Charakterisierung von Proteinen und Lipiden der Glykosomen von Trypanosoma brucei
Zusammenfassung der Projektergebnisse
Peroxisomes, glyoxysomes and glycosomes belong to the family of Microbodies. They are single membrane-bound organelles present in most eukaryotes and contain enzymes involved in detoxification, lipid and energy metabolism. Microbodies share a common apparatus for membrane formation and the import of matrix proteins. The protein and lipid composition of membranes from Microbodies is still enigmatic. Results of genetic studies in both yeast and trypanosomes suggest that the membrane is impermeable to a range of metabolites, whereas purified microbodies are very leaky. Some microbody membrane proteins have been identified genetically and biochemically, but there are no general consensus microbody membrane targeting signals. Microbody matrix proteins enter as folded complexes, but import pores are not evident. Also the lipid contents of the different microbody membranes have received little attention and some of the published results imply that there is insufficient phospholipid in microbodies to create a classical lipid bilayer. Trypanosoma brucei is a model lower eukaryote, which is easy to manipulate by reverse genetics and has abundant “relative easy-to-purify” glycosomes. Glycosomes are specialised peroxisomes that have been identified in different members from the order of Kinetoplastida. Some of these Kinetoplastida, like trypanosomes and the related leishmanias, are major tropical pathogens adversely affecting millions of people each year. Existing drugs for treatment of the predominant lethal diseases they cause are expensive, have toxic side effects or are ineffective due to emerging drug resistance. The kinetoplastid glycosome is regarded as an important source of potential drug targets because of the unique features of the energy metabolism that is essentially compartmentalised in this organelle, and the fact that it is absolute required for the survival of these parasites. Major aim of this DFG project was (1) to identify all major proteins and lipids present in the glycosomes of Trypanosoma brucei brucei, and (2) to functionally characterise potential glycosomal metabolite transporters, with focus on metabolite transporters belonging to the mitochondrial carrier family (MCF). To address (1), T. brucei brucei glycosomes were isolated to a high level of purity from the two main life cycle stages of the parasite, e.g. the mammalian bloodstream-form and the insect (vector) procyclic-form, and were analysed by both protein and lipid mass spectrometry. The obtained results from the protein mass spectrometry provided an important and first time overall picture (metabolic snapshot) of the glycosome and its constituents in the two different main lifecycle stages of T. brucei brucei. Comparative (non-quantitative) analysis resulted further in the validation of already known functional aspects of glycosomes and the identification of novel glycosomal constituents and metabolic pathways. The results for the lipid mass spectrometry of purified glycosomes revealed that the glycosomal (membrane) lipid composition is not significantly different from that of peroxisomes from humans and yeast, and that glycosomes contain sufficient amounts of lipid to form a prototypical lipid bilayer. Aim (2) of the DFG project was addressed by the identification and functional characterization of metabolite transporters belonging to the mitochondrial carrier family (MCF). MCF proteins have predominantly been identified in mitochondria, but are also found in peroxisomes of different eukaryotes. The T. b. brucei genome database encodes for 24 different MCF proteins. Mass spectrometry of glycosomes lead to the identification of at least 3 different MCF proteins. Subsequent immunolocalisation studies confirmed that TbMCP6, a potential nucleotide carrier, is indeed located in bloodstream-form glycosomes. Immunolocalisation studies of the remaining 23 TbMCPs showed that they are mainly located in the mitochondrion of procyclic-form T. b. brucei. Unfortunately, we could not exclude that some of the TbMCPs are maybe also located in the glycosomes, which would support the initial glycosome mass spectrometry results. Additional sequence analysis and phylogenetic reconstruction of the 24 identified TbMCPs gave important insights into their evolution and conservation. For most of TbMCPs, putative metabolite transport functions could be predicted based on conserved substrate binding sites and their significant homology to MCF proteins from other eukaryotes, whose function had been previously characterized. Comparison of the kinetoplastid MCP inventory to those previously reported for other eukaryotes revealed also some remarkable deviations: T. b. brucei lacks genes encoding some prototypical MCF members, such as the citrate carrier and uncoupling proteins.
Projektbezogene Publikationen (Auswahl)
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(2006) Characterisation and developmentally regulated localisation of the mitochondrial carrier protein homologue MCP6 from Trypanosoma brucei. Eukaryotic Cell 5, p1194-1205
Colasante, C., Alibu, V.P., Kirchberger, S., Tjaden, J., Clayton, C., and Voncken, F.
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(2006) Comparative proteomics of glycosomes from bloodstream form and procyclic culture-form Trypanosoma brucei brucei. Proteomics 6, p3275-3293
Colasante, C., Ellis, M., Ruppert, T., and Voncken, F.
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(2009) Mitochondrial Carrier Family Inventory of Trypanosoma brucei brucei: Identification, Expression and Subcellular Localisation. Molecular and Biochemical Parasitology 167, p104-117
Colasante, C., Peña-Diaz, P., Clayton, C., and Voncken, F.
