Inhibition of the Formation of the Spf1p Phosphoenzyme by Ca2+

Abstract

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-...

Reduction of the P5A-ATPase Spf1p phosphoenzyme by a Ca2+-dependent phosphatase

“…In agreement with predictions based on the primary amino acid sequences, all P5A ATPases characterized so far have been shown to hydrolyze ATP and to form an EP phosphoenzyme intermediate [16,[26][27][28]. While EP formation seems to proceed with Mg 2+ as the only required ion, lower levels of EP were detected in the presence of Ca 2+ , an observation that was taken to indicate that Spf1p is possibly regulated by Ca 2+ [26,27]. Intriguingly, Ca 2 + -dependent EP dephosphorylation did not require the endogenous phosphatase activity of the enzyme [26] and the ATPase activity of Spf1p was only marginally affected by Ca 2+ [16,19,28].…”

“…A distinctive characteristic of P-type ATPases is the coupling of the transport process with the formation and decomposition of an aspartyl-phosphoenzyme (EP) during each transport cycle [24,25]. In agreement with predictions based on the primary amino acid sequences, all P5A ATPases characterized so far have been shown to hydrolyze ATP and to form an EP phosphoenzyme intermediate [16,[26][27][28]. While EP formation seems to proceed with Mg 2+ as the only required ion, lower levels of EP were detected in the presence of Ca 2+ , an observation that was taken to indicate that Spf1p is possibly regulated by Ca 2+ [26,27].…”

“…The His-Spf1p and His-Spf1p (D 487 N) proteins were constitutively expressed in Saccharomyces cerevisiae cells as described previously [27,28]. Yeast cells were transformed with the pMP625 vector containing a Leu + marker, the PMAI promoter, and the cDNA encoding either wildtype His-Spf1p or the His-Spf1p (D 487 N) mutant, both containing a 9XHis tag at the N-terminus [16].…”

“…The phosphorylation reaction was performed as previously described [27]. Briefly, 1.5 μg of purified Spf1p was phosphorylated at 4˚C in 0.25 mL reaction buffer containing 50 mM Tris-HCl (pH 7.4), 0.5 mM EGTA, 0.5 μM ATP, MgCl 2 to give a concentration of 2 mM Mg 2+ , and CaCl 2 to give a final concentration of 10 μM Ca 2+ .…”

“…The inhibitory effect of phosphatase substrates on the contaminant ATPase activity ( Fig 3D) was examined at a lower concentration of ATP of 30 μM ATP, closer to that used for the formation of phosphoenzyme. In this case the ATP hydrolysis was estimated as described previously [27] from the release of 32 P from [γ-32 P]ATP at 28˚C in a final volume of 0.25 ml of reaction media containing 50 mM Tris-HCl (pH 7.2 at 28˚C), 5 mM N 3 Na, 0.5 mM EGTA, 5 mM MgCl 2 , 30μM ATP, 1 μg of Spf1p (D 487 N) in 40 μL of elution buffer, and 0.5 mM of CaCl 2 to give a final concentration of 10 μM of free Ca 2+ and the indicated phosphatase substrates. The enzyme was supplemented with 40 μg of C 12 E 10 and 40 μg of PC and this mixture was then transferred to the reaction media, incubated for 5 minutes at 28˚C prior to the addition of ATP to start the reaction.…”