Abstract
The vacuolar-type H+-ATPases (V-ATPases) are multimeric proton pumps involved in a wide variety of physiological processes. We have identified two alternative splicing variants of C2 subunit isoforms: C2-a, a lung-specific isoform containing a 46-amino acid insertion, and C2-b, a kidney-specific isoform without the insert. Immunohistochemistry with isoform-specific antibodies revealed that V-ATPase with C2-a is localized specifically in lamellar bodies of type II alveolar cells, whereas the C2-b isoform is found in the plasma membranes of renal alpha and beta intercalated cells. Immunoprecipitation combined with immunohistological analysis revealed that C2-b together with other kidney-specific isoforms was selectively assembled to form a unique proton pump in intercalated cells. Furthermore, a chimeric yeast V-ATPase with mouse the C2-a or C2-b isoform showed a lower Km(ATP) and lower proton transport activity than that with C1 or Vma5p (yeast C subunit). These results suggest that V-ATPases with the C2-a and C2-b isoform are involved in luminal acidification of lamellar bodies and regulation of the renal acid-base balance, respectively.
Highlights
Differentiated endomembrane organelles, including the Golgi apparatus, lysosomes, endosomes, and secretory vesicles, have a luminal acidic pH, which is required for various cellular functions
Immunohistochemistry with isoform-specific antibodies revealed that V-ATPase with C2-a is localized in lamellar bodies of type II alveolar cells, whereas the C2-b isoform is found in the plasma membranes of renal ␣ and  intercalated cells
In addition to the intracellular organelles, the V-ATPase is localized in the plasma membranes of highly differentiated cells, including osteoclasts [6], kidney intercalated cells [7], and male tract epithelial cells [8], where it is required for bone metabolism, urine acidification, and spermatogenesis, respectively
Summary
V-ATPase, vacuolar Hϩ-ATPase; EST, expressed sequence tag; RT-PCR, reverse-transcribed PCR; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; AMCA, 9-amino-6-chloro-2-methoxyacridine. Immunoprecipitation revealed that the kidney-specific isoforms, including B1, G3, d2, a4, and C2-b, were present in the same complex, whereas the ubiquitously expressed B2, C1, G1, and other a isoforms were found in different complex These results indicated a selective assembly of V-ATPase subunit isoforms in vivo. A chimeric yeast V-ATPase with the mouse C2-a or C2-b isoform was functional in yeast vacuoles but showed a lower Km(ATP) value and lower proton transport activity than that with C1 or yeast C subunit (Vma5p).
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