Identification of a Vacuole-associated Metalloreductase and Its Role in Ctr2-mediated Intracellular Copper Mobilization

Abstract

Copper is an essential trace metal whose biological utility is derived from its ability to cycle between oxidized Cu(II) and reduced Cu(I). Ctr1 is a high affinity plasma membrane copper permease, conserved from yeast to humans, that mediates the physiological uptake of Cu(I) from the extracellular environment. In the baker's yeast Saccharomyces cerevisiae, extracellular Cu(II) is reduced to Cu(I) via the action of the cell surface metalloreductase Fre1, similar to the human gp91 phox subunit of the NADPH oxidase complex, which utilizes heme and flavins to catalyze electron transfer. The S. cerevisiae Ctr2 protein is structurally similar to Ctr1, localizes to the vacuole membrane, and mobilizes vacuolar copper stores to the cytosol via a mechanism that is not well understood. Here we show that Ctr2-1, a mutant form of Ctr2 that mislocalizes to the plasma membrane, requires the Fre1 plasma membrane metalloreductase for Cu(I) import. The conserved methionine residues that are essential for Ctr1 function at the plasma membrane are also essential for Ctr2-1-mediated Cu(I) uptake. We demonstrate that Fre6, a member of the yeast Fre1 metalloreductase protein family, resides on the vacuole membrane and functions in Ctr2-mediated vacuolar copper export, and cells lacking Fre6 phenocopy the Cu-deficient growth defect of ctr2⌬ cells. Furthermore, both CTR2 and FRE6 mRNA levels are regulated by iron availability. Taken together these studies suggest that copper movement across intracellular membranes is mechanistically similar to that at the plasma membrane. This work provides a model for communication between the extracellular Cu(I) uptake and the intracellular Cu(I) mobilization machinery.

Response to Iron Deprivation in Saccharomyces cerevisiae

“…In many ways, these vacuolar proteins duplicate the iron transport systems that are present at the plasma membrane. One member of the FRE family of metalloreductases, Fre6p, is expressed exclusively on the vacuolar membrane, where it is involved in the reduction of vacuolar iron and copper prior to transport into the cytosol (49,54). Paralogues of the plasma membrane high-affinity ferrous iron transport complex are also present on the vacuolar membrane and are expressed under conditions of iron deficiency.…”

“…Molecular oxygen is also required for the activity of Fet3p, and under hypoxic conditions, cells express a low-affinity ferrous iron transporter, Fet4p, that serves as an oxygen-independent system of iron uptake (15,19). A paralogue of Fet3p, termed Fet5p, assembles with a paralogue of Ftr1p, termed Fth1p, to form a ferrous iron transport complex (55,61) that functions in conjunction with Fre6p at the vacuolar membrane (49,54). Although FET5 does not appear to be regulated directly by Aft1p, the remainder of these genes are regulated by iron through Aft1p.…”

Mechanistic Comparison of Human High-Affinity Copper Transporter 1-Mediated Transport between Copper Ion and Cisplatin

“…The encoded hCtr1 is cytoplasmic membrane located and mainly for Cu(I) acquisition (Safaei and Howell, 2005;Kuo et al, 2007;Kim et al, 2008); hCtr2 is primarily localized in the membrane of intracellular vacuoles (Rees and Thiele, 2007) and endosomes/lysosomes (van den Berghe et al, 2007) and primarily for copper storage. The hCtr1 has 190 amino acids spanning across three transmembrane domains (TM1-TM3).…”

Bioquímica da relação patógeno-hospedeiro: a importância biológica, funcional e estrutural do micronutriente cobre para o crescimento e infecção por fungos patogênicos

“…Schizosaccharomyces pombe e funcionalmente caracterizado como transportador vacuolar de cobre (BELLEMARE et al, 2002;PETRIS, 2004;REES e THIELE, 2004;REES e THIELE, 2007).…”