescence. Large (10 nuclei) and small (5 nuclei) osteoclasts differed in that large osteoclasts had a higher basal pHi, their pHi was decreased by bafilomycinA1 addition or removal of extracellular Na+,and the realkalinization upon readdition of Na+ wasbafilomycin A1 sensitive. After acid loading, asubpopulation of large osteoclasts (40%) recovered by V-ATPaseactivity alone, whereas all small osteoclasts recovered byNa+/H+ exchanger activity. Interestingly, in60% of the large osteoclasts, pHi recovery was mediated byboth the Na+/H+ exchanger and V-ATPaseactivity. Our results show a striking difference betweenpHi regulatory mechanisms of large and small osteoclaststhat we hypothesize may be associated with differences in the potentialresorptive activity of these cells. 相似文献
7.
Paunescu TG Jones AC Tyszkowski R Brown D 《American journal of physiology. Cell physiology》2008,295(4):C923-C930
The vacuolar proton-pumping ATPase (V-ATPase) is responsible for the acidification of intracellular organelles and for the pH regulation of extracellular compartments. Because of the potential role of the latter process in olfaction, we examined the expression of V-ATPase in mouse olfactory epithelial (OE) cells. We report that V-ATPase is present in this epithelium, where we detected subunits ATP6V1A (the 70-kDa "A" subunit) and ATP6V1E1 (the ubiquitous 31-kDa "E" subunit isoform) in epithelial cells, nerve fiber cells, and Bowman's glands by immunocytochemistry. We also located both isoforms of the 56-kDa B subunit, ATP6V1B1 ("B1," typically expressed in epithelia specialized in regulated transepithelial proton transport) and ATP6V1B2 ("B2") in the OE. B1 localizes to the microvilli of the apical plasma membrane of sustentacular cells and to the lateral membrane in a subset of olfactory sensory cells, which also express carbonic anhydrase type IV, whereas B2 expression is stronger in the subapical domain of sustentacular cells. V-ATPase expression in mouse OE was further confirmed by immunoblotting. These findings suggest that V-ATPase may be involved in proton secretion in the OE and, as such, may be important for the pH homeostasis of the neuroepithelial mucous layer and/or for signal transduction in CO2 detection. proton secretion; vacuolar H+-ATPase; immunofluorescence; pH homeostasis; olfaction 相似文献
8.
In the aquatic liverwort Riccia fluitans the regulation of theplasma membrane H+/amino acid symport has been investigated.Cytosolic pH (pHc), membrane potential (Em) and membrane conductancehave been measured and related to transport data, (i) The releaseof [14C]amino acids is strongly stimulated by cytosolic acidification,induced by the external addition of acetic acid, a decreasein external K+, and in the change from light to dark. On average,a decrease in pHc of 0.5 to 0.6 units corresponded with a 4-foldstimulation in amino acid efflux. (ii) External pH changes havefar less effect on substrate transport than the cytosolic pHshifts of the same order. (iii) The inwardly directed positivecurrent, induced by amino acids, is severely inhibited by cytosolicacidification. (iv) Fusicoccin (FC) stimulates amino acid uptakewithout considerable change in proton motive force. (v) Whenthe proton motive force is kept constant, the uptake of aminoacids into Riccia thalli is much lower than when the pump isdeactivated. It is suggested that both the proton pump activityand cytosolic pH are the dominant factors in the regulationof the H+/amino acid symport across the plasma membrane of Ricciafluitans, and it is concluded that the proton motive force isnot a reliable quantity to predict and interpret transport kinetics. Key words: Amino acid, cytosolic pH, pH-sensitive electrode, proton motive force, regulation, Riccia fluitans 相似文献
9.
Machen TE Leigh MJ Taylor C Kimura T Asano S Moore HP 《American journal of physiology. Cell physiology》2003,285(1):C205-C214
The influences of the gastric H+/K+ pump on organelle pH during trafficking to and from the plasma membrane were investigated using HEK-293 cells stably expressing the - and -subunits of human H+/K+-ATPase (H+/K+-, cells). The pH values of trans-Golgi network (pHTGN) and recycling endosomes (pHRE) were measured by transfecting H+/K+-, cells with the pH-sensitive GFP pHluorin fused to targeting sequences of either TGN38 or synaptobrevin, respectively. Immunofluorescence showed that H+/K+-ATPase was present in the plasma membrane, TGN, and RE. The pHTGN was similar in both H+/K+-, cells (pHTGN 6.36) and vector-transfected ("mock") cells (pHTGN 6.34); pHRE was also similar in H+/K+-, (pHRE 6.40) and mock cells (pHRE 6.37). SCH28080 (inhibits H+/K+-ATPase) caused TGN to alkalinize by 0.12 pH units; subsequent addition of bafilomycin (inhibits H+ v-ATPase) caused TGN to alkalinize from pH 6.4 up to a new steady-state pHTGN of 7.07.5, close to pHcytosol. Similar results were observed in RE. Thus H+/K+-ATPases that trafficked to the plasma membrane were active but had small effects to acidify the TGN and RE compared with H+ v-ATPase. Mathematical modeling predicted a large number of H+ v-ATPases (8,000) active in the TGN to balance a large, passive H+ leak (with PH 103 cm/s) via unidentified pathways out of the TGN. We propose that in the presence of this effective, though inefficient, buffer system in the Golgi and TGN, H+/K+-ATPases (estimated to be 4,000 active in the TGN) and other transporters have little effect on luminal pH as they traffic to the plasma membrane. pHluorin; H+ v-ATPase; trans-Golgi network; organelle pH; H+ permeability 相似文献
10.
The vacuolar H+ ATPases (V-ATPases) are ATP-driven proton pumps that transport protons across both intracellular and plasma membranes. Previous studies have implicated V-ATPases in the invasiveness of various cancer cell lines. In this study, we evaluated the role of V-ATPases in the invasiveness of two closely matched human breast cancer lines. MCF10a cells are a non-invasive, immortalized breast epithelial cell line, and MCF10CA1a cells are a highly invasive, H-Ras-transformed derivative of MCF10a cells selected for their metastatic potential. Using an in vitro Matrigel assay, MCF10CA1a cells showed a much higher invasion than the parental MCF10a cells. Moreover, this increased invasion was completely sensitive to the specific V-ATPase inhibitor concanamycin. MCF10CA1a cells expressed much higher levels of both a1 and a3 subunit isoforms relative to the parental line. Isoforms of subunit a are responsible for subcellular localization of V-ATPases, with a3 and a4 targeting V-ATPases to the plasma membrane of specialized cells. Knockdown of either a3 alone or a3 and a4 together using isoform-specific siRNAs inhibited invasion by MCF10CA1a cells. Importantly, overexpression of a3 but not the other a subunit isoforms greatly increased the invasiveness of the parental MCF10a cells. Similarly, overexpression of a3 significantly increased expression of V-ATPases at the plasma membrane. These studies suggest that breast tumor cells employ particular a subunit isoforms to target V-ATPases to the plasma membrane, where they function in tumor cell invasion. 相似文献
11.
Human Na(+)/H(+) exchanger isoform 6 is found in recycling endosomes of cells, not in mitochondria 总被引:5,自引:0,他引:5
Brett CL Wei Y Donowitz M Rao R 《American journal of physiology. Cell physiology》2002,282(5):C1031-C1041
Since thediscovery of the first intracellular Na+/H+exchanger in yeast, Nhx1, multiple homologs have been cloned andcharacterized in plants. Together, studies in these organismsdemonstrate that Nhx1 is located in the prevacuolar/vacuolarcompartment of cells where it sequesters Na+ into thevacuole, regulates intravesicular pH, and contributes to vacuolarbiogenesis. In contrast, the human homolog of Nhx1, Na+/H+ exchanger isoform 6 (NHE6), has beenreported to localize to mitochondria when transiently expressed as afusion with green fluorescent protein. This result warrantsreevaluation because it conflicts with predictions from phylogeneticanalyses. Here we demonstrate that when epitope-tagged NHE6 istransiently expressed in cultured mammalian cells, it does notcolocalize with mitochondrial markers. It also does not colocalize withmarkers of the lysosome, late endosome, trans-Golgi network,or Golgi cisternae. Rather, NHE6 is distributed in recyclingcompartments and transiently appears on the plasma membrane. Theseresults suggest that, like its homologs in yeast and plants, NHE6 is anendosomal Na+/H+ exchanger that may regulateintravesicular pH and volume and contribute to lysosomal biogenesis. 相似文献
12.
GYENES M.; ANDRIANOV V. K.; BULYCHEV A. A.; KURELLA G. A. 《Journal of experimental botany》1978,29(5):1185-1195
The effects of light on the pH in the vacuole and the electricpotential difference across the plasmalemma and the tonoplastof Nitellopsis obtusa were investigated by means of conventionaland H+-specific glass or antimony microelectrodes. Illuminationis found to bring about a decrease in the pH of the vacuolarsap by 0.10.5 units concomitant with a depolarizationof the cell. The light-induced changes of the potential differenceand the vacuolar pH depend in different ways on the pH of theexternal medium (pHo). At pHo 9.0 cells exhibit great light-inducedpotential changes (up to 100 mV), but only small pH changesof the vacuolar sap. At neutral or slightly acidic pHo valuesthe amplitude of the light-induced pH changes in the vacuoleincreases up to 0.30.5 pH units, but the amplitudes ofthe potential changes at the plasmalemma are relatively small.At pHo 9.0 a transient acidification of the medium is observedupon illumination whereas at lower pH values light-induced alkalinizationwas only seen. Transfer of the cells from pHo 9.0 to pHo 7.5results in a cell hyperpolarization by 6080 mV and adecrease of the vacuolar pH by 0.40.5 units under lightconditions but has no significant effect on the potential andthe vacuolar pH in the darkness. It is proposed that mechanismsof active H+ extrusion from the cytoplasm are located both inthe plasmalemma and the tonoplast. The observed acidificationin the vacuole appears to be determined by a light-induced increaseof the concentration of H+ in the cytoplasm. The H+ conductionof the plasmalemma seems to increase on illumination. The patternof the light-induced H+ fluxes across the tonoplast and theplasmalemma depends crucially on the extent of the light-inducedchanges in the H+ conductance and on the electrochemical gradientfor H+ at the plasmalemma. 相似文献
13.
Cowley EA Sellers MC Illsley NP 《American journal of physiology. Cell physiology》2005,288(4):C891-C898
Resting or basal intracellular pH (pHi) measured in cultured human syncytiotrophoblast cells was 7.26 ± 0.04 (without HCO3) or 7.24 ± 0.03 (with HCO3). Ion substitution and inhibitor experiments were performed to determine whether common H+-transporting species were operating to maintain basal pHi. Removal of extracellular Na+ or Cl or addition of amiloride or dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H2DIDS) had no effect. Acidification with the K+/H+ exchanger nigericin reduced pHi to 6.25 ± 0.15 (without HCO3) or 6.53 ± 0.10 (with HCO3). In the presence of extracellular Na+, recovery to basal pHi was prompt and occurred at similar rates in the absence and presence of HCO3. Ion substitution and inhibition experiments were also used to identify the species mediating the return to basal pHi after acidification. Recovery was inhibited by removal of Na+ or addition of amiloride, whereas removal of Cl and addition of H2DIDS were ineffective. Addition of the Na+/H+ exchanger monensin to cells that had returned to basal pHi elicited a further increase in pHi to 7.48 ± 0.07. Analysis of recovery data showed that there was a progressive decrease in pH per minute as pHi approached the basal level, despite the continued presence of a driving force for H+ extrusion. These data show that in cultured syncytial cells, in the absence of perturbation, basal pHi is preserved despite the absence of active, mediated pH maintenance. They also demonstrate that an Na+/H+ antiporter acts to defend the cells against acidification and that it is the sole transporter necessary for recovery from an intracellular acid load. sodium/hydrogen antiporter; pH regulation; fluorescence; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein 相似文献
14.
Da Silva N Shum WW El-Annan J Păunescu TG McKee M Smith PJ Brown D Breton S 《American journal of physiology. Cell physiology》2007,293(1):C199-C210
An acidic luminal pH in the epididymis contributes to maintaining sperm quiescent during their maturation and storage. The vacuolar H+ATPase (V-ATPase), located in narrow and clear cells, is a major contributor to luminal acidification. Mutations in one of the V-ATPase subunits, ATP6v1B1 (B1), cause distal renal tubular acidosis in humans but surprisingly, B1/ mice do not develop metabolic acidosis and are fertile. While B1 is located in the apical membrane of narrow and clear cells, the B2 subunit localizes to subapical vesicles in wild-type mouse, rat and human epididymis. However, a marked increase (84%) in the mean pixel intensity of B2 staining was observed in the apical pole of clear cells by conventional immunofluorescence, and relocalization into their apical membrane was detected by confocal microscopy in B1/ mice compared with B1+/+. Immunogold electron microscopy showed abundant B2 in the apical microvilli of clear cells in B1/ mice. B2 mRNA expression, determined by real time RT-PCR using laser-microdissected epithelial cells, was identical in both groups. Semiquantitative Western blots from whole epididymis and cauda epididymidis showed no variation of B2 expression. Finally, the luminal pH of the cauda epididymidis was the same in B1/ mice as in B1+/+ (pH 6.7). These data indicate that whereas overall expression of B2 is not affected in B1/ mice, significant redistribution of B2-containing complexes occurs from intracellular compartments into the apical membrane of clear cells in B1/ mice. This relocation compensates for the absence of functional B1 and maintains the luminal pH in an acidic range that is compatible with fertility. male reproductive tract; male fertility; luminal acidification; proton pump; vacuolar H+ATPase 相似文献
15.
Decrease in rat taste receptor cell intracellular pH is the proximate stimulus in sour taste transduction 总被引:10,自引:0,他引:10
Lyall V Alam RI Phan DQ Ereso GL Phan TH Malik SA Montrose MH Chu S Heck GL Feldman GM DeSimone JA 《American journal of physiology. Cell physiology》2001,281(3):C1005-C1013
Taste receptor cells (TRCs)respond to acid stimulation, initiating perception of sour taste.Paradoxically, the pH of weak acidic stimuli correlates poorly with theperception of their sourness. A fundamental issue surrounding sourtaste reception is the identity of the sour stimulus. We tested thehypothesis that acids induce sour taste perception by penetratingplasma membranes as H+ ions or as undissociated moleculesand decreasing the intracellular pH (pHi) of TRCs. Our datasuggest that taste nerve responses to weak acids (acetic acid andCO2) are independent of stimulus pH but strongly correlatewith the intracellular acidification of polarized TRCs. Taste nerveresponses to CO2 were voltage sensitive and were blockedwith MK-417, a specific blocker of carbonic anhydrase. Strong acids(HCl) decrease pHi in a subset of TRCs that contain apathway for H+ entry. Both the apical membrane and theparacellular shunt pathway restrict H+ entry such that alarge decrease in apical pH is translated into a relatively smallchange in TRC pHi within the physiological range. Weconclude that a decrease in TRC pHi is the proximate stimulus in rat sour taste transduction. 相似文献
16.
Increased vacuolar and plasma membrane H+-ATPase activities in Salicornia bigelovii Torr. in response to NaCl 总被引:3,自引:0,他引:3
The halophyte Salicornia bigelovii Torr. shows optimal growthand Na+ accumulation in 200 mM NaCl and reduced growth underlower salinity conditions. The ability to accumulate and compartmentalizeNa+ may result, in part, from stimulation of the H+ -ATPaseson the plasma membrane (PM-ATPase) and vacuolar membranes (V-ATPase).To determine if these two primary transport systems are involvedin salt tolerance, shoot fresh weight (FW) and activity of thePM- and V-ATPases from shoots in Salicornia grown in 5 and 200mM NaCI were compared. Higher PM-ATPase activity (60%) and FW(60%) were observed in plants grown in 200 mM NaCI and thesestimulations in growth and enzyme activity were specific forNa+ and not observed with Na+ added in vitro. V-ATPase activitywas significantly stimulated in vivo and in vitro (26% and 46%,respectively) after exposure to 200 mM NaCl, and stimulationwas Na+ -specific. Immunoblots indicated that the increasesin activity of the H+ -ATPases from plants grown in 200 mM NaCIwas not due to increases in protein expression. These studiessuggest that the H+-ATPases in Salicornia are important in salttolerance and provide a biochemical framework for understandingmechanisms of salt tolerance in plants. Key words: Salicornia, H+-ATPases, salt tolerance 相似文献
17.
Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast 总被引:1,自引:0,他引:1
Vacuolar proton-translocating ATPases (V-ATPases) play a central role in organelle acidification in all eukaryotic cells. To address the role of the yeast V-ATPase in vacuolar and cytosolic pH homeostasis, ratiometric pH-sensitive fluorophores specific for the vacuole or cytosol were introduced into wild-type cells and vma mutants, which lack V-ATPase subunits. Transiently glucose-deprived wild-type cells respond to glucose addition with vacuolar acidification and cytosolic alkalinization, and subsequent addition of K(+) ion increases the pH of both the vacuole and cytosol. In contrast, glucose addition results in an increase in vacuolar pH in both vma mutants and wild-type cells treated with the V-ATPase inhibitor concanamycin A. Cytosolic pH homeostasis is also significantly perturbed in the vma mutants. Even at extracellular pH 5, conditions optimal for their growth, cytosolic pH was much lower, and response to glucose was smaller in the mutants. In plasma membrane fractions from the vma mutants, activity of the plasma membrane proton pump, Pma1p, was 65-75% lower than in fractions from wild-type cells. Immunofluorescence microscopy confirmed decreased levels of plasma membrane Pma1p and increased Pma1p at the vacuole and other compartments in the mutants. Pma1p was not mislocalized in concanamycin-treated cells, but a significant reduction in cytosolic pH under all conditions was still observed. We propose that short-term, V-ATPase activity is essential for both vacuolar acidification in response to glucose metabolism and for efficient cytosolic pH homeostasis, and long-term, V-ATPases are important for stable localization of Pma1p at the plasma membrane. 相似文献
18.
Gildeíde Aparecida Costa Sávio Bastos de Souza Layz Ribeiro da Silva Teixeira Lev A. Okorokov Andrea Cristina Vetö Arnholdt Anna L. Okorokova-Façanha Arnoldo Rocha Façanha 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(3):684-691
Background
V-ATPase interactions with cholesterol enriched membrane microdomains have been related to metastasis in a variety of cancers, but the underlying mechanism remains at its beginnings. It has recently been reported that the inhibition of this H+ pump affects cholesterol mobilization to the plasma membrane.Methods
Inhibition of melanoma cell migration and invasiveness was assessed by wound healing and Transwell assays in murine cell lines (B16F10 and Melan-A). V-ATPase activity was measured in vitro by ATP hydrolysis and H+ transport in membrane vesicles, and intact cell H+ fluxes were measured by using a non-invasive Scanning Ion-selective Electrode Technique (SIET).Results
Cholesterol depletion by 5 mM MβCD was found to be inhibitory to the hydrolytic and H+ pumping activities of the V-ATPase of melanoma cell lines, as well as to the migration and invasiveness capacities of these cells. Nearly the same effects were obtained using concanamycin A, a specific inhibitor of V-ATPase, which also promoted a decrease of the H+ efflux in live cells at the same extent of MβCD.Conclusions
We found that cholesterol depletion significantly affects the V-ATPase activity and the initial metastatic processes following a profile similar to those observed in the presence of the V-ATPase specific inhibitor, concanamycin.General significance
The results shed new light on the functional role of the interactions between V-ATPases and cholesterol-enriched microdomains of cell membranes that contribute with malignant phenotypes in melanoma. 相似文献19.
Phototropins (phot1 and phot2) are blue light (BL) receptorsthat mediate responses including phototropism, chloroplast movementand stomatal opening, and increased cytosolic Ca2+. BL absorbedby phototropins activates plasma membrane H+-ATPase in guardcells, resulting in membrane hyperpolarization, and drives K+uptake and stomatal opening. However, it is unclear whetherthe phototropin-mediated Ca2+ increase activates the H+-ATPase.Here, we determined cytosolic Ca2+ concentrations in guard cellprotoplasts (GCPs) from Arabidopsis transformed with aequorin.Cytosolic Ca2+ increased rapidly in response to BL in GCPs fromboth the wild type and phot1 phot2 double mutants, but was mostlysuppressed by an inhibitor of photosynthetic electron flow (DCMU).With depleted external K+, we observed another slower Ca2+ increase,which was phototropin- dependent. Fusicoccin, a H+-ATPase activator,mimicked the effect of BL. The slow Ca2+ increase thus appearsto result from membrane hyperpolarization. The slow Ca2+ increasewas suppressed by external K+ and was restored by blockers ofinward-rectifying K+ channels, CsCl and tetraethylammonium,suggesting the preferential uptake of K+ over Ca2+. Such efficientK+ uptake in response to BL was not found in mesophyll cells.Both the fast and the slow Ca2+ increases were inhibited byCa2+ channel blockers (CoCl2 and LaCl3) and a chelating agent(EGTA). These results indicate that the phototropin-mediatedCa2+ increase was not observed prior to H+-ATPase activationin guard cells and that Ca2+ entered guard cells via Ca2+ channelsthrough photosynthesis and phototropin-mediated membrane hyperpolarization. 相似文献
20.
Wieczorek H Huss M Merzendorfer H Reineke S Vitavska O Zeiske W 《Journal of bioenergetics and biomembranes》2003,35(4):359-366
The plasma membrane H+ V-ATPase from the midgut of larval Manduca sexta, commonly called the tobacco hornworm, is the sole energizer of epithelial ion transport in this tissue, being responsible for the alkalinization of the gut lumen up to a pH of more than 11 and for any active ion movement across the epithelium. This minireview deals with those topics of our recent research on this enzyme that may contribute novel aspects to the biochemistry and physiology of V-ATPases. Our research approaches include intramolecular aspects such as subunit topology and the inhibition by macrolide antibiotics, intermolecular aspects such as the hormonal regulation of V-ATPase biosynthesis and the interaction of the V-ATPase with the actin cytoskeleton, and supramolecular aspects such as the interactions of V-ATPase, K+/H+ antiporter, and ion channels, which all function as an ensemble in the transepithelial movement of potassium ions. 相似文献