Molecular cloning, functional complementation in Saccharomyces cerevisiae and enzymatic properties of phosphatidylinositol synthase from the protozoan parasite Toxoplasma gondii.
The obligate intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis, switches between the rapidly dividing tachyzoite and the slowly replicating bradyzoite in intermediate hosts such as humans and domestic animals. We have recently identified a bradyzoite cDNA encoding a putative phosphatidylinositol (PtdIns) synthase using a subtractive library [Yahiaoui, B., Dzierszinski, F., Bernigaud, A., Slomianny, C., Camus, D., and Tomavo, S. (1999) Mol. Biochem. Parasitol. 99, 223-235]. Here, we report the cloning of another cDNA encoding PtdIns synthase that is exclusively expressed in the tachyzoite stage. The two transcripts are encoded by two different genes, which are stage-specifically regulated. The deduced amino-acid sequence (258 amino acids with a calculated total molecular mass of 27.8 kDa) of the tachyzoite-specific cDNA shares a significant degree of identity (between 26.5 and 30.1%) to the PtdIns synthases from human, rat, Arabidopsis thaliana and yeast. Interestingly, the putative protein encompasses an N-terminal extension that is approximately 40 amino-acids longer than that of PtdIns synthases from other organisms. Functional complementation realized by tetrad analysis of segregants of a Saccharomyces cerevisiae PtdIns synthase-deficient mutant (PIS1/pis1:kanMX4) showed that only the T. gondii putative PtdIns synthase truncated at its N-terminal extension is able to restore the viability of the cells. We demonstrate that this protein expressed in yeast transformants is functionally active in the membrane preparation and requires manganese and magnesium ions for activity. To our knowledge, this is the first report on the molecular cloning and functional analysis of a gene encoding a PtdIns synthase in protozoan parasites.
Séron
K
K
Laboratoire de Chimie Biologique, UMR 8576 CNRS-Université des Sciences et Technologies de Lille France.
Dzierszinski
F
F
Tomavo
S
S
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D001483:Base Sequence; D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology; D003001:Cloning, Molecular; D015723:Gene Library; D005816:Genetic Complementation Test; D006801:Humans; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D017855:Transferases (Other Substituted Phosphate Groups); D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase