The SzA mutations of the B subunit of the Drosophila vacuolar H+ ATPase identify conserved residues essential for function in fly and yeast.
V-ATPases play multiple roles in eukaryotes: in Drosophila, null mutations are recessive lethal. Here, mutations underlying five extant lethal alleles of vha55, encoding the B subunit, were identified, including a premature termination codon and two mutations very close to residues thought to participate in the catalytic site of the enzyme. Lethality of these alleles could be reverted by transformation of flies with a wild type vha55::GFP fusion, confirming that the lethal phenotype described for these alleles was due to defects in V-ATPase function. The chimeric protein was correctly localised to the apical domain of the Malpighian (renal) tubule, and restored fluid transport function to wild-type levels. No dominant-negative phenotype was apparent in heterozygotes. When the vha55::GFP fusion was driven ubiquitously, fluorescent protein was only detectable in tissues known to contain high levels of V-ATPase, suggesting that vha55 requires stoichometric co-expression of other subunits to be stable. Yeast (Saccharomyces cerevisiae) deleted for the corresponding gene (Deltavma2) demonstrated a pH-sensitive growth phenotype that was rescued by the vha55::GFP construct. Deltavma2 yeast could not be rescued with fly cDNAs encoding any of the mutant vha55 alleles, confirming the functional significance of the mutated residues. In yeast, bafilomycin-sensitive ATPase activity and growth rate correlated with the ability of different constructs to rescue the pH-sensitive conditional-lethal phenotype. These classical Drosophila mutants thus identify residues that are essential for function in organisms with wide phylogenetic separation.
Du
Juan
J
Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, G11 6NU, UK.
Kean
Laura
L
Allan
Adrain K
AK
Southall
Tony D
TD
Davies
Shireen A
SA
McInerny
Christopher J
CJ
Dow
Julian A T
JA
D000483:Alleles; D000818:Animals; D017124:Conserved Sequence / Q000502:physiology; D029721:Drosophila Proteins / Q000235:genetics*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005804:Genes, Lethal; D049452:Green Fluorescent Proteins / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics* / Q000502:physiology*
D029721:Drosophila Proteins; D049452:Green Fluorescent Proteins; C514181:V-ATPase 55-kDa B subunit, Drosophila; D025262:Vacuolar Proton-Translocating ATPases