Distinct function of Chlamydomonas CTRA-CTR transporters in Cu assimilation and intracellular mobilization

Abstract

ABSTRACTSuccessful acclimation to copper (Cu) deficiency involves a fine balance between Cu import and export. In the unicellular green algaChlamydomonas reinhardtii, Cu import is dependent onCopperResponseRegulator1(CRR1), the master regulator of Cu homeostasis. Among CRR1 target genes are two Cu transporters belonging to the CTR/COPT gene family (CTR1andCTR2) and a related soluble cysteine-rich protein (CTR3). The ancestor of these green algal proteins was likely acquired from an ancient chytrid and contained conserved cysteine-rich domains (named the CTR-associated domains, CTRA) that are predicted to be involved in Cu acquisition. We show by reverse genetics that Chlamydomonas CTR1 and CTR2 are canonical Cu importers albeit with distinct affinities, while loss of CTR3 did not result in an observable phenotype under the conditions tested. Mutation ofCTR1, but notCTR2, recapitulate the poor growth ofcrr1in Cu-deficient medium, consistent with a dominant role for CTR1 in high affinity Cu(I) uptake. Notably, the over-accumulation of Cu(I) in Zinc (Zn)-deficiency (20 times the quota) depends on CRR1 and both CTR1 and CTR2. CRR1-dependent activation ofCTRgene expression needed for Cu over-accumulation can be bypassed by the provision of excess Cu in the growth medium. Over-accumulated Cu is sequestered into the acidocalcisome but can become remobilized by restoring Zn nutrition. This mobilization is also CRR1-dependent, and requires activation ofCTR2expression, again distinguishing CTR2 from CTR1 and is consistent with the lower substrate affinity of CTR2.