Mass-spectrometry based proteomics reveals mitochondrial supercomplexome plasticity

Abstract

Mitochondrial respiratory complex subunits assemble in supercomplexes. Studies of 13 supercomplexes have typically relied upon antibody-based protein quantification, often 14 limited to the analysis of a single subunit per respiratory complex. To provide a deeper 15 insight into mitochondrial and supercomplex plasticity, we combined Blue Native 16Polyacrylamide Gel Electrophoresis (BN-PAGE) and mass spectrometry to determine the 17 supercomplexome of skeletal muscle from sedentary and exercise-trained mice. We 18 quantified 422 mitochondrial proteins within ten supercomplex bands, in which we showed 19 the debated presence of complex II and V. Upon exercise-induced mitochondrial biogenesis, 20 non-stoichiometric changes in subunits and incorporation into supercomplexes was 21 apparent. We uncovered the dynamics of supercomplex-related assembly proteins and 22 mtDNA-encoded subunits within supercomplexes, as well as the complexes of ubiquinone 23 biosynthesis enzymes and Lactb, a mitochondrial-localized protein implicated in obesity. 24Our approach can be applied to broad biological systems. In this instance, comprehensively 25 analyzing respiratory supercomplexes illuminates previously undetectable complexity in 26 mitochondrial plasticity. 27 28 Highlights: 29 • Comprehensive quantification of respiratory subunits within supercomplexes 30 • Complex II and V assemble within supercomplexes 31 • Mitochondrial-encoded subunits display elevated upregulation upon exercise training 32 2 • Exercise increases ubiquinone biosynthesis enzyme complexes 33 34

The structure of the human LACTB filament reveals the mechanisms of assembly and membrane binding

“…Moreover, mammalian LACTB promotes membrane organization 23 , regulates mitochondrial respiratory complex I activity 26 , and participates in cellular metabolic processes [27][28][29] . Given its strong relation to metabolic pathways, LACTB is also validated as an obesity gene capable of modifying adiposity 28 .…”

“…LACTB is widely expressed in different mammalian tissues, most notably in the skeletal muscle, heart, and liver, and was found to negatively affect the proliferation of a variety of tumor cells in vivo , while it exerts little or no effect on growth of non-tumorigenic cells 18, 25 . Moreover, mammalian LACTB promotes membrane organization 23 , regulates mitochondrial respiratory complex I activity 26 , and participates in cellular metabolic processes 27–29 . Given its strong relation to metabolic pathways, LACTB is also validated as an obesity gene capable of modifying adiposity 28 .…”

“…Filaments of mammalian LACTB are reported to have a possible structural function in the spatial organization of mitochondrial IMS 23 . Recently, endurance exercise training has been shown to affect the polymerization of LACTB molecules in mice mitochondria suggesting that polymer assembly may have been directly influenced by metabolic activity in cells 29 . The formation of filaments may serve a regulatory function that results in more rapid activation or inhibition of the enzyme in response to changing cellular conditions.…”

High-Intensity Interval Training Remodels the Proteome and Acetylome of Human Skeletal Muscle

“…Exercise training is the most effective means to improve cardiovascular fitness and metabolic health, both being predominant determinants of life expectancy and all-cause mortality 1 . Alterations to the proteome of skeletal muscle underpin its adaptations to exercise training, including glucose and fatty acid metabolism, insulin sensitivity, mitochondrial respiration, immune function and excitationcontraction coupling [2][3][4][5][6][7][8] . Furthermore, proteome-wide post-translational modifications play an important role in regulating metabolism via modulating signaling, protein stability and enzyme activity 9,10 , and are sensitive to exercise stimuli [11][12][13] .…”