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A Genome Wide Copper-Sensitized Screen Identifies Novel Regulators of Mitochondrial Cytochrome C Oxidase Activity

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A Genome Wide Copper-Sensitized Screen Identifies Novel Regulators of Mitochondrial Cytochrome C Oxidase Activity bioRxiv preprint doi: https://doi.org/10.1101/2020.12.31.424969; this version posted January 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Genetic regulators of mitochondrial copper A genome wide copper-sensitized screen identifies novel regulators of mitochondrial cytochrome c oxidase activity Natalie M. Garza1, Aaron T. Griffin1,2, Mohammad Zulkifli1, Chenxi Qiu1,3, Craig D. Kaplan1,4, Vishal M. Gohil*1 1Department of Biochemistry and Biophysics, MS 3474, Texas A&M University, College Station, TX 77843, USA 2Present Address: Department of Systems Biology, Columbia University, New York, NY 10032, USA 3Present Address: Department of Medicine, Division of Translational Therapeutics, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA 4Present Address: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA *To whom the correspondence should be addressed: Vishal M. Gohil, 301 Old Main Drive, MS 3474, Texas A&M University, College Station, TX 77843 USA; Email: [email protected]; Tel: (979) 847-6138; Fax: (979) 845-9274 Running Title: Genetic regulators of mitochondrial copper Keywords: Copper, mitochondria, vacuole, cytochrome c oxidase, pH, AP-3, Rim20, Rim21 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.31.424969; this version posted January 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Genetic regulators of mitochondrial copper ABSTRACT For example, copper is essential for the activity of cytochrome c oxidase (CcO), the Copper is essential for the activity and evolutionarily conserved enzyme of the stability of cytochrome c oxidase (CcO), the mitochondrial respiratory chain and the terminal enzyme of the mitochondrial main site of cellular respiration (2). CcO respiratory chain. Loss-of-function metalation requires transport of copper to mutations in genes required for copper mitochondria followed by its insertion into transport to CcO result in fatal human Cox1 and Cox2, the two copper-containing disorders. Despite the fundamental subunits of CcO (3). Genetic defects that importance of copper in mitochondrial and prevent copper delivery to CcO disrupt its organismal physiology, systematic assembly and activity resulting in rare but characterization of genes that regulate fatal infantile disorders (4, 5, 6). mitochondrial copper homeostasis is lacking. To identify genes required for Intracellular trafficking of copper poses a mitochondrial copper homeostasis, we challenge because of the high reactivity of performed a genome-wide copper- this transition metal. Copper in an aqueous sensitized screen using DNA barcoded environment of the cell can generate yeast deletion library. Our screen recovered deleterious reactive oxygen species via a number of genes known to be involved in cellular copper homeostasis while revealing Fenton chemistry (7) and can inactivate genes previously not linked to mitochondrial other metalloproteins by mismetallation (8). copper biology. These newly identified Consequently, organisms must tightly genes include the subunits of the adaptor control copper import and trafficking to protein 3 complex (AP-3) and components subcellular compartments to ensure proper of the cellular pH-sensing pathway- Rim20 cuproprotein biogenesis while preventing its and Rim21, both of which are known to toxicity. Indeed, aerobic organisms have affect vacuolar function. We find that AP-3 evolved highly conserved proteins to import and the Rim mutants impact mitochondrial and distribute copper to cuproenzymes in CcO function by maintaining vacuolar cells (9). Extracellular copper is imported by acidity. CcO activity of these mutants could plasma membrane copper transporters and be rescued by either restoring vacuolar pH is immediately bound to metallochaperones or by supplementing growth media with Atx1 and Ccs1 for its delivery to different additional copper. Consistent with these cuproenzymes residing in the Golgi and genetic data, pharmacological inhibition of cytosol, respectively (10). the vacuolar proton pump leads to decreased mitochondrial copper content However, copper transport to the and a concomitant decrease in CcO mitochondria is not well understood. A non- abundance and activity. Taken together, our proteinaceous ligand, whose molecular study uncovered a number of novel genetic identity remains unknown, has been regulators of mitochondrial copper proposed to transport cytosolic copper to homeostasis and provided a mechanism by the mitochondria (3), where it is stored in which vacuolar pH impacts mitochondrial the matrix (11). This mitochondrial matrix respiration through copper homeostasis. pool of copper is the main source of copper ions that are delivered to CcO subunits in a INTRODUCTION particularly complex process requiring multiple metallochaperones and thiol Copper is an essential trace metal that reductases (3, 12, 13). Specifically, copper serves as a cofactor for a number of from the mitochondrial matrix is exported to enzymes in various biochemical processes, the intermembrane space via a yet including mitochondrial bioenergetics (1). 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.31.424969; this version posted January 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Genetic regulators of mitochondrial copper unidentified transporter, where it is inserted RESULTS into the CcO subunits by metallochaperones A genome-wide copper-sensitized Cox17, Sco1, and Cox11 that operate in a screen using barcoded yeast deletion bucket-brigade manner (13). The copper- mutant library transporting function of metallochaperones We chose the yeast, Saccharomyces requires disulfide reductase activities of cerevisiae, to screen for genes that impact Sco2 and Coa6, respectively (14, 15). mitochondrial copper homeostasis because it can tolerate mutations that inactivate In addition to the mitochondria, vacuoles in mitochondrial respiration by surviving on yeast and vacuole-like lysosomes in higher glycolysis. This enables the discovery of eukaryotes have been identified as critical novel regulators of mitochondrial copper storage sites and regulators of cellular metabolism whose knockout is expected to copper homeostasis (16-18). Copper enters result in a defect in aerobic energy the vacuole by an unknown mechanism and is generation (25). Yeast cultured in glucose- proposed to be stored as Cu(II) coordinated to containing media (YPD) uses glycolytic polyphosphate (19). Depending on the cellular fermentation as the primary source for requirement, vacuolar copper is reduced to cellular energy, however in glycerol/ethanol- Cu(I), allowing its mobilization and export containing non-fermentable media (YPGE), through Ctr2 (20, 21). Currently, the yeast must utilize the mitochondrial complete set of factors regulating the respiratory chain and its terminal distribution of copper to mitochondria cuproenzyme, CcO, for energy production. remains unknown. Based on the nutrient-dependent utilization of different energy-generating pathways, we Here, we sought to identify regulators of expect that deletion of genes required for mitochondrial copper homeostasis by respiratory growth will specifically reduce exploiting the copper requirement of CcO in growth in non-fermentable (YPGE) medium a genome-wide screen using a barcoded but will not impair growth of those mutants yeast deletion library. Our screen was in fermentable (YPD) medium. Moreover, if motivated by prior observations that respiratory deficiency in yeast mutants is respiratory growth of yeast mutants such as caused by defective copper delivery to coa6Δ can be rescued by copper mitochondria, then these mutants may be supplementation in the media (22-24). Thus, amenable to rescue via copper we designed a copper-sensitized screen to supplementation in YPGE respiratory identify yeast mutants whose growth can be growth media (Fig. 1). Therefore, to identify rescued by addition of copper in the media. genes required for copper-dependent Our screen recovered Coa6 and other respiratory growth, we cultured the yeast genes with known roles in copper deletion mutants in YPD and YPGE with or metabolism while uncovering genes without 5 μM CuCl supplementation (Fig. involved in vacuolar function as regulators 2 1). Our genome-wide yeast deletion mutant of mitochondrial copper homeostasis. Here, library was derived from the variomics we have highlighted the roles of two cellular library reported previously (26). It is pathways - adaptor protein 3 complex (AP- composed of viable haploid yeast mutants, 3) and the pH-sensing pathway Rim101 – where each mutant has one nonessential that converge on vacuolar function as gene replaced with the selection marker important factors regulating CcO biogenesis KANMX4 and two unique flanking by maintaining mitochondrial copper sequences (Fig. 1). These flanking homeostasis. sequences labeled “UP” and “DN” contains universal priming sites as well as a 20-bp 3 bioRxiv
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