Previously, we demonstrated that the vacuolar-type H(+)-ATPase (V-ATPase) a2-subunit functions as an endosomal pH sensor that interacts with the ADP-ribosylation factor (Arf) guanine nucleotide exchange factor, ARNO. In the present study, we showed that ARNO directly interacts not only with the a2-subunit but with all a-isoforms (a1-a4) of the V-ATPase, indicating a widespread regulatory interaction between V-ATPase and Arf GTPases. We then extended our search for other ARNO effectors that may modulate V-ATPase-dependent vesicular trafficking events and actin cytoskeleton remodeling. Pull-down experiments using cytosol of mouse proximal tubule cells (MTCs) showed that ARNO interacts with aldolase, but not with other enzymes of the glycolytic pathway. Direct interaction of aldolase with the pleckstrin homology domain of ARNO was revealed by pull-down assays using recombinant proteins, and surface plasmon resonance revealed their high avidity interaction with a dissociation constant: K(D) = 2.84 × 10(-10) M. MTC cell fractionation revealed that aldolase is also associated with membranes of early endosomes. Functionally, aldolase knockdown in HeLa cells produced striking morphological changes accompanied by long filamentous cell protrusions and acidic vesicle redistribution. However, the 50% knockdown we achieved did not modulate the acidification capacity of endosomal/lysosomal compartments. Finally, a combination of small interfering RNA knockdown and overexpression revealed that the expression of aldolase is inversely correlated with gelsolin levels in HeLa cells. In summary, we have shown that aldolase forms a complex with ARNO/Arf6 and the V-ATPase and that it may contribute to remodeling of the actin cytoskeleton and/or the trafficking and redistribution of V-ATPase-dependent acidic compartments via a combination of protein-protein interaction and gene expression mechanisms.
"The V-ATPase is a complex enzyme, composed of at least 14 subunits assembled into a transmembrane, proton- (H+-) translocating V0 sector and a cytosolic, catalytic V1 domain (Fig. 1) , , , . In mammalian tissues the 56-kDa B subunit of the enzyme is expressed as two isoforms, B1 and B2. "
[Show abstract][Hide abstract] ABSTRACT: The vacuolar proton-pumping ATPase (V-ATPase) is the main mediator of intracellular organelle acidification and also regulates transmembrane proton (H+) secretion, which is necessary for an array of physiological functions fulfilled by organs such as the kidney, male reproductive tract, lung, bone, and ear. In this study we characterize expression of the V-ATPase in the main olfactory epithelium of the mouse, as well as a functional role for the V-ATPase in odor detection. We report that the V-ATPase localizes to the apical membrane microvilli of olfactory sustentacular cells and to the basolateral membrane of microvillar cells. Plasma membrane V-ATPases containing the B1 subunit isoform are not detected in olfactory sensory neurons or in the olfactory bulb. This precise localization of expression affords the opportunity to ascertain the functional relevance of V-ATPase expression upon innate, odor-evoked behaviors in B1-deficient mice. This animal model exhibits diminished innate avoidance behavior (revealed as a decrease in freezing time and an increase in the number of sniffs in the presence of trimethyl-thiazoline) and diminished innate appetitive behavior (a decrease in time spent investigating the urine of the opposite sex). We conclude that V-ATPase-mediated H+ secretion in the olfactory epithelium is required for optimal olfactory function.
PLoS ONE 09/2012; 7(9):e45395. DOI:10.1371/journal.pone.0045395 · 3.23 Impact Factor
"V-ATPases have also been shown to interact with several glycolytic enzymes [15-19], which are known to also bind microfilaments [20-24]. Recently, interactions between V-ATPases, fructose bisphosphate aldolase and ARNO were described which may signify the emergence of a mechanism by which the spatial localization and activity of V-ATPases are coupled to the metabolic state of the cell . "
[Show abstract][Hide abstract] ABSTRACT: Binding between vacuolar H+-ATPases (V-ATPases) and microfilaments is mediated by an actin binding domain in the B-subunit. Both isoforms of mammalian B-subunit bind microfilaments with high affinity. A similar actin-binding activity has been demonstrated in the B-subunit of yeast. A conserved “profilin-like” domain in the B-subunit mediates this actin-binding activity, named due to its sequence and structural similarity to an actin-binding surface of the canonical actin binding protein profilin. Subtle mutations in the “profilin-like” domain eliminate actin binding activity without disrupting the ability of the altered protein to associate with the other subunits of V-ATPase to form a functional proton pump. Analysis of these mutated B-subunits suggests that the actin-binding activity is not required for the “housekeeping” functions of V-ATPases, but is important for certain specialized roles. In osteoclasts, the actin-binding activity is required for transport of V-ATPases to the plasma membrane, a prerequisite for bone resorption. A virtual screen led to the identification of enoxacin as a small molecule that bound to the actin-binding surface of the B2-subunit and competitively inhibited B2-subunit and actin interaction. Enoxacin disrupted osteoclastic bone resorption in vitro, but did not affect osteoblast formation or mineralization. Recently, enoxacin was identified as an inhibitor of the virulence of Candida
albicans and more importantly of cancer growth and metastasis. Efforts are underway to determine the mechanisms by which enoxacin and other small molecule inhibitors of B2 and microfilament binding interaction selectively block bone resorption, the virulence of Candida, cancer growth, and metastasis.
Current Protein and Peptide Science 10/2011; 13(2):180-91. DOI:10.2174/138920312800493151 · 3.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic vacuolar ATPase (V-ATPase) is regulated by a reversible dissociation mechanism that involves breaking and reforming of protein-protein interactions at the interface of the V(1)-ATPase and V(o)-proton channel domains. We found previously that the head domain of the single copy C subunit (C(head)) binds one subunit EG heterodimer with high affinity (Oot, R.A. and Wilkens, S. (2010) J. Biol. Chem. 285, 24654-24664). Here we generated a water-soluble construct of the N-terminal domain of the V(o) "a" subunit composed of amino acid residues 104-372 (a(NT(104-372))). Analytical gel filtration chromatography and sedimentation velocity analysis revealed that a(NT(104-372)) undergoes reversible dimerization in a concentration-dependent manner. A low-resolution molecular envelope was calculated for the a(NT(104-372)) dimer using small angle x-ray scattering data. Isothermal titration calorimetry experiments revealed that a(NT(104-372)) binds the C(foot) and EG heterodimer with dissociation constants of 22 and 33 μM, respectively. We speculate that the spatial closeness of the a(NT), C(foot), and EG binding sites in the intact V-ATPase results in a high-avidity interaction that is able to resist the torque of rotational catalysis, and that reversible enzyme dissociation is initiated by breaking either the a(NT(104-372))-C(foot) or a(NT(104-372))-EG interaction by an as-yet unknown signaling mechanism.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.