Abstract
The mitochondrial genome is responsible for the production of a handful of polypeptides that are core subunits of the membrane-bound oxidative phosphorylation system. Until now the mechanistic studies of mitochondrial protein synthesis inside cells have been conducted with inhibition of cytoplasmic protein synthesis to reduce the background of nuclear gene expression with the undesired consequence of major disturbances of cellular signaling cascades. Here we have generated a system that allows direct monitoring of mitochondrial translation in unperturbed cells. A recoded gene for superfolder GFP was inserted into the yeast (Saccharomyces cerevisiae) mitochondrial genome and enabled the detection of translation through fluorescence microscopy and flow cytometry in functional mitochondria. This novel tool allows the investigation of the function and regulation of mitochondrial translation during stress signaling, aging and mitochondrial biogenesis.
Highlights
In Saccharomyces cerevisiae the mitochondrial genome encodes eight proteins of which seven are membrane proteins and core subunits of the oxidative phosphorylation system (OXPHOS) [1]
Integration of a superfolder GFP gene into the mitochondrial genome In a pioneering previous study, a gene encoding fluorescence-enhanced GFP was inserted into the mitochondrial genome to replace the open reading frame of COX3 [12]
To avoid the respiratory deficiency associated with inactivation of mitochondrial genes, we engineered a new mitochondrial genome that coded for superfolder GFP (sfGFP) as an additional ninth open reading frame
Summary
In Saccharomyces cerevisiae the mitochondrial genome encodes eight proteins of which seven are membrane proteins and core subunits of the oxidative phosphorylation system (OXPHOS) [1]. A recoded gene for superfolder GFP was inserted into the yeast (Saccharomyces cerevisiae) mitochondrial genome and enabled the detection of translation through fluorescence microscopy and flow cytometry in functional mitochondria. This reporter is compatible with mitochondrial respiratory function and enables the direct detection of mitochondrial translation in vivo as GFP fluorescence.
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