Single Point Mutations in the Small Cytoplasmic Loop of ACA8, a Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana, Generate Partially Deregulated Pumps

Abstract

ACA8 is a type 2B Ca2؉ -ATPase having a regulatory N terminus whose auto-inhibitory action can be suppressed by binding of calmodulin (CaM) or of acidic phospholipids. ACA8 N terminus is able to interact with a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To determine the role of this interaction in auto-inhibition we analyzed single point mutants produced by mutagenesis of ACA8 ) renders an enzyme that is less dependent on CaM for activity. These results highlight the relevance in ACA8 auto-inhibition of a negative charge of the surface area of the small cytoplasmic loop. The most deregulated of these mutants is D291A ACA8, which is less activated by controlled proteolysis or by acidic phospholipids; the D291A mutant has an apparent affinity for CaM higher than wildtype ACA8. Moreover, its phenotype is stronger than that of D291N ACA8, suggesting a more direct involvement of this residue in the mechanism of auto-inhibition. Among the other produced mutants (I284A, N286A, P289A, P322A, V344A, and N345A), only P322A ACA8 is less dependent on CaM for activity than the wild type. The results reported in this study provide the first evidence that the small cytoplasmic loop of a type 2B Ca 2؉

ACA12 is a deregulated isoform of plasma membrane Ca2+-ATPase of Arabidopsis thaliana

“…However, to evaluate this possibility, we purified ACA12 from yeast using CaM-affinity chromatography (Bonza et al 1998; Bonza et al 2000; Fusca et al 2009; Bonza and Luoni 2010), and tested its biochemical properties.…”

“…Microsomal fraction was isolated as previously reported (Fusca et al 2009) except that yeast cells were lysed using a Cell Disruptor (Constant Systems Ltd) operating at 27 psi. Protein concentration was determined using the Bio-Rad assay (Bio-Rad) with γ-globulin as standard.…”

“…CaM-affinity purification was performed as described (Fusca et al 2009) with slight modifications. Proteins (100 mg) from yeast microsomes expressing ACA12 or ACA12-GFP were incubated with n -dodecyl β-D-maltoside (4 mg detergent ml −1 : 4 mg protein ml −1 ) at 25 °C for 30 min in a solubilization medium containing 5% (v/v) glycerol, 50 mM Tris-HCl, pH 7.5, 1 mM p -aminobenzamidine, 2 mM DTT, 1.5 mM ATP, 2 mM CaCl 2 , 1mM MgSO 4 , 125 mM NaCl, supplemented with 5 µg ml −1 leupeptin, 5 µg ml −1 pepstatin, 5 µg ml −1 chymostatin immediately before use.…”

“…The sample was then centrifuged 1 h at 110,000 g at 4°C; the supernatant recovered and incubated overnight under gentle rotation at 4 °C with 0.8 ml of CaM-Sepharose (GeHealthcare) previously equilibrated with solubilization medium supplemented with 37.5 µg ml −1 Brij 58. After removal of the unbound fraction, the resin was washed with 10 ml of washing medium containing 10% (v/v) glycerol, 20 mM Mops-KOH, pH 7.5, 1 mM p -aminobenzamidine, 2 mM DTT, 0.25 mM NaBr, 37.5 µg ml −1 Brij 58, 5 µg ml −1 leupeptin, 5 µg ml −1 pepstatin, 5 µg ml −1 chymostatin, 100 µM CaCl 2 , 100 µM MgSO 4 ; second and third washes were performed in the same medium but in the absence of CaCl 2 and MgSO 4 (Bonza et al 1998; Fusca et al 2009). CaM-bound proteins were eluted in 2.5 ml of 5 mM Mops-KOH, pH 7.5, 37.5 µg ml −1 Brij 58, 10% (v/v) glycerol and 2 mM EGTA, followed by 1.5 ml of the same solution.…”

“…Their N-terminal regions are highly divergent compared to those of other ACAs; moreover they have an Asn (N211 in ACA12) and an Arg (R334 in ACA12) at positions close to transmembrane domain (TM) 2 and 3 respectively, where all other ACAs - as well as animal PM Ca 2+ -ATPases - have an acidic residue. In different ACA isoforms, as well as in an animal pump isoform, mutation of these acidic residues generates deregulated pumps that show near full activity without further activation by CaM (Curran et al 2000; Bredeston and Adamo 2004; Fusca et al 2009). …”