Figure 2. The addition of a mitochondrial localization signal (MLS) and mutation of start codons from GTG to ATG allows some E. coli genes to swap for their respective yeast orthologs.
(A) 14 of the 25 non-replaceable E. coli genes were predicted to function in mitochondria in yeast. 4 of 14 were replaceable after adding the MLS at the N-termini of the E. coli genes. Site-specific mutagenesis of E. coli gene start codon from GTG to ATG allowed two to functionally complement the corresponding yeast genes bringing the total number E. coli genes that functionally replace yeast genes to 31 of 51 (~61%). (B) Haploid yeast gene deletion strains carrying mitochondrially localized E. coli genes rescued the growth defect of the yeast gene (red solid-line) comparable to the wild type yeast (black dashed-line). The empty vector control (grey solid-line) and the yeast cells expressing of E. coli gene without MLS (blue-solid line) showed no such growth rescue in the presence of G418. Mean and standard deviation plotted with N = 3. (C) EGFP-tagged E. coli genes that functionally replaced the yeast gene function were imaged after MitoTracker red staining. EGFP-tagged Ec-MLS-HscB and Ec-MLS-IlvD (green) show colocalization with MitoTracker red stained mitochondria (red).
Figure 2—figure supplement 1. Some E. coli genes require a yeast mitochondrial localization signal to efficiently replace.
