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J. Biol. Chem., Vol. 282, Issue 37, 26775-26785, September 14, 2007

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Distinct Mechanisms for Ctr1-mediated Copper and Cisplatin Transport^*

From the Redox Biology Center, Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664

The Ctr1 family of integral membrane proteins is necessary for high affinity copper uptake in eukaryotes. Ctr1 is also involved in cellular accumulation of cisplatin, a platinum-based anticancer drug. Although the physiological role of Ctr1 has been revealed, the mechanism of action of Ctr1 remains to be elucidated. To gain a better understanding of Ctr1-mediated copper and cisplatin transport, we have monitored molecular dynamics and transport activities of yeast Saccharomyces cerevisiae Ctr1 and its mutant alleles. Co-expression of functional Ctr1 monomers fused with either cyan or yellow fluorescent protein resulted in fluorescence resonance energy transfer (FRET), which is consistent with multimer assembly of Ctr1. Copper near the K_m value of Ctr1 enhanced FRET in a manner that correlated with cellular copper transport. In vitro cross-linking of Ctr1 confirmed that copper-induced FRET reflects conformational changes within pre-existing Ctr1 complexes. FRET assays in membrane-disrupted cells and protein extracts showed that intact cell structure is necessary for Ctr1 activity. Despite Ctr1-dependent cellular accumulation, cisplatin did not change Ctr1 FRET nor did it attenuate copper-induced FRET. A Ctr1 allele defective in copper transport enhanced cellular cisplatin accumulation. N-terminal methionine-rich motifs that are dispensable for copper transport play a critical role for cisplatin uptake. Taken together, our data reveal functional roles for structural remodeling of the Ctr1 multimeric complex in copper transport and suggest distinct mechanisms employed by Ctr1 for copper and cisplatin transport.

Received for publication, May 14, 2007 , and in revised form, July 10, 2007.

^* This work was supported by National Institutes of Health (NIH) Grant P20-RR-17675 (project 3 to J. L.), NIH Grant DK79209 (to J. L.), a Nutricia Foundation pre-doctoral fellowship (to D. S.), and funds provided through the Hatch Act in the University of Nebraska Agricultural Research Division. 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 To whom correspondence should be addressed: Dept. of Biochemistry, University of Nebraska-Lincoln, N210 Beadle Center, Lincoln, NE 68588-0664. Tel.: 402-472-2658; Fax: 402-472-7842; E-mail: jlee7{at}unlnotes.unl.edu.

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