This review discusses the history of discovery and study of the operation of the two rotary ion-translocating ATPase nano-motors: (i) F-ATPase/synthase (holocomplex F1FO) of mitochondria/bacteria and (ii) eukaryotic V-ATPase (holocomplex V1VO). Vacuolar adenosine triphosphatase (V-ATPase) is a transmembrane multisubunit complex found in all eukaryotes from yeast to humans. It is structurally and functionally similar to the F-ATPase/synthase of mitochondria/bacteria and the A-ATPase/synthase of archaebacteria, which indicates a common evolutionary origin of the rotary ion-translocating nano-motors built into cell membranes and invented by Nature billions of years ago. Previously we have published several reviews on this topic with appropriate citations of our original research. This review is focused on the historical analysis of the discovery and study of transmembrane rotary ion-translocating ATPase nano-motors functioning in bacteria, eukaryotic cells and mitochondria of animals.

We have previously identified the interaction between mammalian V-ATPase a2-subunit isoform and cytohesin-2 (CTH2) and studied molecular details of binding between these proteins. In particular, we found that six peptides derived from the N-terminal cytosolic domain of a2 subunit (a2N(1-402)) are involved in interaction with CTH2 (Merkulova, Bakulina, Thaker, Gruber, & Marshansky, 2010). However, the actual 3D binding interface was not determined in that study due to the lack of high-resolution structural information about a-subunits of V-ATPase. Here, using a combination of homology modeling and NMR analysis, we generated the structural model of complete a2N(1-402) and uncovered the CTH2-binding interface. First, using the crystal-structure of the bacterial M. rubber I-cyt-subunit of A-ATPase as a template (Srinivasan, Vyas, Baker, & Quiocho, 2011), we built a homology model of mammalian a2N(1-352) fragment. Next, we combined it with the determined NMR structures of peptides a2N(368-395) and a2N(386-402) of the C-terminal section of a2N(1-402). The complete molecular model of a2N(1-402) revealed that six CTH2 interacting peptides are clustered in the distal and proximal lobe sub-domains of a2N(1-402). Our data indicate that the proximal lobe sub-domain is the major interacting site with the Sec7 domain of first CTH2 protein, while the distal lobe sub-domain of a2N(1-402) interacts with the PH-domain of second CTH2. Indeed, using Sec7/Arf-GEF activity assay we experimentally confirmed our model. The interface formed by peptides a2N(1-17) and a2N(35-49) is involved in specific interaction with Sec7 domain and regulation of GEF activity. These data are critical for understanding of the cross-talk between V-ATPase and CTH2 as well as for the rational drug design to regulate their function.

PTH regulates serum calcium, phosphate, and 1,25-dihydroxyvitamin D (1,25(OH)2D) levels by acting on bone and kidney. In renal proximal tubules (PTs), PTH inhibits reabsorption of phosphate and stimulates the synthesis of 1,25(OH)2D. The PTH receptor couples to multiple G proteins. We here ablated the α-subunit of the stimulatory G protein (Gsα) in mouse PTs by using Cre recombinase driven by the promoter of type-2 sodium-glucose cotransporter (Gsα(Sglt2KO) mice). Gsα(Sglt2KO) mice were normophosphatemic but displayed, relative to controls, hypocalcemia (1.19 ±0.01 vs 1.23 ±0.01 mmol/L; P < .05), reduced serum 1,25(OH)2D (59.3 ±7.0 vs 102.5 ±12.2 pmol/L; P < .05), and elevated serum PTH (834 ±133 vs 438 ±59 pg/mL; P < .05). PTH-induced elevation in urinary cAMP excretion was blunted in Gsα(Sglt2KO) mice (2- vs 4-fold over baseline in controls; P < .05). Relative to baseline in controls, PTH-induced reduction in serum phosphate tended to be blunted in Gsα(Sglt2KO) mice (-0.39 ±0.33 vs -1.34 ±0.36 mg/dL; P = .07). Gsα(Sglt2KO) mice showed elevated renal vitamin D 24-hydroxylase and bone fibroblast growth factor-23 (FGF23) mRNA abundance (∼3.4- and ∼11-fold over controls, respectively; P < .05) and tended to have elevated serum FGF23 (829 ±76 vs 632 ±60 pg/mL in controls; P = .07). Heterozygous mice having constitutive ablation of the maternal Gsα allele (E1(m-/+)) (model of pseudohypoparathyroidism type-Ia), in which Gsα levels in PT are reduced, also exhibited elevated serum FGF23 (474 ±20 vs 374 ±27 pg/mL in controls; P < .05). Our findings indicate that Gsα is required in PTs for suppressing renal vitamin D 24-hydroxylase mRNA levels and for maintaining normal serum 1,25(OH)2D.

The eukaryotic vacuolar-type ATPase (V-ATPase) is a multi -subunit membrane protein complex, which is evolutionarily conserved from yeast to human. It is also functionally conserved and operates as a rotary proton pumping nano motor. In the first part of this chapter we discuss the structure and function of the yeast V-ATPase (ViVo) holoenzyme, We focus on the structural features of its subunits forming both catalytic V1 and proton conducting Vo sectors. Particularly, the recently solved structure of DF-subunit complex is discussed in relation to the energy coupling and regulation of yeast V-ATPase. It is noteworthy that the structure could contribute to understanding the function and regulation of V-ATPases of eukaryotes including human, leading to the rational design of specific inhibitors for medical applications. In addition to the well characterized role as proton pump, V-ATPases have acquired alternative cellular functions during evolution. In the second part we analyze novel roles of V-ATPase in function, signaling, and vesicular trafficking of cellular receptors. Our recent studies have uncovered that V-ATPase itself functions as an evolutionarily conserved pH-sensing and signaling receptor, which forms super-complex with aldolase/cytohesin-2/Arf1,6 small GTPases in early endosomes. On the other hand, V-ATPase forms a super-complex with mTORC1/Ragulator/Rag/Rheb small GTPases in late endosome/lysosomes and is involved in amino -acids sensing and monitoring nutritional state of cells. Finally, we discuss the role of V-ATPase in the development and progression of various diseases including cancer, diabetes, and osteopetrosis among others. We also present emerging approaches and future perspectives for specific drug targeting to V-ATPase and its super -complexes.

V-ATPases (H + ATPases) are multisubunit, ATP-dependent proton pumps that regulate pH homeostasis in virtually all eukaryotes. They are involved in key cell biological processes including vesicle trafficking, endosomal pH sensing, membrane fusion and intracellular signaling. They also have critical systemic roles in renal acid excretion and blood pH balance, male fertility, bone remodeling, synaptic transmission, olfaction and hearing. Furthermore, V-ATPase dysfunction either results in or aggravates various other diseases, but little is known about the complex protein interactions that regulate these varied V-ATPase functions. Therefore, we performed a proteomic analysis to identify V-ATPase associated proteins and construct a V-ATPase interactome. Our analysis using kidney tissue revealed V-ATPase-associated protein clusters involved in protein quality control, complex assembly and intracellular trafficking. ARHGEF7, DMXL1, EZR, NCOA7, OXR1, RPS6KA3, SNX27 and 9 subunits of the chaperonin containing TCP1 complex (CCT) were found to interact with V-ATPase for the first time in this study. Knockdown of two interacting proteins, DMXL1 and WDR7, inhibited V-ATPase-mediated intracellular vesicle acidification in a kidney cell line, providing validation for the utility of our interactome as a screen for functionally important novel V-ATPase-regulating proteins. Our data, therefore, provide new insights and directions for the analysis of V-ATPase cell biology and (patho)physiology.

ABSTRACT Pseudohypoparathyroidism type-Ia (PHP-Ia), characterized by renal proximal tubular resistance to parathyroid hormone (PTH), results from maternal mutations of GNAS that lead to loss of α-subunit of the stimulatory G protein (Gαs) activity. Gαs expression is paternally silenced in the renal proximal tubule, and this genomic event is critical for the development of PTH resistance, as patients display impaired hormone action only if the mutation is inherited maternally. The primary clinical finding of PHP-Ia is hypocalcemia, which can lead to various neuromuscular defects including seizures. PHP-Ia patients frequently do not present with hypocalcemia until after infancy, but it has remained uncertain whether PTH resistance occurs in a delayed fashion. Analyzing reported cases of PHP-Ia with documented GNAS mutations and mice heterozygous for disruption of Gnas, we herein determined that the manifestation of PTH resistance caused by the maternal loss of Gαs, ie, hypocalcemia and elevated serum PTH, occurs after early postnatal life. To investigate whether this delay could reflect gradual development of paternal Gαs silencing, we then analyzed renal proximal tubules isolated by laser capture microdissection from mice with either maternal or paternal disruption of Gnas. Our results revealed that, whereas expression of Gαs mRNA in this tissue is predominantly from the maternal Gnas allele at weaning (3 weeks postnatal) and in adulthood, the contributions of the maternal and paternal Gnas alleles to Gαs mRNA expression are equal at postnatal day 3. In contrast, we found that paternal Gαs expression is already markedly repressed in brown adipose tissue at birth. Thus, the mechanisms silencing the paternal Gαs allele in renal proximal tubules are not operational during early postnatal development, and this finding correlates well with the latency of PTH resistance in patients with PHP-Ia. © 2014 American Society for Bone and Mineral Research.

The eukaryotic V-type adenosine triphosphatase (V-ATPase) is a multi-subunit membrane protein complex that is evolutionarily related to F-type adenosine triphosphate (ATP) synthases and A-ATP synthases. These ATPases/ATP synthases are functionally conserved and operate as rotary proton-pumping nano-motors, invented by Nature billions of years ago. In the first part of this review we will focus on recent structural findings of eukaryotic V-ATPases and discuss the role of different subunits in the function of the V-ATPase holocomplex. Despite structural and functional similarities between rotary ATPases, the eukaryotic V-ATPases are the most complex enzymes that have acquired some unconventional cellular functions during evolution. In particular, the novel roles of V-ATPases in the regulation of cellular receptors and their trafficking via endocytotic and exocytotic pathways were recently uncovered. In the second part of this review we will discuss these unique roles of V-ATPases in modulation of function of cellular receptors, involved in the development and progression of diseases such as cancer and diabetes as well as neurodegenerative and kidney disorders. Moreover, it was recently revealed that the V-ATPase itself functions as an evolutionarily conserved pH sensor and receptor for cytohesin-2/Arf-family GTP-binding proteins. Thus, in the third part of the review we will evaluate the structural basis for and functional insights into this novel concept, followed by the analysis of the potentially essential role of V-ATPase in the regulation of this signaling pathway in health and disease. Finally, future prospects for structural and functional studies of the eukaryotic V-ATPase will be discussed.

Structural model of cytosolic N-terminus of a2-subunit isoform of V-ATPase: Identification and characterization of two binding interfaces for cytohesin-2 Vladimir Marshansky, Maria Merkulova, Hiroyuki Hosokawa, Anastasia Bakulina, Phat Vinh Dip, Youg Raj Thaker, Ashok Khatri, Dennis A. Ausiello, Dennis Brown, Gerhard Grüber Kadmon Corp & CSB/PMB/MGH/Harvard Medical School CSB/PMB/MGH/Harvard Medical School State Research Center “VECTOR” NTU, School of Biological Sciences E-mail: vladimir.marshansky@kadmon.com

Previously, we reported an acidification‐dependent interaction of the endosomal V‐ATPase with cytohesin‐2, a GDP/GTP‐exchange factor (GEF), suggesting that it functions as a pH‐sensing receptor. Here, we have studied the molecular mechanism of signaling between the V‐ATPase, cytohesin‐2 and Arf GTP‐binding proteins. We found that part of the N‐terminal cytosolic tail of the V‐ATPase a2‐subunit (a2N1–402), corresponding to its first seventeen amino acids (a2N1–17), potently modulates the GDP/GTP‐exchange activity of cytohesin‐2. Moreover, this peptide strongly inhibits GEF‐activity via direct interaction with the Sec7 domain of cytohesin‐2. The structure of a2N1–17 and its amino acids F5, M10 and Q14 involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V‐ATPase formed by its a2N1–17 epitope competes with the Switch 2 region of Arf1 and Arf6 for a binding on the Sec7 domain of cytohesin‐2. Moreover, the amino acid sequence alignment and GEF‐activity experiments also uncovered the conserved character of signaling between all four (a1‐a4) a‐subunit isoforms of mammalian V‐ATPase and cytohesin‐2. Thus, here we have uncovered an evolutionarily conserved function of the V‐ATPase as a novel cytohesin‐signaling receptor. Supported by grants NIH DK038452, BADERC DK057521–08 to VM and by A*STAR BMRC09/1/22/19/609 to GG.

We have previously discovered an important interaction between N‐terminal cytosolic tail of the V‐ATPase a2‐subunit (a2N1–402) and cytohesin‐2. We also identified six V‐ATPase derived peptides involved in interaction with cytohesin‐2. However, their localization and binding interfaces remained unknown. Here, we used a combination of homology modeling and NMR analysis to generate the structural model of complete a2N1–402. According to our model all six peptides are clustered in two binding sites, the proximal and distal lobe sub‐domains of a2N1–402. Moreover, our data indicate that the proximal lobe sub‐domain is the major interacting site with the Sec7 domain of cytohesin‐2. In particular, we showed that two V‐ATPase derived peptides a2N1–17 and a2N35–49 are forming a specific Sec‐domain interaction interface and are involved in regulation of its enzymatic GEF‐activity. The distal lobe sub‐domain of a2N1–402 most likely interacts with another cytohesin‐2 via its PH‐domain. Moreover, our model also suggests that both cytoheins‐2 molecules are binding in a close proximity with two G/E‐peripheral stalks of V‐ATPase and may modulate their interaction with a2N1–402. Thus, we hypothesize that recruitment of cytohesin‐2 to V‐ATPase during its function as a pH‐sensing receptor may regulate function of V‐ATPase. Supported by grants NIH DK038452, BADERC DK057521–08 to VM and by A*STAR BMRC09/1/22/19/609 to GG.

Previously, we reported an acidification-dependent interaction of the endosomal vacuolar H(+)-ATPase (V-ATPase) with cytohesin-2, a GDP/GTP exchange factor (GEF), suggesting that it functions as a pH-sensing receptor. Here, we have studied the molecular mechanism of signaling between the V-ATPase, cytohesin-2, and Arf GTP-binding proteins. We found that part of the N-terminal cytosolic tail of the V-ATPase a2-subunit (a2N), corresponding to its first 17 amino acids (a2N(1-17)), potently modulates the enzymatic GDP/GTP exchange activity of cytohesin-2. Moreover, this peptide strongly inhibits GEF activity via direct interaction with the Sec7 domain of cytohesin-2. The structure of a2N(1-17) and its amino acids Phe(5), Met(10), and Gln(14) involved in interaction with Sec7 domain were determined by NMR spectroscopy analysis. In silico docking experiments revealed that part of the V-ATPase formed by its a2N(1-17) epitope competes with the switch 2 region of Arf1 and Arf6 for binding to the Sec7 domain of cytohesin-2. The amino acid sequence alignment and GEF activity studies also uncovered the conserved character of signaling between all four (a1-a4) a-subunit isoforms of mammalian V-ATPase and cytohesin-2. Moreover, the conserved character of this phenomenon was also confirmed in experiments showing binding of mammalian cytohesin-2 to the intact yeast V-ATPase holo-complex. Thus, here we have uncovered an evolutionarily conserved function of the V-ATPase as a novel cytohesin-signaling receptor.

Recently, we uncovered four V‐ATPase a‐subunit isoforms derived peptides a1N 1–17 , a2N 1–17 , a3N 1–17 and a4N 1–17 as a potent inhibitors of the GDP/GTP‐exchange activity of cytohesin‐2 in vitro . Here, we used these peptides to study the role of cytohesin‐2 in regulation of macropinocytosis pathway and cell proliferation in vivo . Treatment of HeLa and MTC cells by cell‐permeable and fluorescently labeled peptides (FITC‐a2N 1–17 ‐TAT or CMTR‐a2N 1–17 ‐TAT) give rise to cell shape remodeling and activation of the macropinocytosis pathway. Uptake of RITC‐dextran but not albumin‐Alexa555 by this pathway was insensitive to inhibitors of NHE‐exchanger (amiloride, DMA, EIPA) and V‐ATPase (bafilomycin A 1 ). Pulse‐chase and confocal time‐lapse imaging revealed that anti‐cytohesin peptides are targeted to endosomal/lysosomal compartments and slow down vesicular trafficking. We also showed that all four anti‐cytohesin peptides are potent inhibitors of the EGFR/cytohesin‐2 dependent proliferation of human lung cancer A549 cells. In summary, here we demonstrated an important role of signaling between V‐ATPase and cytohesin‐2 in regulation of macropinocytosis pathway and cell proliferation. Importantly, these anti‐cytohesin peptides could be used to treat EGFR/cytohesin‐2 dependent cancers and to prevent development of diabetic nephropathy. Supported by grants NIH DK038452 and BADERC DK05752–08 to VM.