Shadows of an Absent Partner: ATP Hydrolysis and Phosphoenzyme Turnover of the Spf1 (Sensitivity to Pichia farinosa killer toxin) P5-ATPase

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

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Lugar: Bethesda, Maryland; Año: 2012 vol. 287 p. 30477 - 30484

0021-9258

The P5-ATPases are important components of eukaryotic cells. They have been showed to influence protein biogenesis, folding and transport. The knowledge of their biochemical properties is however limited and the transported ions are still unknown. We expressed in Saccharomyces cerevisiae the yeast Spf1 P5A-ATPase containing the GFP fused at the N-terminal end. The GFP-Spf1 protein was localized in the yeast endoplasmic reticulum. Purified preparations of GFPSpf1 hydrolyzed ATP at a rate of about 0.3-1 μmol Pi/mg/min and formed a phosphoenzyme in a simple reaction media containing no added metal ions except Mg2+. No significant differences were found between the ATPase activity of GFP-Spf1 and recombinant Spf1. Omission of protease inhibitors from the purification buffers resulted in a high level of endogenous proteolysis at the C-terminal portion of the GFP-Spf1 molecule that abolished phosphoenzyme formation. The Mg2+ dependency of the GFP-Spf1 ATPase was similar to that of other P-ATPases were Mg2+ acts as a cofactor. The addition of Mn2+ to the reaction media decreased the ATPase activity. The enzyme manifested optimal activity at a near neutral pH. When chased by the addition of cold ATP 90 % of the phosphoenzyme remained stable after 5 s. In contrast, the phosphoenzyme rapidly decayed to less than 20% when chased for 3 s by the addition of ADP. The greater effect of ADP accelerating the disappearance of EP suggests that GFP-Spf1 accumulated the E1~P phosphoenzyme. This behavior may reflect a limiting countertransported substrate needed to promote turnover or a missing regulatory factor.