Sample Preparation of Isolated Mitochondria for Cryoelectron Tomography and In Situ Studies of Translation

Cryoelectron tomography is a method to image biological samples three-dimensionally at molecular resolution. This modality provides insights into intracellular processes in their physiological settings. Obtaining a high-quality sample for cryoelectron tomography on mitochondria, however, can be challenging. In this chapter, we describe the crucial steps from sample preparation to data acquisition enabling studies of mitochondrial translation in situ by cryoelectron tomography. We provide detailed protocols for yeast and human mitochondria preparations yielding a high concentration of intact mitochondrial vesicles on cryo-EM grids. In addition, we describe a workflow for particle identification and spatial mapping in context of the organelle

Contactin 2 homophilic adhesion structure and conformational plasticity

The cell-surface attached glycoprotein contactin 2 is ubiquitously expressed in the nervous system and mediates homotypic cell-cell interactions to organize cell guidance, differentiation, and adhesion. Contactin 2 consists of six Ig and four fibronectin type III domains (FnIII) of which the first four Ig domains form a horseshoe structure important for homodimerization and oligomerization. Here we report the crystal structure of the six-domain contactin 2 Ig1-6 and show that the Ig5-Ig6 combination is oriented away from the horseshoe with flexion in interdomain connections. Two distinct dimer states, through Ig1-Ig2 and Ig3-Ig6 interactions, together allow formation of larger oligomers. Combined size exclusion chromatography with multiangle light scattering (SEC-MALS), small-angle X-ray scattering (SAXS) and native MS analysis indicates contactin 2 Ig1-6 oligomerizes in a glycan dependent manner. SAXS and negative-stain electron microscopy reveals inherent plasticity of the contactin 2 full-ectodomain. The combination of intermolecular binding sites and ectodomain plasticity explains how contactin 2 can function as a homotypic adhesion molecule in diverse intercellular environments

Protein S-Bacillithiolation Functions in Thiol Protection and Redox Regulation of the Glyceraldehyde-3-Phosphate Dehydrogenase Gap in Staphylococcus aureus Under Hypochlorite Stress

Aims: Bacillithiol (BSH) is the major low-molecular-weight thiol of the human pathogen Staphylococcus aureus. In this study, we used OxICAT and Voronoi redox treemaps to quantify hypochlorite-sensitive protein thiols in S. aureus USA300 and analyzed the role of BSH in protein S-bacillithiolation.  Results: The OxICAT analyses enabled the quantification of 228 Cys residues in the redox proteome of S. aureus USA300. Hypochlorite stress resulted in >10% increased oxidation of 58 Cys residues (25.4%) in the thiol redox proteome. Among the highly oxidized sodium hypochlorite (NaOCl)-sensitive proteins are five S-bacillithiolated proteins (Gap, AldA, GuaB, RpmJ, and PpaC). The glyceraldehyde-3-phosphate (G3P) dehydrogenase Gap represents the most abundant S-bacillithiolated protein contributing 4% to the total Cys proteome. The active site Cys151 of Gap was very sensitive to overoxidation and irreversible inactivation by hydrogen peroxide (H2O2) or NaOCl in vitro. Treatment with H2O2 or NaOCl in the presence of BSH resulted in reversible Gap inactivation due to S-bacillithiolation, which could be regenerated by the bacilliredoxin Brx (SAUSA300_1321) in vitro. Molecular docking was used to model the S-bacillithiolated Gap active site, suggesting that formation of the BSH mixed disulfide does not require major structural changes.  Conclusion and Innovation: Using OxICAT analyses, we identified 58 novel NaOCl-sensitive proteins in the pathogen S. aureus that could play protective roles against the host immune defense and include the glycolytic Gap as major target for S-bacillithiolation. S-bacillithiolation of Gap did not require structural changes, but efficiently functions in redox regulation and protection of the active site against irreversible overoxidation in S. aureus. Antioxid. Redox Signal. 28, 410–430

Structural insights into protein biogenesis on the mitochondrial surface

Translation on the surface of the endoplasmic reticulum (ER), as well as co-translational import into the ER have been well characterized, while complementary processes on the mitochondrial surface have remained largely enigmatic. In this thesis we aimed to improve our understanding of the cytosolic ribosome-mitochondria association, polyribosome organization and states of translation on the outer mitochondrial membrane (OMM). The first chapter describes the detailed protocols used for the sample preparation of isolated yeast and human mitochondria from cultured cells and gives an overview over the main method used for this thesis, cryo-ET. In the second chapter we describe the molecular organization of the cytosolic translation machinery on the surface of isolated yeast mitochondria. We present spatial, geometrical, and molecular analysis of ribosomes attached to mitochondria using clustering, subtomogram-averaging and 3D classification approaches. We show that polysome organization adapts to the geometrical constraints of the mitochondrial outer membrane (OMM), but within a flexible regime. Most ribosomes are programmed, whereas only 4 % are found hibernating and connected to the OMM in an unexpected manner. Based on the observation that the conformation of ES27L correlates with the presence of OMM density in subtomogram averages, we visualized a molecular anchor connecting ribosomes to the yeast OMM. A final comparison to published data from Schizosaccharomyces pombe reveals clear species-specific differences and suggests translation on the mitochondrial surface needs to be addressed in several species, and likely differs among different eukaryotes. In the third chapter we build on the toolset used and developed in the second chapter and apply it to cryo-electron tomograms of isolated human mitochondria. In contrast to yeast, the translation arresting drug cycloheximide is not strictly necessary to keep ribosomes attached to mitochondria during isolation. The majority of ribosomes are programmed with tRNAs, unexpectedly with the A-tRNA delivering elongation factor eEF1A still present. The inactive proportion is bound by the elongation factor eEF2 associated with hibernating ribosomes. In contrast to yeast there is no correlation between ES27L conformation and distance or orientation to the OMM, since on the human OMM ribosomes predominantly attach in a tilted manner via ES39L. Comparison to ER-bound ribosomes highlights in total three rRNA expansion segments as organelle specificity facilitating modules in humans. Comparison to less complex eukaryotes furthermore illustrates how ribosomal expansion segments contribute to molecular specialization in higher eukaryotes. In the fourth chapter we describe our efforts to reconstitute the ribosome-mitochondria association observed in the third chapter in vitro with the aim to generate a more defined sample, more suitable for averaging techniques. For initial validation of the mitochondrial sample used in the previous chapters, we established an in vitro import assay using HEK cell mitochondria and radiolabeled in vitro translated mitochondrial precursors. After adaptation of the protocol for cryo-ET, we successfully targeted ribosomes to the outer mitochondrial membrane in vitro. However, we found these ribosomes in an unexpected state and associated in a peculiar manner