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J. Biol. Chem., Vol. 279, Issue 51, 53435-53441, December 17, 2004

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Eukaryotic CTR Copper Uptake Transporters Require Two Faces of the Third Transmembrane Domain for Helix Packing, Oligomerization, and Function^*

Stephen G. Aller, Edward T. Eng, Christopher J. De Feo, and Vinzenz M. Unger

From the Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-8024

Members of the copper uptake transporter (CTR) family from yeast, plants, and mammals including human are required for cellular uptake of the essential metal copper. Based on biochemical data, CTRs have three transmembrane domains and have been shown to oligomerize in the membrane. Among individual members of the family, there is little amino acid sequence identity, raising questions as to how these proteins adopt a common fold, oligomerize, and participate in copper transport. Using site-directed mutagenesis, tryptophan scanning, genetic complementation, subcellular localization, chemical cross-linking, and the yeast unfolded protein response, we demonstrated that at least half of the third transmembrane domain (TM3) plays a vital role in CTR structure and function. The results of our analysis showed that TM3 contains two functionally distinct faces. One face bears a highly conserved Gly-X-X-X-Gly (GG4) motif, which we showed to be essential for CTR oligomerization. Moreover, we showed that steric constraints reach past the GG4-motif itself including amino acid residues that are not conserved throughout the CTR family. A second face of TM3 contains three amino acid positions that, when mutated to tryptophan, cause predominantly abnormal localization but are still partially functional in growth complementation experiments. These mutations cluster on the face opposite to the GG4-bearing face of TM3 where they may mediate interactions with the remaining two transmembrane domains. Taken together, our data support TM3 as being buried within trimeric CTR where it plays an essential role in CTR assembly.

Received for publication, August 17, 2004 , and in revised form, September 21, 2004.

^* This work was supported in part by NRSA Predoctoral Fellowship F32 NS45550 (to S. G. A.), a Hellman Family Fellowship, and Public Health Service Grants GM66145 and DA017088 (to V. M. U.). 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.

Supported by National Institutes of Health Training Grant T32 GM07223.

To whom correspondence should be addressed. Tel.: 203-785-5652; Fax: 203-785-6404; E-mail: vinzenz.unger{at}yale.edu.

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