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J. Biol. Chem., Vol. 282, Issue 30, 21629-21638, July 27, 2007

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

From the Department of Pharmacology and Cancer Biology and Sarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center, Durham, North Carolina 27710

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.

Received for publication, April 24, 2007 , and in revised form, June 5, 2007.

^* This work was supported by National Institutes of Health Grant GM41840 (to D. J. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ A trainee of the Duke University Program in Genetics and Genomics.

² To whom correspondence should be addressed: 3813 Research Dr., LSRC C-351, Durham, NC 27710. Tel.: 919-684-5776; Fax: 919-668-6044; E-mail: dennis.thiele{at}duke.edu.

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