A Msp1-containing complex removes orphaned proteins in the mitochondrial outer membrane of T. brucei.

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.

p166 links membrane and intramitochondrial modules of the trypanosomal tripartite attachment complex.

The protist parasite Trypanosoma brucei has a single mitochondrion with a single unit genome termed kinetoplast DNA (kDNA). Faithfull segregation of replicated kDNA is ensured by a complicated structure termed tripartite attachment complex (TAC). The TAC physically links the basal body of the flagellum with the kDNA spanning the two mitochondrial membranes. Here, we characterized p166 as the only known TAC subunit that is anchored in the inner membrane. Its C-terminal transmembrane domain separates the protein into a large N-terminal region that interacts with the kDNA-localized TAC102 and a 34 aa C-tail that binds to the intermembrane space-exposed loop of the integral outer membrane protein TAC60. Whereas the outer membrane region requires four essential subunits for proper TAC function, the inner membrane integral p166, via its interaction with TAC60 and TAC102, would theoretically suffice to bridge the distance between the OM and the kDNA. Surprisingly, non-functional p166 lacking the C-terminal 34 aa still localizes to the TAC region. This suggests the existence of additional TAC-associated proteins which loosely bind to non-functional p166 lacking the C-terminal 34 aa and keep it at the TAC. However, binding of full length p166 to these TAC-associated proteins alone would not be sufficient to withstand the mechanical load imposed by the segregating basal bodies.

Independent evolution of functionally exchangeable mitochondrial outer membrane import complexes.

Assembly and/or insertion of a subset of mitochondrial outer membrane (MOM) proteins, including subunits of the main MOM translocase, require the fungi-specific Mim1/Mim2 complex. So far it was unclear which proteins accomplish this task in other eukaryotes. Here, we show by reciprocal complementation that the MOM protein pATOM36 of trypanosomes is a functional analogue of yeast Mim1/Mim2 complex, even though these proteins show neither sequence nor topological similarity. Expression of pATOM36 rescues almost all growth, mitochondrial biogenesis, and morphology defects in yeast cells lacking Mim1 and/or Mim2. Conversely, co-expression of Mim1 and Mim2 restores the assembly and/or insertion defects of MOM proteins in trypanosomes ablated for pATOM36. Mim1/Mim2 and pATOM36 form native-like complexes when heterologously expressed, indicating that additional proteins are not part of these structures. Our findings indicate that Mim1/Mim2 and pATOM36 are the products of convergent evolution and arose only after the ancestors of fungi and trypanosomatids diverged.

The pseudo-dimeric tyrosyl-tRNA synthetase of T. brucei aminoacylates cytosolic and mitochondrial tRNATyr and requires both monomeric units for activity.

Aminoacyl-tRNA synthetases are essential for protein synthesis. The single-copy tyrosyl-tRNA synthetase (Tb-TyrRS) of T. brucei has an unusual structure and forms a pseudo-dimer. It is therefore twice the size than tyrosyl-tRNA synthetases of most other organisms. Here we show by inducible RNAi that Tb-TyrRS is essential for normal growth of procyclic T. brucei. Furthermore we demonstrate that Tb-TyrRS aminoacylates cytosolic as well as mitochondrial tRNATyr indicating that it is dually localized. Finally we show that individual deletion of the 36 N- or C-terminal amino acids abolishes the function of Tb-TyrRS. This indicates that both monomeric units of the enzyme, the C-terminal one of which is predicted to lack enzymatic activity, are essential for Tb-TyrRS function. In summary our results together with previous studies support the notion that Tb-TyrRS might be a suitable drug target.

Molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei.

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.

Biogenesis of the mitochondrial DNA inheritance machinery in the mitochondrial outer membrane of Trypanosoma brucei.

Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan Trypanosoma brucei has a single mitochondrion with a single-unit genome that is physically connected across the two mitochondrial membranes with the basal body of the flagellum. This connection, termed the tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins-TAC60, TAC42 and TAC40-which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane ß-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal ß-signals. Thus in trypanosomes the highly conserved ß-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.

Charting organellar importomes by quantitative mass spectrometry.

Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analysing whether a protein is transported into an organelle is largely restricted to single constituents. This renders knowledge about imported proteins incomplete, limiting our understanding of organellar biogenesis and function. Here we introduce a method that enables charting an organelle's importome. The approach relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. Furthermore, the method allows for the identification of proteins with dual or multiple locations and the substrates of distinct protein import pathways. We demonstrate the specificity and versatility of this ImportOmics method by targeting import factors in mitochondria and glycosomes, which demonstrates its potential for globally studying protein import and inventories of organelles.

Zinc finger nuclease technology: A stable tool for high efficiency transformation in bloodstream form T. brucei.

In Trypanosoma brucei, the generation of knockout mutants is relatively easy compared to other organisms as transfection methods are well established. These methods have their limitations, however, when it comes to the generation of genome-wide libraries that require a minimum of several hundred thousand transformants. Double-strand breaks with the meganuclease ISce-I dramatically increase transformation efficiency, but are not widely in use as cell lines need to be generated de novo before each transfection. Here we show that zinc finger nucleases are a robust and stable tool that can enhance transformation in bloodstream forms by more than an order of magnitude.

Biogenesis of a Mitochondrial Outer Membrane Protein in Trypanosoma brucei: TARGETING SIGNAL AND DEPENDENCE ON A UNIQUE BIOGENESIS FACTOR.

The mitochondrial outer membrane (OM) contains single and multiple membrane-spanning proteins that need to contain signals that ensure correct targeting and insertion into the OM. The biogenesis of such proteins has so far essentially only been studied in yeast and related organisms. Here we show that POMP10, an OM protein of the early diverging protozoan Trypanosoma brucei, is signal-anchored. Transgenic cells expressing variants of POMP10 fused to GFP demonstrate that the N-terminal membrane-spanning domain flanked by a few positively charged or neutral residues is both necessary and sufficient for mitochondrial targeting. Carbonate extraction experiments indicate that although the presence of neutral instead of positively charged residues did not interfere with POMP10 localization, it weakened its interaction with the OM. Expression of GFP-tagged POMP10 in inducible RNAi cell lines shows that its mitochondrial localization depends on pATOM36 but does not require Sam50 or ATOM40, the trypanosomal analogue of the Tom40 import pore. pATOM36 is a kinetoplastid-specific OM protein that has previously been implicated in the assembly of OM proteins and in mitochondrial DNA inheritance. In summary, our results show that although the features of the targeting signal in signal-anchored proteins are widely conserved, the protein machinery that mediates their biogenesis is not.

TbLOK1/ATOM19 is a novel subunit of the noncanonical mitochondrial outer membrane protein translocase of Trypanosoma brucei.

TbLOK1 has previously been characterized as a trypanosomatid-specific mitochondrial outer membrane protein whose ablation caused a collapse of the mitochondrial network, disruption of the membrane potential and loss of mitochondrial DNA. Here we show that ablation of TbLOK1 primarily abolishes mitochondrial protein import, both in vivo and in vitro. Co-immunprecipitations together with blue native gel analysis demonstrate that TbLOK1 is a stable and stoichiometric component of the archaic protein translocase of the outer membrane (ATOM), the highly diverged functional analogue of the TOM complex in other organisms. Furthermore, we show that TbLOK1 together with the other ATOM subunits forms a complex functional network where ablation of individual subunits either causes degradation of a specific set of other subunits or their exclusion from the ATOM complex. In summary these results establish that TbLOK1 is an essential novel subunit of the ATOM complex and thus that its primary molecular function is linked to mitochondrial protein import across the outer membrane. The previously described phenotypes can all be explained as consequences of the lack of mitochondrial protein import. We therefore suggest that in line with the nomenclature of the ATOM complex subunits, TbLOK1 should be renamed to ATOM19.

Outer membrane protein functions as integrator of protein import and DNA inheritance in mitochondria.

Trypanosomatids are one of the earliest diverging eukaryotes that have fully functional mitochondria. pATOM36 is a trypanosomatid-specific essential mitochondrial outer membrane protein that has been implicated in protein import. Changes in the mitochondrial proteome induced by ablation of pATOM36 and in vitro assays show that pATOM36 is required for the assembly of the archaic translocase of the outer membrane (ATOM), the functional analog of the TOM complex in other organisms. Reciprocal pull-down experiments and immunofluorescence analyses demonstrate that a fraction of pATOM36 interacts and colocalizes with TAC65, a previously uncharacterized essential component of the tripartite attachment complex (TAC). The TAC links the single-unit mitochondrial genome to the basal body of the flagellum and mediates the segregation of the replicated mitochondrial genomes. RNAi experiments show that pATOM36, in line with its dual localization, is not only essential for ATOM complex assembly but also for segregation of the replicated mitochondrial genomes. However, the two functions are distinct, as a truncated version of pATOM36 lacking the 75 C-terminal amino acids can rescue kinetoplast DNA missegregation but not the lack of ATOM complex assembly. Thus, pATOM36 has a dual function and integrates mitochondrial protein import with mitochondrial DNA inheritance.

Trypanosomal TAC40 constitutes a novel subclass of mitochondrial ß-barrel proteins specialized in mitochondrial genome inheritance.

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.

The nuclear mRNA export receptor Mex67-Mtr2 of Trypanosoma brucei contains a unique and essential zinc finger motif.

Trypanosoma brucei is the causative agent of Human African Trypanosomiasis. Trypanosomes are early diverged protozoan parasites and show significant differences in their gene expression compared with higher eukaryotes. Due to a lack of individual gene promoters, large polycistronic transcripts are produced and individual mRNAs mature by trans-splicing and polyadenylation. In the absence of transcriptional control, regulation of gene expression occurs post-transcriptionally mainly by control of transcript stability and translation. Regulation of mRNA export from the nucleus to the cytoplasm might be an additional post-transcriptional event involved in gene regulation. However, our knowledge about mRNA export in trypanosomes is very limited. Although export factors of higher eukaryotes are reported to be conserved, only a few orthologues can be readily identified in the genome of T. brucei. Hence, biochemical approaches are needed to identify the export machinery of trypanosomes. Here, we report the functional characterization of the essential mRNA export factor TbMex67. TbMex67 contains a unique and essential N-terminal zinc finger motif. Furthermore, we could identify two interacting export factors namely TbMtr2 and the karyopherin TbIMP1. Our data show that the general heterodimeric export receptor Mex67-Mtr2 is conserved throughout the eukaryotic kingdom albeit exhibiting parasite-specific features.

Nucleolar proteins regulate stage-specific gene expression and ribosomal RNA maturation in Trypanosoma brucei.

Different life-cycle stages of Trypanosoma brucei are characterized by stage-specific glycoprotein coats. GPEET procyclin, the major surface protein of early procyclic (insect midgut) forms, is transcribed in the nucleolus by RNA polymerase I as part of a polycistronic precursor that is processed to monocistronic mRNAs. In culture, when differentiation to late procyclic forms is triggered by removal of glycerol, the precursor is still transcribed, but accumulation of GPEET mRNA is prevented by a glycerol-responsive element in the 3' UTR. A genome-wide RNAi screen for persistent expression of GPEET in glycerol-free medium identified a novel protein, NRG1 (Nucleolar Regulator of GPEET 1), as a negative regulator. NRG1 associates with GPEET mRNA and with several nucleolar proteins. These include two PUF proteins, TbPUF7 and TbPUF10, and BOP1, a protein required for rRNA processing in other organisms. RNAi against each of these components prolonged or even increased GPEET expression in the absence of glycerol as well as causing a significant reduction in 5.8S rRNA and its immediate precursor. These results indicate that components of a complex used for rRNA maturation can have an additional role in regulating mRNAs that originate in the nucleolus.