116 C. J. De Feo et al.: Tryptophan Scanning of Copper Transporters Fig. 2 Trp mutations cause processing defects in both TM1 and TM2. a Yeast transformed with WT yCTR3-GFP and Trp mutants that displayed a complementation phenotype were subjected to ﬂuorescence microscopy. For each indicated mutant an image was captured in ﬂuorescent (left) and DIC (right) mode. b Crude membranes of yeast transformed with nonfunctional HA-hCTR1 Trp mutants were resuspended in buffer, normalized for total protein and incubated in the absence or presence (– and ?, respectively) of PGNase F to remove N-linked glycosylation. Samples were then subjected to Western blotting for the a-HA epitope. Since the levels of expression varied between mutants, some of the blots needed to be overexposed relative to the WT (*). In addition to the relative level of expression, blots revealed whether the mutants retained any ability to acquire complex N-glycosylation patterns that would indicate exit of the protein from the ER (see brackets). A negative control sample from cells just carrying the vector is labeled V. c Summary of positions whose posttranslational processing and/or trafﬁcking was impaired. As in Fig. 1, all residues assayed in this experiment are underlined and those that were sensitive to Trp are marked with a black circle postimaging processing, intensities shown in Fig. 2a do not reﬂect the actual level of expression. Deglycosylation Assay hCTR1 mutants that exhibited a growth defect by com- plementation were grown overnight (typically to an A600 of \1.0) before normalization of cell densities and lysis using glass beads. Cell debris was pelleted with a low-speed spin, and the supernatant was centrifuged to obtain crude membranes (20 min, 100,000 9 g using a TLA-110 rotor on a tabletop ultracentrifuge; Beckman Coulter, Brea, CA). The pellet was then resuspended in buffer (10 mM MOPS [pH 7.4], 280 mM NaCl, 2 mM EDTA), and the protein concentration was determined (Coomassie blue method [Pierce Protein Science, Waltham, MA]). After normali- zation for protein content, samples were subjected to deglycosylation with PNGase F (New England Biolabs, Ipswich, MA) for 60 min at 37 C. Reactions were stopped with SDS-PAGE sample loading buffer, which contained 5% b-mercaptoethanol, and then separated on polyacryl- amide gels. Western blotting was performed using anti-HA 123 primary antibody (Covance Research Products, Princeton, NJ) and a goat-anti-mouse HRP-coupled secondary anti- body (KPL, Gaithersburg, MA). Since we were interested in qualitative answers only, the scanned images of the Western blots were autoleveled in Photoshop across the entire blot and resized for display. These alterations did not change the interpretation presented in the text. Visualization of Model Helices within the Electron Microscopic Map of hCTR1 A relatively straight and canonical a-helix (residues 125– 155, PDB 2B1E) was used as a template for modeling the CTR TM segments. Using Coot model building tools (Emsley and Cowtan 2004), residues were mutated to conform with those in TMs 1, 2 and 3 of hCTR1 and yCTR3. The helices for hCTR1 were then aligned with the electron densities of the corresponding helices from our previous electron microscopic (EM) map of hCTR1 for one of the monomers. The MetxxxMet region in TM2 was manually positioned to face the center threefold axis, and the GlyxxxGly motif in TM3 was positioned at the closet