Shadows of an absent partner: accumulation of the Spf1 P5-ATPase in the E1P conformational state

ADAMO HUGO PEDRO; CORRADI GERARDO RAUL; DE TEZANOS PINTO FELICITAS

ASILOMAR, CA

Conferencia; 13th International ATPase Conference Na, K-ATPase and Related P-ATPase: Structure, Biology and Medicine Symposium; 2011

American Society for Biochemistry and Molecular Biology

The function of P5 ATPases has been showed to influence protein biogenesis, folding and transport. To advance the biochemical characterization of P5-ATPases we overexpressed in Saccharomyces cerevisiae, solubilized the membranes with detergent C12E10, and purified the yeast His tagged Spf1 P5A-ATPase containing GFP fused at the N-terminal end. When supplemented with lipids, GFP-Spf1 was able to hydrolyze ATP at a rate of about 0.3-1 mmol Pi/mg/min in a simple reaction media containing 500 mM EGTA and only Mg2+ as added ion. No significant differences were found between the ATPase activity of GFP-Spf1 and recombinant Spf1. The Mg2+ dependency of the GFP-Spf1 ATPase was similar to that of other P-ATPases were Mg2+ acts as a cofactor. In the presence of ATP the purified GFP-Spf1 protein formed a stable phosphoenzyme intermediate. The amount of phosphoenzyme did not change in the presence of Ca2+ or a mixture or acidic lipids. When chased by the addition of cold ATP 90 % of the phosphoenzyme remained stable after 5 s suggesting that the forward processing was impaired. In contrast, when chased by the addition of ADP the phosphoenzyme decayed very fast indicating that GFP-Spf1 accumulated the E1P phosphoenzyme. It is suggested that this unusual behavior of Spf1 reflects either the absence of the proper countertransported substrate or the need of an additional regulatory subunit. With grants from CONICET, ANPCyT and UBA.