The unicellular green alga Chlamydomonas reinhardtii is a valuable model for studying metal metabolism in a photosynthetic background. A search of the Chlamydomonas expressed sequence tag database led to the identification of several components that form a copper-dependent iron assimilation pathway related to the high-affinity iron uptake pathway defined originally for Saccharomyces cerevisiae. They include a multicopper ferroxidase (encoded by Fox1), an iron permease (encoded by Ftr1), a copper chaperone (encoded by Atx1), and a copper-transporting ATPase. A cDNA, Fer1, encoding ferritin for iron storage also was identified. Expression analysis demonstrated that Fox1 and Ftr1 were coordinately induced by iron deficiency, as were Atx1 and Fer1, although to lesser extents. In addition, Fox1 abundance was regulated at the posttranscriptional level by copper availability. Each component exhibited sequence relationship with its yeast, mammalian, or plant counterparts to various degrees; Atx1 of C. reinhardtii is also functionally related with respect to copper chaperone and antioxidant activities. Fox1 is most highly related to the mammalian homologues hephaestin and ceruloplasmin; its occurrence and pattern of expression in Chlamydomonas indicate, for the first time, a role for copper in iron assimilation in a photosynthetic species. Nevertheless, growth of C. reinhardtii under copper-and iron-limiting conditions showed that, unlike the situation in yeast and mammals, where copper deficiency results in a secondary iron deficiency, copper-deficient Chlamydomonas cells do not exhibit symptoms of iron deficiency. We propose the existence of a copper-independent iron assimilation pathway in this organism.While iron is abundant in the environment, it is present in the insoluble ferric [Fe(III)] state, so that its bioavailability is low (16). Yet iron is an essential micronutrient for all organisms because it functions as a cofactor in enzymes that catalyze redox reactions in fundamental metabolic processes. Iron exhibits stable, redox-interchangeable ionic states with the potential to generate less stable electron-deficient intermediates during multielectron redox reactions involving oxygen chemistry (16). Therefore, organisms are challenged with the acquisition of sufficient iron to meet cellular metabolic requirements while avoiding uncontrolled intracellular chemistry. This is accomplished via the operation of iron homeostatic mechanisms. The essential features of iron metabolism include assimilation and distribution, storage and sequestration, and utilization and allocation. The assimilatory pathway can be further subdivided into reduction of insoluble ferric species to more soluble ferrous species and uptake into the cell, followed by intracellular transport and intraorganellar distribution. The storage and sequestration of iron involve loading of cellular proteins as well as compartmentalization into organelles like vacuoles and plastids, which in turn requires proteins for transport into and out of these compartm...

Chlamydomonas reinhardtii adapts to copper deficiency by degrading apoplastocyanin and inducing Cyc6 and Cpx1 encoding cytochrome c6 and coproporphyrinogen oxidase, respectively. To identify other components in this pathway, colonies resulting from insertional mutagenesis were screened for copper‐ conditional phenotypes. Twelve crd (copper response defect) strains were identified. In copper‐deficient conditions, the crd strains fail to accumulate photosystem I and light‐harvesting complex I, and they contain reduced amounts of light‐harvesting complex II. Cyc6, Cpx1 expression and plastocyanin accumulation remain copper responsive. The crd phenotype is rescued by a similar amount of copper as is required for repression of Cyc6 and Cpx1 and for maintenance of plastocyanin at its usual stoichiometry, suggesting that the affected gene is a target of the same signal transduction pathway. The crd strains represent alleles at a single locus, CRD1, which encodes a 47 kDa, hydrophilic protein with a consensus carboxylate‐bridged di‐iron binding site. Crd1 homologs are present in the genomes of photosynthetic organisms. In Chlamydomonas, Crd1 expression is activated in copper‐ or oxygen‐deficient cells, and Crd1 function is required for adaptation to these conditions.

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Crd1 (Copper response defect 1), which is required for the maintenance of photosystem I and its associated light-harvesting complexes in copper-deficient (-Cu) and oxygen-deficient (-O(2)) Chlamydomonas reinhardtii cells, is localized to the thylakoid membrane. A related protein, Cth1 (Copper target homolog 1), is shown to have a similar but not identical function by genetic suppressor analysis of gain-of-function sct1 (suppressor of copper target 1) strains that are transposon-containing alleles at CTH1. The pattern of Crd1 versus Cth1 accumulation is reciprocal; Crd1 abundance is increased in -Cu or -O(2) cells, whereas Cth1 accumulates in copper-sufficient (+Cu), oxygenated cells. This expression pattern is determined by a single trans-acting regulatory locus, CRR1 (COPPER RESPONSE REGULATOR 1), which activates transcription in -Cu cells. In +Cu cells, a 2.1-kb Cth1 mRNA is produced and translated, whereas Crd1 is transcribed only at basal levels, leading to Cth1 accumulation in +Cu cells. In -Cu cells, CRR1 function determines the activation of Crd1 expression and the production of an alternative 3.1-kb Cth1 mRNA that is extended at the 5' end relative to the 2.1-kb mRNA. Synthesis of the 3.1-kb mRNA, which encodes six small upstream open reading frames that possibly result in poor translation, blocks the downstream promoter through transcriptional occlusion. Fluorescence analysis of wild-type, crd1, and sct1 strains indicates that copper-responsive adjustment of the Cth1:Crd1 ratio results in modification of the interactions between photosystem I and associated light-harvesting complexes. The tightly coordinated CRR1-dependent regulation of isoenzymes Cth1 and Crd1 reinforces the notion that copper plays a specific role in the maintenance of chlorophyll proteins.

In Chlamydomonas reinhardtii, cytochrome c6 (cyt c6) is synthesized only under conditions of copper deficiency when plastocyanin cannot be synthesized. In previous work, the copper-responsive regulation of cyt c6 synthesis was demonstrated to occur by control of transcription, with no contribution from post-transcriptional processes. To understand the mechanism underlying its regulation, the genomic DNA encoding cyt c6 (Cyc6) was analyzed for the presence of copper-responsive elements. Sequences lying between positions -127 and -7 with respect to the start site of transcription were found to be sufficient to confer copper-responsive expression on either a promoterless or a minimal beta-tubulin promoter-driven (arylsulfatase-encoding) reporter gene. Analysis of this 120-bp fragment indicated that copper-responsive elements lie in two distinct regions (between -110 to -56 and -127 to -109). ATG fusions between copper-insensitive promoters and the coding plus 3' untranslated region of the Cyc6 gene resulted in the accumulation of cyt c6 in copper-supplemented medium; this confirms earlier studies indicating a lack of post-transcriptional control in this copper-responsive pathway. In the context of a constitutive promoter (derived from the beta-tubulin gene), each region was found to function as an activator of transcription in copper-deficient cells, and the metal specificity of the response of reporter genes containing either one or both regions was identical to that of the endogenous Cyc6 gene. The copper-responsive synthesis of cyt c6 is thus attributed to these two 5' upstream sequences.