RESUMEN
The AAA-ATPase Msp1 extracts mislocalised outer membrane proteins and thus contributes to mitochondrial proteostasis. Using pulldown experiments, we show that trypanosomal Msp1 localises to both glycosomes and the mitochondrial outer membrane, where it forms a complex with four outer membrane proteins. The trypanosome-specific pATOM36 mediates complex assembly of α-helically anchored mitochondrial outer membrane proteins such as protein translocase subunits. Inhibition of their assembly triggers a pathway that results in the proteasomal digestion of unassembled substrates. Using inducible single, double, and triple RNAi cell lines combined with proteomic analyses, we demonstrate that not only Msp1 but also the trypanosomal homolog of the AAA-ATPase VCP are implicated in this quality control pathway. Moreover, in the absence of VCP three out of the four Msp1-interacting mitochondrial proteins are required for efficient proteasomal digestion of pATOM36 substrates, suggesting they act in concert with Msp1. pATOM36 is a functional analog of the yeast mitochondrial import complex complex and possibly of human mitochondrial animal-specific carrier homolog 2, suggesting that similar mitochondrial quality control pathways linked to Msp1 might also exist in yeast and humans.
Asunto(s)
Proteómica , Saccharomyces cerevisiae , Animales , Humanos , Saccharomyces cerevisiae/genética , Membranas Mitocondriales , Proteínas de la Membrana , Complejo de la Endopetidasa Proteasomal , Subunidades de Proteína , ATPasas Asociadas con Actividades Celulares Diversas/genéticaRESUMEN
Consistent with other eukaryotes, the Trypanosoma brucei mitochondrial genome encodes mainly hydrophobic core subunits of the oxidative phosphorylation system. These proteins must be co-translationally inserted into the inner mitochondrial membrane and are synthesized by the highly unique trypanosomal mitoribosomes, which have a much higher protein to RNA ratio than any other ribosome. Here, we show that the trypanosomal orthologue of the mitoribosome receptor Mba1 (TbMba1) is essential for normal growth of procyclic trypanosomes but redundant in the bloodstream form, which lacks an oxidative phosphorylation system. Proteomic analyses of TbMba1-depleted mitochondria from procyclic cells revealed reduced levels of many components of the oxidative phosphorylation system, most of which belong to the cytochrome c oxidase (Cox) complex, three subunits of which are mitochondrially encoded. However, the integrity of the mitoribosome and its interaction with the inner membrane were not affected. Pull-down experiments showed that TbMba1 forms a dynamic interaction network that includes the trypanosomal Mdm38/Letm1 orthologue and a trypanosome-specific factor that stabilizes the CoxI and CoxII mRNAs. In summary, our study suggests that the function of Mba1 in the biogenesis of membrane subunits of OXPHOS complexes is conserved among yeast, mammals and trypanosomes, which belong to two eukaryotic supergroups.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Trypanosoma brucei brucei , Animales , Fosforilación Oxidativa , Trypanosoma brucei brucei/metabolismo , Proteómica , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Mamíferos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The endoplasmic reticulum membrane complex (EMC) is a versatile complex that plays a key role in membrane protein biogenesis in the ER. Deletion of the complex has wide-ranging consequences including ER stress, disturbance in lipid transport and organelle tethering, among others. Here we report the function and organization of the evolutionarily conserved EMC (TbEMC) in the highly diverged eukaryote, Trypanosoma brucei. Using (co-) immunoprecipitation experiments in combination with mass spectrometry and whole cell proteomic analyses of parasites after depletion of select TbEMC subunits, we demonstrate that the TbEMC is composed of 9 subunits that are present in a high molecular mass complex localizing to the mitochondrial-endoplasmic reticulum interface. Knocking out or knocking down of single TbEMC subunits led to growth defects of T. brucei procyclic forms in culture. Interestingly, we found that depletion of individual TbEMC subunits lead to disruption of de novo synthesis of phosphatidylcholine (PC) or phosphatidylethanolamine (PE), the two most abundant phospholipid classes in T. brucei. Downregulation of TbEMC1 or TbEMC3 inhibited formation of PC while depletion of TbEMC8 inhibited PE synthesis, pointing to a role of the TbEMC in phospholipid synthesis. In addition, we found that in TbEMC7 knock-out parasites, TbEMC3 is released from the complex, implying that TbEMC7 is essential for the formation or the maintenance of the TbEMC.
Asunto(s)
Trypanosoma brucei brucei , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Fosfolípidos/metabolismo , Proteómica , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMEN
Mitochondrial ribosomes (mitoribosomes) play a central role in synthesizing mitochondrial inner membrane proteins responsible for oxidative phosphorylation. Although mitoribosomes from different organisms exhibit considerable structural variations, recent insights into mitoribosome assembly suggest that mitoribosome maturation follows common principles and involves a number of conserved assembly factors. To investigate the steps involved in the assembly of the mitoribosomal small subunit (mt-SSU) we determined the cryoelectron microscopy structures of middle and late assembly intermediates of the Trypanosoma brucei mitochondrial small subunit (mt-SSU) at 3.6- and 3.7-Å resolution, respectively. We identified five additional assembly factors that together with the mitochondrial initiation factor 2 (mt-IF-2) specifically interact with functionally important regions of the rRNA, including the decoding center, thereby preventing premature mRNA or large subunit binding. Structural comparison of assembly intermediates with mature mt-SSU combined with RNAi experiments suggests a noncanonical role of mt-IF-2 and a stepwise assembly process, where modular exchange of ribosomal proteins and assembly factors together with mt-IF-2 ensure proper 9S rRNA folding and protein maturation during the final steps of assembly.
Asunto(s)
Proteínas Mitocondriales/química , Ribosomas Mitocondriales/química , Fosforilación Oxidativa , ARN Ribosómico/química , Proteínas Ribosómicas/química , Subunidades Ribosómicas/química , Línea Celular , Microscopía por Crioelectrón , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Ribosomas Mitocondriales/metabolismo , Modelos Moleculares , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas/genética , Subunidades Ribosómicas/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMEN
We investigated manifestations of ethnic and gender-based prejudice in a rather understudied high-status environment, that is we studied biased ratings of physicians with a migration background and female physicians. In a preregistered, archival study, we analyzed ratings of more than 140,000 physicians on a German rating website for medical professionals. Results indicate that general practitioners (but not dentists or specialists) with non-German names are rated less favorably than general practitioners with German names. This effect did not vary across regional contexts with varying prosperity and diversity. Our analyses also revealed that female physicians are evaluated less positively than male physicians. Contrary to our assumptions, bias against female physicians was especially strong in medical sub-disciplines that are characterized by a high share of female physicians.
Asunto(s)
Etnicidad , Médicos , Femenino , Humanos , Masculino , Prejuicio , Población BlancaRESUMEN
Mitochondrial tRNA import is widespread, but mechanistic insights of how tRNAs are translocated across mitochondrial membranes remain scarce. The parasitic protozoan T. brucei lacks mitochondrial tRNA genes. Consequently, it imports all organellar tRNAs from the cytosol. Here we investigated the connection between tRNA and protein translocation across the mitochondrial inner membrane. Trypanosomes have a single inner membrane protein translocase that consists of three heterooligomeric submodules, which all are required for import of matrix proteins. In vivo depletion of individual submodules shows that surprisingly only the integral membrane core module, including the protein import pore, but not the presequence-associated import motor are required for mitochondrial tRNA import. Thus we could uncouple import of matrix proteins from import of tRNAs even though both substrates are imported into the same mitochondrial subcompartment. This is reminiscent to the outer membrane where the main protein translocase but not on-going protein translocation is required for tRNA import. We also show that import of tRNAs across the outer and inner membranes are coupled to each other. Taken together, these data support the 'alternate import model', which states that tRNA and protein import while mechanistically independent use the same translocation pores but not at the same time.
Asunto(s)
Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Protozoarias/metabolismo , ARN Protozoario/metabolismo , ARN de Transferencia/metabolismo , Trypanosoma brucei brucei/metabolismo , Transporte Biológico , Citosol/metabolismo , Expresión Génica , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Membranas Mitocondriales/metabolismo , Organismos Modificados Genéticamente , Proteínas Protozoarias/genética , ARN Protozoario/genética , ARN de Transferencia/genética , Trypanosoma brucei brucei/genéticaRESUMEN
In contrast to the bacterial translation machinery, mitoribosomes and mitochondrial translation factors are highly divergent in terms of composition and architecture. There is increasing evidence that the biogenesis of mitoribosomes is an intricate pathway, involving many assembly factors. To better understand this process, we investigated native assembly intermediates of the mitoribosomal large subunit from the human parasite Trypanosoma brucei using cryo-electron microscopy. We identify 28 assembly factors, 6 of which are homologous to bacterial and eukaryotic ribosome assembly factors. They interact with the partially folded rRNA by specifically recognizing functionally important regions such as the peptidyltransferase center. The architectural and compositional comparison of the assembly intermediates indicates a stepwise modular assembly process, during which the rRNA folds toward its mature state. During the process, several conserved GTPases and a helicase form highly intertwined interaction networks that stabilize distinct assembly intermediates. The presented structures provide general insights into mitoribosomal maturation.
Asunto(s)
Ribosomas Mitocondriales/química , ARN Ribosómico/metabolismo , Subunidades Ribosómicas Grandes/química , Trypanosoma brucei brucei/metabolismo , Microscopía por Crioelectrón , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Ribosomas Mitocondriales/metabolismo , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Ribosómico/química , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes/metabolismo , Trypanosoma brucei brucei/genéticaRESUMEN
The aim of this study was to predict self-report data for self-regulated learning with sensor data. In a longitudinal study multichannel data were collected: self-report data with questionnaires and embedded experience samples as well as sensor data like electrodermal activity (EDA) and electroencephalography (EEG). 100 students from a private university in Germany performed a learning experiment followed by final measures of intrinsic motivation, self-efficacy and gained knowledge. During the learning experiment psychophysiological data like EEG were combined with embedded experience sampling measuring motivational states like affect and interest every 270 s. Results of machine learning models show that consumer grade wearables for EEG and EDA failed to predict embedded experience sampling. EDA failed to predict outcome measures as well. This gap can be explained by some major technical difficulties, especially by lower quality of the electrodes. Nevertheless, an average activation of all EEG bands at T7 (left-hemispheric, lateral) can predict lower intrinsic motivation as outcome measure. This is in line with the personality system interactions (PSI) theory of Julius Kuhl. With more advanced sensor measures it might be possible to track affective learning in an unobtrusive way and support micro-adaptation in a digital learning environment.
RESUMEN
Due to its unique biology the mitochondrion of Trypanosoma brucei has attracted a lot of interest since many decades, making it arguably the best studied mitochondrion outside yeast and mammals. Here we describe a method allowing purification of mitochondria from procyclic trypanosomes that yields highly enriched and functional organelles. The method is based on isotonic lysis of cells by nitrogen cavitation, DNase I digestion, differential centrifugation and Nycodenz gradient centrifugation. The method is scalable and can be adapted to culture volumes a small as 100 mL or as large as 24 L.
Asunto(s)
Fraccionamiento Celular/métodos , Mitocondrias , Trypanosoma brucei brucei/citología , Fraccionamiento Celular/instrumentación , Centrifugación por Gradiente de Densidad/instrumentación , Centrifugación por Gradiente de Densidad/métodosRESUMEN
Mitochondrial ribosomes (mitoribosomes) are large ribonucleoprotein complexes that synthesize proteins encoded by the mitochondrial genome. An extensive cellular machinery responsible for ribosome assembly has been described only for eukaryotic cytosolic ribosomes. Here we report that the assembly of the small mitoribosomal subunit in Trypanosoma brucei involves a large number of factors and proceeds through the formation of assembly intermediates, which we analyzed by using cryo-electron microscopy. One of them is a 4-megadalton complex, referred to as the small subunit assemblosome, in which we identified 34 factors that interact with immature ribosomal RNA (rRNA) and recognize its functionally important regions. The assembly proceeds through large-scale conformational changes in rRNA coupled with successive incorporation of mitoribosomal proteins, providing an example for the complexity of the ribosomal assembly process in mitochondria.
Asunto(s)
Proteínas Mitocondriales/ultraestructura , Ribosomas Mitocondriales/ultraestructura , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/ultraestructura , Trypanosoma brucei brucei/química , Microscopía por Crioelectrón , Modelos Moleculares , Conformación de Ácido Nucleico , Estructura Cuaternaria de Proteína , Interferencia de ARN , Estabilidad del ARNRESUMEN
The mitochondrial contact site and cristae organization system (MICOS) mediates the formation of cristae, invaginations in the mitochondrial inner membrane. The highly diverged MICOS complex of the parasitic protist Trypanosoma brucei consists of nine subunits. Except for two Mic10-like and a Mic60-like protein, all subunits are specific for kinetoplastids. Here, we determined on a proteome-wide scale how ablation of individual MICOS subunits affects the levels of the other subunits. The results reveal co-regulation of TbMic10-1, TbMic10-2, TbMic16 and TbMic60, suggesting that these nonessential, integral inner membrane proteins form an interdependent network. Moreover, the ablation of TbMic34 and TbMic32 reveals another network consisting of the essential, intermembrane space-localized TbMic20, TbMic32, TbMic34 and TbMic40, all of which are peripherally associated with the inner membrane. The downregulation of TbMic20, TbMic32 and TbMic34 also interferes with mitochondrial protein import and reduces the size of the TbMic10-containing complexes. Thus, the diverged MICOS of trypanosomes contains two subcomplexes: a nonessential membrane-integrated one, organized around the conserved Mic10 and Mic60, that mediates cristae formation, and an essential membrane-peripheral one consisting of four kinetoplastid-specific subunits, that is required for import of intermembrane space proteins.
Asunto(s)
Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Trypanosoma brucei brucei/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/fisiología , Unión Proteica , Transporte de Proteínas , Trypanosoma/metabolismo , Trypanosoma/fisiología , Trypanosoma brucei brucei/fisiologíaRESUMEN
Ribosomal RNA (rRNA) plays key functional and architectural roles in ribosomes. Using electron microscopy, we determined the atomic structure of a highly divergent ribosome found in mitochondria of Trypanosoma brucei, a unicellular parasite that causes sleeping sickness in humans. The trypanosomal mitoribosome features the smallest rRNAs and contains more proteins than all known ribosomes. The structure shows how the proteins have taken over the role of architectural scaffold from the rRNA: They form an autonomous outer shell that surrounds the entire particle and stabilizes and positions the functionally important regions of the rRNA. Our results also reveal the "minimal" set of conserved rRNA and protein components shared by all ribosomes that help us define the most essential functional elements.
Asunto(s)
Evolución Molecular , Ribosomas Mitocondriales/química , Proteínas Protozoarias/química , Proteínas Ribosómicas/química , Trypanosoma brucei brucei/ultraestructura , Ribosomas Mitocondriales/ultraestructura , Modelos Moleculares , Proteínas Protozoarias/ultraestructura , ARN Ribosómico/química , ARN Ribosómico/ultraestructura , Proteínas Ribosómicas/ultraestructuraRESUMEN
Mitochondrial tRNA import is widespread, but the mechanism by which tRNAs are imported remains largely unknown. The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes, and thus imports all tRNAs from the cytosol. Here we show that in T. brucei in vivo import of tRNAs requires four subunits of the mitochondrial outer membrane protein translocase but not the two receptor subunits, one of which is essential for protein import. The latter shows that it is possible to uncouple mitochondrial tRNA import from protein import. Ablation of the intermembrane space domain of the translocase subunit, archaic translocase of the outer membrane (ATOM)14, on the other hand, while not affecting the architecture of the translocase, impedes both protein and tRNA import. A protein import intermediate arrested in the translocation channel prevents both protein and tRNA import. In the presence of tRNA, blocking events of single-channel currents through the pore formed by recombinant ATOM40 were detected in electrophysiological recordings. These results indicate that both types of macromolecules use the same import channel across the outer membrane. However, while tRNA import depends on the core subunits of the protein import translocase, it does not require the protein import receptors, indicating that the two processes are not mechanistically linked.
Asunto(s)
Membranas Mitocondriales/fisiología , Transporte de Proteínas/fisiología , Transporte de ARN/fisiología , Proteínas Portadoras/metabolismo , Línea Celular , Citosol/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Mitocondrias/fisiología , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/fisiología , Conformación Proteica , ARN de Transferencia/metabolismo , ARN de Transferencia/fisiología , Trypanosoma/genética , Trypanosoma/metabolismo , Trypanosoma brucei brucei/genéticaRESUMEN
Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are among the most abundant phospholipids in biological membranes. In many eukaryotes, the CDP-ethanolamine and CDP-choline branches of the Kennedy pathway represent major and often essential routes for the production of PE and PC, with ethanolamine and choline/ethanolamine phosphotransferases (EPT and CEPT, respectively) catalysing the last reactions in the respective pathways. Although the site of PE and PC synthesis is commonly known to be the endoplasmic reticulum (ER), detailed information on the localization of the different phosphotransferases is lacking. In the unicellular parasite, Trypanosoma brucei, both branches of the Kennedy pathway are essential for cell growth in culture. We have previously reported that T. brucei EPT (TbEPT) catalyses the production of ether-type PE molecular species while T. brucei CEPT (TbCEPT) synthesizes diacyl-type PE and PC molecular species. We now show that the two enzymes localize to different sub-compartments of the ER. By expressing a series of tagged forms of the two enzymes in T. brucei parasites, in combination with sub-cellular fractionation and enzyme activity measurements, TbEPT was found exclusively in the perinuclear ER, a distinct area located close to but distinct from the nuclear membrane. In contrast, TbCEPT was detected in the bulk ER.
Asunto(s)
Vías Biosintéticas , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolaminas/biosíntesis , Trypanosoma brucei brucei/metabolismo , Transporte Biológico , Espacio Intracelular , Proteínas Protozoarias/metabolismoRESUMEN
A subclass of eukaryotic proteins is subject to modification with fatty acids, the most common of which are palmitic and myristic acid. Protein acylation allows association with cellular membranes in the absence of transmembrane domains. Here we examine POMP39, a protein previously described to be present in the outer mitochondrial membrane proteome (POMP) of the protozoan parasite Trypanosoma brucei. POMP39 lacks canonical transmembrane domains, but is likely both myristoylated and palmitoylated on its N-terminus. Interestingly, the protein is also dually localized on the surface of the mitochondrion as well as in the flagellum of both insect-stage and the bloodstream form of the parasites. Upon abolishing of global protein acylation or mutation of the myristoylation site, POMP39 relocates to the cytosol. RNAi-mediated ablation of the protein neither causes a growth phenotype in insect-stage nor bloodstream form trypanosomes.
Asunto(s)
Ácidos Grasos/análisis , Mitocondrias/química , Proteoma/química , Proteínas Protozoarias/química , Trypanosoma brucei brucei/química , Acilación , Animales , Línea Celular , Biología Computacional , Flagelos/química , Ratones , Ácido Mirístico/metabolismo , Sistemas de Lectura Abierta , Ácido Palmítico/metabolismo , Proteoma/genética , Proteínas Protozoarias/genética , Conejos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/ultraestructuraRESUMEN
Elucidating the mechanism of action of trypanocidal compounds is an important step in the development of more efficient drugs against Trypanosoma brucei. In a screening approach using an RNAi library in T. brucei bloodstream forms, we identified a member of the mitochondrial carrier family, TbMCP14, as a prime candidate mediating the action of a group of anti-parasitic choline analogs. Depletion of TbMCP14 by inducible RNAi in both bloodstream and procyclic forms increased resistance of parasites towards the compounds by 7-fold and 3-fold, respectively, compared to uninduced cells. In addition, down-regulation of TbMCP14 protected bloodstream form mitochondria from a drug-induced decrease in mitochondrial membrane potential. Conversely, over-expression of the carrier in procyclic forms increased parasite susceptibility more than 13-fold. Metabolomic analyses of parasites over-expressing TbMCP14 showed increased levels of the proline metabolite, pyrroline-5-carboxylate, suggesting a possible involvement of TbMCP14 in energy production. The generation of TbMCP14 knock-out parasites showed that the carrier is not essential for survival of T. brucei bloodstream forms, but reduced parasite proliferation under standard culture conditions. In contrast, depletion of TbMCP14 in procyclic forms resulted in growth arrest, followed by parasite death. The time point at which parasite proliferation stopped was dependent on the major energy source, i.e. glucose versus proline, in the culture medium. Together with our findings that proline-dependent ATP production in crude mitochondria from TbMCP14-depleted trypanosomes was reduced compared to control mitochondria, the study demonstrates that TbMCP14 is involved in energy production in T. brucei. Since TbMCP14 belongs to a trypanosomatid-specific clade of mitochondrial carrier family proteins showing very poor similarity to mitochondrial carriers of mammals, it may represent an interesting target for drug action or targeting.
Asunto(s)
Resistencia a Múltiples Medicamentos , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Metabolómica , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Pruebas de Sensibilidad Parasitaria , Pentamidina/farmacología , Prolina/metabolismo , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Pirroles/metabolismo , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/metabolismoRESUMEN
Mitochondrial protein import is essential for all eukaryotes and mediated by hetero-oligomeric protein translocases thought to be conserved within all eukaryotes. We have identified and analysed the function and architecture of the non-conventional outer membrane (OM) protein translocase in the early diverging eukaryote Trypanosoma brucei. It consists of six subunits that show no obvious homology to translocase components of other species. Two subunits are import receptors that have a unique topology and unique protein domains and thus evolved independently of the prototype receptors Tom20 and Tom70. Our study suggests that protein import receptors were recruited to the core of the OM translocase after the divergence of the major eukaryotic supergroups. Moreover, it links the evolutionary history of mitochondrial protein import receptors to the origin of the eukaryotic supergroups.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Trypanosoma brucei brucei/genética , Evolución Biológica , Northern Blotting , Proteínas Portadoras/metabolismo , Línea Celular , Kinetoplastida/genética , Espectrometría de Masas , Microscopía Fluorescente , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Filogenia , Trypanosoma brucei brucei/metabolismoRESUMEN
Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a ß-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum-mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a ß-barrel protein of the mitochondrial porin family that mediates a DNA-cytoskeleton linkage that is essential for mitochondrial DNA inheritance.
Asunto(s)
Genes Mitocondriales/genética , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Modelos Biológicos , Porinas/genética , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genética , Secuencia de Bases , Línea Celular , Análisis por Conglomerados , Citoesqueleto/metabolismo , ADN Mitocondrial/metabolismo , Técnica del Anticuerpo Fluorescente , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Organismos Modificados Genéticamente , Filogenia , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei.
Asunto(s)
Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteoma/genética , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/genética , Animales , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Estadios del Ciclo de Vida/genética , Mitocondrias/genética , Membranas Mitocondriales/química , Proteínas Mitocondriales/antagonistas & inhibidores , Proteínas Mitocondriales/metabolismo , Forma de los Orgánulos/genética , Proteoma/antagonistas & inhibidores , Proteoma/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , ARN Interferente Pequeño/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Espectrometría de Masas en Tándem , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/metabolismoRESUMEN
Potentiometric sensing represents the preferred technique in many routine measurements of pH and ions. Unfortunately, the simplicity of the technique has not been exploited so far in high throughput biomolecular sensing. In this work, we demonstrate the capabilities of the hybrid functional material carbon nanotubes/aptamer for the creation of a new generation of nuclease-resistant aptasensors using the potentiometric transduction capabilities of single-walled carbon nanotubes in combination with the recognition capabilities of a protein-specific RNA aptamer. The aptasensor was used to detect and identify disease-related proteins at attomolar concentration values in a rapid and non-expensive way. The variable surface glycoprotein from African Trypanosomes was chosen as an ideal model system for a pathogenic exoantigen protein in a clinical sample. Variations in the electromotive force are achieved in real-time upon the direct addition of diluted real blood samples containing the target protein thus eliminating the need of preliminary matrix removal. This work would open the door to real-time diagnostic assays for a wide range of diseases, but also to the rapid molecular detection of several proteins in truly customizable protein biosensing platforms.