Subcellular localization of adenine and xanthine phosphoribosyltransferases in Leishmania donovani.

The subcellular location of a protein is a critical factor in its physiological function and an important consideration in therapeutic paradigms that target the protein. Because Leishmania donovani cannot synthesize purine nucleotides de novo, they rely predominantly upon therapeutically germane phosphoribosyltransferase (PRT) enzymes, hypoxanthine-guanine PRT (HGPRT), adenine PRT (APRT), and xanthine PRT (XPRT), for purine acquisition from the host. Previous studies have shown that the L. donovani HGPRT is localized to the glycosome, a fuel-metabolizing microbody that is unique to kinetoplastid parasites [J. Biol. Chem. 273 (1998) 1534]. The sequences of the other two PRTs indicate that XPRT, but not APRT, possesses a COOH-terminal tripeptide that mediates protein targeting to the glycosome. To determine definitively the intracellular milieu of APRT and XPRT, polyclonal antibodies were raised to each recombinant protein. APRT and XPRT were then shown by immunofluorescence to be localized to the cytosol and glycosome, respectively. The glycosomal milieu for XPRT was also verified by immunoelectron microscopy. Amputation of the glycosomal targeting signal from XPRT resulted in protein mislocalization to the cytosol, but the cytosolic xprt was still functional with respect to purine salvage. These studies establish that APRT is cytosolic and XPRT, like the homologous HGPRT, is glycosomal and demonstrate that a mutant xprt protein that mislocalizes to the cytosol is still functional and supports parasite viability.

Peroxisomal targeting protein 14 (PEX14) from Leishmania donovani. Molecular, biochemical, and immunocytochemical characterization.

Pathogens of the Leishmania and Trypanosoma genera compartmentalize glycolytic and other nutritional pathways in glycosomes, unique subcellular organelles related to the peroxisomes of mammals and yeasts. Most glycosomal proteins are targeted to the glycosomes by a COOH-terminal tripeptide signal similar to the peroxisomal targeting signal-1 (PTS-1). It has been proposed that PTS-1 forms a complex with the PEX5 receptor protein which then docks to the glycosomal membrane through interactions with the membrane associated PEX14 protein. To analyze the role of PEX14 in glycosomal protein import, the gene encoding the L. donovani PEX14 (LdPEX14) was isolated and shown to encode a 464 amino acid protein that exhibited very limited sequence homology with peroxisomal PEX14 proteins. In vitro binding experiments with purified recombinant LdPEX14 and LdPEX5 confirmed that LdPEX14-LdPEX5 interacted with a K(d) of 2.75 microM. When LdPEX5 was preloaded with a PTS-1 peptide, the affinity of the LdPEX14-LdPEX5 interaction affinity increased. Furthermore, binding experiments with truncated forms of LdPEX5 and LdPEX14 showed that the interaction domains localized to the amino terminal region of both proteins. Finally, confocal microscopy, subcellular fractionation, and differential extraction experiments indicated that LdPEX14 is a soluble protein that associates tightly with the glycosomal membrane and further support the role of LdPEX14 in forming a docking complex involved in glycosome biogenesis.