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Abstract: The plasma membrane Ca2+ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca2+ at a rate near 1.5% of that attained at saturating concentrations of Ca2+. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca2+-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca2+-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca2+ and is catalyzed by E2-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis. [Copyright &y& Elsevier]

The yeast Spf1p P5A-ATPase actively translocates membrane spanning peptides of mislocalized proteins from the endoplasmic reticulum. Loss of Spf1p function causes a pleiotropic ER stress-phenotype associated with alterations of homeostasis of metal ions, lipids, protein folding, glycosylation, and membrane insertion. A unique characteristic of P5A-ATPases is the presence of an extended insertion which was called the "arm-like" domain connecting the phosphorylation domain (P) with transmembrane segment M5 near the peptidyl-substrate binding pocket. Here we have constructed and characterized a Δarm mutant of Spf1p lacking a segment of 117 amino acids of the "arm-like" domain. The Δarm mutant was capable of hydrolyzing ATP at maximal rates of 50% of that of the wild type enzyme. With the non-nucleotide substrate analog pNPP, the hydrolytic activity of the mutant dropped to 10%. The mutant showed an apparent affinity for ATP similar to the wild type. When incubated with ATP the Δarm mutant produced a lower level of the catalytic phosphoenzyme in amounts proportionate to the ATPase activity. These results indicate that the "arm-like" domain is not essential for hydrolytic activity and suggest that it is needed for the stabilization of Spf1p in a phosphorylation-ready conformation. • P5A-ATPases have been recently shown to be a new class of P-type ATPases with polypeptides as transported substrates. • P5A-ATPases exhibit a unique structural feature of P5A-ATPases described "arm-like domain" of unknown function. • The "arm-like domain" is not essential for ATP hydrolysis by the yeast Spf1p P5A-ATPase. • Deletion of the "arm-like domain" decreases autophosphorylation. [ABSTRACT FROM AUTHOR]