RESUMO
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.
Assuntos
Rim/enzimologia , ATPases Vacuolares Próton-Translocadoras/isolamento & purificação , Equilíbrio Ácido-Base , Sequência de Aminoácidos , Animais , Membrana Celular/enzimologia , Polaridade Celular , Biblioteca Gênica , Córtex Renal/citologia , Córtex Renal/enzimologia , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/enzimologia , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Subunidades Proteicas , Homologia de Sequência de Aminoácidos , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
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.
Assuntos
Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , ATPases Translocadoras de Prótons/genética , ATPases Vacuolares Próton-Translocadoras , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/embriologia , Proteínas de Caenorhabditis elegans , Transtornos do Desenvolvimento Sexual , Embrião não Mamífero , Feminino , Genes Reporter , Proteínas de Fluorescência Verde , Intestinos/citologia , Intestinos/enzimologia , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Luminescentes/genética , Masculino , Dados de Sequência Molecular , Sistema Nervoso/citologia , Sistema Nervoso/enzimologia , Especificidade de Órgãos , Subunidades Proteicas , Bombas de Próton/química , Bombas de Próton/genética , Bombas de Próton/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , RNA de Cadeia Dupla/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Pele/citologia , Pele/enzimologia , Útero/citologia , Útero/enzimologiaRESUMO
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.