Inhibitors of V-ATPase Proton Transport Reveal Uncoupling Functions of Tether Linking Cytosolic and Membrane Domains of V0 Subunit a (Vph1p)

Chan, Chun-Yuan; Prudom, Catherine E.; Raines, Summer M.; Charkhzarrin, Sahba; Melman, Sandra D.; Haro, Leyma P. De; Allen, Chris; Lee, Samuel A.; Sklar, Larry A.; Parra, Karlett J.

doi:10.1074/jbc.m111.321133

Cited by 49 publications

(55 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In order to quantitatively confirm this phenotype, cells were stained with BCECF, a pH-sensitive fluorophore that accumulates in the vacuole. By comparing the fluorescence profile of BCECF-treated cells to that of a calibration curve, vacuolar pH can be quantitatively determined (10,25,45). We previously studied vacuolar pH in the THE1-CIp10 strain both with and without DOX; the vacuolar pH was approximately 6.25 under both conditions (4).…”

Section: Resultsmentioning

confidence: 99%

“…4B). Proton transport was measured fluorometrically using ACMA (25,26). Repression of VMA2 with DOX in tetR-VMA2 completely abrogated proton transport (101.2%) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Proton transport of 30 g purified vacuolar vesicles was measured via quenching of 1 M 9-amino-6-chloro-2-methoxyacridine (ACMA) upon the addition of 0.5 mM ATP-1 mM MgSO 4 (MgATP) as described previously (25,26). Fluorescence at excitation and emission wavelengths of 410 nm and 490 nm, respectively, was monitored for 60 s prior to MgATP addition and for 40 s after.…”

Section: Methodsmentioning

confidence: 99%

“…In order to quantify ATP hydrolysis, 15 g purified vacuolar vesicles was used in an enzymatic assay in which the rate of ATP hydrolysis is coupled to the oxidation of NADH, measured as a loss of A 340 over time (24) in the presence and absence of the V-ATPase inhibitor concanamycin A (12,25). Proton transport of 30 g purified vacuolar vesicles was measured via quenching of 1 M 9-amino-6-chloro-2-methoxyacridine (ACMA) upon the addition of 0.5 mM ATP-1 mM MgSO 4 (MgATP) as described previously (25,26).…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

et al. 2014

Self Cite

View full text Add to dashboard Cite

Candida albicans vacuoles are central to many critical biological processes, including filamentation and in vivo virulence. The V-ATPase proton pump is a multisubunit complex responsible for organellar acidification and is essential for vacuolar biogenesis and function. To study the function of the V 1 B subunit of C. albicans V-ATPase, we constructed a tetracycline-regulatable VMA2 mutant, tetR-VMA2. Inhibition of VMA2 expression resulted in the inability to grow at alkaline pH and altered resistance to calcium, cold temperature, antifungal drugs, and growth on nonfermentable carbon sources. Furthermore, V-ATPase was unable to fully assemble at the vacuolar membrane and was impaired in proton transport and ATPase-specific activity. VMA2 repression led to vacuolar alkalinization in addition to abnormal vacuolar morphology and biogenesis. Key virulence-related traits, including filamentation and secretion of degradative enzymes, were markedly inhibited. These results are consistent with previous studies of C. albicans V-ATPase; however, differential contributions of the V-ATPase V o and V 1 subunits to filamentation and secretion are observed. We also make the novel observation that inhibition of C. albicans V-ATPase results in increased susceptibility to osmotic stress. Notably, V-ATPase inhibition under conditions of nitrogen starvation results in defects in autophagy. Lastly, we show the first evidence that V-ATPase contributes to virulence in an acidic in vivo system by demonstrating that the tetR-VMA2 mutant is avirulent in a Caenorhabditis elegans infection model. This study illustrates the fundamental requirement of V-ATPase for numerous key virulence-related traits in C. albicans and demonstrates that the contribution of VATPase to virulence is independent of host pH.T he fungal pathogen Candida albicans is the fourth most common cause of hospital-acquired bloodstream infections and is a major cause of catheter-associated infections, sepsis, and devicerelated infections. It is also an extremely common cause of urinary and mucosal infections. Despite its clinical significance, the diagnosis and treatment of disseminated candidiasis remain limited by an incomplete understanding of its molecular pathogenesis. The fungal vacuole, a degradative organelle roughly equivalent to the mammalian lysosome, plays an important role in numerous biological processes in C. albicans, including key aspects of pathogenesis. Previous studies have established that C. albicans mutants compromised in vacuolar function are defective in yeast-to-hypha transitioning, a major virulence-related trait, and exhibit reduced virulence in vivo (1, 2).An essential component of vacuolar biogenesis and function is the vacuolar H ϩ -ATPase (V-ATPase) proton pump, which is a multisubunit complex responsible for the acidification of internal organelles. V-ATPase is located at the vacuolar membrane and throughout the endomembrane system, including prevacuolar compartments and the Golgi complex (1). The acidification of these organelles has sever...

show abstract

Section: Resultsmentioning

confidence: 99%

“…4B). Proton transport was measured fluorometrically using ACMA (25,26). Repression of VMA2 with DOX in tetR-VMA2 completely abrogated proton transport (101.2%) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…Proton transport of purified vacuolar vesicles (30 g) was measured via quenching of 1 M 9-amino-6-chloro-2-methoxyacridine (ACMA) upon the addition of 0.5 mM ATP-1 mM MgSO 4 (MgATP) as described previously (42,43). Fluorescence at 410-nm excitation/490-nm emission was monitored for 1 min prior to MgATP addition and for an CMAC was added to a concentration of 100 M, and cells were incubated at room temperature for 15 min and examined via microscopy using Texas Red (FM4-64) and 4=,6-diamidino-2-phenylindole (DAPI) (CMAC) filters.…”

Section: Identification Of Vma3pmentioning

confidence: 99%

Candida albicans VMA3 Is Necessary for V-ATPase Assembly and Function and Contributes to Secretion and Filamentation

et al. 2013

Self Cite

View full text Add to dashboard Cite

The vacuolar membrane ATPase (V-ATPase) is a protein complex that utilizes ATP hydrolysis to drive protons from the cytosol into the vacuolar lumen, acidifying the vacuole and modulating several key cellular response systems in Saccharomyces cerevisiae. To study the contribution of V-ATPase to the biology and virulence attributes of the opportunistic fungal pathogen Candida albicans, we created a conditional mutant in which VMA3 was placed under the control of a tetracycline-regulated promoter (tetR-VMA3 strain). Repression of VMA3 in the tetR-VMA3 strain prevents V-ATPase assembly at the vacuolar membrane and reduces concanamycin A-sensitive ATPase-specific activity and proton transport by more than 90%. Loss of C. albicans VATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. Candida albicans is a major opportunistic human fungal pathogen and is responsible for 6.8% of hospital-acquired infections in the United States (1). Despite the availability of several classes of antifungal drugs, attributable mortality, cost of care, and length of stay due to invasive candidiasis remain unacceptably high (2, 3). In addition, resistance to currently available antifungal drugs is emerging (see reference 4 for a review). Therefore, development of new antifungal drug targets remains a critical need. A diverse set of factors contributing to C. albicans virulence have been identified, including the secretion of aspartyl proteases and lipases, filamentation, and biofilm formation (5-8). Understanding the biology and regulation of these processes and pathways may illuminate new candidates for antifungal therapy.The vacuole is a dynamic acidic organelle found in yeast and plants that is analogous to the mammalian lysosome. It functions in an array of cellular homeostasis processes and thereby plays an important role in stress response, adaptation to novel environments, and cell differentiation (9-13). Furthermore, in C. albicans, intact vacuolar function is important for filamentation and virulence (12)(13)(14)(15). Vacuolar function depends on the maintenance of acidic pH by the vacuolar H-ATPase (V-ATPase), an enzyme complex that functions in organelle acidification across eukaryotes (16,17). The V-ATPase utilizes hydrolysis of ATP to transport protons from the cytosol into a variety of organelles. V-ATPase-mediated acidification and membrane energization are necessary for important vacuolar functions, including calcium and metal homeostasis (18), cargo sorting and membrane trafficking in endocytic and secretory pathways (19), and drug resistance (20). In Saccharomyces cerevisiae, the V-ATPase is expressed at the vacuolar membrane and the membrane of prevacuolar compartments and the Golgi compartment.The V-ATPase complex consists of the V 1 and V o subcomplexes (16). The V 1 subcomplex is composed of peripherally associated subunits that form the sites of ATP binding and hydrolysis on the cytosolic side of the membrane. The V o su...

show abstract

Proton electrochemical gradient: Driving and regulating neurotransmitter uptake

2017

View full text Add to dashboard Cite

Accumulation of neurotransmitters in the lumen of synaptic vesicles (SVs) relies on the activity of the vacuolar-type H -ATPase. This pump drives protons into the lumen, generating a proton electrochemical gradient (Δμ ) across the membrane. Recent work has demonstrated that the balance between the chemical (ΔpH) and electrical (ΔΨ) components of Δμ is regulated differently by some distinct vesicle types. As different neurotransmitter transporters use ΔpH and ΔΨ with different relative efficiencies, regulation of this gradient balance has the potential to influence neurotransmitter uptake. Nevertheless, the underlying mechanisms responsible for this regulation remain poorly understood. In this review, we provide an overview of current neurotransmitter uptake models, with a particular emphasis on the distinct roles of the electrical and chemical gradients and current hypotheses for regulatory mechanisms.

show abstract

Inhibitors of V-ATPase Proton Transport Reveal Uncoupling Functions of Tether Linking Cytosolic and Membrane Domains of V0 Subunit a (Vph1p)

Cited by 49 publications

References 57 publications

The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

Candida albicans VMA3 Is Necessary for V-ATPase Assembly and Function and Contributes to Secretion and Filamentation

Proton electrochemical gradient: Driving and regulating neurotransmitter uptake

Contact Info

Product

Resources

About

Inhibitors of V-ATPase Proton Transport Reveal Uncoupling Functions of Tether Linking Cytosolic and Membrane Domains of V0 Subunit a (Vph1p)

Cited by 49 publications

References 57 publications

The Contribution of Candida albicans Vacuolar ATPase Subunit V 1 B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

The Contribution of Candida albicans Vacuolar ATPase Subunit V 1 B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

Candida albicans VMA3 Is Necessary for V-ATPase Assembly and Function and Contributes to Secretion and Filamentation

Proton electrochemical gradient: Driving and regulating neurotransmitter uptake

Contact Info

Product

Resources

About

The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH

The Contribution of Candida albicans Vacuolar ATPase Subunit V ₁ B, Encoded by VMA2 , to Stress Response, Autophagy, and Virulence Is Independent of Environmental pH