A parafusin-related Toxoplasma protein in Ca2+-regulated secretory organelles

Authors: Matthiesen S.H.1; Shenoy S.M.1; Kim K.2; Singer R.H.1; Satir B.H.1

Source: European Journal of Cell Biology, Volume 80, Number 12, December 2001 , pp. 775-783(9)

Publisher: Urban & Fischer

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Abstract:

We cloned a gene, PRP1, of Toxoplasma gondii encoding a 637-amino-acids protein having a calculated mass of 70 kDa. The sequence showed high homology to parafusin, a protein that in Paramecium tetraurelia participates in Ca2+-regulated exocytosis and is a paralog of phosphoglucomutase. We show that Toxoplasma gondii homogenate and an expressed recombinant PRP1 fusion protein cross-react with a specific peptide-derived antibody to parafusin in Western blots. Antibodies to the recombinant PRP1 showed cross-reaction with parafusin and recognized PRP1, as bands at Mr 63×103 and 68×103, respectively. PRP1 is labeled when Toxoplasma gondii cells are incubated with inorganic 32P and appears as the major band on autoradiograms of SDS-PAGE gels. The localization of PRP1 was examined in secretory organelles of Toxoplasma gondii by deconvolution light microscopy followed by three dimensional reconstruction using pairwise combinations of specific antibodies. PRP1 localized to the apical third of the cell. It co-localized with micronemes, the only secretory organelle the secretion of which is Ca2+ dependent. Quantification of the co-localized stain suggests that only mature micronemes ready for exocytosis have PRP1. These findings suggest that PRP1, parafusin and other members of the phosphoglucomutase superfamily have a conserved role in Ca2+-regulated exocytic processes.

Keywords: Exocytosis; PGM superfamily; PRP1; Toxoplasma gondii; 3-D deconvolution

Language: English

Document Type: Original article

DOI: 10.1078/0171-9335-00214

Affiliations: 1: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY/USA 2: Department of Medicine and Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY/USA

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