Mitochondrial proteomics: analysis of a whole mitochondrial extract with two-dimensional electrophoresis

Mitochondria are complex organelles, and their proteomics analysis requires a combination of techniques. The emphasis in this chapter is made first on mitochondria preparation from cultured mammalian cells, then on the separation of the mitochondrial proteins with two-dimensional electrophoresis (2DE), showing some adjustment over the classical techniques to improve resolution of the mitochondrial proteins. This covers both the protein solubilization, the electrophoretic part per se, and the protein detection on the gels, which makes the interface with the protein identification part relying on mass spectrometry

Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein

The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins

In Vivo Dissection of the Intrinsically Disordered Receptor Domain of Tim23

In the intermembrane space (IMS) of mitochondria, the receptor domain of Tim23 has an essential role during translocation of hundreds of different proteins from the cytosol via the TOM and TIM23 complexes in the outer and inner membranes, respectively. This intrinsically disordered domain, which can even extend into the cytosol, was shown, mostly in vitro, to interact with several subunits of the TOM and TIM23 complexes. To obtain molecular understanding of this organizational hub in the IMS, we dissected the IMS domain of Tim23 in vivo. We show that the interaction surface of Tim23 with Tim50 is larger than previously thought and reveal an unexpected interaction of Tim23 with Pam17 in the IMS, impairment of which influences their interaction in the matrix. Furthermore, mutations of two conserved negatively charged residues of Tim23, close to the inner membrane, prevented dimerization of Tim23. The same mutations increased exposure of Tim23 on the mitochondrial surface, whereas dissipation of membrane potential decreased it. Our results reveal an intricate network of Tim23 interactions in the IMS, whose influence is transduced across two mitochondrial membranes, ensuring efficient translocation of proteins into mitochondria. (C) 2020 The Author(s). Published by Elsevier Ltd

Role of Magmas in protein transport and human mitochondria biogenesis

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of Δpam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process

Compromised Mitochondrial Protein Import Acts as a Signal for UPRmt

The induction of the mitochondrial unfolded protein response (UPRmt) results in increased transcription of the gene encoding the mitochondrial chaperone HSP70. We systematically screened the C. elegans genome and identified 171 genes that, when knocked down, induce the expression of an hsp-6 HSP70 reporter and encode mitochondrial proteins. These genes represent many, but not all, mitochondrial processes (e.g., mitochondrial calcium homeostasis and mitophagy are not represented). Knockdown of these genes leads to reduced mitochondrial membrane potential and, hence, decreased protein import into mitochondria. In addition, it induces UPRmt in a manner that is dependent on ATFS-1 but that is not antagonized by the kinase GCN-2. We propose that compromised mitochondrial protein import signals the induction of UPRmt and that the mitochondrial targeting sequence of ATFS-1 functions as a sensor for this signal

Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

Tom40 is an integral protein of the mitochondrial outer membrane, which as the central component of the Translocase of the Outer Membrane (TOM) complex forms a channel for protein import. We characterize the diffusion properties of individual Tom40 molecules fused to the photoconvertable fluorescent protein Dendra2 with millisecond temporal resolution. By imaging individual Tom40 molecules in intact isolated yeast mitochondria using photoactivated localization microscopy with sub-diffraction limited spatial precision, we demonstrate that Tom40 movement in the outer mitochondrial membrane is highly dynamic but confined in nature, suggesting anchoring of the TOM complex as a whole

Pre-microRNA and Mature microRNA in Human Mitochondria

Chantier qualité GAInternational audienceBACKGROUND: Because of the central functions of the mitochondria in providing metabolic energy and initiating apoptosis on one hand and the role that microRNA (miRNA) play in gene expression, we hypothesized that some miRNA could be present in the mitochondria for post-transcriptomic regulation by RNA interference. We intend to identify miRNA localized in the mitochondria isolated from human skeletal primary muscular cells. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the potential origin of mitochondrial miRNA, we in-silico searched for microRNA candidates in the mtDNA. Twenty five human pre-miRNA and 33 miRNA aligments (E-value35) for the smallest RNA input concentration and 204 miRNA for the maximum RNA input concentration. In silico analysis predicted 80 putative miRNA target sites in the mitochondrial genome (E-value<0.05). CONCLUSIONS/SIGNIFICANCE: The present study experimentally demonstrated for the first time the presence of pre-miRNA and miRNA in the human mitochondria isolated from skeletal muscular cells. A set of miRNA were significantly detected in mitochondria fraction. The origin of these pre-miRNA and miRNA should be further investigate to determine if they are imported from the cytosol and/or if they are partially processed in the mitochondria

The Minimal Proteome in the Reduced Mitochondrion of the Parasitic Protist Giardia intestinalis

The mitosomes of Giardia intestinalis are thought to be mitochondria highly-reduced in response to the oxygen-poor niche. We performed a quantitative proteomic assessment of Giardia mitosomes to increase understanding of the function and evolutionary origin of these enigmatic organelles. Mitosome-enriched fractions were obtained from cell homogenate using Optiprep gradient centrifugation. To distinguish mitosomal proteins from contamination, we used a quantitative shot-gun strategy based on isobaric tagging of peptides with iTRAQ and tandem mass spectrometry. Altogether, 638 proteins were identified in mitosome-enriched fractions. Of these, 139 proteins had iTRAQ ratio similar to that of the six known mitosomal markers. Proteins were selected for expression in Giardia to verify their cellular localizations and the mitosomal localization of 20 proteins was confirmed. These proteins include nine components of the FeS cluster assembly machinery, a novel diflavo-protein with NADPH reductase activity, a novel VAMP-associated protein, and a key component of the outer membrane protein translocase. None of the novel mitosomal proteins was predicted by previous genome analyses. The small proteome of the Giardia mitosome reflects the reduction in mitochondrial metabolism, which is limited to the FeS cluster assembly pathway, and a simplicity in the protein import pathway required for organelle biogenesis

The Essentials of Protein Import in the Degenerate Mitochondrion of Entamoeba histolytica

Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica

Granzyme B-induced mitochondrial ROS are required for apoptosis

Caspases and the cytotoxic lymphocyte protease granzyme B (GB) induce reactive oxygen species (ROS) formation, loss of transmembrane potential and mitochondrial outer membrane permeabilization (MOMP). Whether ROS are required for GB-mediated apoptosis and how GB induces ROS is unclear. Here, we found that GB induces cell death in an ROS-dependent manner, independently of caspases and MOMP. GB triggers ROS increase in target cell by directly attacking the mitochondria to cleave NDUFV1, NDUFS1 and NDUFS2 subunits of the NADH: ubiquinone oxidoreductase complex I inside mitochondria. This leads to mitocentric ROS production, loss of complex I and III activity, disorganization of the respiratory chain, impaired mitochondrial respiration and loss of the mitochondrial cristae junctions. Furthermore, we have also found that GB-induced mitocentric ROS are necessary for optimal apoptogenic factor release, rapid DNA fragmentation and lysosomal rupture. Interestingly, scavenging the ROS delays and reduces many of the features of GB-induced death. Consequently, GB-induced ROS significantly promote apoptosis