The general questions: In 1979, Paul Wood noted that Chlamydomonas cells could use either plastocyanin (a blue copper protein) or cytochrome c6 (a heme protein) for photosynthesis in vivo; the choice between them is determined by the supply of copper in the growth medium. If copper is provided, Chlamydomonas cells synthesize and accumulate plastocyanin; if the medium is copper-deficient, cytochrome c6 is produced instead. The organism remains photosynthetically competent regardless of the supply of copper in its growth environment. This is one of the most elegant examples of metal-responsive adaptation of a biochemical pathway, and my group set out to understand how it works. What is the signal? Is the cell measuring copper availability directly, or is it responding to a deficiency in photosynthetic electron flow? What are the targets of the signal transduction pathway (besides plastocyanin and cytochrome c6)? How is the response implemented? What is the sensor and how does it communicate with the targets? Can we understand the gene regulation phenomena in a cellular/physiological context? Dissection of this regulatory circuit in a system where it is possible to link molecular events with metabolic physiology is key to the development of fundamental principles underlying trace element homeostasis and metabolism.

        Some answers: Plastocyanin and cytochrome c6 are each encoded by a single nuclear gene in Chlamydomonas reinhardtii (13,15). When copper availability is adequate to allow the synthesis of the full complement of plastocyanin (~ 8 x 10E6 molecules per cell), the Cyc6 gene encoding cytochrome c6 is transcriptionally silent (18). In copper-depleted medium (< 8 x 10E6 Cu per cell), the Cyc6 gene is induced to allow cytochrome c6 synthesis to the extent - and only to the extent -- required to compensate for the plastocyanin deficiency (23). Plastocyanin deficiency in copper-depleted medium results from a block at the level of holoplastocyanin formation in the thylakoid lumen. The pre-protein is synthesized, targeted to the organelle and processed in the usual two-step pathway but lack of copper in the lumen prevents holoplastocyanin formation. Under these conditions, the apoprotein is degraded rapidly. The short half-life (under 20 minutes) is attributed not only to the increased proteolytic susceptibility of the apoprotein, which results probably from the reduced stability of the apopolypeptide, but also to activation of a protease in copper-deficient cells (34). Induction of the Cyc6 gene occurs by transcriptional activation in direct response to copper-deficiency (33). This is mediated by at least two copper-response element (CuREs) - each contains at its core the sequence GTAC and each can function independently in the context of a reporter gene (Ars2). It is possible that the dynamic range of the response in vivo is ensured by the occurrence of multiple elements.

        Besides plastocyanin and cytochrome c6 biosynthesis, there are several other adaptations to copper-deficiency (32,37). Each of these -- transcriptional activation of the Cpx1 gene encoding coproporphyrinogen oxidase, activation of copper transport and a cell surface cupric reductase -- occurs in coordination with Cyc6 induction. Specifically, these responses display the same metal selectivity (Cu > Hg >> Ag) and sensitivity (nanomolar range) as does the Cyc6 gene and are targets of the same copper-sensing signal transduction pathway. The product of the Cpx1 gene is required in copper-supplemented as well as copper-deficient cells; therefore, unlike Cyc6, the gene is transcribed constitutively. In copper-replete cells, two transcripts accumulate; in copper-depleted cells, a third transcript with a distinct 5’ end accumulates. Depending on the severity of copper deficiency, the -Cu form of the transcript can accumulate to 35-times the abundance of the two constitutive forms resulting in a large increase in the amount of coproporphyrinogen oxidase in -Cu cells. Transcriptional activation occurs in direct response to copper-sensing rather than to feedback regulation from the tetrapyrrole pathway. Analysis of Cpx1-Ars2 reporter gene reveals only a single CuRE: the sequence GTAC is critical for its activity (48).

        With a view to identifying additional targets of the copper-sensing system and also the signal transduction components, we began a genetic screen for copper-conditional growth defects, leading to the discovery of the CRT1 (for copper response target) and CRR1 (for copper response regulator) loci. The crt1 mutants display a chlorophyll deficiency and loss of photosystem I exclusively under copper-deficient conditions. The pattern of plastocyanin, cytochrome c6 and coprogen oxidase accumulation is normal in all crt1 strains indicating that the CRT1 does not encode a regulatory component; rather we propose that it defines a hitherto unknown aspect of photosystem I biochemistry.  The crr1 mutants are likely to be regulatory mutants because they do not induce Cyc6 and Cpx1 and are unable to grow in copper-deficient medium.

        The reciprocal copper-responsive accumulation of plastocyanin and cytochrome c6 is viewed as an adaptive mechanism, which allows the organism to survive occasional copper-deficiency - for instance, during an algal bloom. Since plastocyanin is the major sink for copper in the green algae (which lack a Cu/Zn superoxide dismutase and other abundant copper proteins), its degradation in copper-deficiency would ensure the re-distribution of this essential trace element to cytochrome oxidase in the respiratory pathway. Indeed, loss of plastocyanin precedes loss of cytochrome oxidase as Chlamydomonas reinhardtii cells become copper-deficient.

        Specific questions for the immediate future: The immediate objective of our ongoing studies in Chlamydomonas is to understand the molecular basis of copper measurement by the cell through the identification of the copper-sensor and the signal transduction components. Our work suggests that copper utilization and distribution is controlled tightly and by specific regulatory mechanisms so that it is provided where it is most needed for biochemical metabolism. We will identify copper-handling components in Chlamydomonas (such as assimilatory transporters, delivery molecules, and chaperones) through functional complementation of appropriate Saccharomyces mutants with the intent of monitoring intracellular copper flux in response to supply. The repertoire of molecular and genetic tools at our disposal will be applied to build a dynamic picture of homeostatic copper metabolism in a eukaryotic cell. The recent discovery of copper chaperones and delivery mechanisms in Saccharomyces cerevisiae enables us to test the general applicability of our model. Specifically, we will determine the hierarchy of distribution of copper to the respiratory pathway (cytochrome oxidase) vs. the oxidative stress pathway (superoxide dismutase) in Saccharomyces. More recently, we have discovered an oxygen-responsive signal transduction pathway in Chlamydomonas, which may use copper chemistry to sense the intracellular redox level. We are interested in understanding the physiological basis for the response as well as the molecular constituents of what could be a unique oxygen sensor.

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