A co-purifying contaminant lowers the phosphoenzyme level of Spf1p

CORRADI, GERARDO R.; DE TEZANOS PINTO, FELICITAS; PETROVICH, GUIDO; ADAMO HUGO P.; MAZZITELLI, LUCIANA R.; GRENON, PAULA

Salta

Congreso; LV Annual SAIB Meeting and XIV PABMB Congress; 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

P5-ATPases are eukaryotic proteins believed to actively transport a yet not identified substrate. They are necessary for cellular functions associated with the ER and the endo-lysosomal membranes, and in humans, mutations in the P5-ATPases genes are associated with neurological disorders. At the molecular level the best characterized P5-ATPase is the Spf1 from Saccharomyces cerevisiae. We have previously shown that purified micellar preparations of recombinant Spf1 can hydrolyze ATP and produce the phosphoenzyme intermediate (EP) characteristic of the transport reaction cycle of P-ATPases (Corradi et al., 2016). Moreover, Spf1 was proposed to be modulated by Ca2+ by decreasing the level of EP. Here we present results suggesting that at least part of the effect of Ca2+ is mediated by traces of contaminant proteins that co-purify with Spf1. When the reaction media contained EGTA the preparation exhibited a low ATPase activity that was either increased or inhibited by the addition of CaCl2 depending on each particular preparation. The addition of 1 mM of the phosphatase inhibitor ammonium molybdate abolished the stimulation of the ATPase. The catalytic death mutant Spf1-D487N showed a marginal ATPase activity in EGTA but was highly stimulated by Ca2+. These results suggest that the increase of ATPase did not result from a direct effect of Ca2+ on Spf1. Moreover, the stimulation of the ATPase activity was also produced by other metals like Mn, and Zn suggesting that the increase in ATPase was not a specific effect of Ca2+ but the result of decreasing the free EGTA. The level of EP formed by Spf1 was higher in the presence of EGTA and decreased with the increase in the Ca2+ of the media. The magnitude of this Ca2+ effect on the EP level showed a positive correlation with the stimulation of the ATPase activity in each preparation of Spf1 tested. Altogether these results suggest that the catalytic function of Spf1 may be regulated by a protein phosphatase Analysis by mass spectrometry of the Spf1 preparation after SDS-PAGE detected the presence of several kinases and phosphatases proteins.