Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP

Even if a myriad of approaches has been developed to identify the subcellular localization of a protein, the easiest and fastest way remains to fuse the protein to Green Fluorescent Protein (GFP) and visualize its location using fluorescence microscopy. However, this strategy is not well suited to visualize the organellar pools of proteins that are simultaneously localized both in the cytosol and in organelles because the GFP signal of a cytosolic pool of the protein (cytosolic echoform) will inevitably mask or overlay the GFP signal of the organellar pool of the protein (organellar echoform). To solve this issue, we engineered a dedicated yeast strain expressing a Bi-Genomic Mitochondrial-Split-GFP. This split-GFP is bi-genomic because the first ten ß-strands of GFP (GFP_ß1-10) are encoded by the mitochondrial genome and translated by mitoribosomes whereas the remaining ß-strand of GFP (GFP_ß11) is fused to the protein of interest encoded by the nucleus and expressed by cytosolic ribosomes. Consequently, if the GFP_ß11-tagged protein localizes into mitochondria, GFP will be reconstituted by self-assembly GFP_ß1-10 and GFP_ß11 thereby generating a GFP signal restricted to mitochondria and detectable by regular fluorescence microscopy. In addition, because mitochondrial translocases and import mechanisms are evolutionary well conserved, the BiG Mito-Split-GFP yeast strain can be used to probe mitochondrial importability of proteins regardless of their organismal origins and can thus serve to identify unsuspected mitochondrial echoforms readily from any organism.