Regulation Of The Auto-inhibition Activity Of Plasma Membrane H⁺-ATPase By Ca²⁺Sensor SCaBP3 In Arabidopsis Thaliana

Plasma membrane(PM)H+-ATPases play an important role in the establishment of trans-plasma membrane electrochemical proton gradient to support ion and metabolite transport and maintain the intra-and extracellular pH balance.It belongs to a large family of ion transporters named P-type ATPases.In plants,PM H+-ATPases are also one of the most critical regulators in responding to hormones and environment stimuli,including,for example,abscisic acid(ABA),calcium,blue light,fungal elicitors and increased soil salinization.Their C-terminal autoinhibitory domain(R domain)is pivotal for the regulation of PM H+-ATPase activity,which involves in the(de)phosphorylation of different sites within the R-domain and interacting proteins.Besides,based on genetic studies in yeast,it has been hypothesized that the autoinhibition of the PM H-ATPase activity is achieved by intramolecular interaction of the R-domain with other domain(s)of the pump,and that somehow mutual relationships between the N terminus and C terminus are required for this autoinhibition.However,the detailed mechanism that would convey such intramolecular regulation has remained elusive.Here,we identified a SOS3-like calcium binding protein,SCaBP3,as a negative regulator of the PM H'-ATPase,which repressed the enzyme activity by physically interacting with its C terminus.Consistently,Arabidopsis SCaBP3 loss-of-function plants exhibited increased PM H-ATPase activity.Subsequently,we found that the C-terminal autoinhibition of the PM H+-ATPase activity was partially achieved by the interaction between its C terminus and Centerloop,and the interaction of SCaBP3 with the RI domain of the C terminus further enhanced this interaction in firefly luciferase complementation imaging(LCI)and yeast three-hybrid assays,which in turn promoted this autoinhibition to the PM H-ATPase activity.Furthermore,SCaPB3 interacted with the SOS2-like protein kinase 5(PKS5),a negative regulator of PM H-ATPase,and also enhanced the interaction between PM H+-ATPase AHA2 and PKS5 thereby further enhancing or stabilizing PKS5 mediated repression of PM H+-ATPase activity in yeast.In order to determine a possible physiological function of SCaBP3 in regulating PM H+-ATPase activity and verify whether SCaBP3 augments PKS5 mediated inhibition of the PM H+-ATPase in plants,we generated and analyzed the tolerance of the scabp3 and pks5 scabp3 mutants under saline-alkaline conditions and further determined the change of PM H-ATPase activity.These results showed that deficiency of SCaBP3 enhanced the saline-alkaline resistance in the knockout mutant pks5-1 and rescued the saline-alkaline sensitive in constitutively active kinase mutants pks5-3 and pks5-4,which supports the notion that SCaBP3 regulates plant saline-alkaline resistance in both PKS5 dependent and independent processes.Taken together,our results suggest that the C-terminal autoinhibition of PM H+-ATPase is at least partially established through directly interacting with the Centerloop region,and that the SCaBP3 plays a critical role in enhancing or stabilizing the repression of PM H+-ATPase activity by two distinct mechanisms which simultaneously promotes the interaction between the C terminus and its Centerloop region and between the C terminus and PKS5 via a intramolecular or intermolecular manner respectively.