This Is AuburnElectronic Theses and Dissertations

Investigation of the Impact of Copper Homeostasis on Cellular Physiology




Zhu, Xinyu

Type of Degree

PhD Dissertation


Biological Sciences

Restriction Status


Restriction Type

Auburn University Users

Date Available



The copper pool within the mitochondrial matrix is required for assembly and activity of cytochrome c oxidase and a portion of copper/zinc superoxide dismutase. The mitochondrial copper exists bound to an anionic, fluorescent molecule known as the copper ligand (CuL). The CuL is imported into mammalian mitochondria through mitochondrial carrier family (MCF) protein SLC25A3, while in Saccharomyces cerevisiae Pic2 mediates the translocation. SLC25A3 has promiscuity for phosphate and CuL, but yeast has dedicated MCFs to transport these two substrates, Pic2 transports copper and Mir1 transports phosphate. To better understand the mechanism for substrate selection, we adopted phylogenetic approach to identify the critical residues dictate the substrate specificity of MCF. We tested the identified candidates by site directed mutations and expression of the mutants in Lactococcus lactis for copper and silver uptake. In SLC25A3, we were able to demonstrate that transport specificity could be modulated by mutating leucine 175 to alanine. The mutant transporter retained copper transporting capacity but lost its ability to import phosphate. We speculated that mutating the leucine residue caused changes in protein structure which most likely resulted in movement of residues with positive charges used for substrate binding. It is estimated that only 10%-20% of the mitochondrial copper is associated with mitochondria-localized cupro-enzymes. To identify novel targets of which the mitochondrial copper binds to, we performed synthetic genetic array in yeast and found a copper repressible lysine auxotroph phenotype. ACO2 is a paralog of ACO1 that is required in the de novo lysine synthesis pathway. We observed that the lysine auxotroph of an aco2∆ yeast can be rescued by copper if ACO1 is present and intermediate rescue can be achieved by modulating the transcriptional level of ACO1. We hypothesized the copper binds to Aco1 at a remote allosteric site, changes the active site of the enzyme so that it can accommodate the larger substrate, homoaconitate, thus bypassing the requirement for Aco2. We demonstrated the copper bound to the mitochondrial aconitase and switched its substrate specificity.