C. J. De Feo et al.: Tryptophan Scanning of Copper Transporters 115 unfolded protein response. Transformants were selected by plating on minimal dextrose (MD) plates containing a 50 lM copper sulfate supplement, dextrose (2%), adenine, lysine and Trp (all 20 mg/l) but lacking histidine and uracil. b Fig. 1 Effect of Trp mutations in TM1 and TM2 on yCTR3 and hCTR1 function. The Dctr1,3 S. cerevisiae strain, deﬁcient in high- afﬁnity copper uptake, was transformed with vector alone (V), wild- type (WT) yCTR3-GFP, WT HA-tagged hCTR1 (HA-hCTR1) or one of the Trp mutants. Yeast were plated in serial 1:10 dilutions on YPG, supplemented with either 50 lM copper sulfate or 80 lM of the copper chelator BCS. a, b Complementation results for TM1 (a) and TM2 (b) of yCTR3-GFP (left) and HA-hCTR1 (right). *Mutants exhibited clonal variation (see ‘‘Experimental Procedures’’ section). To facilitate visual comparison of spatially correlated positions, panels were mounted such that residues at the extracellular end of each helix are displayed ﬁrst. c Summary of positions sensitive to Trp under the coppe-chelating conditions in the complementation assay. All residues that were assayed in this experiment are underlined and those that were sensitive to Trp are marked with a black dot Functional Complementation The Dctr1, 3 yeast strain transformed with the plasmids carrying either just the p423GPD vector (V), wild type (WT) yCTR3-GFP, WT HA-hCTR1 or Trp mutants were grown in MD (described above) to A600 of * 1.0. Com- plementation assays were set up essentially as before (Aller et al. 2004). Yeast ODs were normalized to A600 of 1.0 and washed with sterile doubled distilled (dd) H2O, and ﬁve serial dilutions (each 10-fold) were spotted onto plates containing glycerol (3% v/v), yeast extract (1% w/v), bactopeptone (2% w/v) and kanamycin (30 lg/ml), sup- plemented with either 50 lM of copper sulfate or 80 lM of the copper chelator bathocuproinedisulfonic acid (BCS; Sigma, St; Louis, MO). Plates were incubated at 30 C for 5–8 days, and images were taken using digital photogra- phy. Images were contrast-inverted, autoleveled and re- sized using Photoshop (Adobe, San Jose, CA) to produce the ﬁnal presented images. At least three different clones were complemented for each mutant. However, some mutants displayed variability in their ability to complement the host strain under copper chelating conditions. In these cases, up to nine different clones were analyzed; the most common phenotype served to represent the behavior of this mutant. Florescence Microscopy Yeast expressing yCTR3-GFP Trp mutants were grown overnight to A600 of *1.0. Cells were washed once in ddH2O and placed on a glass slide for visualization on a Nikon (Tokyo, Japan) TE2000 using epi-illumination. Images were captured with a Hamamatsu (Shizuoka, Japan) ORCA-ER CCD camera. After acquisition, the au- tolevels option in Photoshop was used to adjust brightness for variations in expression levels. Because of this 123