Let's make it clear: systematic exploration of mitochondrial DNA- and RNA-protein complexes by complexome profiling.

Complexome profiling (CP) is a powerful tool for systematic investigation of protein interactors that has been primarily applied to study the composition and dynamics of mitochondrial protein complexes. Here, we further optimized this method to extend its application to survey mitochondrial DNA- and RNA-interacting protein complexes. We established that high-resolution clear native gel electrophoresis (hrCNE) is a better alternative to preserve DNA- and RNA-protein interactions that are otherwise disrupted when samples are separated by the widely used blue native gel electrophoresis (BNE).

Uncharacterized protein C17orf80 - a novel interactor of human mitochondrial nucleoids.

Molecular functions of many human proteins remain unstudied, despite the demonstrated association with diseases or pivotal molecular structures, such as mitochondrial DNA (mtDNA). This small genome is crucial for the proper functioning of mitochondria, the energy-converting organelles. In mammals, mtDNA is arranged into macromolecular complexes called nucleoids that serve as functional stations for its maintenance and expression.

MRPS36 provides a structural link in the eukaryotic 2-oxoglutarate dehydrogenase complex.

The tricarboxylic acid cycle is the central pathway of energy production in eukaryotic cells and plays a key part in aerobic respiration throughout all kingdoms of life. One of the pivotal enzymes in this cycle is 2-oxoglutarate dehydrogenase complex (OGDHC), which generates NADH by oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. OGDHC is a megadalton protein complex originally thought to be assembled from three catalytically active subunits (E1o, E2o, E3).

Top3α is the replicative topoisomerase in mitochondrial DNA replication.

Mitochondrial DNA has been investigated for nearly fifty years, but many aspects of the maintenance of this essential small genome remain unknown. Like any genome, mammalian mitochondrial DNA requires the function of topoisomerases to counter and regulate the topological tension arising during replication, transcription, segregation, and repair. However, the functions of the different mitochondrial topoisomerases are poorly understood.

Complexome Profiling-Exploring Mitochondrial Protein Complexes in Health and Disease.

Complexome profiling (CP) is a state-of-the-art approach that combines separation of native proteins by electrophoresis, size exclusion chromatography or density gradient centrifugation with tandem mass spectrometry identification and quantification. Resulting data are computationally clustered to visualize the inventory, abundance and arrangement of multiprotein complexes in a biological sample. Since its formal introduction a decade ago, this method has been mostly applied to explore not only the composition and abundance of mitochondrial oxidative phosphorylation (OXPHOS) complexes in several species but also to identify novel protein interactors involved in their assembly, maintenance and functions.

Mitochondrial RNA granules are critically dependent on mtDNA replication factors Twinkle and mtSSB.

Newly synthesized mitochondrial RNA is concentrated in structures juxtaposed to nucleoids, called RNA granules, that have been implicated in mitochondrial RNA processing and ribosome biogenesis. Here we show that two classical mtDNA replication factors, the mtDNA helicase Twinkle and single-stranded DNA-binding protein mtSSB, contribute to RNA metabolism in mitochondria and to RNA granule biology. Twinkle colocalizes with both mitochondrial RNA granules and nucleoids, and it can serve as bait to greatly enrich established RNA granule proteins, such as G-rich sequence factor 1, GRSF1.