122 copper delivery copper chaperone for superoxide dismutase, CCS (Barry and Blackburn 2008). However, even if one were inclined to reject the idea of possible CTR–CTR interactions, the observation that TM1 harbors functionally important Trp-sensitive positions on the surface of the molecule (i.e., pointing toward the lipid) clearly indicated that the role of TM1 in copper uptake is more complex than previously anticipated. For instance, if not engaging other CTR trimers, TM1 may participate in molecular interactions of CTRs with ferric reductases that play a critical role in high-afﬁnity copper uptake (Georgatsou et al. 1997; Hassett and Kosman 1995; Rees and Thiele 2007). Alternatively, TM1 may represent a link that allows incorporation of CTR1 into larger macromolecular signaling complexes, where it functions independently of its role in copper uptake (Hare- maki et al. 2007). At this point in time, none of these pos- sibilities can be rejected, but any of them seems more likely than radical conformational changes that would result in an almost 180 rotation of TM1 about its long axis. Clearly, much mechanistic detail needs to be worked out to understand CTR-dependent copper uptake. The data presented here provide the largest ensemble of observations related to structure–function aspects of the membrane- embedded domain of CTR copper transporters. Moreover, the results of the comparative Trp scan provide strong support for our previous assignment of the TM helices and leave little doubt that the ‘‘chemistry’’ of copper uptake occurs along the central threefold axis of the trimer where TM2, with some contributions from TM1 and TM3, create a copper-permeable pore through the membrane. Taken together, these data for the ﬁrst time put results obtained for individual residues into a larger and coherent context, which will be useful for the design of experiments that explore the conformational dynamics of copper transport. Acknowledgement We thank Dr. Peter Takizawa and Brian Dunn for help with ﬂorescence microscopy and Dr. Stephen G. Aller for initial help with this work. This work was supported by PHS grant GM071590 to V. M. U. C. J. D. was supported by PHS grants T32 GM07223 and F31 NS56825. Molecular graphics images in Figs. 3–5 were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). 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