Overlap of copper and iron uptake systems in mitochondria in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, the mitochondrial carrier family protein Pic2 imports copper into the matrix. Deletion of PIC2 causes defects in mitochondrial copper uptake and copper-dependent growth phenotypes owing to decreased cytochrome c oxidase activity. However, copper import is not completely eliminated in this mutant, so alternative transport systems must exist. Deletion of MRS3, a component of the iron import machinery, also causes a copper-dependent growth defect on non-fermentable carbon. Deletion of both PIC2 and MRS3 led to a more severe respiratory growth defect than either individual mutant. In addition, MRS3 expressed from a high copy number vector was able to suppress the oxygen consumption and copper uptake defects of a strain lacking PIC2. When expressed in Lactococcus lactis, Mrs3 mediated copper and iron import. Finally, a PIC2 and MRS3 double mutant prevented the copper-dependent activation of a heterologously expressed copper sensor in the mitochondrial intermembrane space. Taken together, these data support a role for the iron transporter Mrs3 in copper import into the mitochondrial matrix.

The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis

“…As an additional assessment of copper transport, we used silver in place of copper as silver is more toxic to L. lactis and is widely used to demonstrate copper transport. In this assay, expression of a functional copper transporter increases the silver sensitivity of L. lactis (18,31). Silver was more toxic to L. lactis expressing human SLC25A3-A when compared with L. lactis harboring an empty expression vector, as indicated by a lack of growth at 24 h in silver-containing medium (Fig.…”

“…Uptake was reported as the increase in copper over time. To determine silver toxicity in L. lactis strains containing vector, or human SLC25A3-A, or human SLC25A3-B, cells were grown in a 96-well plate containing M17 medium plus 1 ng/ml nisin and increasing concentrations of silver (0 -250 M) or arsenate (0 -2.5 mM) (31). Controls containing M17 without nisin or M17 plus silver or arsenate without nisin were included.…”

“…Fluorescence anisotropy was performed using mitochondrial anionic copper complex (CuL) isolated from yeast and recombinant SLC25A3-A reconstituted into liposomes (31). Equal amounts of control liposome without protein were assayed and subtracted from each point to account for increase viscosity.…”

“…Equal amounts of control liposome without protein were assayed and subtracted from each point to account for increase viscosity. CuL was isolated as described previously (5,17,31) and then diluted to give a fluorescence intensity of ϳ30 using an excitation at 320 nm and monitoring emission at 400 nm. SLC25A3 protein incorporated into liposomes was added in 1-5-l increments.…”

The mitochondrion: a central architect of copper homeostasis

“…Recent studies in yeast have shown that the mitochondrial carrier family proteins PIC2, a phosphate transporter, 34 and MRS3, an iron transporter, 35 also move copper across the inner membrane. 36, 37 Both proteins transport copper in a heterologous expression system, and their simultaneous deletion in yeast further decreases the amount of bioavailable copper in the matrix and leads to greater deficiencies in the activity of COX and IMS-localized SOD1. 37 There are currently no known cupro-enzymes localized to the matrix.…”