FUNCTIONAL COMPLEMENTATION BETWEEN TRANSMEMBRANE LOOPS OF SACCHAROMYCES-CEREVISIAE AND CANDIDA-ALBICANS PLASMA-MEMBRANE

Saccharomyces cerevisiae PMA1 sequences encoding a putative antifungal target site comprising transmembrane loops 1 + 2 and/or 3 + 4 were re placed with the homologous sequences from Candida albicans PMA1 by usi ng PCR-mediated domain transfer. The chimeric pma1 mutants and an isog enic wild type S. cerevisiae strain had similar growth rates, growth y ields, glucose-dependent proton pumping rates, acid-activated omeprazo le sensitivities, salt tolerances and antifungal sensitivities. The yi elds and kinetic properties of H+-ATPases in plasma membranes of mutan t and wild type strains were comparable. Single heterologous transmemb rane loops caused deleterious phenotypes at low pH and elevated temper ature. Inclusion of both heterologous transmembrane loops fully suppre ssed the temperature sensitivity caused by heterologous transmembrane loop 1 + 2, partially suppressed the pH sensitivity and gave Candida-l ike in vitro sensitivity to vanadate, suggesting that the loops operat e as a domain. The fully functional chimeric H+-ATPase containing C. a lbicans 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 s creening target.