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

HUGO P. ADAMO; GERARDO R. CORRADI; FELICITAS DE TEZANOS PINTO

Conferencia; Na,K-ATPasa and Related P-ATPasas: Structure, Biology and Medicine; 2011

The P5-ATPases are important components of eukaryotic cells. They have been showed toinfluence protein biogenesis, folding and transport. The knowledge of their biochemicalproperties is however limited and the transported ion is not known. We have expressed inSaccharomyces cerevisiae the yeast Spf1 P5A-ATPase containing the GFP fused at the Nterminalend. Purified preparations of the GFP-Spf1 protein were able to hydrolyze ATP ata rate of about 0.3-1 mol Pi/mg/min and to form a phosphoenzyme in a simple reactionmedia containing no added metal ions except Mg2+. Omission of protease inhibitors from thepurification buffers resulted in a high level of endogenous proteolysis at the C-terminalportion of the GFP-Spf1 molecule that abolished phosphoenzyme formation. No significantdifferences were found between the ATPase activity of GFP-Spf1 and recombinant Spf1. TheMg2+ dependency of the GFP-Spf1 ATPase was similar to that of other P-ATPases wereMg2+ acts as a cofactor. Ca2+ was not required for activity while Mn2+ decreased it. Theenzyme manifested optimal activity at a near neutral pH. When chased by the addition ofcold ATP 90 % of the phosphoenzyme remained stable after 5 s suggesting that the forwardprocessing was impaired. In contrast, the phosphoenzyme rapidly decayed when chased bythe addition of ADP, indicating that GFP-Spf1 accumulated E1P phosphoenzyme. It issuggested that this behavior of Spf1 reflects either the absence of the propercountertransported substrate or a regulatory “specificity” factor needed for accelerating theswitching of Spf1 to the E2 part of the reaction cycle.