bims-mitran 2024-11-03 papers

Issue of 2024–11–03
two papers selected by
Andreas Kohler, Umeå University

Methods Enzymol. 2024 ;pii: S0076-6879(24)00384-7. [Epub ahead of print]706 533-547

Analysis of mitochondrial translation using click chemistry.

Mitochondria contain their own gene expression machinery, which synthesizes core subunits of the oxidative phosphorylation system. Monitoring mitochondrial translation within spatial compartments of cells is difficult. Here we describe a method to visualize mitochondrial translation within defined parts of cells, using a click chemistry approach. This method can be applied to different cell types such as neurons and allows detection of newly synthesized mitochondrial proteins in spatial resolution using microscopy techniques. Furthermore, using click chemistry, mitochondrial translation can also be monitored by standard SDS-PAGE. The described method avenues the analysis of newly synthesized mitochondrial encoded proteins in the cellular context, by avoiding the usage of radioactive components.

Keywords: Microscopy; Mitochondria; Mitochondrial translation

DOI: https://doi.org/10.1016/bs.mie.2024.07.044

Methods Enzymol. 2024 ;pii: S0076-6879(24)00385-9. [Epub ahead of print]706 519-532

Assays to monitor mitochondrial translation.

Sung-Jun Jung, Martin Ott.

The complexes of the oxidative phosphorylation (OXPHOS) system found in the mitochondrial inner membrane comprises nuclear and mitochondrial-encoded proteins. The mitochondrial-encoded subunits of the OXPHOS complexes play vital catalytic roles for OXPHOS. These subunits are inserted co-translationally into the inner membrane, where they are matured and assembled with nuclear encoded subunits, requiring a set of OXPHOS assembly and quality control factors. Hence, monitoring the fate of newly synthesized mitochondrial-encoded polypeptides is a basic and essential approach for exploring OXPHOS biogenesis and the related protein quality control processes. Here, we describe a detailed protocol for labeling mitochondrial encoded proteins with 35S-methionine for pulse and pulse/chase experiments, both in vivo and in organello, using the yeast Saccharomyces cerevisiae as the model. These methods enable analyses of the early steps during the biogenesis and turnover of mitochondrial-encoded proteins.

Keywords: 35S-methionine; Mitochondrial translation; isolated mitochondria; protein stability; protein synthesis; yeast

DOI: https://doi.org/10.1016/bs.mie.2024.07.045