ABSTRACT
The vacuolar-type H+ -ATPase (V-ATPase) translocates protons across membranes. Here, we have identified a mouse cDNA coding for a fourth isoform (a4) of the membrane sector subunit a of V-ATPase. This isoform was specifically expressed in kidney, but not in the heart, brain, spleen, lung, liver, muscle, or testis. Immunoprecipitation experiments, together with sequence similarities for other isoforms (a1, a2, and a3), indicate that the a4 isoform is a component of V-ATPase. Moreover, histochemical studies show that a4 is localized in the apical and basolateral plasma membranes of cortical alpha- and beta-intercalated cells, respectively. These results suggest that the V-ATPase, with the a4 isoform, is important for renal acid/base homeostasis.
Subject(s)
Kidney/enzymology , Vacuolar Proton-Translocating ATPases/isolation & purification , Acid-Base Equilibrium , Amino Acid Sequence , Animals , Cell Membrane/enzymology , Cell Polarity , Gene Library , Kidney Cortex/cytology , Kidney Cortex/enzymology , Kidney Tubules, Collecting/cytology , Kidney Tubules, Collecting/enzymology , Mice , Molecular Sequence Data , Protein Isoforms/genetics , Protein Isoforms/isolation & purification , Protein Subunits , Sequence Homology, Amino Acid , Vacuolar Proton-Translocating ATPases/geneticsABSTRACT
We have identified four genes (vha-5, vha-6, vha-7, and unc-32) coding for vacuolar-type proton-translocating ATPase (V-ATPase) subunit a in Caenorhabditis elegans, the first example of four distinct isoforms in eukaryotes. Their products had nine putative transmembrane regions, exhibited 43-60% identity and 62-84% similarity with the bovine subunit a1 isoform, and retained 11 amino acid residues essential for yeast V-ATPase activity (Leng, X. H., Manolson, M. F., and Forgac, M. (1998) J. Biol. Chem. 273, 6717-6723). The similarities, together with the results of immunoprecipitation, suggest that these isoforms are components of V-ATPase. Transgenic and immunofluorescence analyses revealed that these genes were strongly expressed in distinct cells; vha-5 was strongly expressed in an H-shaped excretory cell, vha-6 was strongly expressed in intestine, vha-7 was strongly expressed in hypodermis, and unc-32 was strongly expressed in nerve cells. Furthermore, the vha-7 and unc-32 genes were also expressed in the uteri of hermaphrodites. RNA interference analysis showed that the double-stranded RNA for unc-32 caused embryonic lethality similar to that seen with other subunit genes (vha-1, vha-4, and vha-11) (Oka, T., and Futai, M. (2000) J. Biol. Chem. 275, 29556-29561). The progenies of worms injected with the vha-5 or vha-6 double-stranded RNA became died at a specific larval stage, whereas the vha-7 double-stranded RNA showed no effect on development. These results suggest that V-ATPases with these isoforms generate acidic compartments essential for worm development in a cell-specific manner.
Subject(s)
Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Proton-Translocating ATPases/genetics , Vacuolar Proton-Translocating ATPases , Amino Acid Sequence , Animals , Animals, Genetically Modified , Caenorhabditis elegans/embryology , Caenorhabditis elegans Proteins , Disorders of Sex Development , Embryo, Nonmammalian , Female , Genes, Reporter , Green Fluorescent Proteins , Intestines/cytology , Intestines/enzymology , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Luminescent Proteins/genetics , Male , Molecular Sequence Data , Nervous System/cytology , Nervous System/enzymology , Organ Specificity , Protein Subunits , Proton Pumps/chemistry , Proton Pumps/genetics , Proton Pumps/metabolism , Proton-Translocating ATPases/chemistry , Proton-Translocating ATPases/metabolism , RNA, Double-Stranded/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Sequence Alignment , Sequence Homology, Amino Acid , Skin/cytology , Skin/enzymology , Uterus/cytology , Uterus/enzymologyABSTRACT
Vacuolar H(+)-ATPase (V-ATPase) is a multi-subunit enzyme with a membrane peripheral catalytic (V(1)) and an intrinsic (V(o)) sector. We have identified three cDNA clones coding for isoforms of mouse V(o) subunit a (a1, a2, and a3). They exhibit 48-52% identity with each other and high similarity to subunit a of other species. The a1 isoform was mainly expressed in brain and liver. The a2 isoform was observed in heart and kidney in addition to brain and liver. Transcripts for the a3 isoform were strongly expressed in heart and liver. The a3 isoform was induced during osteoclast differentiation, and localized in the plasma membrane and cytoplasmic filamentous structures. In contrast to a3, the a1 isoform was constitutively expressed and localized in the cytoplasmic endomembrane compartments of the same cells. These findings suggest that the a3 isoform is a component of the plasma membrane V-ATPase essential for bone resorption.
