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

Phosphatidylinositol 3-phosphate, PtdIns(3)P, 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, PtdIns(3,5)P2, 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, PtdIns(3)P and PtdIns(3,5)P2 were found to comprise 16% and 30-40% of their relevant phosphoinositide pools, respectively. Treatment of Tetrahymena cells with wortmannin (0.1-10 μM) resulted in the depletion of PtdIns(3)P and PtdIns(3,5)P2 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.