P5-ATPases are important for processes associated with the endosomal-lysosomal system of eukaryotic cells. In humans, the loss of function of P5-ATPases causes neurodegeneration. In the yeast Saccharomyces cerevisiae, deletion of P5-ATPase Spf1p gives rise to endoplasmic reticulum stress. The reaction cycle of P5-ATPases is poorly characterized. Here, we showed that the formation of the Spf1p catalytic phosphoenzyme was fast in a reaction medium containing ATP, Mg 2؉ , and EGTA. . Halfmaximal phosphorylation was attained at 8 M Mg 2؉, but higher concentrations partially protected from Ca 2؉ inhibition. In conditions similar to those used for phosphorylation, Ca 2؉ had a small effect accelerating dephosphorylation and minimally affected ATPase activity, suggesting that the formation of the phosphoenzyme was not the limiting step of the ATP hydrolytic cycle.P5-ATPases comprise a group of proteins that are classified as P-ATPases based on the presence of the characteristic PATPase motifs in their primary sequence (1, 2). P5-ATPases have been found only in eukaryotes and have been recently proposed to play an essential role in the endosomal-lysosomal system (3, 4). The yeast Saccharomyces cerevisiae contains two genes coding for P5-ATPases: YEL031W, coding for Spf1p (also called Cod1p), and YOR291W, coding for Ypk9. Spf1p (sensitivity to Pichia farinosa killer toxin) was initially isolated from a mutation protecting Saccharomyces from the effect of a Pichia toxin (5). The protein was also independently identified as required for the controlled degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the endoplasmic reticulum (ER) 3 (6). These and later studies have shown that Spf1p is located in the yeast ER and that deletion of Spf1p leads to phenotypes related to ER stress (7-9). In humans, five genes (ATP13A1-A5) code for P5-ATPases (10). Mutations in ATP13A2 have been linked to an early onset autosomal recessive form of Parkinson disease (Kufor-Rakeb syndrome) and neuronal ceroid lipofuscinosis, whereas mutations in ATP13A4 have been associated with autism spectrum disorder (11-13). P-ATPases are a large group of enzymes that couple the hydrolysis of ATP with the active transport of ions (14, 15). During the transport cycle, they transiently form a phosphoenzyme (EP) that plays a key role in the active transport mechanism. P-ATPases comprise a membrane domain (M) and a soluble portion with nucleotide binding (N), phosphorylation (P), and actuator (A) domains. These domains are involved in a kinasephosphatase reaction cycle through two major conformations, E 1 -E 2 , and the transient formation of a catalytic EP. The binding of the transported ion to the E 1 form prompts the assembly of the phosphorylation site between the ATP-bound N domain and the P domain, whereas the A domain directs the occlusion of the bound ion. When the phosphorylation reaction occurs, it initially generates the high energy E 1 ϳP intermediate and releases ADP. E 1 ϳP then changes to E 2 P, and the A domain associates with the N-...