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Functional characterization of the T. gondii ortholog of parafusin by an in vivo Paramecium ortholog assay
Author(s) -
TUCKER S. C.,
LIU L.,
SATIR B. H.
Publication year - 2005
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.2005.05202003_1_77.x
Subject(s) - biology , paramecium , toxoplasma gondii , secretion , microbiology and biotechnology , cytoplasm , recombinant dna , electroporation , microneme , biochemistry , gene , genetics , apicomplexa , malaria , antibody , immunology , plasmodium falciparum
Parafusin (PFUS) is a phosphoglycoprotein of Paramecium that functions in Ca 2+ ‐regulated exocytosis. The ortholog of this protein in Toxoplasma gondii , called P arafusin R elated P rotein 1 or PRP1, is associated with micronemes and functions in Ca 2+ –regulated secretion (Matthiesen et al., 2003). We have demonstrated that PRP1 colocalizes with PFUS to the Paramecium dense core vesicles (trichocysts) by means of a new assay. In this assay, recombinant, fluorescently tagged PRP1 was electroporated directly into either wild type or mutant live Paramecium cells. After a brief recovery period, the protein was observed on trichocysts docked below the cell membrane. In control experiments using labeled actin and pyruvate kinase, no localization to trichocysts was seen. Additionally, PRP1 could only be detected on undocked trichocysts that had accumulated in the cytoplasm of tam8 , a Paramecium exo‐mutant that fails to transport trichocysts to the surface. Furthermore, PRP1 fluorescence disappeared from the trichocysts of wild‐type Paramecium that were stimulated with lysozyme for synchronous secretion, in a manner similar to that observed for PFUS. Cumulatively these data suggest that PRP1 of T. gondii can be properly modified, transported and localized after electroporation and that fluorochrome labeling of recombinant PRP1 did not adversely affect localization. The assay is ideal for assessing strategic residues and domains within PRP1 and other orthologs for their structural and functional relevance in exocytic processes in living cells.