Mitochondria are the powerhouses of eukaryotic cells and the site of essential metabolic reactions. Complex I or NADH:ubiquinone oxidoreductase is the main entry site for electrons into the mitochondrial respiratory chain and constitutes the largest of the respiratory complexes. Its structure and composition vary across eukaryote species. However, high resolution structures are available only for one group of eukaryotes, opisthokonts. In plants, only biochemical studies were carried out, already hinting at the peculiar composition of complex I in the green lineage. Here, we report several cryo-electron microscopy structures of the plant mitochondrial complex I. We describe the structure and composition of the plant respiratory complex I, including the ancestral mitochondrial domain composed of the carbonic anhydrase. We show that the carbonic anhydrase is a heterotrimeric complex with only one conserved active site. This domain is crucial for the overall stability of complex I as well as a peculiar lipid complex composed of cardiolipin and phosphatidylinositols. Moreover, we also describe the structure of one of the plant-specific complex I assembly intermediates, lacking the whole PD module, in presence of the maturation factor GLDH. GLDH prevents the binding of the plant specific P1 protein, responsible for the linkage of the PP to the PD module.
The vast majority of eukaryotic cells contain mitochondria, essential powerhouses and metabolic hubs 1 . These organelles have a bacterial origin and were acquired during an early endosymbiosis event 2 . Mitochondria possess specialized gene expression systems composed of various molecular machines including the mitochondrial ribosomes (mitoribosomes). Mitoribosomes are in charge of translating the few essential mRNAs still encoded by mitochondrial genomes 3 . While chloroplast ribosomes strongly resemble those of bacteria 4,5 , mitoribosomes have diverged significantly during evolution and present strikingly different structures across eukaryotic species [6][7][8][9][10] . In contrast to animals and trypanosomatides, plants mitoribosomes have unusually expanded ribosomal RNAs and conserved the short 5S rRNA, which is usually missing in mitoribosomes 11 . We have previously characterized the composition of the plant mitoribosome 6 revealing a dozen plant-specific proteins, in addition to the common conserved mitoribosomal proteins. In spite of the tremendous recent advances in the field, plant mitoribosomes remained elusive to high-resolution structural investigations, and the plant-specific ribosomal features of unknown structures. Here, we present a cryoelectron microscopy study of the plant 78S mitoribosome from cauliflower at near-atomic resolution. We show that most of the plant-specific ribosomal proteins are pentatricopeptide repeat proteins (PPR) that deeply interact with the plant-specific rRNA expansion segments. These additional rRNA segments and proteins reshape the overall structure of the plant mitochondrial ribosome, and we discuss their involvement in the membrane association and mRNA recruitment prior to translation initiation. Finally, our structure unveils an rRNAconstructive phase of mitoribosome evolution across eukaryotes.Previously, we determined the full composition as well as the overall architecture of the Arabidopsis thaliana mitoribosome 6 . However, due to the difficulty to purify large amounts of A. thaliana mitoribosomes, mainly because of the low quantities of plant material usable for mitochondrial extraction, only a low-resolution cryo-EM reconstruction was derived. In order to obtain a highresolution structure of the plant mitochondrial ribosome, we purified mitoribosome from a closely related specie, Brassica oleracea var. botrytis, or cauliflower (both Arabidopsis and cauliflower belong to the group of Brassicaceae plants), as previously described 6 (see Methods). We have recorded cryo-EM images for ribosomal complexes purified from two different sucrose gradient peaks (see Methods), corresponding to the small ribosomal subunit (SSU) and the full 78S mitoribosome. After extensive particle sorting (see Methods) we have obtained cryo-EM reconstructions for both types of complexes. The SSU reconstruction displayed an average resolution of 4.36Å (Extended Data Fig. 1). After multibody refinement (3 bodies) and particle polishing in RELION3 12 (see Methods), reconstructions were de...
Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed inTrypanosoma bruceialready highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoansLeishmania tarentolaeandTrypanosoma cruzimitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.
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