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Identification and analysis of novel mitochondrial proteins encoded by small open reading frames

Periodic Reporting for period 3 - MITOsmORFs (Identification and analysis of novel mitochondrial proteins encoded by small open reading frames)

Reporting period: 2018-09-01 to 2020-02-29

Mitochondria are essential organelles with crucial roles in the cellular metabolism, cofactor biogenesis, signaling and programmed cell death. Due to their important function in the energy metabolism, mitochondria are considered as cellular power stations. Mitochondria convert the energy from nutrition to generate more than 90% of the cellular adenosine triphosphate (ATP), which is the major driver for the cellular metabolism. Mitochondrial protein dysfunction causes severe encephalomyopathy often associated with multiple organ dysfunction and neurodegenerative diseases.
Mitochondria possess a remarkably high content of small proteins compared to other cellular compartments. About one third of the cellular proteins ≤15 kDa characterized to date are located in mitochondria. Functional examples of such small proteins cover the whole mitochondrial biology like cristae morphology, Fe-S cluster formation, metabolite transport, protein biogenesis and respiration. However, the intracellular localization and function of many small proteins is unknown. At the beginning of the project these small proteins constitute one third of the uncharacterized open reading frames in the model organism baker’s yeast. We predict that more than 10% of the mitochondrial proteome deserves to be discovered.
To understand mitochondrial functions, it is absolutely crucial to identify the mitochondrial protein composition. MITOsmORFs aims to identify over 100 novel mitochondrial proteins. In addition, MITOsmORFs will determine the submitochondrial localization and the functional role of mitochondrial proteins. This includes interaction mapping and characterization of mitochondrial activities in vivo and in organello to explore the unknown biology mitochondrial proteins.
The major focus of large scale organelle proteomic studies was the identification of proteins from highly purified organelle samples. Summing up high throughput and single protein studies for the model organism baker’s (budding) yeast (Saccharomyces cerevisiae) about 1000 proteins have a mitochondrial annotation, but only ~700 proteins were considered by single protein studies to belong the mitochondrial proteome. Thus, for many proteins it is not clear if they are authentic mitochondrial proteins. In addition, due to the increased sensitivity of mass spectrometry equipment MITOsmORFs identified and quantified ~3500 proteins in purified mitochondria. To classify all these proteins, MITOsmORFs implemented a ratio of protein abundance in purified versus crude mitochondria. Therefore, MITOsmORFs was able to identify 83 novel mitochondrial proteins without previous mitochondrial annotation in yeast and assign 119 high confidence mitochondrial proteins from over 400 previous mitochondrial high throughput annotations in S. cerevisiae (Morgenstern et al., 2017). Taken together, by combination of quantitative mass spectrometry, fluorescence microscopy, subcellular fractionation and mitochondrial protein import assays we were able to assign 202 high confidence mitochondrial proteins representing 22% of the total mitochondrial proteome in yeast. Thus, the MITOsmORFs project consortium achieved a so far unprecedented characterization of the mitochondrial proteome.
The MITOsmORFs mitochondrial proteome study will impact future large scale proteomics studies, by its rigid data analysis and its integrative approach. In the long term we expect, that the MITOsmORFs mitochondrial proteome and protein function analysis will contribute to our understanding of mitochondrial dysfunction and most important mitochondrial disease. Moreover, MITOsmORFs described the relevance of the mitochondrial biogenesis pathway combining the eukaryotic mitochondrial protein import system into mitochondria with the bacterial inherited protein export pathway. The general relevance of this ‘conservative sorting’ pathway changed the view of mitochondrial biogenesis. In addition, MITOsmORFs dissected the functional mechanism of the beta-barrel membrane protein insertion. This represents a milestone in the biochemistry for membrane proteins.