bims-cytox1 2021-05-23 papers

Issue of 2021–05–23
three papers selected by
Gavin McStay, Staffordshire University

J Cell Sci. 2020 Jan 01. pii: jcs.240374. [Epub ahead of print]

Cardiolipin is required for membrane docking of mitochondrial ribosomes and protein synthesis.

Richard G Lee, Junjie Gao, Stefan J Siira, Anne-Marie Shearwood, Judith A Ermer, Vinzenz Hofferek, James C Mathews, Minghao Zheng, Gavin E Reid, Oliver Rackham, Aleksandra Filipovska.

The mitochondrial inner membrane contains a unique phospholipid known as cardiolipin (CL), which stabilises the protein complexes embedded in the membrane and supports its overall structure. Recent evidence indicates that the mitochondrial ribosome may associate with the inner membrane to facilitate co-translational insertion of the hydrophobic oxidative phosphorylation (OXPHOS) proteins into the inner membrane. We generated three mutant knockout cell lines for the cardiolipin biosynthesis gene Crls1 to investigate the effects of cardiolipin loss on mitochondrial protein synthesis. Reduced CL levels caused altered mitochondrial morphology and transcriptome-wide changes that were accompanied by reduced uncoordinated mitochondrial translation rates and impaired respiratory supercomplex formation. Aberrant protein synthesis was caused by impaired formation and distribution of mitochondrial ribosomes. Reduction or loss of cardiolipin resulted in divergent mitochondrial and endoplasmic reticulum stress responses. We show that cardiolipin is required to stabilise the interaction of the mitochondrial ribosome with the membrane via its association with OXA1 during active translation. This interaction facilitates insertion of newly synthesised mitochondrial proteins into the inner membrane and stabilises the respiratory supercomplexes.

Keywords: Mitochondrial membranes; Mitochondrial ribosomes; Protein synthesis

DOI: https://doi.org/10.1242/jcs.240374

J Cell Sci. 2020 Jan 01. pii: jcs.248492. [Epub ahead of print]

Increased supra-organization is a dynamic multistep remodelling of respiratory complexes against proteostasis stress.

Shivali Rawat, Suparna Ghosh, Debodyuti Mondal, Valpadashi Anusha, Swasti Raychaudhuri.

Proteasome-mediated degradation of misfolded proteins prevents aggregation inside and outside mitochondria. But how do cells safeguard mitochondrial proteome and function despite increased aggregation during proteasome-inactivation? Here, using a novel two-dimensional complexome profiling strategy, we report increased supra-organizations of respiratory complexes (RCs) in proteasome-inhibited cells simultaneous to pelletable aggregation of RC-subunits inside mitochondria. Complex-II (CII) and CV-subunits are increasingly incorporated into oligomers. CI, CIII and CIV-subunits are engaged into supercomplex formation. We unravel unique quinary-states of supercomplexes at early-stress that exhibit plasticity and inequivalence of constituent RCs. Core stoichiometry of CI and CIII is preserved whereas CIV-composition varies. These partially disintegrated supercomplexes remain functionally competent via conformational optimization. Subsequently, increased stepwise integration of RC-subunits into holocomplex and supercomplexes re-establish steady-state stoichiometry. Overall, the mechanism of increased supra-organization of RCs mimics the cooperative unfolding and folding pathways for protein-folding, restricted to RCs only and not observed for any other mitochondrial protein complexes.

Keywords: Increased supercomplex; Multistep proteome remodelling; Proteostasis; Quinary supercomplex; Respiratory complex biogenesis; Two-dimensional complexome profiling

DOI: https://doi.org/10.1242/jcs.248492

Biochim Biophys Acta Bioenerg. 2021 May 17. pii: S0005-2728(21)00081-5. [Epub ahead of print] 148448

Multiplexed complexome profiling using tandem mass tags.

Sergio Guerrero-Castillo, Christoph Krisp, Katrin Küchler, Susanne Arnold, Hartmut Schlüter, Søren W Gersting.

Complexome profiling is a rapidly spreading, powerful technique to gain insight into the nature of protein complexes. It identifies and quantifies protein complexes separated into multiple fractions of increasing molecular mass using mass spectrometry-based, label-free bottom-up proteomics. Complexome profiling enables a sophisticated and thorough characterization of the composition, molecular mass, assembly, and interactions of protein complexes. However, in practice, its application is limited by the large number of samples it generates and the related time of mass spectrometry analyses. Here, we report an improved process workflow that implements tandem mass tags for multiplexing complexome profiling. This workflow substantially reduces the number of samples and measuring time without compromising protein identification or quantification reliability. In profiles from mitochondrial fractions of cells recovering from chloramphenicol treatment, tandem mass tags-multiplexed complexome profiling exhibited migration patterns of mature ATP synthase (complex V) and assembly intermediates that were consistent in composition and abundance with profiles obtained by the label-free approach. Reporter ion quantifications of proteins and complexes unaffected by the chloramphenicol treatment presented less variation in comparison to the label-free method. Incorporation of tandem mass tags enabled an efficient and robust complexome profiling analysis and may foster broader application for protein complex profiling in biomedical research and diagnostics.

Keywords: ATP synthase.; Complexome profiling; Multiplexing; OXPHOS; Protein complexes; Tandem mass tags

DOI: https://doi.org/10.1016/j.bbabio.2021.148448