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.
Zarella-Boitz
Jan M
JM
Department of Biochemistry and Molecular Biology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239-3098, USA.
Rager
Nicolle
N
Jardim
Armando
A
Ullman
Buddy
B
D000228:Adenine Phosphoribosyltransferase / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D017125:Genes, Protozoan; D007893:Leishmania donovani / Q000201:enzymology* / Q000254:growth & development / Q000648:ultrastructure; D008830:Microbodies / Q000201:enzymology; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D009154:Mutation; D010430:Pentosyltransferases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D019820:Xanthine / Q000378:metabolism
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D019820:Xanthine; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D000228:Adenine Phosphoribosyltransferase