Vacuolar proton-translocating ATPases (V-ATPases) are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although V-ATPases are ...involved in a number of cellular processes, how the proton pumps are regulated under physiological conditions is not well understood. We have reported that the glycolytic enzyme aldolase mediates V-ATPase assembly and activity by physical association with the proton pump (Lu, M., Holliday, L. S., Zhang, L., Dunn, W. A., and Gluck, S. L. (2001) J. Biol. Chem. 276, 30407–30413 and Lu, M., Sautin, Y., Holliday, L. S., and Gluck, S. L. (2004) J. Biol. Chem. 279, 8732–8739). In this study, we generate aldolase mutants that lack binding to the B subunit of V-ATPase but retain normal catalytic activities. Functional analysis of the aldolase mutants shows that disruption of binding between aldolase and the B subunit of V-ATPase results in disassembly and malfunction of V-ATPase. In contrast, aldolase enzymatic activity is not required for V-ATPase assembly. Taken together, these findings strongly suggest an important role for physical association between aldolase and V-ATPase in the regulation of the proton pump.
Hypokalemia is a relatively common electrolyte disorder usually resulting from gastrointestinal wasting. Transient hyperkalemia in those treated for hypokalemia has been previously described to occur ...in 16% of hospitalized patients. The majority of those patients had acute, hospital-acquired hypokalemia. Here, we report a case of a young man with alcohol use disorder and chronic hypokalemia who was hospitalized for muscle weakness, abdominal pain, and intractable emesis. His potassium was 2.5 mEq/L on the day of admission. Four days later, with a creatinine at baseline (0.9 mg/dL), potassium abruptly increased to 6.7 mEq/L. He did not have evidence of hyperaldosteronism. In cases of chronic hypokalemia, we propose that the adaptive mechanisms of the distal tubule with total body potassium deficits require time to revert back to a nonactive state and that transient hyperkalemia may be observed during these “refractory” periods during which potassium supplementation is continued. The time required for disassembly of with no lysine kinases following resolution of hypokalemia is unknown. Hyperkalemia is an important consideration when treating patients with chronic hypokalemia.
The primary hyperoxalurias (PHs) are a group of diseases characterized by kidney stones, nephrocalcinosis, and chronic kidney disease. At stages of advanced kidney disease, glomerular filtration of ...oxalate becomes insufficient, plasma levels increase, and tissue deposition may occur. Hemodialysis is often unable to overcome the excess hepatic oxalate production. The current surgical management of primary hyperoxaluria type 1 (PH1) is combined liver kidney transplantation. In a subset of PH1 patients who respond to pyridoxine, kidney-only transplantation has been successfully performed. Recently, kidney-only transplantation has also been performed in PH1 patients receiving a small interfering RNA therapy called lumasiran. This drug targets the hepatic overproduction of oxalate, making kidney-only transplantation a potentially practical novel approach for managing PH1 patients with advanced kidney disease. It is unknown if similar effects could be seen with a different small interfering RNA agent called nedosiran. This article will briefly review PH1, describe the small interfering RNA therapies being used to treat PH, summarize the reported cases of kidney-only transplantation performed with lumasiran, and detail a case of kidney-only transplantation performed in a PH1 patient receiving nedosiran.
Vacuolar H+-ATPases (V-ATPases) are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. How ATP is supplied for V-ATPase-mediated ...hydrolysis and for coupling of proton transport is poorly understood. We have reported that the glycolytic enzyme aldolase physically associates with V-ATPase (Lu, M., Holliday, L. S., Zhang, L., Dunn, W. A., and Gluck, S. L. (2001) J. Biol. Chem. 276, 30407–30413). Here we show that aldolase interacts with three different subunits of V-ATPase (subunits a, B, and E). The binding sites for the V-ATPase subunits on aldolase appear to be on distinct interfaces of the glycolytic enzyme. Aldolase deletion mutant cells were able to grow in medium buffered at pH 5.5 but not at pH 7.5, displaying a growth phenotype similar to that observed in V-ATPase subunit deletion mutants. Abnormalities in V-ATPase assembly and protein expression observed in aldolase deletion mutant cells could be fully rescued by aldolase complementation. The interaction between aldolase and V-ATPase increased dramatically in the presence of glucose, suggesting that aldolase may act as a glucose sensor for V-ATPase regulation. Taken together, these findings provide functional evidence that the ATP-generating glycolytic pathway is directly coupled to the ATP-hydrolyzing proton pump through physical interaction between aldolase and V-ATPase.
Vacuolar H+-ATPase (V-ATPase) binds actin filaments with high affinity (Kd = 55 nm; Lee, B. S., Gluck, S. L., and Holliday, L. S. (1999) J. Biol. Chem. 274, 29164–29171). We have proposed that this ...interaction is an important mechanism controlling transport of V-ATPase from the cytoplasm to the plasma membrane of osteoclasts. Here we show that both the B1 (kidney) and B2 (brain) isoforms of the B subunit of V-ATPase contain a microfilament binding site in their amino-terminal domain. In pelleting assays containing actin filaments and partially disrupted V-ATPase, B subunits were found in greater abundance in actin pellets than were other V-ATPase subunits, suggesting that the B subunit contained an F-actin binding site. In overlay assays, biotinylated actin filaments also bound to the B subunit. A fusion protein containing the amino-terminal half of B1 subunit bound actin filaments tightly, but fusion proteins containing the carboxyl-terminal half of B1 subunit, or the full-length E subunit, did not bind F-actin. Fusion proteins containing the amino-terminal 106 amino acids of the B1 isoform or the amino-terminal 112 amino acids of the B2 isoform bound filamentous actin withKd values of 130 and 190 nm, respectively, and approached saturation at 1 mol of fusion protein/mol of filamentous actin. The B1 and B2 amino-terminal fusion proteins competed with V-ATPase for binding to filamentous actin. In summary, binding sites for F-actin are present in the amino-terminal domains of both isoforms of the B subunit, and likely are responsible for the interaction between V-ATPase and actin filaments in vivo.
Vacuolar H+-ATPases (V-ATPases) are highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although it is generally believed that V-ATPases ...transport protons by a rotary catalytic mechanism analogous to that used by F1F0-ATPases, the structure and subunit composition of the central or peripheral stalk of the multisubunit complex are not well understood. We searched for proteins that bind to the E subunit of V-ATPase using the yeast two-hybrid assay and identified the H subunit as an interacting partner. Physical association between the E and H subunits of V-ATPase was confirmedin vitro by precipitation assays. Deletion mapping analysis revealed that a 78-amino acid fragment at the amino terminus of the E subunit was sufficient for binding to the H subunit. Expression of the amino-terminal fragments of the E subunits from human and yeast as dominant-negative mutants resulted in dramatic decreases in bafilomycin A1-sensitive ATP hydrolysis and proton transport activities of V-ATPase. Our data demonstrate the physiological significance of the interaction between the E and H subunits of V-ATPase and extend previous studies on the arrangement of subunits on the peripheral stalk of V-ATPase.
Vacuolar H + -ATPases (V-ATPases) are essential for acidification of intracellular compartments and for proton secretion from the plasma
membrane in kidney epithelial cells and osteoclasts. The ...cellular proteins that regulate V-ATPases remain largely unknown.
A screen for proteins that bind the V-ATPase E subunit using the yeast two-hybrid assay identified the cDNA clone coded for
aldolase, an enzyme of the glycolytic pathway. The interaction between E subunit and aldolase was confirmed in vitro by precipitation assays using E subunit-glutathione S -transferase chimeric fusion proteins and metabolically labeled aldolase. Aldolase was isolated associated with intact V-ATPase
from bovine kidney microsomes and osteoclast-containing mouse marrow cultures in co-immunoprecipitation studies performed
using an anti-E subunit monoclonal antibody. The interaction was not affected by incubation with aldolase substrates or products.
In immunocytochemical assays, aldolase was found to colocalize with V-ATPase in the renal proximal tubule. In osteoclasts,
the aldolase-V-ATPase complex appeared to undergo a subcellular redistribution from perinuclear compartments to the ruffled
membranes following activation of resorption. In yeast cells deficient in aldolase, the peripheral V 1 domain of V-ATPase was found to dissociate from the integral membrane V 0 domain, indicating direct coupling of glycolysis to the proton pump. The direct binding interaction between V-ATPase and
aldolase may be a new mechanism for the regulation of the V-ATPase and may underlie the proximal tubule acidification defect
in hereditary fructose intolerance.
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