7. Project Summary / Abstract Copper (Cu) is an essential micronutrient for nearly all forms of aerobic life because of its participation in key redox reactions and reactions of O2 chemistry. Cu deficiency, accordingly, has an impact on multiple metabolic and developmental pathways. Cu deficiency in animals and humans occurs because of genetic defects or in situations of malnutrition. In previous work, Merchant and co-workers, using a model organism, Chlamydomonas (an alga in the green lineage), discovered fundamental mechanisms and regulatory circuits for maintaining Cu homeostasis that are broadly relevant. Specifically, the group showed that an abundant Cu protein, plastocyanin, is replaced by an iron-containing heme protein, cytochrome (Cyt) c6, when Cu is scarce. The Cu that is spared by this replacement is “upcycled” for use in Cyt oxidase in respiration. This switch allows maintenance of two important bioenergetic pathways – photosynthesis and respiration. Genetic analysis identified a Cu-response regulator (CRR1), a transcription factor that activates the CYC6 gene encoding Cyt c6 through associated Cu response elements (CuREs) as well as ~64 other genes that form the nutritional Cu regulon, including assimilatory Cu(I) transporters CTR1 and CTR2, enzymes of heme biosynthesis, and other factors that enable Cu allocation to Cyt oxidase and that adjust thylakoid membrane properties to accommodate the substitute protein. In this project period, the focus is on how CRR1 is turned off for tight homeostatic regulation of Cu metabolism. In the working model, supported by substantial preliminary data, CRR1 activates the Cu regulon in deficiency, while upon Cu supplementation, the protein is post-translationally modified, which marks CRR1 for recognition and ubiquitylation by CEHC1/FBXO3, a newly discovered F-box protein, followed by proteasome- dependent degradation. In this context, there are 3 aims. 1) Post-translational modifications of CRR1 in +Cu cells will be captured by LC-MS following immunoprecipitation of CRR1 under specific conditions where the protein accumulates, as in the cehc1 mutant. The site(s) of modification will be tested for their impact on Cu- responsive degradation of CRR1, individually and in combination, by site-directed mutagenesis of CRR1. 2) Physical interaction between CEHC1 and CRR1 will be tested by co-localization and captured by co- immunoprecipitation, and the substrate binding domain of CEHC1 will be identified by mutation and tested in vitro for binding to the Cu dependent degron. Candidate CRR1 modification enzymes or other components in this regulatory circuit, such as a Cu sensor, will be discovered by exploiting a powerful gain-of-function classical genetic screen for constitutively active CRR1 using CuRE-reporter constructs. 3) The operation of a post- translational mechanism to regulate Cu uptake by CTRs will be evaluated by using quantitative proteomics to measure Cu-dependent changes in half-life and by...