Autoinhibition of a Calmodulin-dependent Calcium Pump Involves a Structure in the Stalk That Connects the Transmembrane Domain to the ATPase Catalytic Domain

Abstract

The regulation of Ca 2؉ -pumps is important for controlling [Ca 2؉ ] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca 2؉ -pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca 2؉ -pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys 23 and Arg 54 . Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V max , K mATP , and K mCa 2؉ similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp 219 (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H ؉ -ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse Ptype ATPases.

Identification of a gain-of-function mutation in a Golgi P-type ATPase that enhances Mn 2+ efflux and protects against toxicity

“…There are a few known mutations of P-type ATPases that increase activity but these mutations relieve negative regulation to restore constitutive activity (34)(35)(36). The best-characterized example is for plasma membrane calcium ATPase (PMCA), which has an autoinhibitory domain that binds calmodulin (35,36).…”

Domain Organization and Movements in Heavy Metal Ion Pumps

“…Pmr1, a Golgi Ca 2ϩ /Mn 2ϩ -ATPase, has an EF-hand motif in the N-terminal part of the A-domain, presumably working as a Ca 2ϩ -dependent regulator of the ATPase (49). A plant Ca 2ϩ -ATPase (ACA2 from Arabidopsis) has an N-terminal regulatory domain, which interacts with the stalk region (50). Some heavy metal pumps have additional metal ion binding domains at the C terminus (5).…”

Calcium Pumps and Interacting BON1 Protein Modulate Calcium Signature, Stomatal Closure, and Plant Immunity

“…1D), likely because the N-terminal part of BON1 targets to the PM and inhibits the fusion protein from going to the nucleus to activate gene expression. Because the segment I of ACA10 contains the putative autoinhibitory motif that confers autoinhibition of many ACA proteins (Bonza et al, 2000;Curran et al, 2000;Geisler et al, 2000), BON1 may regulate ACA10 activity by binding to this motif or its nearby sequences. We found that ACA10, like BON1, is a negative regulator of plant immunity.…”