Department of Chemistry, Ryerson University, Toronto, Ontario, Canada.
The Molecular Science Graduate Program, Ryerson University, Toronto, Ontario, Canada.
PLoS One. 2018 Jun 5;13(6):e0198454. doi: 10.1371/journal.pone.0198454. eCollection 2018.
Phosphoinositide (PtdInsP) 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/late endosomes, 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 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 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.
磷脂酰肌醇（PtdInsP）脂质将效应蛋白募集到膜上，以介导各种功能，包括信号转导和膜运输。每种 PtdInsP 通过特征性蛋白结构域（如 PH、FYVE 和 PX 结构域）与特定的效应蛋白结合。与单一 PtdInsP 物种具有高亲和力的结构域可用作探针，以可视化该 PtdInsP 的分布和动态。内体溶酶体系统由两种主要的 PtdInsP 控制：磷脂酰肌醇 3-磷酸[PtdIns(3)P]和磷脂酰肌醇 3,5-二磷酸[PtdIns(3,5)P2]，它们分别被认为定位于和控制早期内体和溶酶体/晚期内体。虽然已经使用哺乳动物衍生的 PX 和 FYVE 结构域分析了 PtdIns(3)P，但 PtdIns(3,5)P2 指示剂仍然存在争议。因此，需要针对这些 PtdInsP 的互补探针，包括来自非哺乳动物蛋白的探针。在这里，我们在哺乳动物细胞中表征了来自寄生虫刚地弓形虫（Toxoplasma gondii）的含 PH 结构域蛋白-1 的 PH 结构域（TgPH1）的动力学，该结构域先前在体外被证明与 PtdIns(3,5)P2 结合。然而，我们表明 TgPH1 在 PIKfyve 抑制的细胞中保留膜结合，表明 TgPH1 不是哺乳动物细胞中可行的 PtdIns(3,5)P2 标记物。相反，使用药理学和基于酶的测定法耗尽 PtdIns(3)P 会使 TgPH1 从膜上解离。事实上，TgPH1 与 Rab5 阳性早期内体共定位。此外，TgPH1 与 p40phox 的 PX 结构域和 EEA1 的 FYVE 结构域共定位且行为相似，它们通常被用作 PtdIns(3)P 指示剂。总之，TgPH1 提供了一种来自非哺乳动物蛋白的 PtdIns(3)P 的互补报告器，与常用的基于 PX 和 FYVE 结构域的探针不同。
