Characterization of the role of the receptors PEX5 and PEX7 in the import of proteins into glycosomes of Trypanosoma brucei

Abstract

Peroxins 5 and 7 are receptors for protein import into the peroxisomal matrix. We studied the involvement of these peroxins in the biogenesis of glycosomes in the protozoan parasite Trypanosoma brucei. Glycosomes are peroxisome-like organelles in which a major part of the glycolytic pathway is sequestered. We here report the characterization of the T. brucei homologue of PEX7 and provide several data strongly suggesting that it can bind to PEX5. Depletion of PEX5 or PEX7 by RNA interference had a severe effect on the growth of both the bloodstream-form of the parasite, that relies entirely on glycolysis for its ATP supply, and the procyclic form representative of the parasite living in the tsetse-fly midgut and in which also other metabolic pathways play a prominent role. The role of the two receptors in import of glycosomal matrix proteins with different types of peroxisome/glycosome-targeting signals (PTS) was analyzed by immunofluorescence and subcellular fractionation studies. Knocking down the expression of either receptor gene resulted, in procyclic cells, in the mislocalization of proteins with both a type 1 or 2 targeting motif (PTS1, PTS2) located at the C- and N-termini, respectively, and proteins with a sequence-internal signal (I-PTS) to the cytosol. Electron microscopy confirmed the apparent integrity of glycosomes in these procyclic cells. In bloodstream-form trypanosomes, PEX7 depletion seemed to affect only the subcellular distribution of PTS2-proteins. Western blot analysis suggested that, in both life-cycle stages of the trypanosome, the levels of both receptors are controlled in a coordinated fashion, by a mechanism that remains to be determined. The observation that both PEX5 and PEX7 are essential for the viability of the parasite indicates that the respective branches of the glycosome-import pathway in which each receptor acts might be interesting drug targets.

Three Mitochondrial DNA Polymerases Are Essential for Kinetoplast DNA Replication and Survival of Bloodstream Form Trypanosoma brucei

“…S1 in the supplemental material). Sustained sensitivity to induction for RNAi is noteworthy here, since RNAi-resistant "revertant" parasites have been widely reported in both PF and BF T. brucei (8,15,22,30,31,49). The reasons why revertants were not produced when silencing POLIB, POLIC, or POLID is beyond the scope of our analyses.…”

A PEX7-Centered Perspective on the Peroxisomal Targeting Signal Type 2-Mediated Protein Import Pathway

“…In contrast to the PTS1, the PTS2 is generally cleaved when the protein reaches the organelle matrix (5)(6)(7). In mammals and many other organisms, both PTS1 and PTS2 proteins are transported to the organelle by PEX5, the peroxisomal shuttling receptor (8)(9)(10)(11). The interaction of PEX5 with PTS1 proteins is direct (12)(13)(14)(15)(16), whereas the interaction between PEX5 and PTS2 proteins requires the adaptor protein PEX7 (17)(18)(19).…”

Environmentally Regulated Glycosome Protein Composition in the African Trypanosome

“…Silencing of trypanosome PEX proteins involved in glycosome matrix protein import (TbPEX5, causes the mislocalization of glycosome proteins to the cytosol and compromised growth (8)(9)(10)(11)(12). Reduction in the expression of PEX genes involved in glycosome biogenesis (TbPEX11and TbPEX19) through RNA interference (13, 14) results in parasites harboring fewer, larger glycosomes, while overexpression of TbPEX11 results in cells with many smaller glycosomes (14).…”