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.

Figures:

Fig. 1. Subtomogram analysis applied to the ER-associated mammalian ribosome. A: Principle of subtomogram analysis. A tomogram of a ribosome-studded microsome is reconstructed from projections. Particles (ribo-somes) are detected using automated methods and the corresponding subtomograms are aligned. Classification allows resolving different populations of particles. B: Architecture of the major complexes constituting the na-tive translocon (protein-conducting channel Sec61, TRAP complex, and oligosaccharyl transferase complex, OST) resolved by subtomogram averaging to ~8 Å.

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EMC Abstracts - https://emc-proceedings.com/abstract/native-machinery-of-membrane-associated-protein-synthesis/