Cells require a number of metal ions such as copper, iron, zinc as cofactors for enzymes and proteins. We are interested in how these metal ions are distributed in cells to sites of utilization. One major focus concerns mechanisms by which cells provide these metal ions within the mitochondrion for assembly of metalloproteins. We are trying to identify pools of matrix zinc and copper that are used for biosynthesis of mitochondrial zinc and copper proteins. Eukaryotic cells contain a matrix pool of copper that we showed is utilized for assembly of cytochrome c oxidase within the intermitochondrial membrane space (IMS), a process mediated by the following yeast proteins: Cox11, Cox17, Cox19, Cox23 and Sco1. We are trying to structurally identify the matrix copper ligand. We are trying to identify the transporter that delivers copper to the IMS from the matrix pool. We are studying the mechanism of copper insertion into the CuA and CuB sites of cytochrome c oxidase. Our data suggest that Cox17 is a specific metallochaperone within the IMS for delivery of Cu(I) to both Sco1 and Cox11 for insertion into the CuA and CuB sites, respectively. A scheme of the copper insertion pathway is shown. We are trying to map interfaces on these proteins that mediate specific protein:protein interactions necessary for the Cu(I) delivery step.

We have active projects relating to the characterization of Cox11, Cox17, Cox19, Cox23, Sco1, Shy1 and Pet191 in the copper assembly of cytochrome oxidase (CcO). We are using genetic screens to identify the transporters that distribute Cu(I) to the matrix for storage and back to the IMS for use by Cox17 for the assembly pathway. In studies to elucidate the function of two novel proteins within the mitochondria that are essential for CcO biogenesis, we have learned that one of these proteins Coa1 functions at an early step in Cox1 (subunit 1 of CcO) maturation, but also affects the matrix copper pool. We are testing the postulate that Cox1 assembly is linked to transport of Cu(I) from the matrix pool to the IMS for distribution by Cox17. A second novel protein Coa2 was found to be important for stabilization of Cox1 enabling Shy1-chaperoned heme a insertion. We are interested in the mechanism of early steps in the maturation of CcO subunits and how Coa1 functions with Mss51 and Cox14 in an early step in Cox1 assembly. We are interesting in the mechanism of heme a insertion in Cox1 and the roles of Cox10 and Cox15 in this process.

We are interested in the process of mitochondrial metallation of proteins using biochemical, cellular and genetic approaches. For example, one project focuses on identifying the molecules that regulate the matrix pool of zinc used for assembly of zinc-metalloenzymes within the mitochondrion. We are taking a biochemical approach to identify the ligand that stabilizes the zinc pool and a genetic approach to identify genes that modulate the concentration of this pool.

We are interested in mechanisms by which cells sense the intracellular concentration of copper, iron and zinc ions and transduce these signals into regulation of gene expression. Yeast contain two transcriptional activators, Aft1 and Aft2, regulated by iron. Aft1 is inhibited in iron-replete cells. The signal for iron inhibition of Aft1 and Aft2 originates within the mitochondrion and the iron sulfur cluster biogenesis pathway. The signal is transmitted to the cytoplasm through the mitochondrial inner membrane transporter Atm1 and signal transduction involves two monothiol glutaredoxins. The nature of the signal is being pursued.