Mol Gen Genet (1987) 207:38-46 MGG © Springer-Verlag 1987 Genetic and molecular mapping of the prom mutation conferring vanadate resistance to the plasma membrane ATPase from Saccharomyces cerevisiae* Stanislaw Ulaszewski**, Elisabetta Balzi, and Andr~ Goffeau Laboratoire d'Enzymologie, Universit6 Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium Summary. In the yeast Saccharomyces cerevisiae, the pmal mutations confers vanadate-resistance to H +-ATPase activ- ity when measured in isolated plasma membranes. In vivo, the growth ofpmal mutants is resistant to Dio-9, ethidium bromide and guanidine derivatives. This phenotype was used to map the pmal mutation adjacent to LEU1 gene on chromosome VII. From a cosmid library of a wild-type Saceharomyces cerevisiae genome, a large 30 kb DNA frag- ment was isolated by complementation of a l e u l - p m a l double mutant. A 5 kb HindIII fragment was subcloned and it restored both Leu + and Pma + phenotypes after inte- grative transformation. The restriction map of the 5 kb Hin- dIII fragment and Southern blot analysis reveal that the cloned fragment contains the entire structural gene for the plasma membrane ATPase and the 5' end of the adjacent LEU1 gene. The pmal mutation conferring vanadate-resis- tance is thus located in the structural gene for the plasma membrane ATPase. Key words: H +-ATPase gene - Cloning - Vanadate resis- tance - Yeast plasma membrane Introduction The yeast plasma membrane contains an ATPase which functions as an electrogenic proton pump in vivo and in vitro. The enzyme is sensitive to a variety of inhibitors among which Dio-9 and vanadate are the most powerful (see review by Goffeau and Slayman 1981). Mutations af- fecting the plasma membrane ATPase of Saccharomyces cerevisiae were previously obtained by selecting mutants resistantto Dio-9 (Roon etal. 1978; Ulaszewskietal. 1983). Four allelic nuclear mutants defining the PMA1 lo- cus of S. cerevisiae were modified in several ATPase cata- lytic properties, including low specific activity, high Km for MgATP, increased resistance to temperature and mercur- ials, increased stimulation by monovalent cations, and strong vanadate-resistance of the ATPase activity measured in plasma membranefractions (Ulaszewski et al. 1983). Furthermore, as expected for a mutation in a structural * Publication n ° 2456 from the Biology Directorate of the Com- mission of European Communities ** Permanent address." Wroclaw University, Institute of Microbiol- ogy, Przybyszewskiego 63/77, PL-51-t48 Wroclaw, Poland Offprint requests to. A. Goffeau gene the resistance of ATPase to its specific inhibitors was dominant or semi-dominant in vitro and in vivo. It was concluded that the PMA1 locus belongs to a structural gene coding either for the 100000 Mr subunit of the plasma mem- brane ATPase or for a protein responsible for a stable modi- fication of the ATPase that was conserved during purifica- tion of the membranes (Ulaszewski et al. 1983). In this paper we report on the genetic mapping and cloning of the PMA1 gene which is shown to be identical to the H +-ATPase structural gene recently sequenced (Ser- rano et al. 1986). Materials and methods Strains and genetic methods. The strains used are listed in Table 1. Conventional genetic procedures for yeast cross- ing, sporulation, and tetrad analysis were used. The recipi- ent pmal, leul, ura3 strains for transformation (US62-24B and US62-46B) were obtained as follows: First, mitotic re- combination between pmal and leul was induced by gamma-irradiation of the diploid US13 heterozygous for the above markers. Leu colonies were isolated and sporu- lated. The haploid US13-25-6B ~pmal leul trp5 strain was selected. The ura3 and pdrl-1 markers were introduced by crossing with US53-19A. After segregation (Table 1), the presence of pmal mutation was checked by in vitro mea- surement of vanadate resistance of the ATPase activity. The genetic mapping was performed according to Mortimer and Hawthorne (1975). From the results of the tetrad analy- sis, the distance between two markers was calculated in centimorgans as described by Snow (1979). The gene-cen tromere distances were estimated with their standard errors using the second-division segregation values with trpl as centromere marker (Snow 1979). Media and growth conditions. Rich medium contained 1% yeast extract Difco, 2% bactopeptone Difco and 2% glu- cose (YEPD) or 4% glycerol (YEPGly). Minimal medium (SD) contained 2% yeast nitrogen base Difco without ami- no acids and 2% glucose. In supplemented SD medium, the final concentration of amino acids and adenine sulfate was 20 gg/ml, except for L-tryptophan (10 gg/ml) and L- lysine (40 ~tg/ml). For plating, the media were solidified with 2% bacto-agar Difco. Drug resistance was evaluated on solid media according to Saunders and Rank (•982). The strains were tested on