Localization and Targeting of the Leishmania donovaniHypoxanthine-Guanine Phosphoribosyltransferase to the Glycosome

Abstract

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme in the purine salvage pathway of many protozoan parasites. The predicted amino acid sequences of certain HGPRT proteins from parasites of the Trypanosomatidae family reveal a COOH-terminal tripeptide signal that is consistent with the degenerate topogenic signal targeting proteins to the glycosome, a fuel-metabolizing microbody unique to these parasites. To determine definitively the intracellular milieu of HG-PRT in these pathogens, polyclonal antiserum to the purified recombinant HGPRT from Leishmania donovani was generated in rabbits, and confocal and immunoelectron microscopy were employed to establish that the L. donovani HGPRT is localized exclusively to the glycosome. No HGPRT protein was detected in ⌬hgprt null mutants in which both alleles of the HGPRT locus had been replaced by a drug-resistance cassette. Transfectants of the ⌬hgprt knockout strain in which a wildtype HGPRT was amplified on an expression plasmid contained augmented amounts of HGPRT, all of which was localized to the glycosome. ⌬hgprt transfectants containing amplified copies of a mutated HGPRT construct in which the Ser-Lys-Val COOH-terminal targeting signal had been deleted expressed HGPRT throughout the parasite, including subcellular organelles such as the nucleus and flagellum. These data demonstrate that the L. donovani HGPRT is compartmentalized exclusively within the glycosome and that the COOH-terminal tripeptide of the protein is necessary to achieve targeting to this organelle.

Arginase Plays a Pivotal Role in Polyamine Precursor Metabolism in Leishmania

“…2A). Co-localization experiments with antibodies against the L. donovani HGPRT, a known glycosomal marker (35), indicated that ARG and HGPRT inhabited the same subcellular milieu (Fig. 2, B and C).…”

“…2). Thus, the enzymes involved in the synthesis of polyamines, like those for purine and pyrimidine nucleotide synthesis (35,(62)(63)(64), are compartmentalized between the glycosome and cytosol in Leishmania. The rationale for the sequestering of ARG from the cytosolic ODC, SPDSYN, and ADOMETDC is not apparent, but the glycosomal milieu is not essential for ARG function in promastigotes because of the ability of cytosolic arg (arg⌬skl) to complement ⌬arg parasites (Fig.…”

Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

“…Leishmania express four enzymes that are capable of converting host or extracellular purines into nucleotides: 1) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) xanthine phosphoribosyltransferase (XPRT); 3) adenine phosphoribosyltransferase (APRT); 4) and adenosine kinase (2,5,9,10). HGPRT and XPRT are confined within the glycosome (11,12), a membrane-bound microbody organelle that is found exclusively among trypanosomatid parasites (13)(14)(15), whereas APRT has been definitively localized to the cytosol (12). Genetic studies in L. donovani reveal that none of the four enzymes by itself is essential for parasite survival because promastigotes deficient in the activity of any one of the four purine salvage enzymes are viable and do not exhibit a fitness deficit (16 -20).…”

Glucose is toxic to glycosome-deficient trypanosomes

“…Each parasite possesses numerous glycosomes that house not only glycolysis (2) but also enzymes of ether-lipid biosynthesis (3,4), ␤-oxidation of fatty acids (5), and purine salvage (6). The spectrum of proteins contained within the glycosome changes during parasite development.…”