Autoinhibition of a calmodulin-dependent calcium pump involves a structurein the stalk that connects the transmembrane domain to the ATPase catalytic domain

The regulation of Ca2+-pumps is important for controlling [Ca2+] in the cyt osol and organelles of all eukaryotes. Here, we report a genetic strategy t o identify residues that function in autoinhibition of a novel calmodulin-a ctivated Ca2+-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by co mplementation of a yeast (K616) deficient in two Ca2+-pumps. Fifteen mutati ons were found that disrupted a segment of the N-terminal autoinhibitor loc ated between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341R) were found associated with the stalk that connects the ATPase catalytic do main (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V-ma x K-mATP, and K(mCa)2+ similar to that of a pump in which the N-terminal au toinhibitor had been deleted. A highly conservative substitution at Asp(219 ) (D219E) still produced a deregulated pump, indicating that the autoinhibi tory structure in the stalk is highly sensitive to perturbation. In plasma membrane H+-ATPases from yeast and plants, similarly positioned mutations r esulted in hyperactive pumps. Together, these results suggest that a struct ural feature of the stalk is of general importance in regulating diverse P- type ATPases.