Peroxisomal Targeting Signal-1 Receptor Protein PEX5 fromLeishmania donovani

Abstract

The human pathogens of the Leishmania and Trypanosoma genera compartmentalize glycolytic and other key metabolic pathways in unique subcellular microbodies called glycosomes, organelles related to the peroxisomes of mammals and yeast. The molecular machinery that carries out the specific targeting of glycosomal proteins to the organelle has not been characterized, although the bulk of glycosomal proteins contain the COOH-terminal tripeptide glycosomal peroxisomal targeting signal-1 (PTS-1) similar to the mammalian and fungal peroxisomal targeting signal. To characterize the mechanisms of glycosomal targeting, the gene encoding PEX5, designated LdPEX5, has been isolated 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 COOH-terminal half proposed to mediate PTS-1 binding and three copies of a WXXX(Y/F) motif in its NH 2 terminus conjectured to be essential for protein translocation into the organelle. LdPEX5 was overexpressed in Escherichia coli and purified to homogeneity for binding experiments and generation of antibodies. Recombinant LdPEX5 bound xanthine phosphoribosyltransferase (XPRT), 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 studies 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 essential gene.

Pex5p, the Peroxisomal Cycling Receptor, Is a Monomeric Non-globular Protein

“…Indeed, all the available data indicate that this domain displays "normal" hydrodynamic properties as can also be predicted by the tridimensional structure of the human TPR domains (11,28,44). Considering the high frictional ratio of human Pex5p observed in this work, it is thus evident that the N-terminal domain of Pex5p dominates the hydrodynamic properties of the full-length protein.…”

“…In this context, the results available for Pex5p from L. donovani are particularly interesting. This protein also displays an abnormal behavior upon size exclusion chromatography, an observation that led to the conclusion that Pex5p from this organism is a tetramer (44). However, as emphasized in a later work, binding assays using a PTS1-containing peptide failed to demonstrate the existence of those tetramers (26).…”

Domain Mapping of Human PEX5 Reveals Functional and Structural Similarities to Saccharomyces cerevisiae Pex18p and Pex21p

“…A further comparison of all other known PEX5 sequences revealed that this conserved motif and, therefore, the long form of PEX5 are only found in mammals; plants (18,84) (Arabidopsis thaliana: AF074843); and protozoa Leishmania donovani (85) and Trypanosoma brucei (86) but not in Caenorhabditis elegans (C34C6.6) (87); Drosophila melanogaster (AAF45676.1); or different yeast species. For all species that share the common PEX7 binding motif there is evidence for PEX7 as in A. thaliana (AAF88113), or at least evidence for proteins with a PTS2 targeting signal as in plants and T. brucei (88).…”

Glucose is toxic to glycosome-deficient trypanosomes

“…Among such proteins are PEX5, the receptor for the type 1 peroxisomal targeting sequence (10,11), and PEX2 (12), a protein required for peroxisomal protein import but whose exact function remains elusive. Also identified is a homologue of PEX11 (13), a peroxisomal membrane protein thought to function in fatty acid metabolism (14) as well as regulation of peroxisomal abundance (15).…”