Background

Mitochondria are essential organelles that act as both energy conversion powerhouses and metabolic hubs in eukaryotic cells. Mitochondria contain a genome and fully functional gene expression machinery. These gene expression complexes combine traits inherited from prokaryotic ancestors and specific features acquired during eukaryotic evolution (Gray, 2015; Waltz and Giegé, 2020). Mitochondrial research has huge societal implications given that numerous diseases are linked to mitochondrial dysfunctions in humans and many other eukaryotes (Nunnari and Suomalainen, 2012; De Silva et al., 2015). For instance, in plants, male sterility resulting from recombined mitochondrial DNA is a trait widely used in agronomy (Chen and Liu, 2014). Among mitochondrial gene expression processes, translation is considered the most important in terms of regulation since it is clear that transcriptional processes are not limiting for mitochondrial gene expression. Still, the mechanistic details of translational regulation, and even the basic composition of the translational machinery, remain poorly understood for many eukaryotes. Recent functional, biochemical, and structural data have revealed an unexpected diversity of mitochondrial translation systems, in particular of their key players, the mitochondrial ribosomes (mitoribosomes) in diverse eukaryotes (Waltz and Giegé, 2020). In particular, the mitoribosomes of the land plant Arabidopsis thaliana and cauliflower were recently characterized at the biochemical and structural levels, revealing their unique and unexpected features such as the occurrence of novel domains and additional plant-specific ribosomal proteins (Waltz et al., 2019, 2020a and 2020b). This represents the first examples of mitoribosome characterization in photosynthetic organisms. These major advances have only been possible as a result of the recent advances in cryo-electron microscopy (cryo-EM) techniques, acknowledged by the Nobel Prize in Chemistry to J. Frank, J. Dubochet, and R. Henderson in 2017 (Cressey and Callaway, 2017). Cryo-EM is an electron microscopy technique that is used to investigate the structure of various types of biomolecules from single proteins to large complexes such as ribosomes. The technique relies on imaging isolated macromolecules and complexes that are embedded in vitreous water on a microscopy grid. To do so, the liquid sample containing the biomolecules is flash-frozen to cryogenic temperatures in liquid ethane. Thanks to this process, the complexes can conserve their native organization and structure. Near atomic resolution structures, and even atomic structures (Nakane et al., 2020), of biological complexes, can be obtained through computational processing and averaging of multiple images, which ultimately allow 3D reconstruction of the complexes.

While ribosome purification and analysis by cryo-EM have been described elsewhere (Aibara et al., 2018), the protocol described herein details the specific procedures required to prepare the individual plant mitoribosome large subunit (LSU) and small subunit (SSU) and to single out the intact 78S plant mitoribosome monosomes. In addition to the specific steps for the biochemical preparation of plant mitoribosome samples, the protocol also describes the specific bioinformatics pipeline used for the cryo-EM analysis of plant mitoribosome samples. Although significant progress has recently been made in this field, numerous questions remain completely unexplored, such as how the mitoribosome interacts with the mitochondrial membrane insertase systems, how translation initiation functions, and how translation is regulated. The protocol described herein will be instrumental in helping to solve these key questions regarding plant mitochondrial translation.