Florent Waltz

Chloroplast ribosomes (chlororibosomes) synthesize the core protein components of the photosynthetic apparatus, yet their structural diversity outside flowering plants remains largely unexplored. Here we combine in situ cryo-electron tomography with single-particle cryo-electron microscopy to determine the structure of the chlororibosome from the unicellular green alga Chlamydomonas reinhardtii . Subtomogram averaging of chlororibosomes in their native environment, resolved to ~5 Å and in distinct translational states, reveals particles both free in the stroma and flexibly tethered to thylakoid membranes. These in situ reconstructions uncover an additional ‘arm’ domain on the small subunit. High-resolution single-particle structures of isolated chlororibosomes resolved to ~2.5 Å, in states bound either to the inhibitory translation factor pY or to a nascent chain-linked P-site transfer RNA, reveal that this domain is built primarily from extensive chloroplast-encoded insertions and extensions of conserved small subunit proteins, supported by chlororibosome-specific ribosomal proteins. The arm domain is located around the mRNA entry and exit channels, suggesting a role in stabilizing the mRNA trajectory through the small subunit and organizing chloroplast polysomes. Together, these data reveal the unexpected structural variation of algal chlororibosomes and suggest that chloroplast translation has diversified substantially even among relatively closely related photosynthetic lineages.

Regulated biogenesis of photosynthetic proteins by the chloroplast ribosomes is central to plant development and environmental adaptation. Here, we describe changes to the ribosome that occurred during evolution of chloroplasts from cyanobacteria in the photosynthetic lineage based on structural, proteomic and bioinformatic analyses. We identify structural features common to oxygenic photosynthetic organisms and distinct from other bacteria, as well as the gain and loss of ribosomal proteins, rRNA modifications and hibernation factors upon endosymbiosis. We uncover structural variation among land plant lineages at sites that remodel the peptide exit tunnel and features associated with translation initiation. The data challenge the prevailing view of ribosomal features that are specific to the chloroplast by demonstrating that many originated earlier, in cyanobacteria, or later, during land plant evolution.

Caractérisation de la machinerie de traduction mitochondriale chez Arabidopsis thaliana: Dans les cellules eucaryotes, différents types de ribosomes coexistent. Les ribosomes mitochondriaux synthétisent les quelques protéines codées par l’ADN mitochondrial, qui sont essentielles au fonctionnement de l’organisme. Ces ribosomes sont particulièrement divergents des ribosomes procaryotes, mais sont également très différents entre les eucaryotes. Mon travail de thèse s'est concentré sur la caractérisation de la structure et de la composition en protéines du ribosome mitochondrial de la plante modèle Arabidopsis thaliana. Des approches biochimiques complémentaires ont permis d’identifier 19 protéines uniquement trouvées dans le mitoribosome de plante, parmi lesquelles 10 sont des protéines PPR, des protéines particulièrement abondantes chez les plantes. Les mutations des gènes codant pour ces PPR ribosomales (rPPR) mènent à l’apparition de phénotypes macroscopiques distincts, notamment une létalité ou des retards de croissance importants. L'analyse moléculaire du mutant rppr1 par profilage des ribosomes, ainsi que l'analyse du taux de protéines mitochondriales, révèlent que la protéine rPPR1 est un facteur de traduction générique, ce qui constitue une nouvelle fonction des protéines PPR. De plus, la cryo-électron microscopie a été utilisée pour déterminer l’architecture tridimensionnelle de ce mitoribosome. Cette approche a révélé la structure unique du mitoribosome de plante, caractérisée par une très grande petite sous-unité ribosomale ayant un domaine additionnel jamais décrit jusqu’à présent. Globalement, mes résultats ont montré que le mitoribosome d’Arabidopsis est complètement différent des ribosomes bactériens et des autres mitoribosomes eucaryotes, à la fois en terme de structure mais aussi de composition, permettant ainsi de mieux comprendre l’évolution de ce composant central de l’expression génétique.

Chloroplast ribosomes (chlororibosomes) synthesize the core protein components of the photosynthetic apparatus, yet their structural diversity outside flowering plants remains largely unexplored. Here, we combine in situ cryo-electron tomography (cryo-ET) with single-particle cryo-electron microscopy (cryo-EM) to determine the structure of the chlororibosome from the unicellular green alga Chlamydomonas reinhardtii . Subtomogram averaging of chlororibosomes in their native environment, resolved to ∼5 Å resolution and in distinct translational states, reveals particles both free in the stroma and loosely tethered to thylakoid membranes. These in situ reconstructions uncover an additional “arm” domain on the small subunit. High-resolution single-particle reconstruction of isolated chlororibosomes to ∼2.5 Å, in states bound either to the inhibitory translation factor pY or to a nascent chain-linked P-site tRNA, reveals that this domain is built primarily from extensive chloroplast-encoded insertions and extensions of conserved small subunit proteins, supported by chlororibosome-specific ribosomal proteins. The arm domain is located around the mRNA entry and exit channels, suggesting a role in stabilizing the mRNA trajectory through the small subunit and organizing chloroplast polysomes. Together, these data reveal unexpected structural variation of algal chlororibosomes and suggest that chloroplast translation has diversified substantially even among relatively closely related photosynthetic lineages.

Ribosomes are key molecular machines that translate mRNA into proteins. Mitoribosomes are specific ribosomes found in mitochondria, which have been shown to be remarkably diverse across eukaryotic lineages. In plants, they possess unique features, including additional rRNA domains stabilized by plant-specific proteins. However, the structural specificities of plant mitoribosomes in translation state remained unknown. We used cryo-electron microscopy to provide a high-resolution structural characterization of the cauliflower mitoribosome, in translating and maturation states. The structure reveals the mitoribosome bound with a tRNA in the peptidyl site, along with a segment of mRNA and a nascent polypeptide. Moreover, using structural data, nanopore sequencing and mass spectrometry, we identify a set of 19 ribosomal RNA modifications. Additionally, we observe a late assembly intermediate of the small ribosomal subunit, in complex with the RsgA assembly factor. This reveals how a plant-specific extension of RsgA blocks the mRNA channel to prevent premature mRNA association before complete small subunit maturation. Our findings elucidate key aspects of translation in angiosperm plant mitochondria, revealing its distinct features compared to other eukaryotic lineages.

The mitochondrial NADH:ubiquinone oxidoreductase, or complex I, is composed of a hydrophobic arm comprising the P module and a hydrophilic arm comprising the N and Q modules. The assembly of complex I is well characterized in humans and is catalyzed by a series of assembly factors that join the Q, P, and N modules sequentially. The complex I of protists and plants, however, contains additional ancestral features, namely a ferredoxin bridge that connects the matrix and the membrane arms and a γ carbonic anhydrase domain, whose mechanisms of assembly are unknown. In this work, a strain where the complex I assembly factor NDUFAF3 has been tagged with a 3×FLAG at the C‐terminus is investigated in the green microalga Chlamydomonas reinhardtii . Like its human homolog, NDUFAF3 interacts strongly with the classical subunits of the Q and P modules, but also with the γ carbonic anhydrase domain and C1‐FDX, a subunit of the ferredoxin bridge. The predicted structural positioning of NDUFAF3 within the Q module suggests a role in the formation of this bridge. In contrast, subunits of the N module are only loosely associated with NDUFAF3. We further demonstrate that the N module is attached at a later stage of assembly, suggesting that Chlamydomonas complex I assembles in a human‐like sequence. This contrasts with what is documented in Angiosperms, where the N and Q modules are attached together before anchoring to the P module. Altogether, these results highlight a conserved and ancestral role of NDUFAF3 in complex I manufacture.

Mitochondria regenerate adenosine triphosphate (ATP) through oxidative phosphorylation. This process is carried out by five membrane-bound complexes collectively known as the respiratory chain, working in concert to transfer electrons and pump protons. The precise organization of these complexes in native cells is debated. We used in situ cryo–electron tomography to visualize the native structures and organization of several major mitochondrial complexes in Chlamydomonas reinhardtii cells. ATP synthases and respiratory complexes segregate into curved and flat crista membrane domains, respectively. Respiratory complexes I, III, and IV assemble into a respirasome supercomplex, from which we determined a native 5-angstrom (Å) resolution structure showing binding of electron carrier cytochrome c . Combined with single-particle cryo–electron microscopy at 2.4-Å resolution, we model how the respiratory complexes organize inside native mitochondria.

In situ cryo-electron tomography (cryo-ET) has emerged as the method of choice to investigate structures of biomolecules in their native context. However, challenges remain in the efficient production of large-scale cryo-ET datasets, as well as the community sharing of this information-rich data. Here, we applied a cryogenic plasma-based focused ion beam (cryo-PFIB) instrument for high-throughput milling of the green alga Chlamydomonas reinhardtii , a useful model organism for in situ visualization of numerous fundamental cellular processes. Combining cryo-PFIB sample preparation with recent advances in cryo-ET data acquisition and processing, we generated a dataset of 1829 reconstructed and annotated tomograms, which we provide as a community resource to drive method development and inspire biological discovery. To assay the quality of this dataset, we performed subtomogram averaging (STA) of both soluble and membrane-bound complexes ranging in size from >3 MDa to ∼200 kDa, including 80S ribosomes, Rubisco, nucleosomes, microtubules, clathrin, photosystem II, and mitochondrial ATP synthase. The majority of these density maps reached sub-nanometer resolution, demonstrating the potential of this C. reinhardtii dataset, as well as the promise of modern cryo-ET workflows and open data sharing towards visual proteomics.

Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here, we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a “one CLSY per Pol IV” model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null and clsy quadruple mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.