Arabidopsis thaliana plasma membrane Ca2+-ATPase isoform 8: molecular analysis of the autoinhibitory mechanism using chimeras

ACA8 is a plasma membrane (PM) Ca2+-ATPase of A. thaliana which has an extended N-te containing both an autoinhibitory domain and a calmodulin (CaM) binding site: CaM binding suppresses autoinhibition. This regulatory mechanism is similar to that of animal PM Ca2+ ATPase and plant PM H+-ATPase, but in these proteins the regulatory site is localised in the C-te. To analyse in details the autoinbitory mechanism, mutants in which the N-te of ACA8 is inserted at the C-te of the protein have been constructed using the cDNA of CaM insensitive mutant ï „74-ACA8. Moreover, chimeras in which ï „74-ACA8 is fused to the C-te domains of AHA1 (isoform 1 of A. thaliana PM proton pump) or of PMCA4b (isoform 4b of human PM Ca2+-ATPase) have been produced. All mutants expressed in the S. cerevisiae strain K616 are functional. Results show that repositioning the N-te region of ACA8 to the C-te of the protein does not interfere with its ability to autoinhibit the pump, thus the regulatory function of the terminal domain is independent from its position in ACA8. A detailed characterisation of the chimeras analysing their activity in presence of 14-3-3 and fusicoccin or CaM is in progress