Christensenkryger8998

Cryo-electron tomography (cryo-ET) enables the three-dimensional (3D) visualization of macromolecular complexes in their native environment (in situ). The ability to visualize macromolecules in situ is in particular advantageous for complex, membrane-associated processes, such as mitochondrial translation. Mitochondrial translation occurs almost exclusively associated with the inner mitochondrial membrane, giving rise to the mitochondrial DNA-encoded subunits of oxidative phosphorylation machinery. In cryo-ET, the 3D volume is reconstructed from a set of 2D projections of a frozen-hydrated specimen, which is sequentially tilted and imaged at different angles in a transmission electron microscope. In combination with subtomogram analysis, cryo-ET enables the structure determination of macromolecular complexes and their 3D organization. In buy Tolinapant , we summarize all steps required for structural characterization of mitochondrial ribosomes in situ, ranging from data acquisition to tomogram reconstruction and subtomogram analysis.The mitochondrial genome encodes only a handful of proteins, but methods to track their synthesis are highly limited. Saccharomyces cerevisiae is a model organism that offers possibilities to expand the classical systems to analyze mitochondrial translation. In this chapter, we present two approaches of monitoring mitochondrial protein synthesis. Labeling of mitochondrially translated products with radioactive amino acids can be performed either in intact cells or in isolated mitochondria. However, these classical methods have disadvantages that can affect cell physiology and hence are not suitable for all types of research questions. Some of these limitations can be overcome by the use of reporter genes that are inserted into yeast genetic screens mitochondrial DNA via biolistic transformation. These reporter genes can be used for yeast genetic screen and to monitor regulation and efficiency of mitochondrial translation with a variety of methods.Mitochondria contain ribosomes (mitoribosomes) specialized in the synthesis of a handful of proteins essential for oxidative phosphorylation. Therefore, mitoribosome integrity and function are essential for the life of eukaryotic cells and lesions that affect them result in devastating human disorders. To broadly analyze the integrity and assembly state of mitoribosomes it is useful to start by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts. During centrifugation, mitoribosome subunits, monosomes and polysomes, and potentially accumulated assembly intermediates will sediment through the gradient at different rates. #link# Sedimentation will depend on the centrifugal force applied and the density and viscosity of the gradient. Importantly, it will also depend on the size, shape, and density of the mitoribosome particles present in the samples under study. Variations of this technique, often coupled with additional downstream approaches, have been used to analyze the process of mitoribosome biogenesis, the composition of assembly intermediates, or to monitor the interaction of extraribosomal proteins with individual mitoribosome subunits or monosomes.Mitochondrial ribosomes (mitoribosomes) are specialized machineries that carry out the synthesis of a limited number of proteins encoded in the mitochondrial genome, including components of the oxidative phosphorylation pathway. They have incorporated several structural features distinguishing them from bacterial and eukaryotic cytosolic counterparts. Our current understanding of the assembly and functioning of mitoribosomes is limited, and recent developments in cryo-EM provide promising directions for detailed investigation. Here we describe methods to purify mitoribosomes from human embryonic kidney cells for cryo-EM studies.Ribosome profiling (Ribo-Seq) is a technique that allows genome-wide, quantitative analysis of translation. In recent years, it has found multiple applications in studies of translation in diverse organisms, tracking protein synthesis with single codon resolution. Traditional protocols applied for generating Ribo-Seq libraries from mammalian cell cultures are not suitable to study mitochondrial translation due to differences between eukaryotic cytosolic and mitochondrial ribosomes. Here, we present an adapted protocol enriching for mitoribosome footprints. In addition, we describe the preparation of small RNA sequencing libraries from the resultant mitochondrial ribosomal protected fragments (mtRPFs).Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear. Methods are being developed to determine whether all members of a particular complex are translated in close proximity, whether protein synthesis is clustered in submitochondrial factories, whether these align with incoming polypeptides, and if there is evidence for co-translational translation that is regulated and limited by the interaction of the incoming proteins with synthesis of their mtDNA-encoded partners. Two methods are described in this chapter to visualize the distribution of mitochondrial ribosomal RNAs in conjunction with newly synthesized mitochondrial proteins. The first combines RNA Fluorescent In Situ Hybridization (FISH) and super-resolution immunocytochemistry to pinpoint mitochondrial ribosomal RNA. The second localizes nascent translation within the mitochondrial network through non-canonical amino acid labeling, click chemistry and fluorescent microscopy.Even though the mammalian mitochondrial genome (mtDNA) is very small and only codes for 13 proteins, all being subunits of the oxidative phosphorylation system, it requires several hundred nuclear encoded proteins for its maintenance and expression. These include replication and transcription factors, approximately 80 mitoribosomal proteins and many proteins involved in the posttranscriptional modification, processing, and stability of mitochondrial RNAs. In recent years, many of these factors have been identified and functionally characterized, but the complete mtRNA-interacting proteome is not firmly established. Shotgun proteomics has been used successfully to define whole-cell polyadenylated RNA (poly(A)-RNA) interacting proteomes using the nucleotide analogue 4-thiouridine (4SU) combined with UV crosslinking, poly(A)-RNA isolation and mass spectrometry to identify all poly(A)-RNA bound proteins. Although in this case also a considerable number of mitochondrial proteins were identified, the method was not specifically directed at the mitochondrial poly(A)-RNA bound proteome.