CONICET | Buscador de Institutos y Recursos Humanos

Despite the efforts made during the last years, the putative transported substrate of the P5-ATPases remains a mystery. From the beginning, Ca2+ was an interesting candidate to play this role. Indeed, the phenotypic changes produced by the KO of the Spf1 P5-ATPase of S. cerevisiae seemed consistent with this idea. Disappointingly, the first report did not detect any Ca2+ stimulation of the ATPase activity of the purified Spf1 (1). Latter, an intriguing non-phosphatase dependent destabilizing effect of Ca2+ on the phosphoenzyme formed by Spf1 was described (2). Furthermore we reported that Ca2+ also inhibited the formation of the Spf1 phosphoenzyme when reacting with ATP (3). Altogether these results indicate that somehow Ca2+ modulates the formation and/or the decomposition of the EP formed by Spf1. However, until now, the basis of the Ca2+ modulation of Spf1 and its correlation with the global functioning of Spf1 as estimated by its ATP hydrolytic activity has not been clarified. Our His-tagg purified GFP-Spf1 preparation has an ATPase activity of 0.4 umol/mg/min when measured at 28 ○C in the presence of excess of EGTA. In the presence of 100 uM Ca2+ the ATPase increases 2-4 fold. An even higher stimulatory effect of Ca2+ was observed on the ATPase activity of the Spf1 mutant containing the substitution of the phosphate acceptor D487 by N. This result suggests that the increase of ATPase did not result from a direct effect of Ca2+ on the Spf1. Moreover, the Ca2+ dependent stimulation of the ATPase required the presence of mM concentrations of ATP and it was particularly evident at reaction times of 5 min and longer. One possible interpretation of these results is that a kinase or phosphatase accounting for less than 5% the total amount of protein co-purifies with GFP-Spf1, hydrolyzes ATP at low rate in EGTA and becomes highly active in the presence of Ca2+.