Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis

Abstract

+) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. We present a previously unidentified Cu + chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu + chaperone delivers Cu + with specificity for PAA1, which is flipped in the envelope relative to prototypical bacterial ATPases, compatible with a role in Cu + import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu + -delivery mechanism and provides a unique snapshot into the evolution of a Cu + distribution pathway. We also provide evidence for an interaction between PAA2, the Cu + -ATPase in thylakoids, and the Cu + -chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu + network that has evolved to fine-tune Cu + distribution.Cu-transfer | inner envelope | metal transporter | Arabidopsis thaliana | Atx1

Identification of Two Conserved Residues Involved in Copper Release from Chloroplast PIB-1-ATPases

“…In these photosynthetic bacteria, from which chloroplasts originated, CtaA is found at the plasma membrane, whereas PacS is located at the thylakoids respectively. In plants, HMA6 and HMA8 are functional homologs of CtaA and PacS but exclusively co-orthologs of CtaA (7,27). In agreement, our focus on the sole TMA-L1-TMB region shows that with a short HX 2 H motif in TMA, CtaA belongs to group C, whereas PacS, lacking this motif, is found in the group A.…”

“…Copper delivery to apoplastocyanin involves two transporters of the P-ATPases family, HMA6 and HMA8, located at the chloroplast envelope (3,4) and the thylakoid membranes (5,6), respectively. Both transporters are high affinity transporters for monovalent copper (7)(8)(9). HMA6 and HMA8 loss of function mutations strongly reduce chloroplast copper content, in the stroma for the hma6 mutant and in thylakoids for the hma8 mutant.…”

“…Several steps of copper transport from the cytosol to the lumen of thylakoids have been identified. The copper chaperone PCH1, located in the intermembrane space of the chloroplast envelope, could bring copper to HMA6 (7). HMA6 would then transfer copper in the stroma to the chaperone CCS for the activation of the copper/zinc superoxide dismutase (4,10,11).…”

“…However, this interaction has never been experimentally validated. Although CCS can interact and transfer copper to HMA8 in vitro (7), in planta studies of a ccs mutant suggest that another chaperone might be involved in the transport of copper from HMA6 to HMA8 (12). So far, the mechanism of copper transfer from HMA8 to plastocyanin is not known.…”

Membrane Anchoring and Ion-Entry Dynamics in P-type ATPase Copper Transport

“…Mutagenesis and copper binding experiments have suggested two intramembranous ion-binding sites for AfCopA formed by a set of invariant residues (site I: Cys382, Cys384, and Tyr688; site II: Tyr688, Asn689, Met717, and Ser721) (16). Similarly, a two-ion stoichiometry per protein was observed in plant Cu þ ATPases (28). However, a recent study of LpCopA suggested a combination of a transient site partially exposed to the cytoplasmic membrane-water interface and a subsequent single TM high-affinity metal-binding site (14).…”