Vacuolar ATPase: Insights into the Structure & Function

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Md Murad Khan

Vacuolar ATPase: Insights into the Structure & Function

 

Abstract

Vacuolar ATPase (V-ATPase), a dedicated proton pump, is regulated by mechanism called reversible disassembly that results in autoinhibited free cytosolic V1-ATPase and membrane integrated V0-proton pump. Autoinhibited state of V1-ATPase is partly due to interaction of H subunit, associated with the a subunit of V0 in holo V-ATPase, with the bottom part of catalytic hexamer. To better understand the role of H subunit, we determined the affinity of interaction between Hchim with a4 by isothermal titration calorimetry (ITC) and found that they interact with each other by weak affinity. Here we also studied the mechanism of C subunit dissociation during disassembly of V-ATPase by biolayer interferometry (BLI). BLI experiment suggested that hydrolysis of MgATP by V1Hchim -ATPase is essential for C subunit to come off. Moreover, during the reconstitution of V0 into lipid nanodisc, we incorporated ergosterol to study its effect in maximizing the activity of reconstituted V-ATPase. From the ATPase assay study, we can say that ergosterol does not have any effect on the reconstituted V-ATPase activity under the experimental conditions.

Introduction

Vacuolar ATPase (V-ATPase), present in the endomembrane system of all eukaryotic organisms, is multisubunit protein complexes that acidify the lumen of different subcellular organelles including lysosome, endosome, Golgi apparatus, clathrin coated vesicles and extracellular space of certain tissues (1-3). Its function involves the maintenance of pH and ion homeostasis, autophagy, endocytosis, protein trafficking, mTOR and Notch signaling, bone remodeling, urine acidification, sperm maturation, neurotransmitter release and hormone secretion(1,2,4-8). V-ATPases become potential drug target due to their involvement in  multiple human disease including osteoporosis, renal tubular acidosis, microbial infection, sensorineural deafness, infertility, AIDS, cancer, and diabetes associated mainly with the hypo or hyper activity of the proton pump (8-16). V-ATPases are organized into cytosolic V1 domain (subunits A, B, C, D, E, F, G and H) which is responsible for ATP hydrolysis and membrane integrated V0 domain (subunits a, d, e, c, c′ and c′′) which is responsible for pumping proton from cytosolic side to the extracellular space or lumen of subcellular organelles (17,18).  The rotary mechanism of energy coupling by V-ATPase is highly related to other proton pumps like A-, F-, and A/V-type ATPases/ATP synthases (19-21). However, unlike other proton pumps, V-ATPase is regulated by a unique mechanism called reversible disassembly which results in free cytosolic V1 and C and membrane integrated V0 (22). Upon dissociation, both domain become silenced i.e., isolated V1 does not hydrolyze ATP largely due to the inhibitory interaction of subunit H with catalytic hexamer, possibly together with inhibitory magnesium ADP and membrane integrated V0 doesnot allow the transport of proton across the membrane (23,24).  Moreover, C subunit also comes off into the cytoplasm following the disassembly of V1V0 while H subunit remain associated with V1(25). But how C subunit comes off is poorly understood. In this study, we analyzed the role of different nucleotides in its dissociation from V-ATPase disassembly.

While the C-terminal domain of subunit H (HCT) is essential for both proton pumping by V-ATPAse and inhibition of MgATPase activity in isolated V1 that involves around 1500 rotation of HCT from its holo V1V0 to the bottom of V1 catalytic hexamer, the N-terminal domain of subunit H (HNT) is required only for the MgATPase activity in the holo V-ATPase (23,26,27). This lead us to find out the interaction of HCT with subunit a of V0. But previous study showed that HCT does not co-purify with yeast V1 (27), so used the chimeric H construct (Hchim; in which C-terminal of yeast H subunit replaced by the C-terminal of human H subunit) to study its interaction with its binding partner a4 (isoform of human a subunit) by isothermal titration calorimetry (ITC). Here we also studied the effect of ergosterol in maximizing the activity of reconstituted V-ATPase based on the report that ergosterol biosynthesis mutant failed to acidify the yeast vacuole (28).

Results

Purification of V0 and Reconstitution into Lipid Nanodiscs (ND)

V0 from solubilized yeast vacuolar membranes were purified by calmodulin affinity column that binds to the CBP fused to the C terminus of the vacuole-specific isoform of subunit a (Vph1p). Escherichia coli polar lipids and the recombinant membrane scaffold protein (MSP) were added and mixed well with purified detergent-solubilized V0 for reconstitution. Following the removal of detergent by polystyrene beads, reconstituted proteins were passed through the calmodulin affinity column and Superdex S-200 size-exclusion column chromatography to remove the empty nanodisc. Peaked fractions from the column were analyzed by SDS-PAGE and pooled together to get a final concentration of 0.20 mg/ml.

FIGURE 1. Purification of V0 and reconstitution into lipid nandisc. Upper left panel shows the SDS-PAGE analysis of MBP cleaved Hchim obtained from the CM-cellulose column; Upper middle panel is the elution profile of Hchim in size exclusion chromatography; Upper right panel shows SDS-PAGE of purified Hchim fractions obtained in size-exclusion chromatography. Lower left panel shows the SDS-PAGE analysis of subunit C obtained from the amy.

Expression and Purification of Hchim and C

Both Hchim and C subunits, fused with mannose binding protein (MBP) in their N-terminal, were expressed in Escherichia coli Rosetta2 cell line. After capturing them in amylose affinity chromatography, MBP fusion was cleaved by treating the proteins with precision protease. After MBP cleavage, proteins were purified by ion-exchange chromatography (Carboxymethyl-cellulose column for Hchim and DEAE column for C) and size-exclusion chromatography (Superdex S-200 column). Both proteins were eluted near their expected molecular weight in the size-exclusion chromatography. The final concentrations of the purified Hchim and C subunits were 1.85 and 3.30 mg/ml, respectively.

FIGURE 2. Purification and elution profile of Hchim and C. Upper left panel shows the SDS-PAGE analysis of MBP cleaved Hchim obtained from the CM-cellulose column; Upper middle panel is the elution profile of Hchim in size exclusion chromatography; Upper right panel shows SDS-PAGE of purified Hchim fractions obtained in size-exclusion chromatography. Lower left panel shows the SDS-PAGE analysis of subunit C obtained from the amylose affinity and DEAE columns; Lower middle panel is the elution profile of C in size exclusion chromatography; Lower right panel shows SDS-PAGE of purified C fractions obtained in size-exclusion chromatography.

V1∆H Purification and Reconstitution of V1 Hchim

A yeast strain, deleted for genes encoding endogenous subunits H (VMA13) and G (VMA10) but transformed with a pRS315 plasmid containing N‐terminally FLAG tagged G subunit, was used to express the V1∆H. After purifying with FLAG column, V1∆H was mixed with Hchim. V1 Hchim was purified by size-exclusion chromatography (Superdex S-200 column) and concentrated using Vivaspin Centrifuge Concentrator (MWCO: 50000) to achieve a final concentration of 2.30 mg/ml.

1   2   3   4   5   6   7  8  9  10  11  12  13  14

FIGURE 3. Characterization of reconstituted V1Hchim. Left panel shows the SDS-PAGE of different fractions obtained from the FLAG column; Middle panel is the elution profile of V1Hchim (2nd peak) in size exclusion chromatography; Right panel shows SDS-PAGE of different fractions obtained in size-exclusion chromatography (lane 1-2 are the fractions from 1st peak, lane 3-13 are the fraction from the 2nd peak and lane 14 is the top fraction from 3rd peak).

Reconstitution of V1Hchim V0C and Observation under Electron Microscope

V1 reconstituted with equimolar concentration of chimeric H subunit (Hchim) and V0ND and three-fold excess subunit C.  In ATP regenerating system, reconstituted V1Hchim V0C showed the MgATPase activity of  ̴10 U/mg, more than 90% of which was inhibited by concanamycin A. Reconstituted V-ATPase appeared as a typical dumbbell shell structure under the electron microscope.

1     2      3     4     5     6     7     8      9

FIGURE 4. Characterization of reconstituted V-ATPase. Upper left panel shows the elution profile of reconstituted V-ATPase in size-exclusion chromatography; Upper right panel shows the ATPase activity measurement of the reconstituted V-ATPase in ATP regenerating system; Lower left panel shows the silver stained SDS-PAGE of different fractions obtained in size-exclusion chromatography (lane 1-8 are the fractions from left peak and lane 9 is the top fraction from the middle peak); Lower right panel represents the negative stained electron micrograph showing typical dumbbell shaped reconstituted V-ATPase.

Effect of Ergosterol in V-ATPase Activity

Comparative ATPase activity between V1Hchim V0C reconstituted in E. coli lipid in absence and presence of ergosterol showed that under the experimental conditions, ergosterol didn’t have any significant effect in maximizing the catalytic activity of the reconstituted V-ATPase.

FIGURE 5. Effect of ergosterol in reconstituted V-ATPase activity is non-significant. The experiment has been carried out at different V0ND concentration by taking different amount (volume) from the stock 0.2 mg/ml while keeping the concentration of other components at fixed value.

Role of MgATP Hydrolysis in V1Hchim-C Dissociation

Dissociation kinetic between V1Hchim and MBP tagged C subunit was monitored using the biolayer interferometry (BLI) technique. MBP tagged C was immobilized to the anti-Fc mouse Ig biosensor using anti-MBP antibody and dissociation of C from the V1Hchim was monitored in presence of Mg-ATP, Mg-AMP-PNP, EDTA-ATP, Mg-ADP-P and Mn-ATP. In presence of Mg-ATP and Mn-ATP, the rate of dissociation was very fast.

FIGURE 6. Dissociation of C subunit requires the hydrolysis of MgATP (or MnATP) by reconstituted V1HchimATPase. The figure represents the raw (without curve fitting and subtracting non-specific association) BLI data.

Interaction between Subunit Hchim and a4

To determine the affinity and enthalpy of interaction, we titrated the a4 with Hchim using isothermal titration calorimetry (ITC). The ITC data showed no conclusive exothermic or endothermic type reaction that might be due to the weak interaction between Hchim and a4.

       

FIGURE 7. Isothermal titration calorimetry of interaction between Hchim and a4. Left panel shows the titration of a4 into buffer and right panel shows titration of a4 into Hchim.

Experimental Procedures

Purification of V0 and Reconstitution into Lipid Nanodiscs (ND)

Previously stored (at -800C) vacuolar membranes thawed, supplemented with protease inhibitor cocktail (2 µg/ml of each leupeptin, pepstatin, aprotinin and chymostatin and 1 M PMSF) and mixed with lysis buffer (25 mM Tris pH 7.4, 500 mM Sorbitol, 2 mM CDTA) to reach a final membrane concentration of 10 mg/ml. For each milligram of membrane, 0.6 mg of undecyl-β-d-maltoside (UnDM) was added and mixed gently using rotator for 45 minutes at 40C. 4 mM CaCl2 was added into the mixture and incubated for another 15 minutes under the same conditions. Detergent solubilized membranes were centrifuged at 42000 rmp, 40C for 60 minutes. The supernatant was supplemented with protease inhibitor cocktail (without PMSF) again and passed through the calmodulin column. The column was washed with 10 column volume calmodulin binding buffers (10 mM Tris pH 8.0, 10 mM beta-mercaptoethanol, 2 mM CaCl2 and 0.06% UnDM) sequentially in presence and absence of NaCl (150 mM) and eluted with elution buffer (10 mM Tris pH 8.0, 10 mM beta-mercaptoethanol, 10 mM CDTA and 0.06% UnDM). After SDS-PAGE analysis, peak fraction was concentrated using VivaSpin Concentrator (MWCO 10000) and V0 concentration was measured by BCA (BSA?) method. V0 was then reconstituted in lipid nanodisc by mixing 1.5 mg of V0 with 3.0 mg of membrane scaffold protein (MSP), 4.5 mg Escherichia coli lipid and 1.5% UnDM. The mixture was supplemented with protease inhibitor cocktail (without MPSF) and rotated for 60 minutes at room temperature. Detergent was removed afterward by addition of 1.5 g of biobeads and rotation of the sample for 60 minutes at room temperature. After short spin, supernatant was passed through the size-exclusion (Superdex S-200) column to remove the empty nanodisc.

Expression and Purification of Hchim and C

For the expression of Hchim and C subunits, fused with mannose binding protein (MBP) in their N-terminal, Escherichia coli Rosetta2 cells were grown to midlog phase in rich broth (LB + 0.2% glucose) supplemented with 50 µg/ml of carbenicillin and 34 µg/ml of ampicillin and induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 300C for 5.5 hours. Cells were harvested by centrifuging the culture at 3000 rmp for 25 minutes, resuspended in amylose column buffer (200 mM Tris-HCl, 2 M NaCl, 10 mM EDTA, pH 7.4) and stored at -200C until use. For purification, cells were treated with 20 µg/ml of DNase, 1 mg/ml of lysozyme and 100 mM of PMSF before sonication. After centrifugation at 13000 x g for 40 minutes, cell lysates were passed thorough the amylose column at a rate of 1 ml/min. Column was washed with 10 column volume of amylose column buffer and proteins were eluted with 25 ml of 10 mM maltose in amylose column buffer. MBP tag was cleaved by treating the proteins with Precission protease for 2 hours in presence 25 mM DTT. For Hchim, the pH was adjusted to its isoelectric point by dialysis overnight in 25 mM NaP, 5 mM beta-mercaptoethanol, 0.5 mM EDTA at pH 7.0 and protease cleaved product was passed over the carboxymethyl (cation exchange) column to get rid of the MBP. Eluted protein was then concentrated by VivaSpin Centrifuge Concentrator (MWCO: 50000) and passed through size-exclusion (Superdex-200) column. For C subunit, the pH was adjusted to its isoelectric point by dialysis overnight and protease cleaved product was passed over the DEAE (anion exchange) column to get rid of the MBP. The isolated protein was then dialyzed to readjust the pH away from the pI and concentrated by VivaSpin Centrifuge Concentrator (MWCO: 50000) and passed through size-exclusion (Superdex-200) column for further purification.

V1∆H Purification and Reconstitution of V1 Hchim

Yeast cells, deleted for genes encoding H and G subunits and transformed with a pRS315 plasmid encoding subunit G with an N-terminal FLAG tag, were grown to an OD of  ̴3.0 in synthetic drop out medium (SD minus Leu) and harvested by centrifuging the culture at 4000 x g for 20 minutes and resuspended in TBSE buffer (200 mM Tris, 1.5 M NaCl, 5mM EDTA, pH 7.2) and stored at -800C until use. Cells were thawed and supplemented with 5 mM beta-mercaptoethanol, 1 mM PMSF, and 2 µg/ml each of pepstatin and leupeptin before lysis by 12-15 passes through a microfluidizer with intermittent cooling on ice. After lysis, cell lysate was initially centrifuged at 4000 x g for 30 minutes and supernatant was centrifuged again at 13000 x g for 40 minutes. For purification, supernatant was passed through the FLAG column and protein was eluted with FLAG peptide (0.1 mg/ml of TBSE buffer). Purified V1∆H was then mixed with Hchim and passed thorough the size-exclusion (Superdex-200) column and concentrated using VivaSpin Centrifuge Concentrator (MWCO: 100000).

Reconstitution of V1Hchim V0C

For reconstitution, equimolar concentration of V1Hchim and V0ND and three molar excess C were mixed together and incubated for at least 60 minutes at room temperature. The activity of the reconstituted V-ATPase was measured in ATP regenerating system.

Effect of Ergosterol in V-ATPase Activity

To study the effect of ergosterol in maximizing the reconstituted V-ATPase activity, 20% ergosterol was added during V0 reconstitution in lipid nanodisc. Then we set up V-ATPase reconstitution using different concentration of V0ND (in presence and absence of ergosterol) with fixed amount of f V1Hchim and C.

Component

Volume (µl)

V1Hchim (2.3 mg/ml)

4

4

4

4

4

V0ND (0.2 mg/ml)

32

35

38

41

44

C (3.3 mg/ml)

1

1

1

1

1

Buffer

13

10

7

4

1

Total

50

50

50

50

50

V1Hchim– C Dissociation Kinetic Study

Biolayer interferometry (BLI) was used to measure the dissociation kinetic of interaction between V1Hchim and MBP tagged C (MBP-C) in a 96 well microtiter plate. Initially anti-Fc mouse antibody containing biosensor tips were pre-equilibrated in BLI buffer (10 mg/ml BSA in 200 mM Tris, 1.5 M NaCl, 5mM EDTA, pH 7.2) and then immobilized with anti-MBP antibody (1 µg/ml). Biosensor tips immobilized with anti-MBP antibodies then dipped into wells containing 5 µg/ml MBP-C followed by dipping into wells containing 40 mM V1Hchim for association with MBP-C. Then dissociation of V1Hchim and MBP-C were monitored in presence of different types of nucleotides (Mg-ATP, Mg-AMP-PMP, Mg-EDTA, Mg-ADP-P, Mn-ATP). All steps were done at 220C and 1000 rpm in microtiter plate with 220 µl of sample in each well. Biosensor tips were washed with BLI buffer after every step in the experiment. One reference (only BLI buffer in dissociation phase instead of nucleotides) and two controls (one with no MBP-C and another with no V1Hchim) were included in the experiment.

Interaction between Subunit Hchim and a4

The interaction between Hchim and a4 was quantified using isothermal titration calorimeter (ITC) in a buffer solution (0.5 mM EDTA, 20 mM NaCl, 1 mM Tris, pH 7.0) at 100C. 0.115 mM a4 was injected periodically into the cell containing 0.0184 mM Hchim. To negate the effect of heat due to dilution, we subtracted the blank (a4 titrated against buffer) from the titrated experiment (a4 and Hchim). ITC data were analyzed using VP-ITC programs in OriginLab.

ATPase Assay

ATPase assay tube containing 5 mM ATP, 30 units/ml each of lactate dehydrogenase and pyruvate kinase, 0.5 mM NADH, 2 mM phosphoenolpyruvate, 50 mM HEPES, pH 7.5 was warmed up for 10 minutes at 370C. Then 1 mM MgCl2 and 10 µg of reconstituted V-ATPase was added sequentially into the assay tube and decrease in absorbance was monitored at 340 nm wavelength. 

Acknowledgment

I would like to express my sincere gratitude to my PI Professor Dr. Stephan Wilkens for allowing me to work in his lab and guiding me to successfully complete my rotation project. I am also thankful to Dr. Rebecca Oot for her continuous support and teaching me the different techniques that I have used during my rotation. I would also like to extend my thanks to Zane Suttmore and Nicholas J Stam. I also acknowledge the SUNY ESF and funding agency NIH.

References:

  1. Forgac, M. (2007) Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nature reviews Molecular cell biology 8, 917
  2. Kane, P. M. (2006) The where, when, and how of organelle acidification by the yeast vacuolar H+-ATPase. Microbiology and Molecular Biology Reviews 70, 177-191
  3. Marshansky, V., and Futai, M. (2008) The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function. Current opinion in cell biology 20, 415-426
  4. Zoncu, R., Bar-Peled, L., Efeyan, A., Wang, S., Sancak, Y., and Sabatini, D. M. (2011) mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H+-ATPase. Science 334, 678-683
  5. Yan, Y., Denef, N., and Schüpbach, T. (2009) The vacuolar proton pump, V-ATPase, is required for notch signaling and endosomal trafficking in Drosophila. Developmental cell 17, 387-402
  6. Xu, Y., Parmar, A., Roux, E., Balbis, A., Dumas, V., Chevalier, S., and Posner, B. I. (2012) EGF-induced Vacuolar (H+)-ATPase Assembly: A Role in Signaling via mTORC1 Activation. Journal of Biological Chemistry, jbc. M112. 352229
  7. Vavassori, S., and Mayer, A. (2014) A new life for an old pump: V-ATPase and neurotransmitter release. J Cell Biol 205, 7-9
  8. Sun-Wada, G.-H., Toyomura, T., Murata, Y., Yamamoto, A., Futai, M., and Wada, Y. (2006) The a3 isoform of V-ATPase regulates insulin secretion from pancreatic β-cells. Journal of cell science 119, 4531-4540
  9. Kartner, N., and Manolson, M. F. (2014) Novel techniques in the development of osteoporosis drug therapy: the osteoclast ruffled-border vacuolar H+-ATPase as an emerging target. Expert opinion on drug discovery 9, 505-522
  10. Smith, A. N., Skaug, J., Choate, K. A., Nayir, A., Bakkaloglu, A., Ozen, S., Hulton, S. A., Sanjad, S. A., Al-Sabban, E. A., and Lifton, R. P. (2000) Mutations in ATP6N1B, encoding a new kidney vacuolar proton pump 116-kD subunit, cause recessive distal renal tubular acidosis with preserved hearing. Nature genetics 26, 71
  11. Karet, F. E., Finberg, K. E., Nelson, R. D., Nayir, A., Mocan, H., Sanjad, S. A., Rodriguez-Soriano, J., Santos, F., Cremers, C. W., and Di Pietro, A. (1999) Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nature genetics 21, 84
  12. S Thudium, C., K Jensen, V., A Karsdal, M., and Henriksen, K. (2012) Disruption of the V-ATPase functionality as a way to uncouple bone formation and resorption-a novel target for treatment of osteoporosis. Current Protein and Peptide Science 13, 141-151
  13. Wong, D., Bach, H., Sun, J., Hmama, Z., and Av-Gay, Y. (2011) Mycobacterium tuberculosis protein tyrosine phosphatase (PtpA) excludes host vacuolar-H+–ATPase to inhibit phagosome acidification. Proceedings of the National Academy of Sciences 108, 19371-19376
  14. Brown, D., Smith, P., and Breton, S. (1997) Role of V-ATPase-rich cells in acidification of the male reproductive tract. Journal of Experimental Biology 200, 257-262
  15. Sennoune, S. R., Bakunts, K., Martínez, G. M., Chua-Tuan, J. L., Kebir, Y., Attaya, M. N., and Martínez-Zaguilán, R. (2004) Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity. American Journal of Physiology-Cell Physiology 286, C1443-C1452
  16. Geyer, M., Yu, H., Mandic, R., Linnemann, T., Zheng, Y. H., Fackler, O. T., and Peterlin, B. M. (2002) Subunit H of the V-ATPase binds to the medium chain of AP-2 and connects Nef to the endocytic machinery. Journal of Biological Chemistry
  17. Kitagawa, N., Mazon, H., Heck, A. J., and Wilkens, S. (2008) Stoichiometry of the peripheral stalk subunits E and G of yeast V1-ATPase determined by mass spectrometry. Journal of Biological Chemistry 283, 3329-3337
  18. Zhao, J., Benlekbir, S., and Rubinstein, J. L. (2015) Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Nature 521, 241
  19. Wilkens, S. (2005) Rotary molecular motors. Advances in protein chemistry 71, 345-382
  20. Muench, S. P., Trinick, J., and Harrison, M. A. (2011) Structural divergence of the rotary ATPases. Quarterly reviews of biophysics 44, 311-356
  21. Futai, M., Nakanishi-Matsui, M., Okamoto, H., Sekiya, M., and Nakamoto, R. K. (2012) Rotational catalysis in proton pumping ATPases: from E. coli F-ATPase to mammalian V-ATPase. Biochimica et Biophysica Acta (BBA)-Bioenergetics 1817, 1711-1721
  22. Kane, P. M. (1995) Disassembly and reassembly of the yeast vacuolar H+-ATPase in vivo. Journal of Biological Chemistry 270, 17025-17032
  23. Oot, R. A., Kane, P. M., Berry, E. A., and Wilkens, S. (2016) Crystal structure of yeast V1‐ATPase in the autoinhibited state. The EMBO journal 35, 1694-1706
  24. Zhang, J., Myers, M., and Forgac, M. (1992) Characterization of the V0 domain of the coated vesicle (H+)-ATPase. Journal of Biological Chemistry 267, 9773-9778
  25. Sharma, S., Oot, R. A., and Wilkens, S. (2018) MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H’s role in V-ATPase regulation by reversible disassembly. Journal of Biological Chemistry, jbc. RA118. 002951
  26. Liu, M., Tarsio, M., Charsky, C. M., and Kane, P. M. (2005) Structural and functional separation of the N-and C-terminal domains of the yeast V-ATPase subunit H. Journal of Biological Chemistry
  27. Diab, H., Ohira, M., Liu, M., Cobb, E., and Kane, P. M. (2009) Subunit interactions and requirements for inhibition of the yeast V1-ATPase. Journal of Biological Chemistry
  28. Zhang, Y.-Q., Gamarra, S., Garcia-Effron, G., Park, S., Perlin, D. S., and Rao, R. (2010) Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs. PLoS pathogens 6, e1000939

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