Two Chlamydomonas CTR Copper Transporters with a Novel Cys-Met Motif Are Localized to the Plasma Membrane and Function in Copper Assimilation

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Abstract
Inducible high-affinity copper uptake is key to copper homeostasis in Chlamydomonas reinhardtii. We generated cDNAs and updated gene models for four genes, CTR1, CTR2, CTR3, and COPT1, encoding CTR-type copper transporters in Chlamydomonas. The expression of CTR1, CTR2, and CTR3 increases in copper deficient cells and in response to hypoxia or Ni2+ supplementation; this response depends on the transcriptional activator CRR1. A copper response element was identified by mutational analysis of the 5′ upstream region of CTR1. Functional analyses identify CTR1 and CTR2 as the assimilatory transporters of Chlamydomonas based on localization to the plasma membrane and ability to rescue a Saccharomyces cerevisiae mutant defective in high-affinity copper transport. The Chlamydomonas CTRs contain a novel Cys-Met motif (CxxMxxMxxC-x5/6-C), which occurs also in homologous proteins in other green algae, amoebae, and pathogenic fungi. CTR3 appears to have arisen by duplication of CTR2, but CTR3 lacks the characteristic transmembrane domains found in the transporters, suggesting that it may be a soluble protein. Thus, Chlamydomonas CTR genes encode a distinct subset of the classical CTR family of Cu(I) transporters and represent new targets of CRR1-dependent signaling.