Localization and targeting of the Leishmania donovani hypoxanthine- guanine phosphoribosyltransferase to the glycosome

Sarah Shih, Ho Yon Hwang, Darrick Carter, Paula Stenberg, Buddy Ullman

Research output: Contribution to journalArticle

57 Scopus citations

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 milleu of HGPRT 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 No HGPRT protein was detected in Δhgprt null mutants in which both alleles of the HGPRT locus had been replaced by a drug- resistance cassette. Transfectants of the Δhgprt 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. Δhgprt 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 organeries 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.

Original languageEnglish (US)
Pages (from-to)1534-1541
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number3
DOIs
StatePublished - Jan 16 1998

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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