Alexey Amunts

Structure of the Yeast Mitochondrial Large Ribosomal Subunit

Mitoribosomes Mitochondria—found in all eukaryotic cells—have transferred most of their genes to the nuclear genome. The nuclear-localized mitochondrial genes are expressed and translated in the cytoplasm and the resulting mitochondrial proteins are imported into the mitochondria. Nevertheless, a few genes remain within mitochondria in the mitochondrial genome, and these genes are translated by mitochondrial ribosomes (mitoribosomes). Amunts et al. (p. 1485; see the Perspective by Kühlbrandt) determined the structure of mitoribosomes from yeast using single-particle cryo–electron microscopy. The mitoribosome is highly diverged from the bacterial and eukaryotic ribosomes with, for example, a distinctive exit tunnel for the newly synthesized peptide, and a membrane facing protuberance that might help to anchor the mitoribosome to the mitochondrial membrane. The mitochondrial ribosome structure has substantially diverged from that of bacterial and eukaryotic ribosomes. [Also see Perspective by Kühlbrandt] Mitochondria have specialized ribosomes that have diverged from their bacterial and cytoplasmic counterparts. We have solved the structure of the yeast mitoribosomal large subunit using single-particle cryo–electron microscopy. The resolution of 3.2 angstroms enabled a nearly complete atomic model to be built de novo and refined, including 39 proteins, 13 of which are unique to mitochondria, as well as expansion segments of mitoribosomal RNA. The structure reveals a new exit tunnel path and architecture, unique elements of the E site, and a putative membrane docking site.

The structure of the human mitochondrial ribosome

The whole mitoribosome at high resolution Mitochondria are thought to be the descendents of a prokaryotic cell that took up residence in a protoeukaryotic cell. Mitochondria retain a few genes involved in oxidative phosphorylation. To translate these genes, mitochondria contain highly divergent mitochondrial ribosomes, or mitoribosomes. Amunts et al. determined the high-resolution structures of complete mammalian mitoribosomes using cryoelectron microscopy. Mitoribosomes include an unusual mRNA binding channel. The findings elucidate how aminoglycoside antibiotics can inadvertently inhibit mitoribosomes and how mutations in mitoribosomes can lead to disease. Science, this issue p. 95 Structures of mammalian mitoribosomes reveal details underlying the function of this divergent class of ribosome. The highly divergent ribosomes of human mitochondria (mitoribosomes) synthesize 13 essential proteins of oxidative phosphorylation complexes. We have determined the structure of the intact mitoribosome to 3.5 angstrom resolution by means of single-particle electron cryogenic microscopy. It reveals 80 extensively interconnected proteins, 36 of which are specific to mitochondria, and three ribosomal RNA molecules. The head domain of the small subunit, particularly the messenger (mRNA) channel, is highly remodeled. Many intersubunit bridges are specific to the mitoribosome, which adopts conformations involving ratcheting or rolling of the small subunit that are distinct from those seen in bacteria or eukaryotes. An intrinsic guanosine triphosphatase mediates a contact between the head and central protuberance. The structure provides a reference for analysis of mutations that cause severe pathologies and for future drug design.

Structure of the large ribosomal subunit from human mitochondria

Making mitochondrial hydrophobic proteins Mitochondria produce chemical energy for the cell. Human mitochondria have their own specific ribosomes—mitoribosomes, which are distinct from cytoplasmic ribosomes. Mitoribosomes synthesize the mitochondrial membrane proteins that generate the chemical energy. Brown et al. used cryo–electron microscopy to determine the high-resolution structure of the large subunit of the human mitoribosome. The mitoribosome has a number of unique features, including an exit tunnel lined with hydrophobic amino acid residues. Science, this issue p. 718 The structure of the human mitochondrial ribosome reveals how it is adapted to synthesize mitochondrial membrane proteins. Human mitochondrial ribosomes are highly divergent from all other known ribosomes and are specialized to exclusively translate membrane proteins. They are linked with hereditary mitochondrial diseases and are often the unintended targets of various clinically useful antibiotics. Using single-particle cryogenic electron microscopy, we have determined the structure of its large subunit to 3.4 angstrom resolution, revealing 48 proteins, 21 of which are specific to mitochondria. The structure unveils an adaptation of the exit tunnel for hydrophobic nascent peptides, extensive remodeling of the central protuberance, including recruitment of mitochondrial valine transfer RNA (tRNAVal) to play an integral structural role, and changes in the tRNA binding sites related to the unusual characteristics of mitochondrial tRNAs.

Organization and Regulation of Mitochondrial Protein Synthesis

Mitochondria are essential organelles of endosymbiotic origin that are responsible for oxidative phosphorylation within eukaryotic cells. Independent evolution between species has generated mitochondrial genomes that are extremely diverse, with the composition of the vestigial genome determining their translational requirements. Typically, translation within mitochondria is restricted to a few key subunits of the oxidative phosphorylation complexes that are synthesized by dedicated ribosomes (mitoribosomes). The dramatically rearranged mitochondrial genomes, the limited set of transcripts, and the need for the synthesized proteins to coassemble with nuclear-encoded subunits have had substantial consequences for the translation machinery. Recent high-resolution cryo-electron microscopy has revealed the effect of coevolution on the mitoribosome with the mitochondrial genome. In this review, we place the new structural information in the context of the molecular mechanisms of mitochondrial translation and focus on the novel ways protein synthesis is organized and regulated in mitochondria.

The structure of the yeast mitochondrial ribosome.

The yeast mitoribosome Mitochondria are eukaryotic organelles that produce ATP, the energy source of the cell. They have dedicated ribosomes (mitoribosomes) that encode some of the membrane proteins that are essential to ATP production. Desai et al. present a high-resolution structure of the 75-component yeast mitoribosome, determined by electron cryomicroscopy. Mitoribosomes share an ancestor with modern bacterial ribosomes. Comparing the structure of the yeast mitoribosome with mammalian mitoribosomes suggests how they have evolved differently to perform species-specific functions. Science, this issue p. 528 An electron cryomicroscopy structure gives insight into how mitoribosomes have evolved for species-specific function. Mitochondria have specialized ribosomes (mitoribosomes) dedicated to the expression of the genetic information encoded by their genomes. Here, using electron cryomicroscopy, we have determined the structure of the 75-component yeast mitoribosome to an overall resolution of 3.3 angstroms. The mitoribosomal small subunit has been built de novo and includes 15S ribosomal RNA (rRNA) and 34 proteins, including 14 without homologs in the evolutionarily related bacterial ribosome. Yeast-specific rRNA and protein elements, including the acquisition of a putatively active enzyme, give the mitoribosome a distinct architecture compared to the mammalian mitoribosome. At an expanded messenger RNA channel exit, there is a binding platform for translational activators that regulate translation in yeast but not mammalian mitochondria. The structure provides insights into the evolution and species-specific specialization of mitochondrial translation.

Mitochondrial ribosome assembly in health and disease

The ribosome is a structurally and functionally conserved macromolecular machine universally responsible for catalyzing protein synthesis. Within eukaryotic cells, mitochondria contain their own ribosomes (mitoribosomes), which synthesize a handful of proteins, all essential for the biogenesis of the oxidative phosphorylation system. High-resolution cryo-EM structures of the yeast, porcine and human mitoribosomal subunits and of the entire human mitoribosome have uncovered a wealth of new information to illustrate their evolutionary divergence from their bacterial ancestors and their adaptation to synthesis of highly hydrophobic membrane proteins. With such structural data becoming available, one of the most important remaining questions is that of the mitoribosome assembly pathway and factors involved. The regulation of mitoribosome biogenesis is paramount to mitochondrial respiration, and thus to cell viability, growth and differentiation. Moreover, mutations affecting the rRNA and protein components produce severe human mitochondrial disorders. Despite its biological and biomedical significance, knowledge on mitoribosome biogenesis and its deviations from the much-studied bacterial ribosome assembly processes is scarce, especially the order of rRNA processing and assembly events and the regulatory factors required to achieve fully functional particles. This article focuses on summarizing the current available information on mitoribosome assembly pathway, factors that form the mitoribosome assembly machinery, and the effect of defective mitoribosome assembly on human health.

Bactobolin A Binds to a Site on the 70S Ribosome Distinct from Previously Seen Antibiotics

The ribosome is the target of a large number of antibiotics. Here, we report a 3.4-Å-resolution crystal structure of bactobolin A bound to 70S ribosome–tRNA complex. The antibiotic binds at a previously unseen site in the 50S subunit and displaces tRNA bound at the P-site. It thus likely has a similar mechanism of action as blasticidin S despite binding to a different site. The structure also rationalizes previously identified resistance mutations.

Structures of the human mitochondrial ribosome in native states of assembly

Mammalian mitochondrial ribosomes (mitoribosomes) have less rRNA content and 36 additional proteins compared with the evolutionarily related bacterial ribosome. These differences make the assembly of mitoribosomes more complex than the assembly of bacterial ribosomes, but the molecular details of mitoribosomal biogenesis remain elusive. Here, we report the structures of two late-stage assembly intermediates of the human mitoribosomal large subunit (mt-LSU) isolated from a native pool within a human cell line and solved by cryo-EM to ∼3-Å resolution. Comparison of the structures reveals insights into the timing of rRNA folding and protein incorporation during the final steps of ribosomal maturation and the evolutionary adaptations that are required to preserve biogenesis after the structural diversification of mitoribosomes. Furthermore, the structures redefine the ribosome silencing factor (RsfS) family as multifunctional biogenesis factors and identify two new assembly factors (L0R8F8 and mt-ACP) not previously implicated in mitoribosomal biogenesis.

Publisher Correction: Structure of the chloroplast ribosome with chl-RRF and hibernation-promoting factor

In the version of this Article originally published, the name of co-author Annemarie Perez Boerema was coded wrongly, resulting in it being incorrect when exported to citation databases. This has been corrected, though no visible changes will be apparent.

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart. Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.