The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells

Phosphoinositide (PtdlnsP) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdlnsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdlnsP species are useful as probes to visualize the distribution and dynamics of that PtdlnsP. The endolysosomal system is governed by two primary PtdlnsPs: phosphatidylinositol 3-phosphate [Ptdlns(3)P] and phosphatidylinositol 3,5-bisphosphate [Ptdlns(3,5)P-2], which are thought to localize and control early endosomes and lysosomes/late endosomes, respectively. While Ptdlns(3)P has been analysed with mammalian-derived PX and FYVE domains, Ptdlns(3,5)P-2 indicators remain controversial. Thus, complementary probes against these PtdlnsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind Ptdlns(3,5)P-2 in vitro. However, we show that TgPH1 retains membrane-binding in PlKfyve-inhibited cells, suggesting that TgPH1 is not a viable Ptdlns(3,5)P-2 marker in mammalian cells. Instead, Ptdlns (3)P depletion using pharmacological and enzyme-based assays dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized with Rab5-positive early endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and FYVE domain of EEA1, which are commonly used as Ptdlns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for Ptdlns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.