Organization of the mitochondrial translation machinery studied in situ by cryoelectron tomography

Abstract

Whereas the structure and function of cytosolic ribosomes have been studied in great detail, we know surprisingly little about the structural basis of mitochondrial protein synthesis. Here we used cryoelectron tomography and subtomogram analysis to visualize mitoribosomes in isolated yeast mitochondria, avoiding perturbations during ribosomal purification. Most mitoribosomes reside in immediate proximity to the inner mitochondrial membrane, in line with their specialization in the synthesis of hydrophobic membrane proteins. The subtomogram average of membrane-associated mitoribosomes reveals two distinct membrane contact sites, formed by the 21S rRNA expansion segment 96-ES1 and the inner membrane protein Mba1. On the basis of our data, we further hypothesize that Mba1 is not just a passive mitoribosome receptor on the inner membrane, but that it spatially aligns mitoribosomes with the membrane insertion machinery. This study reveals detailed insights into the supramolecular organization of the mitochondrial translation machinery and its association with the inner membrane in translation-competent mitochondria.

A focused ion beam milling and lift-out approach for site-specific preparation of frozen-hydrated lamellas from multicellular organisms

“…The ultimate goal of cryo-ET is the visualization of macromolecular complexes in their native environment -the cell -and to extract information on their spatial distribution and functional state (Asano et al, 2015;Pfeffer et al, 2015). Recent advances in the field tackle the inherent difficulties associated with low dose imaging of vitrified, low contrast, biological material.…”

“…Vitrification, the freezing of water in an amorphous state, ensures the preservation of biological structures in their near-native state and thus allows the study of macromolecular complexes in their functional environment (Dubochet and Mcdowall, 1981;McDowall et al, 1983). The unique combination of cryogenic sample preservation and low temperature ET has provided unprecedented insights into the supramolecular structure of cells at a resolution of 3-6 nm (Asano et al, 2015;Brandt et al, 2010;Fernandez-Busnadiego et al, 2010;Medalia et al, 2002;Nicastro et al, 2006;Ortiz et al, 2010) and with thin samples, sub-nanometer resolution can be obtained (Pfeffer et al, 2015;Schur et al, 2015).…”

Subtomogram analysis using the Volta phase plate

“…In combination with advanced image processing methods ('subtomogram analysis'), the location, orientation and, in particular, the structure of larger macromolecular complexes can be extracted from tomographic data (Briggs, 2013;Forster and Hegerl, 2007). CET is an excellent method for studying the molecular machine responsible for cellular protein synthesis, the ribosome, in a native context (Brandt et al, 2009;Myasnikov et al, 2014;Pfeffer et al, 2015;Pfeffer et al, 2012;Pfeffer et al, 2014). The ribosome is a universally conserved RNA-protein complex of 3-4 MDa size consisting of two subunits, which in concert mediate decoding of messenger RNA and formation of peptide bonds in the growing polypeptide chain (Steitz, 2008).…”

Three-dimensional CTF correction improves the resolution of electron tomograms

“…Reconstructions of mitochondrial ribosomes (Pfeffer et al, 2015a), and (b) The tobacco mosaic virus (TMV). For the mitochondrial reconstruction the recording parameters are described in detail elsewhere (Pfeffer et al, 2015a).…”