Abstract
Mitochondria contain their own gene expression systems, including membrane-bound ribosomes dedicated to synthesizing a few hydrophobic subunits of the oxidative phosphorylation (OXPHOS) complexes. We used a proximity-dependent biotinylation technique, BioID, coupled with mass spectrometry to delineate in baker's yeast a comprehensive network of factors involved in biogenesis of mitochondrial encoded proteins. This mitochondrial gene expression network (MiGENet) encompasses proteins involved in transcription, RNA processing, translation, or protein biogenesis. Our analyses indicate the spatial organization of these processes, thereby revealing basic mechanistic principles and the proteins populating strategically important sites. For example, newly synthesized proteins are directly handed over to ribosomal tunnel exit-bound factors that mediate membrane insertion, co-factor acquisition, or their mounting into OXPHOS complexes in a special early assembly hub. Collectively, the data reveal the connectivity of mitochondrial gene expression, reflecting a unique tailoring of the mitochondrial gene expression system.
Highlights
The mitochondrial proteome consists of about 1,000 proteins of dual genetic source (Morgenstern et al, 2017)
Detection of Proximal Proteins in Mitochondria by biotin identification (BioID) Analyses In order to acquire a comprehensive understanding of how mitochondrial gene expression is organized, we used a proximitybased labeling method, BioID (Li et al, 2019; Roux et al, 2018), to create a protein-proximity map of mitochondrial gene expression
Biotinylated proteins were purified under stringent denaturing conditions from isolated mitochondria and analyzed by quantitative label-free liquid chromatography-mass spectrometry (LC-MS)
Summary
The mitochondrial proteome consists of about 1,000 proteins of dual genetic source (Morgenstern et al, 2017). The mitochondrial genetic system performs DNA replication, transcription, RNA processing, translation, and post-translation maturation of the newly synthesized polypeptides. How these activities are organized and coordinated is largely unknown. Comparable complexes, which gather post-transcriptional events, exist in mammalian mitochondria and are termed RNA granules (Jourdain et al, 2016; Antonicka et al, 2013). These recent discoveries illustrate that mitochondrial gene expression is more organized than previously anticipated
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