HMA6 and HMA8 are two chloroplast Cu+-ATPases with different enzymatic properties.

Copper (Cu) plays a key role in the photosynthetic process as cofactor of the plastocyanin (PC), an essential component of the chloroplast photosynthetic electron transfer chain. Encoded by the nuclear genome, PC is translocated in its apo-form into the chloroplast and the lumen of thylakoids where it is processed to its mature form and acquires Cu. In Arabidopsis, Cu delivery into the thylakoids involves two transporters of the PIB-1 ATPases family, heavy metal associated protein 6 (HMA6) located at the chloroplast envelope and HMA8 at the thylakoid membrane. To gain further insight into the way Cu is delivered to PC, we analysed the enzymatic properties of HMA8 and compared them with HMA6 ones using in vitro phosphorylation assays and phenotypic tests in yeast. These experiments reveal that HMA6 and HMA8 display different enzymatic properties: HMA8 has a higher apparent affinity for Cu(+) but a slower dephosphorylation kinetics than HMA6. Modelling experiments suggest that these differences could be explained by the electrostatic properties of the Cu(+) releasing cavities of the two transporters and/or by the different nature of their cognate Cu(+) acceptors (metallochaperone/PC).

HMA1 and PAA1, two chloroplast-envelope PIB-ATPases, play distinct roles in chloroplast copper homeostasis.

Copper is an essential micronutrient but it is also potentially toxic as copper ions can catalyse the production of free radicals, which result in various types of cell damage. Therefore, copper homeostasis in plant and animal cells must be tightly controlled. In the chloroplast, copper import is mediated by a chloroplast-envelope PIB-type ATPase, HMA6/PAA1. Copper may also be imported by HMA1, another chloroplast-envelope PIB-ATPase. To get more insights into the specific functional roles of HMA1 and PAA1 in copper homeostasis, this study analysed the phenotypes of plants affected in the expression of both HMA1 and PAA1 ATPases, as well as of plants overexpressing HMA1 in a paa1 mutant background. The results presented here provide new evidence associating HMA1 with copper homeostasis in the chloroplast. These data suggest that HMA1 and PAA1 behave as distinct pathways for copper import and targeting to the chloroplast. Finally, this work also provides evidence for an alternative route for copper import into the chloroplast mediated by an as-yet unidentified transporter that is neither HMA1 nor PAA1.