bims-cytox1 2022-07-03 papers

Issue of 2022–07–03
three papers selected by
Gavin McStay, Staffordshire University

Methods Mol Biol. 2022 ;2497 107-115

Characterizing the Electron Transport Chain: Structural Approach.

Ting Liang, Janice Deng, Bijaya Nayak, Xin Zou, Yuji Ikeno, Yidong Bai.

The mitochondrial respiratory chain which carries out the oxidative phosphorylation (OXPHOS) consists of five multi-subunit protein complexes. Emerging evidences suggest that the supercomplexes which further consist of multiple respiratory complexes play important role in regulating OXPHOS function. Dysfunction of the respiratory chain and its regulation has been implicated in various human diseases including neurodegenerative diseases and muscular disorders. Many mouse models have been established which exhibit mitochondrial defects in brain and muscles. Protocols presented here aim to help to analyze the structures of mitochondrial respiratory chain which include the preparation of the tissue samples, isolation of mitochondrial membrane proteins, and analysis of their respiratory complexes by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) in particular.

Keywords: Assembly; Blue Native Gel; Brain; Muscle; Respiratory complex

DOI: https://doi.org/10.1007/978-1-0716-2309-1_7

Methods Mol Biol. 2022 ;2497 339-348

Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Blue Native PAGE.

Kritarth Singh, Michael R Duchen.

Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a well-established technique for the isolation and separation of mitochondrial membrane protein complexes in a native conformation with high resolution. In combination with histochemical staining methods, BN-PAGE has been successfully used as clinical diagnostic tool for the detection of oxidative phosphorylation (OXPHOS) defects from small tissue biopsies from patients with primary mitochondrial disease. However, its application to patient-derived primary fibroblasts is difficult due to limited proliferation and high background staining. Here, we describe a rapid and convenient method to analyze the organization and activity of OXPHOS complexes from cultured skin fibroblasts.

Keywords: In-gel activity; Mitochondria; Oxidative phosphorylation; Primary fibroblasts; Supercomplex

DOI: https://doi.org/10.1007/978-1-0716-2309-1_25

Methods Mol Biol. 2022 ;2497 243-254

In Vivo Analysis of Mitochondrial Protein Synthesis in Saccharomyces cerevisiae Mitochondrial tRNA Mutants.

Arianna Montanari.

I describe here a protocol for the analysis of mitochondrial protein synthesis as a useful tool to characterize the mitochondrial defects associated with mutations in mitochondrial tRNA genes. The yeast Saccharomyces cerevisiae mutants, bearing human equivalent pathogenic mutations, were used as a simple model for analysis. The mitochondrial proteins were labeled by L[35S]-methionine incorporation in growing cells, extracted from purified mitochondria, and fractionated by SDS-polyacrylamide gel electrophoresis followed by autoradiography. By this method, it is possible to distinguish different protein synthesis profiles in the analyzed mitochondrial tRNA mutants.

Keywords: Human equivalent mutations; In vivo L[35S]-methionine labeling; Mitochondria; Mitochondrial protein synthesis; Mitochondrial tRNA mutants; Saccharomyces cerevisiae

DOI: https://doi.org/10.1007/978-1-0716-2309-1_15