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Plastocyanin and cytochrome c6, abundant proteins in photosynthesis, are readouts for cellular copper status in Chlamydomonas and other algae. Their accumulation is controlled by a transcription factor copper response regulator (CRR1). The replacement of copper-containing plastocyanin with heme-containing cytochrome c6 spares copper and permits preferential copper (re)-allocation to cytochrome oxidase. Under copper-replete situations, the quota depends on abundance of various cuproproteins and is tightly regulated, except under zinc-deficiency where acidocalcisomes over-accumulate Cu(I). CRR1 has a transcriptional activation domain, a Zn-dependent DNA binding SBP-domain with a nuclear localization signal, and a C-terminal Cys-rich region that represses the zinc regulon. CRR1 activates >60 genes in Chlamydomonas through GTAC-containing CuREs; transcriptome differences are recapitulated in the proteome. The differentially-expressed genes encode assimilatory copper transporters of the CTR/SLC31 family including a novel soluble molecule, redox enzymes in the tetrapyrrole pathway that promote chlorophyll biosynthesis and photosystem 1 accumulation, and other oxygen-dependent enzymes, which may influence thylakoid membrane lipids, specifically polyunsaturated galactolipids and γ-tocopherol. CRR1 also down-regulates 2 proteins in Chlamydomonas: for plastocyanin, by activation of proteolysis, while for the di‑iron subunit of the cyclase in chlorophyll biosynthesis, through activation of an upstream promoter that generates a poorly-translated 5' extended transcript containing multiple short ORFs that inhibit translation. The functions of many CRR1-target genes are unknown, and the copper protein inventory in Chlamydomonas includes several whose functions are unexplored. The comprehensive picture of cuproproteins and copper homeostasis in this system is well-suited for reverse genetic analyses of these under-investigated components in copper biology.

Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retention of genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper.

We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.