Mitochondria maintain their own genome and its corresponding protein synthesis machine, the mitochondrial ribosome (mitoribosome). Mitoribosomes primarily synthesize highly hydrophobic proteins of the inner mitochondrial membrane. Recent studies revealed the complete structure of the isolated mammalian mitoribosome, but its mode of membrane association remained hypothetical. In this study, we used cryoelectron tomography to visualize human mitoribosomes in isolated mitochondria. The subtomogram average of the membrane-associated human mitoribosome reveals a single major contact site with the inner membrane, mediated by the mitochondria-specific protein mL45. A second rRNA-mediated contact site that is present in yeast is absent in humans, resulting in a more variable association of the human mitoribosome with the inner membrane. Despite extensive structural differences of mammalian and fungal mitoribosomal structure, the principal organization of peptide exit tunnel and the mL45 homolog remains invariant, presumably to align the mitoribosome with the membrane-embedded insertion machinery.

Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Additional proteins, mainly OPR, PPR and mTERF helical repeat proteins, are found in Chlamydomonas mitoribosome, revealing the structure of an OPR protein in complex with its RNA binding partner. Targeted amiRNA silencing indicates that these ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the inner mitochondrial membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of mitoribosome diversity and the various strategies these specialized molecular machines adopt for membrane tethering.

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A large fraction of ribosomal synthesis occurs at organellar membranes. At the endoplasmic reticulum (ER), the inner mitochondrial, and the thylakoid membrane, nascent proteins are co‐translationally inserted or transported into other compartments. Here, we structurally study membrane‐bound ribosomes and their associated machineries in their native settings using cryo‐electron tomography (cryo‐ET) in combination with subtomogram analysis (Fig. 1). Studies of ribosomes associated to isolated rough ER vesicles reveal the structure of the native ER translocon, as well as its compositional variability. Core components of the ER translocon are the protein‐conducting channel Sec61, the translocon associated protein complex (TRAP), and the sub‐stoichiometric oligosaccharyl transferase complex (OST), which all bind to the ribosome. Subnanometer resolution subtomogram averages indicate that the ribosome alone, even without a nascent chain, is sufficient for lateral opening of Sec61, contrary to recent mechanistic models. To elucidate the structures and functions of TRAP and OST in detail, we make use of mutations involved in congenital disorders, as well as their evolutionary diversity across different organisms. Analysis of cryo‐tomograms from focused‐ion‐beam‐milled whole cells allows studying the compositional variability of the ER‐translocon and the relative arrangement of ER‐associated ribosomes in vivo , which reveals a highly characteristic polysome organization. Mitochondrial ribosomes specialize on the synthesis of few, very hydrophobic membrane proteins. Cryo‐electron tomographic analysis of mitochondria isolated from Saccharomyces cerevisiae reveals the binding mode of mitoribosomes to the inner mitochondrial membrane, as well as their molecular organization into polysomes. The structures of mammalian mitoribosomes differ dramatically from their fungal counterparts and we study the consequences of these differences on membrane association and polysome organization. State‐of‐the‐art phase plate imaging helps to overcome the contrast limitations set by the extremely dense and optically barely electron transparent mammalian mitochondria. Chloroplast ribosomes constitute the third realm of eukaryotic ribosomes. We analyzed the in situ structure and intracellular distribution in green algae. The interaction mode of ribosomes with the thylakoid membrane appears to be much less defined than those of their cytoplasmic and mitochondrial counterparts. In summary, in situ studies using cryoelectron tomography put atomic‐level structural information of ribosomal complexes into context with their associated organellar membranes and their respective co‐translational processing machineries, revealing high evolutionary diversity for organelles and organisms.

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