Peroxisomes are present in nearly every eukaryotic cell and compartmentalize a wide range of important metabolic processes. Glycosomes of Kinetoplastid parasites are peroxisome-like organelles, characterized by the presence of the glycolytic pathway. The two replicating stages of Trypanosoma brucei brucei, the mammalian bloodstream form (BSF) and the insect (procyclic) form (PCF), undergo considerable adaptations in metabolism when switching between the two different hosts. These adaptations involve also substantial changes in the proteome of the glycosome. Comparative (non-quantitative) analysis of BSF and PCF glycosomes by nano LC-ESI-Q-TOF-MS resulted in the validation of known functional aspects of glycosomes and the identification of novel glycosomal constituents.

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Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3). Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide ‘SNL’. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90%) of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed.

SummaryIt is well established that the heart is strongly dependent on fatty acid metabolism. In cardiomyocytes there are two distinct sites for the β-oxidisation of fatty acids: the mitochondrion and the peroxisome. Although the metabolism of these two organelles is believed to be tightly coupled, the nature of this relationship has not been fully investigated. Recent research has established the significant contribution of mitochondrial function to cardiac ATP production under normal and pathological conditions. In contrast, limited information is available on peroxisomal function in the heart. This is despite these organelles harbouring metabolic pathways that are potentially cardioprotective, and findings that patients with peroxisomal diseases, such as adult Refsum´s disease, can develop heart failure. In this article, we provide a comprehensive overview on the current knowledge of peroxisomes and the regulation of lipid metabolism by PPARs in cardiomyocytes. We also present new experimental evidence on the differential expression of peroxisome-related genes in the heart chambers and demonstrate that even a mild peroxisomal biogenesis defect (Pex11α -/-) can induce profound alterations in the cardiomyocyte´s peroxisomal compartment and related gene expression, including the concomitant deregulation of specific PPARs. The possible impact of peroxisomal dysfunction in the heart is discussed and a model for the modulation of myocardial metabolism via a peroxisome/PPAR-loop is proposed.

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In kinetoplastid protists, several metabolic pathways, including glycolysis and purine salvage, are located in glycosomes, which are microbodies that are evolutionarily related to peroxisomes. With the exception of some potential transporters for fatty acids, and one member of the mitochondrial carrier protein family, proteins that transport metabolites across the glycosomal membrane have yet to be identified. We show here that the phosphatidylcholine species composition of Trypanosoma brucei glycosomal membranes resembles that of other cellular membranes, which means that glycosomal membranes are expected to be impermeable to small hydrophilic molecules unless transport is facilitated by specialized membrane proteins. Further, we identified 464 proteins in a glycosomal membrane preparation from Leishmania tarentolae. The proteins included approximately 40 glycosomal matrix proteins, and homologues of peroxisomal membrane proteins - PEX11, GIM5A and GIM5B; PXMP4, PEX2 and PEX16 - as well as the transporters GAT1 and GAT3. There were 27 other proteins that could not be unambiguously assigned to other compartments, and that had predicted trans-membrane domains. However, no clear candidates for transport of the major substrates and intermediates of energy metabolism were found. We suggest that, instead, these metabolites are transported via pores formed by the known glycosomal membrane proteins.

The order Kinetoplastida includes among its members a number of important human and veterinary pathogens, such as the African trypanosome Trypanosoma brucei (83). This parasite has a complex life cycle that alternates between the bloodstream form found in the blood and tissue fluids of mammals and the procyclic (insect) form in the midgut of the tsetse fly (46, 83). T. brucei undergoes a series of metabolic and morphological changes in order to adapt to these distinct host environments (45, 46). Bloodstream-form T. brucei metabolizes glucose to pyruvate and glycerol, obtaining ATP by substrate-level phosphorylation during glycolysis (48, 55). The mitochondria of this life form are highly reduced and lack key enzymes and components of the tricarboxylic acid cycle; their role in energy metabolism is probably restricted to the reoxidation of glycerol-3 phosphate by the mitochondrial alternative oxidase (20,48). This is in contrast to the procyclic form, which metabolizes amino acids and has a well-developed mitochondrion in which ATP is generated by a combination of substratelevel and oxidative phosphorylation (16,48,73,82).In both life forms of T. brucei, most of the glycolytic enzymes are found within a peroxisome-like organelle called the glycosome (14,48,60,61). This unique compartmentation of glycolytic enzymes has been shown to be essential for trypanosome survival (5,11,22,25,51). It has been proposed that the glycosomal membrane forms a barrier to ATP and ADP, insulating the enzymes of the glycosomal matrix from the ADP/ ATP ratio in the cytosol (5).So far, no glycosomal or mitochondrial metabolite carriers have been identified for T. brucei, although they most probably play a key role in the regulation of energy metabolism. The characterization of these carriers should make it possible to build more-accurate biochemical and mathematical models of trypanosome energy metabolism (5).The mitochondrial carrier family (MCF) was initially defined as a group of proteins that are located in the inner mitochondrial membrane and mediate the transport of a large range of metabolic intermediates (3,32,35,(57)(58)(59)86). Recently, several structurally and functionally related carrier proteins were also found in the membranes of peroxisomes (52,81,88,90). We therefore wondered whether such proteins might also be found in kinetoplastid glycosomes. The recently completed genome sequence of T. brucei (8) contains about 29 genes encoding different proteins of the mitochondrial carrier family. In this paper, we describe the characterization of MCP6, a novel mitochondrial carrier protein homologue from T. brucei. MATERIALS AND METHODSCulture and transfection. Trypanosoma brucei cell lines were cultured in either HMI-9 medium for bloodstream cell lines (29) or MEM-PROS medium for procyclic cell lines (56), supplemented with 10% (vol/vol) fetal calf serum (Sigma-Aldrich). Cells were transfected as described previously (9). In all experiments, "wild type" refers to bloodstream or procyclic T. brucei strain

Background:TbMCP5 was predicted to function as a mitochondrial ADP/ATP carrier. Results: TbMCP5 functionally complemented ANC-deficient S. cerevisae, has biochemical properties comparable with those of ScAnc2p, and is essential for mitochondrial ADP/ATP exchange in T. brucei. Conclusion: TbMCP5 is a conserved and essential mitochondrial ADP/ATP carrier in T. brucei. Significance: TbMCP5 is the first functionally characterized mitochondrial ADP/ATP carrier from a kinetoplastid parasite.