Abstract
A single nuclear gene can be translated into a dual localized protein that distributes between the cytosol and mitochondria. Accumulating evidences show that mitoproteomes contain lots of these dual localized proteins termed echoforms. Unraveling the existence of mitochondrial echoforms using current GFP (Green Fluorescent Protein) fusion microscopy approaches is extremely difficult because the GFP signal of the cytosolic echoform will almost inevitably mask that of the mitochondrial echoform. We therefore engineered a yeast strain expressing a new type of Split-GFP that we termed Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). Because one moiety of the GFP is translated from the mitochondrial machinery while the other is fused to the nuclear-encoded protein of interest translated in the cytosol, the self-reassembly of this Bi-Genomic-encoded Split-GFP is confined to mitochondria. We could authenticate the mitochondrial importability of any protein or echoform from yeast, but also from other organisms such as the human Argonaute 2 mitochondrial echoform.
Highlights
Mitochondria provide aerobic eukaryotes with adenosine triphosphate (ATP), which involves carbohydrates and fatty acid oxidation (Saraste, 1999), as well as numerous other vital functions like lipid and sterol synthesis (Horvath and Daum, 2013) and formation of iron-sulfur cluster (Lill et al, 2012)
We used the scaffold of the self-assembling Superfolder Split-green fluorescent protein (GFP) fragments designed by Cabantous and coworkers (Cabantous et al, 2005b; Pedelacq et al, 2006), where the 11 beta strands forming active Superfolder GFP are separated in a fragment encompassing the 10 first beta strands (GFPb1-10) and a smaller one consisting of the remaining beta strand (GFPb11)
Our objective was to integrate the gene encoding the GFPb1-10 fragment into the mtDNA so that it will only be translated inside the mitochondrial matrix, while the GFPb11ch fragment is fused to the nuclear-encoded protein of interest and translated by cytosolic ribosomes (Figure 1A)
Summary
Mitochondria provide aerobic eukaryotes with adenosine triphosphate (ATP), which involves carbohydrates and fatty acid oxidation (Saraste, 1999), as well as numerous other vital functions like lipid and sterol synthesis (Horvath and Daum, 2013) and formation of iron-sulfur cluster (Lill et al, 2012). In the yeast S. cerevisiae, about a third of the mitochondrial proteins (mitoproteome) have been suggested to be dual localized (Ben-Menachem et al, 2011; Dinur-Mills et al, 2008; Kisslov et al, 2014), and have been named echoforms (or echoproteins) to accentuate the fact that two identical or nearly identical forms of a protein, can reside in the mitochondria and another compartment (Ben-Menachem and Pines, 2017).
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