Abstract
Phosphoinositides (PtdInsPs) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP 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 PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol-3-phosphate [PtdIns(3)P] and phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs 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 PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological treatments dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized to EEA1-positive endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and tandem FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.
Highlights
Phosphoinositides (PtdInsPs) are signaling lipids that modulate many important cellular functions including signal transduction, cell proliferation, cell migration, and membrane trafficking
T. gondii PH-domain containing protein-1 (TgPH1) binds to intracellular membranes independently of PtdIns(3,5)P2 in mammalian cells
We generated various mammalian expression vectors encoding fluorescent chimeras of single TgPH1 domain (GFP-TgPH1, GFPNES-TgPH1, iRFPNES-TgPH1) and tandem TgPH1 domains (GFP-2x-TgPH1); the GFPNES and iRFPNES were engineered to include a nuclear export sequence (NES) to reduce the tendency of some fluorescent proteins to accumulate in the nucleus
Summary
Phosphoinositides (PtdInsPs) are signaling lipids that modulate many important cellular functions including signal transduction, cell proliferation, cell migration, and membrane trafficking. The spatial restriction of PtdInsPs coupled to the recruitment of species-specific PtdInsP effector proteins endows the host membrane or organelle with their attributable functions and identity [1–4]. Given these broad roles, dysregulation of PtdInsPs can drive many diseases such as cancer, diabetes, obesity and rare genetic disorders [3,5–7]. The subcellular distribution of PtdInsPs is partly informed through effector proteins and their inositol-lipid binding domains [4,8,9]. There is a large array of widely employed PtdInsP reporters based on this strategy [8,9,18] These protein domain-based probes are not without caveats since they may interact with additional endogenous ligands, preferentially detecting PtdInsP pools that co-exist with the additional ligand [19–25]. There is a need for additional complementary PtdInsP reporters
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