The import of proteins containing the peroxisomal targeting signal 1 (PTS1) into the Leishmania glycosome is dependent on the docking of the PTS1-loaded LdPEX5 cytosolic receptor with LdPEX14 on the glycosome surface. Here we show that, in the absence of PTS1, LdPEX5 is a tetramer that is stabilized by two distinct interaction domains; the first is a coiled-coil motif encompassing residues 277 to 310, whereas the second domain is localized to residues 1 to 202. By using microcalorimetry, surface plasmon resonance, and size exclusion chromatography techniques, we show that PTS1 peptide binding to LdPEX5 tetramers promotes their dissociation into dimeric structures, which are stabilized by a coiled-coil interaction. Moreover, we demonstrated that the resulting LdPEX5-PTS1 complex is remarkably stable and exhibits extremely slow dissociation kinetics. However, binding of LdPEX14 to LdPEX5 modulates the LdPEX5-PTS1 affinity as it decreases the thermodynamic dissociation constant for this latter complex by 10-fold. These changes in the oligomeric state of LdPEX5 and in its affinity for PTS1 ligand upon LdPEX14 binding may explain how, under physiological conditions, LdPEX5 can function to deliver and unload its cargo to the protein translocation machinery on the glycosomal membrane.

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Import of proteins with a PTS1 (peroxisomal targeting signal 1) into the Leishmania glycosomal organelle involves docking of a PTS1-laden LdPEX5 [Leishmania donovani PEX5 (peroxin 5)] receptor to LdPEX14 on the surface of the glycosomal membrane. In higher eukaryotes, the PEX5-PEX14 interaction is mediated by a conserved diaromatic WXXXY/F motif. Site-directed and deletion mutageneses of the three WXXXY/F repeats in LdPEX5 did not abolish the LdPEX5-LdPEX14 association. Analysis of the equilibrium dissociation constant (K(d)) revealed that ldpex5-W53A (Trp53-->Ala), ldpex5-W293A, ldpex5-W176,293A and ldpex5-W53,176,293A mutant receptors were capable of binding LdPEX14 with affinities comparable with wild-type LdPEX5. That the diaromatic motifs were not required for the LdPEX5-LdPEX14 interaction was further verified by deletion analysis that showed that ldpex5 deletion mutants or ldpex5 fragments lacking the WXXXY/F motifs retained LdPEX14 binding activity. Mapping studies of LdPEX5 indicated that the necessary elements required for LdPEX14 association were localized to a region between residues 290 and 323. Finally, mutational analysis of LdPEX14 confirmed that residues 23-63, which encompass the conserved signature sequence AX2FLX7SPX6FLKGKGL/V present in all PEX14 proteins, are essential for LdPEX5 binding.

The import of PTS1 proteins into the glycosome or peroxisome requires binding of a PTS1-laden PEX5 receptor to the membrane-associated protein PEX14 to facilitate translocation of PTS1 proteins into the lumen of these organelles. Quaternary structure analysis of protozoan parasite Leishmania donovani PEX14 (LdPEX14) revealed that this protein forms a homomeric complex with a size >670 kDa. Moreover, deletion mapping indicated that disruption of LdPEX14 oligomerization correlated with the elimination of the hydrophobic region and coiledcoil motif present in LdPEX14. Analysis of the LdPEX5-LdPEX14 interaction by isothermal titration calorimetry revealed a molar binding stoichiometry of 1:4 (LdPEX5: LdPEX14) and an in-solution dissociation constant (K d ) of ϳ74 nM. Calorimetry, circular dichroism, intrinsic fluorescence, and analytical ultracentrifugation experiments showed that binding of LdPEX5 resulted in a dramatic conformational change in the LdPEX14 oligomeric complex that involved the reorganization of the hydrophobic segment in LdPEX14. Finally, limited tryptic proteolysis assays established that in the presence of LdPEX5, LdPEX14 became more susceptible to proteolytic degradation consistent with this protein interaction triggering a significant conformational change in the recombinant and native LdPEX14 structures. These structural changes provide essential clues to how LdPEX14 functions in the translocation of folded proteins across the glycosomal membrane.Leishmania and Trypanosoma protozoan parasites represent organisms that branched off early from the eukaryotic cell lineage (1-3). Consequently, these organisms have retained a myriad of unique metabolic, biochemical, and structural features that are distinctive from other eukaryotic cells. Prominent among these features is the glycosome, an organelle that is distantly related to the peroxisomes in mammalian, yeast, fungi, and plant cells (4 -6). The glycosome compartmentalizes a multitude of indispensable metabolic and biosynthetic pathways that include glycolysis, purine salvage, pyrimidine and ether-lipid biosynthesis, and ␤-oxidation of fatty acids (6, 7). Glycosomal function is essential for parasite viability as mistargeting of glycolytic enzymes to the cytosol or disruption of glycosome biogenesis leads to a lethal phenotype (8 -12) making the glycosome and glycosome biogenesis machinery attractive chemotherapeutic targets (13,14).Glycosomal and peroxisomal matrix proteins are post-translationally trafficked from cytosolic ribosomes to these microbodies by utilizing primarily one of two topogenic signals termed peroxisomal targeting signal 1 and 2 (PTS1 and PTS2) located at the C or N termini of proteins, respectively (15-20). In Leishmania newly synthesized proteins containing PTS1 or PTS2 signals are bound by the receptors peroxin 5 (LdPEX5) 3 and peroxin 7 (LdPEX7), respectively, and these cargo-laden receptors traffic to the glycosome surface where they bind to the membrane-associated protein peroxin 14 (LdPEX14). This latter protein-protein...

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