Figure 6.

Random mutagenesis identifies Ctr2 mutants capable of rescuing respiration-dependent growth in yeast. A, scheme depicting the strategy for isolating Ctr2 mutants capable of copper-dependent growth. Ctr2 expression vectors were transformed into XL-1 E. coli and propagated for 24 h before plasmid harvest. The plasmid pool was transformed into S. cerevisiae ctr1Δctr3Δ cells and plated on YPEG medium. Colonies were isolated, and plasmids were sequenced. B, table displaying the six unique Ctr2 mutations capable of conferring growth on YPEG. C, cells containing the indicated mouse genes were spotted onto the indicated medium. MXXXM indicates mutation of transmembrane domain two methionine residues critical for Cu⁺ transport activity to leucine. D, mutations that were identified in the mouse Ctr2-coding region were mutated in the human Ctr2 gene and assayed for growth in a manner similar as in C.