RESUMO
Fluorescence and light microscopy are important tools in the history of natural science. However, the resolution of microscopes is limited by the diffraction of light. One possible method to circumvent this physical restriction is the recently developed expansion microscopy (ExM). However, the original ultrastructure ExM (U-ExM) protocol is very time-consuming, and some epitopes are lost during the process. In this study, we developed a shortened pre-gelation staining ExM (PS-ExM) protocol and tested it to investigate the Plasmodium liver stage. The protocol presented in this study allows expanding of pre-stained samples, which results in shorter incubation times, better preservation of some epitopes and the advantage that non-expanded controls can be performed alongside using the same staining protocol. The protocol applicability was accessed throughout the Plasmodium liver stage, showing isotropic five-fold expansion. Furthermore, we used PS-ExM to visualise parasite mitochondria as well as the association of lysosomes to the parasitophorous vacuole membrane (PVM) as an example of visualising host-pathogen interaction. We are convinced that this new tool will be helpful for a deeper understanding of the biology of the Plasmodium liver stage.
Assuntos
Parasitos , Plasmodium , Animais , Microscopia , Fígado , EpitoposRESUMO
Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC.
Assuntos
DNA Mitocondrial , Trypanosoma brucei brucei , DNA Mitocondrial/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , DNA de Cinetoplasto/genética , DNA de Cinetoplasto/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Protozoários/metabolismo , Replicação do DNARESUMO
The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body (BB) of the flagellum. Here, we studied the architecture of the exclusion zone filament (EZF) of the TAC, the only known component of which is p197, that connects the BB with the mitochondrial outer membrane (OM). We show that p197 has three domains that are all essential for mitochondrial DNA inheritance. The C terminus of p197 interacts with the mature and probasal body (pro-BB), whereas its N terminus binds to the peripheral OM protein TAC65. The large central region of p197 has a high α-helical content and likely acts as a flexible spacer. Ultrastructure expansion microscopy (U-ExM) of cell lines exclusively expressing p197 versions of different lengths that contain both N- and C-terminal epitope tags demonstrates that full-length p197 alone can bridge the â¼270-nm distance between the BB and the cytosolic face of the OM. Thus U-ExM allows the localization of distinct domains within the same molecules and suggests that p197 is the TAC subunit most proximal to the BB. In addition, U-ExM revealed that p197 acts as a spacer molecule, as two shorter versions of p197, with the repeat domain either removed or replaced by the central domain of the Trypanosoma cruzi p197 ortholog reduced the distance between the BB and the OM in proportion to their predicted molecular weight.
Assuntos
Replicação do DNA , DNA Mitocondrial , Genoma Mitocondrial , Membranas Mitocondriais , Proteínas de Protozoários , Trypanosoma brucei brucei , Corpos Basais/química , DNA Mitocondrial/genética , Epitopos/química , Flagelos/química , Membranas Mitocondriais/química , Proteínas de Protozoários/química , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genéticaRESUMO
Proper mitochondrial genome inheritance is important for eukaryotic cell survival. Trypanosoma brucei, a protozoan parasite, contains a singular mitochondrial genome, the kinetoplast (k)DNA. The kDNA is anchored to the basal body via the tripartite attachment complex (TAC) to ensure proper segregation. Several components of the TAC have been described; however, the connection of the TAC to the kDNA remains elusive. Here, we characterize the TAC-associated protein TAP110. We find that both depletion and overexpression of TAP110 leads to a delay in the separation of the replicated kDNA networks. Proteome analysis after TAP110 overexpression identified several kDNA-associated proteins that changed in abundance, including a TEX-like protein that dually localizes to the nucleus and the kDNA, potentially linking replication and segregation in the two compartments. The assembly of TAP110 into the TAC region seems to require the TAC but not the kDNA itself; however, once TAP110 has been assembled, it also interacts with the kDNA. Finally, we use ultrastructure expansion microscopy in trypanosomes for the first time, and reveal the precise position of TAP110 between TAC102 and the kDNA, showcasing the potential of this approach.This article has an associated First Person interview with the first author of the paper.
Assuntos
Genoma Mitocondrial , Trypanosoma brucei brucei , DNA de Cinetoplasto/genética , Genoma Mitocondrial/genética , Mitocôndrias , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genéticaRESUMO
Kinetoplastids are protists defined by one of the most complex mitochondrial genomes in nature, the kinetoplast. In the sleeping sickness parasite Trypanosoma brucei, the kinetoplast is a chain mail-like network of two types of interlocked DNA molecules: a few dozen â¼23-kb maxicircles (homologs of the mitochondrial genome of other eukaryotes) and thousands of â¼1-kb minicircles. Maxicircles encode components of respiratory chain complexes and the mitoribosome. Several maxicircle-encoded mRNAs undergo extensive post-transcriptional RNA editing via addition and deletion of uridines. The process is mediated by hundreds of species of minicircle-encoded guide RNAs (gRNAs), but the precise number of minicircle classes and gRNA genes was unknown. Here we present the first essentially complete assembly and annotation of the kinetoplast genome of T. brucei. We have identified 391 minicircles, encoding not only â¼930 predicted 'canonical' gRNA genes that cover nearly all known editing events (accessible via the web at http://hank.bio.ed.ac.uk), but also â¼370 'non-canonical' gRNA genes of unknown function. Small RNA transcriptome data confirmed expression of the majority of both categories of gRNAs. Finally, we have used our data set to refine definitions for minicircle structure and to explore dynamics of minicircle copy numbers.
Assuntos
Genoma Mitocondrial , Anotação de Sequência Molecular , Análise de Sequência de DNA , Trypanosoma brucei brucei/genética , Animais , Sequência de Bases , Sequência Conservada , DNA Circular/análise , DNA Circular/genética , DNA de Cinetoplasto/genética , Ordem dos Genes , Genoma de Protozoário , RNA Guia de Cinetoplastídeos/genética , Trypanosoma brucei brucei/ultraestruturaRESUMO
In almost all eukaryotes, mitochondria maintain their own genome. Despite the discovery more than 50 y ago, still very little is known about how the genome is correctly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome, the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure correct segregation of the mitochondrial genome via the basal bodies movement, during the cell cycle. Using superresolution microscopy, we precisely localize each of the currently known TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum toward the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on the biochemical analysis, the TAC consists of several nonoverlapping subcomplexes, suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC is required for correct mitochondrial organelle positioning but not for organelle biogenesis or segregation.
Assuntos
Regulação da Expressão Gênica/fisiologia , Genoma Mitocondrial/fisiologia , Genoma de Protozoário/fisiologia , Trypanosoma brucei brucei/genética , DNA de Cinetoplasto/genética , Modelos BiológicosRESUMO
Unlike most other model eukaryotes, Trypanosoma brucei and its relatives have a single mitochondrion with a single-unit mitochondrial genome that is termed kinetoplast DNA (kDNA). Replication of the kDNA is coordinated with the cell cycle. During binary mitochondrial fission and prior to cytokinesis, the replicated kDNA has to be faithfully segregated to the daughter organelles. This process depends on the tripartite attachment complex (TAC) that physically links the kDNA across the two mitochondrial membranes with the basal body of the flagellum. Thus, the TAC couples segregation of the replicated kDNA with segregation of the basal bodies of the old and the new flagellum. In this Review, we provide an overview of the role of the TAC in kDNA inheritance in T. brucei We focus on recent advances regarding the molecular composition of the TAC, and discuss how the TAC is assembled and how its subunits are targeted to their respective TAC subdomains. Finally, we will contrast the segregation of the single-unit kDNA in trypanosomes to mitochondrial genome inheritance in yeast and mammals, both of which have numerous mitochondria that each contain multiple genomes.
Assuntos
Genoma Mitocondrial/genética , Trypanosoma brucei brucei/patogenicidade , AnimaisRESUMO
The translationally controlled tumor protein (TCTP; also known as TPT1 in mammals) is highly conserved and ubiquitously expressed in eukaryotes. It is involved in growth and development, cell cycle progression, protection against cellular stresses and apoptosis, indicating the multifunctional role of the protein. Here, for the first time, we characterize the expression and function of TCTP in the human and animal pathogen, Trypanosoma brucei We identified two paralogs (TCTP1 and TCTP2) that are differentially expressed in the life cycle of the parasite. The genes have identical 5' untranslated regions (UTRs) and almost identical open-reading frames. The 3'UTRs differ substantially in sequence and length, and are sufficient for the exclusive expression of TCTP1 in procyclic- and TCTP2 in bloodstream-form parasites. Furthermore, we characterize which parts of the 3'UTR are needed for TCTP2 mRNA stability. RNAi experiments demonstrate that TCTP1 and TCTP2 expression is essential for normal cell growth in procyclic- and bloodstream-form parasites, respectively. Depletion of TCTP1 in the procyclic form cells leads to aberrant cell and mitochondrial organelle morphology, as well as enlarged, and a reduced number of, acidocalcisomes.
Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas de Protozoários/biossíntese , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Regiões 3' não Traduzidas , Biomarcadores Tumorais/genética , Estágios do Ciclo de Vida/fisiologia , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
The unicellular parasite Trypanosoma brucei harbors one mitochondrial organelle with a singular genome called the kinetoplast DNA (kDNA). The kDNA consists of a network of concatenated minicircles and a few maxicircles that form the kDNA disc. More than 30 proteins involved in kDNA replication have been described. However, several mechanistic questions are only poorly understood. Here, we describe and characterize minicircle replication factor 172 (MiRF172), a novel mitochondrial genome replication factor that is essential for cell growth and kDNA maintenance. By performing super-resolution microscopy, we show that MiRF172 is localized to the kDNA disc, facing the region between the genome and the mitochondrial membranes. We demonstrate that depletion of MiRF172 leads to a loss of minicircles and maxicircles. Detailed analysis suggests that MiRF172 is involved in the reattachment of replicated minicircles to the kDNA disc. Furthermore, we provide evidence that the localization of the replication factor MiRF172 not only depends on the kDNA itself, but also on the mitochondrial genome segregation machinery, suggesting an interaction between the two essential entities.This article has an associated First Person interview with the first author of the paper.
Assuntos
Replicação do DNA , DNA de Cinetoplasto/metabolismo , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/genética , Genoma Mitocondrial , Trypanosoma brucei brucei/genética , Animais , DNA de Cinetoplasto/genética , DNA Mitocondrial/genética , Proteínas de Ligação a DNA/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMO
Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization.
Assuntos
Segregação de Cromossomos/fisiologia , Genoma Mitocondrial , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , DNA de Cinetoplasto/metabolismo , Flagelos , Imunofluorescência , Immunoblotting , Microscopia Eletrônica de Transmissão , Trypanosoma brucei brucei/ultraestruturaRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1005586.].
RESUMO
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.
Assuntos
Genes Mitocondriais/genética , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Modelos Biológicos , Porinas/genética , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Sequência de Bases , Linhagem Celular , Análise por Conglomerados , Citoesqueleto/metabolismo , DNA Mitocondrial/metabolismo , Imunofluorescência , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Organismos Geneticamente Modificados , Filogenia , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas's disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile.
Assuntos
Antiparasitários/farmacologia , Evolução Molecular , Genômica , Parasitos/genética , Parasitos/metabolismo , Esteróis/biossíntese , Animais , Linhagem Celular , Análise por Conglomerados , Simulação por Computador , Ergosterol/biossíntese , Cadeias de Markov , Parasitos/efeitos dos fármacos , Parasitos/enzimologiaRESUMO
Aminoacyl-tRNA synthetases catalyze the aminoacylation of tRNAs with their cognate amino acids. They are an essential part of each translation system and in eukaryotes are therefore found in both the cytosol and mitochondria. Thus, eukaryotes either have two distinct genes encoding the cytosolic and mitochondrial isoforms of each of these enzymes or a single gene encoding dually localized products. Trypanosomes require trans-splicing of a cap containing leader sequence onto the 5'-untranslated region of every mRNA. Recently we speculated that alternative trans-splicing could lead to the expression of proteins having amino-termini of different lengths that derive from the same gene. We now demonstrate that alternative trans-splicing, creating a long and a short spliced variant, is the mechanism for dual localization of trypanosomal isoleucyl-tRNA synthetase (IleRS). The protein product of the longer spliced variant possesses an amino-terminal presequence and is found exclusively in mitochondria. In contrast, the shorter spliced variant is translated to a cytosol-specific isoform lacking the presequence. Furthermore, we show that RNA stability is one mechanism determining the differential abundance of the two spliced isoforms.
Assuntos
Isoleucina-tRNA Ligase/genética , Proteínas de Protozoários/genética , Trans-Splicing , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Processamento Alternativo , Sequência de Aminoácidos , Células Cultivadas , Citosol/enzimologia , Isoleucina-tRNA Ligase/análise , Isoleucina-tRNA Ligase/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismoRESUMO
Understanding cellular ultrastructure is tightly bound to microscopic resolution and the ability to identify individual components at that resolution. Expansion microscopy has revolutionised this topic. Here we present and compare two protocols of ultrastructure expansion microscopy that allow for 4.5-fold mostly isotropic expansion and the use of antibodies, metabolic labelling, and DNA stains to demarcate individual regions such as the endoplasmic reticulum, the nuclei, the peripheral endocytic compartments as well as the ventral disc and the cytoskeleton in Giardia lamblia. We present an optimised, shortened, and modular protocol that can be swiftly adjusted to the investigators needs in this important protozoan model organism.
RESUMO
Mitochondrial DNA replication is an essential process in most eukaryotes. Similar to the diversity in mitochondrial genome size and organization in the different eukaryotic supergroups, there is considerable diversity in the replication process of the mitochondrial DNA. In this review, we summarize the current knowledge of mitochondrial DNA replication and the associated factors in trypanosomes with a focus on Trypanosoma brucei, and provide a new model of minicircle replication for this protozoan parasite. The model assumes the mitochondrial DNA (kinetoplast DNA, kDNA) of T. brucei to be loosely diploid in nature and the replication of the genome to occur at two replication centers at the opposing ends of the kDNA disc (also known as antipodal sites, APS). The new model is consistent with the localization of most replication factors and in contrast to the current model, it does not require the assumption of an unknown sorting and transport complex moving freshly replicated DNA to the APS. In combination with the previously proposed sexual stages of the parasite in the insect vector, the new model provides a mechanism for maintenance of the mitochondrial genetic diversity.
Assuntos
DNA de Cinetoplasto , Genoma Mitocondrial , DNA de Cinetoplasto/genética , Genoma Mitocondrial/genética , Replicação do DNA/genética , DNA Mitocondrial/genética , Mitocôndrias/genética , Proteínas de Protozoários/genéticaRESUMO
BACKGROUND: Trypanosoma brucei is the causative agent of human African sleeping sickness and Nagana in cattle. In addition to being an important pathogen T. brucei has developed into a model system in cell biology. RESULTS: Using Stable Isotope Labelling of Amino acids in Cell culture (SILAC) in combination with mass spectrometry we determined the abundance of >1600 proteins in the long slender (LS), short stumpy (SS) mammalian bloodstream form stages relative to the procyclic (PC) insect-form stage. In total we identified 2645 proteins, corresponding to ~30% of the total proteome and for the first time present a comprehensive overview of relative protein levels in three life stages of the parasite. CONCLUSIONS: We can show the extent of pre-adaptation in the SS cells, especially at the level of the mitochondrial proteome. The comparison to a previously published report on monomorphic in vitro grown bloodstream and procyclic T. brucei indicates a loss of stringent regulation particularly of mitochondrial proteins in these cells when compared to the pleomorphic in vivo situation. In order to better understand the different levels of gene expression regulation in this organism we compared mRNA steady state abundance with the relative protein abundance-changes and detected moderate but significant correlation indicating that trypanosomes possess a significant repertoire of translational and posttranslational mechanisms to regulate protein abundance.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Estágios do Ciclo de Vida/genética , Proteínas Mitocondriais/metabolismo , Proteoma/genética , Trypanosoma brucei brucei/crescimento & desenvolvimento , Processamento Alternativo/genética , Aminoácidos/metabolismo , Animais , Western Blotting , Biologia Computacional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Marcação por Isótopo , Espectrometria de Massas , Análise de Componente Principal , Trypanosoma brucei brucei/genéticaRESUMO
Trans-splicing of leader sequences onto the 5'ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5'splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT). The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5' splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.
Assuntos
Processamento Alternativo/genética , Perfilação da Expressão Gênica/métodos , Genes de Protozoários/genética , RNA Líder para Processamento/genética , Trypanosoma brucei brucei/genética , Regiões 5' não Traduzidas/genética , Sequência de Bases , Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
It has long been known that trypanosomes regulate mitochondrial biogenesis during the life cycle of the parasite; however, the mitochondrial protein inventory (MitoCarta) and its regulation remain unknown. We present a novel computational method for genome-wide prediction of mitochondrial proteins using a support vector machine-based classifier with â¼90% prediction accuracy. Using this method, we predicted the mitochondrial localization of 468 proteins with high confidence and have experimentally verified the localization of a subset of these proteins. We then applied a recently developed parallel sequencing technology to determine the expression profiles and the splicing patterns of a total of 1065 predicted MitoCarta transcripts during the development of the parasite, and showed that 435 of the transcripts significantly changed their expressions while 630 remain unchanged in any of the three life stages analyzed. Furthermore, we identified 298 alternatively splicing events, a small subset of which could lead to dual localization of the corresponding proteins.
Assuntos
Processamento Alternativo , Biologia Computacional/métodos , Proteínas Mitocondriais/genética , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Inteligência Artificial , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/metabolismo , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMO
Mitochondrial protein import in the parasitic protozoan Trypanosoma brucei is mediated by the atypical outer membrane translocase, ATOM. It consists of seven subunits including ATOM69, the import receptor for hydrophobic proteins. Ablation of ATOM69, but not of any other subunit, triggers a unique quality control pathway resulting in the proteasomal degradation of non-imported mitochondrial proteins. The process requires a protein of unknown function, an E3 ubiquitin ligase and the ubiquitin-like protein (TbUbL1), which all are recruited to the mitochondrion upon ATOM69 depletion. TbUbL1 is a nuclear protein, a fraction of which is released to the cytosol upon triggering of the pathway. Nuclear release is essential as cytosolic TbUbL1 can bind mislocalised mitochondrial proteins and likely transfers them to the proteasome. Mitochondrial quality control has previously been studied in yeast and metazoans. Finding such a pathway in the highly diverged trypanosomes suggests such pathways are an obligate feature of all eukaryotes.