Structural and Functional Association of
            <i>Trypanosoma brucei</i>
            MIX Protein with Cytochrome
            <i>c</i>
            Oxidase Complex

Zı́ková, Alena; Panigrahi, Aswini K.; Uboldi, Alessandro D.; Dalley, Rachel; Handman, Emanuela; Stuart, Kenneth

doi:10.1128/ec.00204-08

Cited by 32 publications

(43 citation statements)

References 42 publications

(50 reference statements)

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“…The interactions are consistent with the known structure of the respiratory complexes, e.g. complex I organized in subcomplexes, association of "MIX" protein with complex IV (17).The interaction map is consistent with our finding that RNAi knockdown of Tb10.70.7760 in complex V, resulted in the loss of the F o F 1 complex interactions that contains the interactor, but the F 1 moiety was retained (8). Also, we report the first evidence of MS identification of mt proteins encoded by edited RNAs in T. brucei.…”

Section: Discussionsupporting

confidence: 84%

“…The .out files (SEQUEST output) were compiled and filtered using the DTASelect program (28) to identify proteins in each analysis. Proteins identified with at least two tryptic peptides and with minimum peptide and protein identification probability of Ն0.9 were included in the list except for known non-mt proteins and highly abundant mt protein that are often seen in various TAP-tag experiments (17). The .raw files were converted to mzXML format and the Census program (29) was used to determine the spectral count of each of the peptides.…”

Section: Methodsmentioning

confidence: 99%

“…However, some respiratory complexes have been partially characterized in other trypanosomatids such as Crithidia fasciculata, T. cruzi, and Leishmania tarentolae (12)(13)(14)(15). In recent studies, we have determined the protein composition of complexes IV and V, and part of complex I purified from mitochondria of T. brucei PF cells (8,16,17,25). These analyses revealed the uniqueness of respiratory complexes in trypanosomes, where large numbers of component proteins have no homologs outside of the Kinetoplastida.…”

mentioning

confidence: 99%

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Trypanosoma brucei Mitochondrial Respiratome: Composition and Organization in Procyclic Form

Acestor

Zı́ková

Dalley

et al. 2011

Molecular & Cellular Proteomics

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The mitochondrial respiratory chain is comprised of four different protein complexes (I-IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F o F 1 -ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen.

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Section: Discussionsupporting

confidence: 84%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Trypanosoma brucei Mitochondrial Respiratome: Composition and Organization in Procyclic Form

Acestor

Zı́ková

Dalley

et al. 2011

Molecular & Cellular Proteomics

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“…mt vesicles isolated from 5 ϫ 10 8 trypanosomes were lysed with 2% dodecyl maltoside, and the cytochrome c oxidase activity was determined in vitro by measuring the change in absorbance of cytochrome c as it became oxidized after passing its electrons to cytochrome c oxidase (40). Cytochrome c reductase activity was determined in a similar way; this time, the reduction of cytochrome c was measured when reduced decylubiquinone (Sigma) was added as an electron donor and cytochrome c reductase transferred those electrons to cytochrome c (33).…”

Section: Methodsmentioning

confidence: 99%

The ADP/ATP Carrier and Its Relationship to Oxidative Phosphorylation in Ancestral Protist Trypanosoma brucei

et al. 2015

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The highly conserved ADP/ATP carrier (AAC) is a key energetic link between the mitochondrial (mt) and cytosolic compartments of all aerobic eukaryotic cells, as it exchanges the ATP generated inside the organelle for the cytosolic ADP. Trypanosoma brucei, a parasitic protist of medical and veterinary importance, possesses a single functional AAC protein (TbAAC) that is related to the human and yeast ADP/ATP carriers. However, unlike previous studies performed with these model organisms, this study showed that TbAAC is most likely not a stable component of either the respiratory supercomplex III؉IV or the ATP synthasome but rather functions as a physically separate entity in this highly diverged eukaryote. Therefore, TbAAC RNA interference (RNAi) ablation in the insect stage of T. brucei does not impair the activity or arrangement of the respiratory chain complexes. Nevertheless, RNAi silencing of TbAAC caused a severe growth defect that coincides with a significant reduction of mt ATP synthesis by both substrate and oxidative phosphorylation. Furthermore, TbAAC downregulation resulted in a decreased level of cytosolic ATP, a higher mt membrane potential, an elevated amount of reactive oxygen species, and a reduced consumption of oxygen in the mitochondria. Interestingly, while TbAAC has previously been demonstrated to serve as the sole ADP/ATP carrier for ADP influx into the mitochondria, our data suggest that a second carrier for ATP influx may be present and active in the T. brucei mitochondrion. Overall, this study provides more insight into the delicate balance of the functional relationship between TbAAC and the oxidative phosphorylation (OXPHOS) pathway in an early diverged eukaryote. The origination event of an ADP/ATP carrier (AAC) was crucial in the evolution of the present-day mitochondrion that enabled it to become the powerhouse of the eukaryotic cell. Due to the activity of AAC, the energy-producing mitochondria can supply the cytosol with ATP molecules, which fuel most cellular reactions. AAC belongs to the well-defined family of mitochondrial (mt) carrier proteins that are located in the inner mt membrane and are involved in the transport of a wide range of metabolites (1). Under physiological aerobic conditions, AAC is responsible for the 1:1 counterexchange of mt ATP for cytosolic ADP (2). mt ATP is produced mainly by the evolutionarily conserved biochemical pathway of oxidative phosphorylation (OXPHOS), which employs respiratory complexes I through IV to convert the redox energy of various mt substrates into an electrochemical proton gradient (⌬m) across the mt inner membrane. Respiratory complexes I (NADH-ubiquinone oxidoreductase) and II (succinate-ubiquinone oxidoreductase) are responsible for the oxidation of reduced NADH and FADH 2 , respectively. The electrons derived from these biochemical processes continue along the electron transport chain as they are sequentially passed to coenzyme Q, complex III (ubiquinol-cytochrome c oxidoreductase), cytochrome c, complex IV (cytochrome c-O 2 oxid...

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“…However, with the use of mass spectrometry, 43 proteins, in addition to TbPRMT6, were identified by two or more peptides. Of these, almost half were presumed to be common contaminants present in TAP-based purifications, based on mass spectrometry studies from other laboratories (85,101,104,105) and our previous experience (see Table S1 in the supplemental material). It was striking that the remaining TbPRMT6-associated proteins could be readily classified into three major groups (nucleocytoplasmic transport proteins, histones, and flagellar proteins) ( Table 1).…”

Section: Downloaded Frommentioning

confidence: 99%

TbPRMT6 Is a Type I Protein Arginine Methyltransferase That Contributes to Cytokinesis in Trypanosoma brucei

et al. 2010

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Arginine methylation is a widespread posttranslational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). In Saccharomyces cerevisiae and mammals, this modification affects multiple cellular processes, such as chromatin remodeling leading to transcriptional regulation, RNA processing, DNA repair, and cell signaling. The protozoan parasite Trypanosoma brucei possesses five putative PRMTs in its genome. This is a large number of PRMTs relative to other unicellular eukaryotes, suggesting an important role for arginine methylation in trypanosomes. Here, we present the in vitro and in vivo characterization of a T. brucei enzyme homologous to human PRMT6, which we term TbPRMT6. Like human PRMT6, TbPRMT6 is a type I PRMT, catalyzing the production of monomethylarginine and asymmetric dimethylarginine residues. In in vitro methylation assays, TbPRMT6 utilizes bovine histones as a substrate, but it does not methylate several T. brucei glycine/arginine-rich proteins. As such, it exhibits a relatively narrow substrate specificity compared to other T. brucei PRMTs. Knockdown of TbPRMT6 in both procyclic form and bloodstream form T. brucei leads to a modest but reproducible effect on parasite growth in culture. Moreover, upon TbPRMT6 depletion, both PF and BF exhibit aberrant morphologies indicating defects in cell division, and these defects differ in the two life cycle stages. Mass spectrometry of TbPRMT6-associated proteins reveals histones, components of the nuclear pore complex, and flagellar proteins that may represent TbPRMT6 substrates contributing to the observed growth and morphological defects.Posttranslational methylation of proteins on arginine residues has multiple roles in a wide array of cellular functions, such as chromatin remodeling leading to transcription activation or repression, RNA processing, DNA repair, and various forms of cell signaling (5,6,8,9,52,70,98). The process of arginine methylation involves the transfer of methyl groups from S-adenosyl-methionine (AdoMet) to arginine residues of proteins and is catalyzed by a group of enzymes known as protein arginine methyltransferases (PRMTs). PRMTs themselves are further divided into four classes, depending on the type of methylated arginine generated. The largest PRMT class comprises the type I enzymes, as characterized by the first discovered PRMT, PRMT1. Type I PRMTs initially catalyze the formation of monomethylated arginine (MMA) on the terminal -nitrogen, followed by addition of a second methyl group on the same -nitrogen, which yields asymmetric dimethylarginine (ADMA). The type II PRMTs are a smaller group, presently consisting only of PRMT5 and its homologues. These enzymes also catalyze the synthesis of MMA on the terminal -nitrogen, but in contrast to the type I enzymes, type II PRMTs add a second methyl group to the adjacent terminal -nitrogen, resulting in symmetric dimethylarginine (SDMA). Almost all known eukaryotic cells possess at least one type I PRMT and one type II PRMT in the form of PRM...

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Structural and Functional Association of Trypanosoma brucei MIX Protein with Cytochrome c Oxidase Complex

Cited by 32 publications

References 42 publications

Trypanosoma brucei Mitochondrial Respiratome: Composition and Organization in Procyclic Form

Trypanosoma brucei Mitochondrial Respiratome: Composition and Organization in Procyclic Form

The ADP/ATP Carrier and Its Relationship to Oxidative Phosphorylation in Ancestral Protist Trypanosoma brucei

TbPRMT6 Is a Type I Protein Arginine Methyltransferase That Contributes to Cytokinesis in Trypanosoma brucei

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