LOCALIZATION AND TARGETING OF THE LEISHMANIA-DONOVANI HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE TO THE GLYCOSOME

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme in the purine salvage pathway of many protozoan parasites, The predic ted amino acid sequences of certain HGPRT proteins from parasites of t he Trypanosomatidae family reveal a COOH-terminal tripeptide signal th at is consistent with the degenerate topogenic signal targeting protei ns to the glycosome, a fuel metabolizing microbody unique to these par asites. To determine definitively the intracellular milieu of HGPRT in these pathogens, polyclonal antiserum to the purified recombinant HGP RT from Leishmania donovani was generated in rabbits, and confocal and immunoelectron microscopy were employed to establish that the L. dono vani HGPRT is localized exclusively to the glycosome, No HGPRT protein was detected in Delta hgprt null mutants in which both alleles of the HGPRT locus had been replaced by a drug-resistance cassette, Transfec tants of the Delta hgprt knockout strain in which a wildtype HGPRT was amplified on an expression plasmid contained augmented amounts of HGP RT, all of which was localized to the glycosome, Delta hgprt transfect ants containing amplified copies of a mutated HGPRT construct in which the Ser-Lys-Val COOH-terminal targeting signal had been deleted expre ssed HGPRT throughout the parasite, including subcellular organelles s uch as the nucleus and flagellum, These data demonstrate that the L. d onovani HGPRT is compartmentalized exclusively within the glycosome an d that the COOH-terminal tripeptide of the protein is necessary to ach ieve targeting to this organelle.