Skip to main content

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

Periodic Reporting for period 4 - MITOsmORFs (Identification and analysis of novel mitochondrial proteins encoded by small open reading frames)

Reporting period: 2020-03-01 to 2021-08-31

Mitochondria are specialized domains of eukaryotic cells, called cell organelles, which are found e.g. in the research model organism baker’s yeast and in human cells. Mitochondria are crucial for the cellular energy metabolism and therefore considered as cellular power stations. Mitochondria convert the energy from nutrition - like carbohydrates and fat - to generate more than 90% of the cellular energy molecule ATP (adenosine triphosphate) through reactions associated with cellular respiration. Therefore mitochondrial dysfunction causes predominantly defects in organs, which require a lot of energy for their function, like the nervous system and muscles. All the crucial functions, which mitochondria perform within the cell, are catalyzed by proteins residing within the organelle. To understand all mitochondrial functions, it is absolutely essential to identify the mitochondrial protein composition and we speculated that the mitochondrial protein composition was not fully described before.
Previously proteins were annotated as mitochondrial in case they were detected in purified mitochondria. However, the detection systems to identify proteins became very sensitive such that one can identify virtually most cellular proteins in purified mitochondria no matter if it is a true mitochondrial protein or a protein, which is in generally located to a different subcellular area and only present as a contamination. To overcome this problem and to identify novel high-confidence mitochondrial proteins we determined the quantitative enrichment of proteins between purified and crude mitochondria, the quantitative profile of proteins by separation into many different organellar fractions and their quantitative abundance after specific inhibition of protein transport into mitochondria.
We determined the mitochondrial protein composition for model organism baker’s yeast and for human mitochondria. Mitochondria contain roughly 1000 proteins and we were able to identify more than 100 novel high-confidence mitochondrial proteins. Many of these were confirmed by single proteins studies involving e.g. fluorescence microscopy or import into isolated mitochondria. In addition, we determined mitochondrial protein copy numbers, half-lives, submitochondrial protein localizations, membrane protein topologies and mitochondrial proteins with multiple cellular localization. To analyze the functional role of novel mitochondrial proteins we identified their interacting proteins and we were able to identify many novel interactors of complexes required for cellular respiration.
By additional single protein studies we were e.g. able to identify proteins required for cellular respiration. We determined general relevance of the ‘conservative sorting’ pathway for the biogenesis of mitochondrial proteins combining the eukaryotic mitochondrial protein import system into mitochondria with the bacterial inherited protein export pathway. In addition, we determined the basic cell biology mechanism for the transport of hydrophobic membrane proteins through an aqueous mitochondrial subcompartment and for the assembly of membrane protein pores in the mitochondrial outer membrane. Moreover a systematic mapping of functional categories of 460 mitochondrial disease-related proteins with their associated disease observations provides a rich framework for defining the role of mitochondria in the pathogenesis of human diseases.
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.
Overview of mitochondrial functions