D-3 phosphoinositides of the ciliate Tetrahymena: Characterization and study of their regulatory role in lysosomal enzyme secretion

Abstract

Phosphatidylinositol 3-phosphate, PtdIns3P, is a phosphoinositide which is implicated in regulating membrane trafficking in both mammalian and yeast cells. It also serves as a precursor for the synthesis of phosphatidylinositol 3,5-bisphosphate, PtdIns3,5P2, a phosphoinositide, the exact functions of which remain unknown. In this report, we show that these two phosphoinositides are constitutive lipid components of the ciliate Tetrahymena. Using HPLC analysis, PtdIns3P and PtdIns3,5P2 were found to comprise 16% and 30-40% of their relevant phosphoinositide pools, respectively. Treatment of Tetrahymena cells with wortmannin (0.1-10 microM) resulted in the depletion of PtdIns3P and PtdIns3,5P2 without any effect on D-4 phosphoinositides. Wortmannin was further used for the investigation of D-3 phosphoinositide involvement in the regulation of lysosomal vesicular trafficking. Incubation of Tetrahymena cells with wortmannin resulted in enhanced secretion of two different lysosomal enzymes without any change in their total activities. Experiments performed with a T. thermophila secretion mutant strain verified that the wortmannin-induced secretion is specific and it is not due to a diversion of lysosomal enzymes to other secretory pathways. Moreover, experiments performed with a phagocytosis-deficient T. thermophila strain showed that a substantial fraction of wortmannin-induced secretion was dependent on the presence of functional phagosomes/phagolysosomes.

Biochemical and Genetic Evidence for the Presence of Multiple Phosphatidylinositol- and Phosphatidylinositol 4,5-Bisphosphate-Specific Phospholipases C in Tetrahymena

“…4B). HPLC analysis of deacylated phosphoinositides according to the method of Leondaritis et al (29) verified that this increase was primarily due to the presence of PtdIns(4,5)P 2 and not to that of other PtdInsP 2 isomers (data not shown). Importantly, under conditions of accelerated PtdIns(4,5)P 2 synthesis, i.e., treatment of starved T. thermophila cells with insulin (T. Prevedoros and D. Galanopoulou, unpublished data), pretreatment with U73122 resulted in even more pronounced increases in PtdIns(4,5)P 2 levels (approximately 5-fold compared to nontreated control samples; Fig.…”

“…Aliquots of the cell suspension were treated with U73122 in the presence or absence of insulin (2.5 M) for a total of 10 to 15 min. Incubations were terminated by the addition of ice-cold perchloric acid, and phosphoinositides were extracted and quantified by TLC or high-performance liquid chromatography (HPLC) as described previously (9,29).…”

Conservation and Innovation in Tetrahymena Membrane Traffic: Proteins, Lipids, and Compartments

“…Work by Galanopoulou and colleagues on phosphoinositides in Tetrahymena pyriformis and thermophila identified PtdIns(3)P, PtdIns(4)P, PtdIns(3,5)P2 and PtdIns(4,5)P2, but not PtdIns(5)P, PtdIns(3,4)P2 or PtdIns(3,4,5)P3(Deli et al, 2008; Leondaritis et al, 2005). One major source of interest in phosphoinositides in mammalian cells comes from their role as compartment-specific determinants, based on the ability of individual phosphoinositide species to recruit proteins with the corresponding phosphoinositide-binding domains.…”

Role of class III phosphatidylinositol 3-kinase during programmed nuclear death ofTetrahymena thermophila

“…- 33 These reports suggest that PtdIns3K activity is important for regulation of proper phagocytotic activity and vesicular trafficking in the asexual stage. Yakisich and Kapler 31 suggest that during conjugation, wortmannin treatment blocks the final resorption of several types of nuclei such as pronuclei and the parental macronucleus, resulting in the accumulation of extra nuclei in the progeny cell.…”