TY - JOUR
T1 - Subcellular localization of adenine and xanthine phosphoribosyltransferases in Leishmania donovani
AU - Zarella-Boitz, Jan M.
AU - Rager, Nicolle
AU - Jardim, Armando
AU - Ullman, Buddy
N1 - Funding Information:
This work was supported in part by grant RO1 AI23682 from the National Institute of Allergy and Infectious Disease. J.Z.-B. has received financial support from the N.L. Tartar Research Fellowship from the Oregon Health & Science University. We would like to thank Ms. Aurelie Snyder of the Molecular Microbiology and Immunology Department of the Oregon Health & Science University for her technical assistance with the deconvolution microscopy and Mr. Jaime Dante and Dr. Wandy Beatty of the Washington University Molecular Microbiology Imaging Facility for their help with the immunoelectronmicroscopy.
PY - 2004/3
Y1 - 2004/3
N2 - 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.
AB - 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.
KW - Adenine phosphoribosyltransferase
KW - Glycosome
KW - Leishmania donovani
KW - Localization
KW - Purines
KW - Xanthine phosphoribosyltransferase
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U2 - 10.1016/j.molbiopara.2003.08.016
DO - 10.1016/j.molbiopara.2003.08.016
M3 - Article
C2 - 14747142
AN - SCOPUS:1642513404
SN - 0166-6851
VL - 134
SP - 43
EP - 51
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1
ER -