118 Some, but not All, Nonfunctional Trp-Replacement Mutants Undergo Aberrant Posttranslational Processing While classiﬁcation of Trp-replacement mutations based on genetic complementation was useful for assessing glo- bal functionality, it did not provide insight at the molecular scale for why certain mutants did not function in vivo. A determination of the precise reason why each of the non- functional mutants is defective would represent a gargan- tuan task, with signiﬁcant likelihood of producing answers as ambiguous as some of the scanning results themselves. Nevertheless, analyses of the levels of expression and subcellular localization were tractable in both systems and likely to provide some, albeit limited, feedback on why certain mutants failed to complement. In the case of the yCTR3-GFP Trp mutants, ﬂuorescence microscopy served to determine the cellular localization (Fig. 2a).With the exception of yCTR3S47W-GFP, all nonfunctional mutants of yCTR3-GFP (TM1: A45W, C48W, I49W, G50W, C51W, V55W; TM2: S181W, I184W, M185W, L187W, M189W) showed clear mislocalization, which in many cases deviated from the characteristic pattern observed for perinuclear endoplasmic reticulum (ER) localization (Aller et al. 2004). This suggested that many of the GFP-tagged yCTR3 mutants did not properly trafﬁc through the trans- Golgi network and, hence, that these mutants were likely to be defective at the level of protein folding and/or assembly of the trimer. In the case of HA-hCTR1, determination of cellular localization was not as straightforward since hCTR1 did not tolerate addition of a GFP at its C terminus, and immunostaining of whole or permeabilized cells did not return clear-cut answers (data not shown). Fortunately, hCTR1 differs from yCTR3 in that it is a glycoprotein (Dancis et al. 1994; Eisses and Kaplan 2002; Klomp et al. 2002, 2003; Maryon et al. 2007), which allowed us to exploit the pattern of N-linked glycosylation as an indirect measure for posttranslational processing and trafﬁcking (Aller et al. 2004). Using N-terminally HA-tagged WT hCTR1 as a template for comparison, N-glycosylation was assessed for all 14 mutants that were nonfunctional by complementation (Fig. 2b). Based on glycosylation, non- functional hCTR1 Trp mutants fell into one of three clas- ses: about half of the mutants (TM1: A70W, G71W, A75W, E84W; TM2: S146W, M150W, M154W) attained at least some fully processed, WT-like complex glycosyl- ation, which indicated that, at a minimum, these mutants had been exposed to the glycosylation machinery of the Golgi and, hence, were the most likely of all mutants to have been properly assembled into a trimer. Therefore, the failure of these mutants to complement copper-uptake defects under copper-limiting conditions may not have been related to defects in protein folding and/or oligomer 123 C. J. De Feo et al.: Tryptophan Scanning of Copper Transporters assembly but may have been caused by a genuine inter- ference with protein function. This view is supported by previous biochemical studies showing that mutations of the methionine residues on TM2 affect the kinetics and efﬁ- ciency of copper transport (Eisses and Kaplan 2005). The second type of nonfunctional mutants (TM1: A80W, Y83W, K87W; TM2: Q142W) failed to undergo complex glycosylation, which suggested that these mutant trans- porters failed quality control and therefore may have been retained in the ER. Finally, the third subset of mutants, all contained on TM2 (H139W, I140W, Y147W), were not expressed at detectable levels, suggesting that these mutants may have failed to fold properly and potentially were targeted for rapid degradation through the ER-asso- ciated protein degradation pathway (Hampton 2002; Romisch 2005). In support of this view, we previously demonstrated that a less severe disturbance of one of these positions (H139C) affected trimerization of hCTR1 (De Feo et al. 2009) while, surprisingly, a H139R mutant has been described to be functionally more active than WT hCTR1 (Eisses and Kaplan 2005). Mapping of the Trp-Scanning Data Reveals that the Majority of Sensitive Positions Are Along the Pore of CTRs or Participate in Helix–Helix Packing Interactions Trp-scanning mutagenesis screens performed on other membrane proteins have been successful at identifying helix–helix packing interactions and/or TM conduits (Choe et al. 1995; Cohen et al. 2003; Hasler et al. 2001; Irizarry et al. 2002; Sharp et al. 1995). Combined with our previous data on TM3 (Aller et al. 2004), the scanning data pre- sented here provided a comprehensive library of experi- mentally determined positions that were sensitive to the presence of steric bulk. To put these data into perspective, we retrieved the pdb coordinates of a straight, canonical a- helix (see ‘‘Experimental Procedures’’ section) and muta- ted it so that its residues corresponded to those found in TM1–3 from hCTR1 and yCTR3. The helices were then placed into the density map of hCTR1 based on our pre- viously published helix assignment. For TM2 and TM3, the azimuthal orientation of each helix was based on previ- ously published biochemical data which demonstrated that the methionine residues in TM2 face the central pore (De Feo et al. 2009) and that the GlyxxxGly motif on TM3 points into the packing interface with TM1 (Aller et al. 2004; De Feo et al. 2009). TM1 was oriented such that Ala80 (hCTR1)/Val55 (yCTR3), which was nonfunctional in both transporters, faced the GlyxxxGly motif on TM3. This seemed reasonable because this position in the sequence of both CTRs would be located at similar depth in the membrane as the GlyxxxGly motif in TM3. To guide