Functional complementation between transmembrane loops of Saccharomyces cerevisiae and Candida albicans plasma membrane H(+)-ATPases.
Saccharomyces cerevisiae PMA1 sequences encoding a putative antifungal target site comprising transmembrane loops 1 + 2 and/or 3 + 4 were replaced with the homologous sequences from Candida albicans PMA1 by using PCR-mediated domain transfer. The chimeric pma1 mutants and an isogenic wild type S. cerevisiae strain had similar growth rates, growth yields, glucose-dependent proton pumping rates, acid-activated omeprazole sensitivities, salt tolerances and antifungal sensitivities. The yields and kinetic properties of H(+)-ATPases in plasma membranes of mutant and wild type strains were comparable. Single heterologous transmembrane loops caused deleterious phenotypes at low pH and elevated temperature. Inclusion of both heterologous transmembrane loops fully suppressed the temperature sensitivity caused by heterologous transmembrane loop 1 + 2, partially suppressed the pH sensitivity and gave Candida-like in vitro sensitivity to vanadate, suggesting that the loops operate as a domain. The fully functional chimeric H(+)-ATPase containing C. albicans transmembrane loops 1 + 2 and 3 + 4 demonstrates this domain's complementarity to the equivalent region of the S. cerevisiae enzyme and validates the wild type S. cerevisiae H(+)-ATPase as an antifungal screening target.
Mason
A B
AB
Experimental Oral Biology Laboratory, School of Dentistry, University of Otago, Dunedin, New Zealand.
Kardos
T B
TB
Perlin
D S
DS
Monk
B C
BC
D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D011993:Recombinant Fusion Proteins; D006180:Proton-Translocating ATPases