Characterization of proteins involved in turnover of glycosomes in Trypanosoma brucei
(2012)
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- Authors
- Supervisors
- Michels, Paul
- Abstract
- (en) Sleeping sickness is an infectious disease affecting humans in sub-Saharan Africa. It is caused by the protist Trypanosoma brucei which is transmitted by blood-feeding tsetse flies. This parasite possesses many unique features, including the organisation of the major part of the glycolytic pathway in peroxisome-like organelles known as glycosomes. This compartmentalisation has been shown to be essential for the viability of the parasite. During its life cycle, T. brucei proceeds through several stages. In its mammalian host, the parasite presents itself as a bloodstream form that, for its ATP supply, is dependent on glucose from the host’s blood and in the tsetse fly’s midgut it occurs as the procyclic form. In this latter stage the parasite prefers the less available glucose, but can also feed, for its energy, on amino acids such as proline and glutamine. The enzyme composition of glycosomes from each of these two stages is associated to the parasite’s metabolic needs. Bloodstream trypanosomes have 90% of the glycosomal enzyme content dedicated to glycolysis, while in glycosomes from procyclic cells this number drops to 40-50%, and the parasites develop a more diversified metabolism. Experiments performed in our laboratory have shown an increase in colocalisation of markers for glycosomes and the lysosome during the steps by which trypanosomes differentiate from the bloodstream to the procyclic form. This indicates that autophagy plays a role in the metabolic adaptation of the parasites during differentiation. It has been shown in yeast cells that peroxisomes reach a certain stage where they are no longer able to import matrix proteins from the cytosol. These organelles then, can be considered unable to adapt their enzyme content to changes in carbon source. We propose that a similar situation may happen in T. brucei where mature glycosomes that are rich in glycolytic enzymes and no longer able to import matrix proteins will be specifically degraded when the parasite differentiates into the procyclic form and requires the expression of a new set of glycosomal enzymes. Our project had two objectives: the characterization of proteins that participate in the degradation as well as a protein implicated in the growth of glycosomes, being respectively, autophagy-related proteins (ATGs) involved in the organelle degradation and a peroxin (PEX), involved in the organelle’s biogenesis, more specifically in matrix protein import. Here we present the characterisation of a T. brucei protein whose yeast counterpart participates in specific autophagy, ATG24, also present in mammalian cells where it is known as SNX4. This protein is part of a putative complex that is involved in autophagy induction and the switch between general autophagy and specific autophagy, including pexophagy, mitophagy, the cytoplasm-to-vacuole pathway and piecemeal microautophagy of the nucleus. TbATG24 seems to be required for the maintenance of the lysosomal morphology in bloodstream and procyclic cells and also participates in endocytosis, as shown by the effect of its RNA interference-dependent partial depletion on transferrin import in a bloodstream-form cell line. This latter function is evolutionary conserved, as it is also found for its orthologues in mammalian cells and yeasts. Moreover the interaction of TbATG24 with endosomal membranes has been shown to be dependent on TbVPS34 and to be sensitive to wortmannin treatment, in agreement with observations reported for its yeast counterpart. Interestingly, its downregulation leads to an increase in autophagosome numbers in autophagy-inducing conditions and an increase of the rate of in vitro differentiation of monomorphic T. brucei cells. Another ATG protein characterized in this study is ATG8, the autophagy marker. Using GFP-tagged constructs, we observed that two different isoforms of ATG8, TbATG8.1 and TbATG8.2, both locate to puncta under different autophagy-inducing conditions and therefore can both be considered as autophagosomal markers. Moreover both isoforms of the protein colocalise with the glycosomal marker triosephosphate isomerase during induction of in vitro differentiation, indicating that both are involved in pexophagy for glycosome turnover. Intriguingly, we observed a different rate of differentiation for cells expressing the GFP-tagged ATG8.2 or cells with GFP-ATG8.1. The T. brucei glycosomal matrix-protein import machinery is similar to that described for peroxisomal protein import in yeasts, with some of the proteins conserved. So far about a dozen proteins that participate in glycosome biogenesis and matrix protein import (called peroxins or PEX proteins) have previously been characterised in our laboratory. Among the peroxins characterised is TbPEX13, a transmembrane component of a complex on which dock TbPEX5 and TbPEX7, cytosolic receptors for proteins with a peroxisomal-targeting signal (PTS). In addition to binding the receptors, TbPEX13 interacts also with TbPEX14, another component of the docking complex. TbPEX13 has been shown to be essential for the parasites and to be involved in the import of PTS-containing proteins. Despite its conserved role and some structural features in common with PEX13s from other organisms, this protein also lacks important PEX13 signatures and has other remarkable features. We describe the identification and characterisation of a second TbPEX13, named TbPEX13.2, that also lacks some PEX13 traits, such as a SH3 domain, but in other respects is more similar to the PEX13 of other organisms than the previously found TbPEX13 (now called TbPEX13.1). We show that this second TbPEX13 is associated with glycosomes, where it interacts with PEX13.1 and other peroxins. It is essential for bloodstream-form cells and participates in the import of PTS proteins. Moreover, as seen for TbPEX13.1, this second isoform is also involved in the association of PEX14 to the glycosomal membrane. Trypanosomatids are the first organisms where two distinct isoforms of PEX13 have been described.
- Affiliations
Citations
Paes Barreto Brennand, A. E. (2012). Characterization of proteins involved in turnover of glycosomes in Trypanosoma brucei. https://hdl.handle.net/2078.5/158505