114 In contrast to an abundance of structural and functional information about copper chaperones that intracellularly deliver copper to its ﬁnal target sites (Banci et al. 2006; Barry and Blackburn 2008; Boal and Rosenzweig 2009; Huffman and O’Halloran 2000; Lamb et al. 1999, 2001; Rae et al. 1999), much less is known about the membrane proteins that are involved in copper transport across membranes. For the past decade, copper-transporting ATPases and the copper uptake transporter (CTR) family of membrane proteins were thought to serve as corner- stones for copper transport. However, recent studies sug- gest that other, less speciﬁc transport mechanisms may account for mobilization of copper from the diet (Arre- dondo et al. 2006; Zimnicka et al. 2007). Focusing on the copper-transporting ATPases and CTR proteins, the for- mer have been studied more extensively because of their causal linkage to human disorders. Nevertheless, research on CTR proteins has recently gained momentum, in part because CTR proteins were linked to the cellular uptake of Pt-based anticancer drugs like cisplatin (Holzer et al. 2006; Ishida et al. 2002; Kuo et al. 2007; Safaei 2006; Song et al. 2004). Moreover, CTR proteins were shown to be essential for development in Drosophila melanogaster (Turski and Thiele 2007; Zhou et al. 2003) and seem to partake in cellular signal transduction (Haremaki et al. 2007). Adding to this, homozygous knockout of the high- afﬁnity transporter CTR1 is embryonic-lethal in mice, further emphasizing the universal importance of these proteins for cellular physiology (Kuo et al. 2001; Lee et al. 2001). Found ubiquitously in eukaryotes but lacking from bacteria, CTR proteins are small (*23–40 kDa) integral membrane proteins, demonstrated to contain three trans- membrane (TMs) domains, an extracellular N terminus and an intracellular C terminus (Eisses and Kaplan 2002; Klomp et al. 2003; Puig et al. 2002). Clues to a transport mechanism have come from studies showing that CTRs form oligomers (Aller et al. 2004; Dancis et al. 1994; Klomp et al. 2003; Lee et al. 2002; Pena et al. 2000; Puig et al. 2002; Zhou and Thiele 2001) and require metal binding motifs, in the N terminus as well as close to the extracellular end of the second TM domain (TM2) for function (Beaudoin et al. 2006; Eisses and Kaplan 2005; Guo et al. 2004; Puig et al. 2002). Extending the biochemical characterization of CTR proteins, our recent 7A˚ structure of the human copper transporter hCTR revealed a putative copper-permeable pore along the interface of a symmetric hCTR1 trimer (De Feo et al. 2009). Moreover, clues in the structure supported a tentative assignment for the TM helices, based on which TM2 represents the principal pore lining helix. These ﬁndings represented an important advance in understanding the structural basis of copper transport but failed to reveal 123 C. J. De Feo et al.: Tryptophan Scanning of Copper Transporters what residues are likely to be involved in helix–helix packing and to what extent the structures of different CTR proteins are equivalent. Despite sharing conserved mech- anistic features, CTRs lack global sequence identity and display differences in posttranslational regulation (Puig et al. 2002), which raises the question as to whether there are also structural differences across family members (Eisses and Kaplan 2002; Pena et al. 2000; Puig and Thiele, 2002). Addressing both these issues, we pursued a trypto- phan (Trp)-scanning analysis to identify helix–helix inter- actions and to establish structural similarities and differences. Over the past 10 years Trp scanning has been validated as a tool to establish regions of direct helix–helix packing contacts (Choe et al. 1995; Irizarry et al. 2002; Monks et al. 1999) and has taken its place alongside other scanning approaches such as those using alanine (Li- Smerin et al. 2000; Mingarro et al. 1996; Panchenko et al. 2001) and cysteine (Falke and Koshland 1987; Frillingos et al. 1998). By applying this approach to two distantly related members of the CTR family, we show that steric constraints are asymmetrically distributed with respect to the membrane and that different CTRs display differences in their ability to accommodate steric bulk inside the putative copper-permeable pore. Furthermore, the data are consistent with a model in which TM1 serves as an adaptor that allows evolutionarily distant CTRs to adopt a similar, but not identical, overall structure. Moreover, the Trp- scanning data suggest that TM1 may also mediate packing interactions between CTR trimers or with other function- ally relevant membrane proteins, which represents a novel aspect in the structure and function of these important transporters. Experimental Procedures Expression Constructs and Yeast Transformation The QuickChange Site-Directed Mutagenesis Kit (Strat- agene, La Jolla, CA) was used to introduce single Trp mutations into Saccharomyces cerevisiae Ctr3p fused to green ﬂorescent protein (yCTR3-GFP) or an N-terminally hemagglutinin (HA)-tagged hCTR1 construct, both of which had already been subcloned into the vector p423GPD (Aller et al. 2004; Mumberg et al. 1995). CTR open reading frames were veriﬁed by nucleotide sequenc- ing. To assure consistency with our previous study (Aller et al. 2004), plasmids were transformed, using the lithium acetate technique, into Dctr1, 3 S. cerevisiae strain (MATa ura3 lys2 ade2 trp1 his3 leu2 Dctr1::LEU2), which in addition to a transposon interfering with Ctr3p expres- sion also harbored the pSZ1 plasmid for measuring the