Toxoplasma PRP1 is an ortholog of parafusin (PFUS) in vesicle scaffold assembly in Ca2+-regulated exocytosis

Li Liu, Stephanie C. Tucker, Birgit H. Satir

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

The Paramecium tetraurelia protein parafusin (PFUS) and the Toxoplasma gondii protein parafusin-related protein 1 (PRP1) both have two covalent modifications (phosphorylation and phosphoglucosylation) and both are members of the phosphoglucomutase superfamily, associating with secretory vesicle scaffolds in their respective cells. This study tests the hypothesis that PRP1 is a functional ortholog of PFUS, functioning identically in Ca2+-regulated exocytosis. Electroporation of fluorescently labeled recombinant His-PRP1 into live Paramecium cells resulted in its localization to docked, dense-core secretory vesicles (DCSVs) in a pattern identical to endogenous PFUS. In tam8 mutants, defective in transport of DCSVs, the fluorescently labeled protein was restricted to the un-transported DCSVs. Specificity of PRP1 localization was demonstrated by electroporating labeled actin or pyruvate kinase, which both failed to localize to either docked or undocked vesicles. In wild-type Paramecium, electroporated His-PRP1 dissociated from DCSVs upon exocytosis, and re-associated as new organelles formed. Mutagenized His-PRP1 species (S146A or S146E) cannot be phosphorylated by P2 calcium-dependent kinase in vitro. Upon electroporation, these molecules remained cytoplasmic and un-associated with DCSVs, while mutation of another PRP1 serine residue (S560A) did neither affect the localization to the DCSVs nor the phosphorylation pattern. Therefore, in this heterologous system, localization, transport and dissociation/re-association of PRP1 substituted for PFUS, supporting the conclusion that the proteins are functional orthologs. The assay also identified a strategic residue S146 within the PFUS ortholog (S138 in PFUS by extrapolation) required for post-translational modification, DCSV scaffold association and for exocytosis.

Original languageEnglish (US)
Pages (from-to)301-313
Number of pages13
JournalEuropean Journal of Cell Biology
Volume88
Issue number5
DOIs
Publication statusPublished - May 1 2009

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Keywords

  • Ca-regulated exocytosis
  • Membrane fusion
  • Ortholog assay
  • PFUS/PRP1
  • Parafusin
  • Site-specific mutagenesis

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

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