Peroxisomal targeting signal-1 receptor protein PEX5 from Leishmania donovani - Molecular, biochemical, and immunocytochemical characterization

The human pathogens of the Leishmania and Trypanosoma genera compartmentali ze glycolytic and other key metabolic pathways in unique subcellular microb odies called glycosomes, organelles related to the peroxisomes of mammals a nd yeast. The molecular machinery that carries out the specific targeting o f glycosomal proteins to the organelle has not been characterized, although the bulk of glycosomal proteins contain the COOH-terminal tripeptide glyco somal peroxisomal targeting signal-1 (PTS-1) similar to the mammalian and f ungal peroxisomal targeting signal. To characterize the mechanisms of glyco somal targeting, the gene encoding PEX5, designated LdPEX5, has been isolat ed from Leishmania donovani. LdPEX5 encodes a 625-amino acid protein with a molecular mass of 69.7 kDa. Like its homologs in yeast and humans, LdPEX5 predicts a protein with seven copies of a tetratricopeptide repeat in its C OOH-terminal half proposed to mediate PTS-1 binding and three copies of a W XXX(Y/F) motif in its NH2 terminus conjectured to be essential for protein translocation into the organelle. LdPEX5 was overexpressed in Escherichia c oli and purified to homogeneity for binding experiments and generation of a ntibodies. Recombinant LdPEX5 bound xanthine phosphoribosyltransferase (XPR T), a PTS-1 containing glycosomal protein with a K-D of 4.2 nM, but did not bind an XPRT in which the PTS-1 had been deleted. Moreover, binding studie s with the COOH-terminal half of the LdPEX5 confirmed that this portion of the PEX5 protein was capable of binding the XPRT PTS-1 with an affinity of 17.3 nM. Confocal microsocopy revealed that LdPEX5 was predominantly in the cytosolic milieu, and genetic analysis implied that LdPEX5 was an essentia l gene.