CONICET | Buscador de Institutos y Recursos Humanos

The effect of P5-ATPases and spermidine on the proliferation of S. cerevisiae. RESERVADO SAB María Clara Sellés, Nicolás Sarbia, Hugo P. Adamo and Gerardo R. Corradi IQUIFIB-Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113, Buenos Aires, Argentina. P-type ATPases comprise a large family of proteins, present in prokaryotic as well as in eukaryotic cells, which catalyze active transport of inorganic cations and other substrates through cell membranes. They have been divided into five sub-families named P1-P5 according to their sequence similarities. The least studied one is the P5-ATPase sub-family which is only expressed in eukaryotes. The yeast Saccharomyces cerevisiae is an inferior eukaryote and it contains two genes that code for P5-ATPases: Yel031w codes for the Spf1 protein or Cod1p, which corresponds to the group of P5A-ATPases; and Yor291w encode Ypk9 protein, which belongs to the P5B-ATPases group. The substrate transported by this sub-family of P-type ATPases is still unknown although it has been shown that their activity modifies cellular uptake of polyamines, such as spermidine. In this study, we investigated the effect of the inactivation of the genes that encode SPF1 and YPK9 in S. cerevisiae and the overexpression of SPF1 regarding cellular proliferation under stress conditions and in the presence of the polycation spermidine. At 28 C in YPD medium, the OD of the wild type culture reached a maximal value in about 10 days. A similar growth curve was observed for the ΔSpf1 and ΔYpk9 strains. However when treated with 4 mM spermidine for 24 hours, an OD 20-40% higher was reached by the wild type and ΔSpf1 while ΔYpk9 was not affected. The ΔSpf1 strain was transformed with a vector coding for Spf1 or a mutant Spf1D487N (devoid of ATPase activity) both fused to GFP. In SC medium (without Leu) the yeasts containing the active protein grew much better than those transformed with Spf1D487N. The incubation in water for 24 hours with 10mM spermidine increased the GFP fluorescence. Altoghether, these results suggest that the YPK9 is necessary for the stimulating effect of spermidine and that overexpression of a functional Spf1 increases proliferation.