ABSTRACT Phosphatidylinositol-4-phosphate (PI4P) plays a crucial role in cellular functions, including protein trafficking, and is mainly located in the cytoplasmic surface of intracellular membranes, which include the trans-Golgi network (TGN) and the plasma membrane. However, many PI4P-binding domains of membrane-associated proteins are localized only to the TGN because of the requirement of a second binding protein such as ADP-ribosylation factor 1 (ARF1) in order to be stably localized to the specific membrane. In this study, we developed new probes that were capable of detecting PI4P at the plasma membrane using the known TGN-targeting PI4P-binding domains. The PI4P-specific binding pleckstrin homology (PH) domain of various proteins including CERT, OSBP, OSH1, and FAPP1 was combined with the N-terminal moderately hydrophobic domain of the short-form of Aplysia phosphodiesterase 4 (S(N30)), which aids in plasma membrane association but cannot alone facilitate this association. As a result, we found that the addition of S(N30) to the N-terminus of the GFP-fused PH domain of OSBP (S(N30)-GFP-OSBP-PH), OSH1 (S(N30)-GFP-OSH1-PH), or FAPP1 (S(N30)-GFP-FAPP1-PH) could induce plasma membrane localization, as well as retain TGN localization. The plasma membrane localization of S(N30)-GFP-FAPP1-PH is mediated by PI4P binding only, whereas those of S(N30)-GFP-OSBP-PH and S(N30)-GFP-OSH1-PH are mediated by either PI4P or PI(4,5)P2 binding. Taken together, we developed new probes that detect PI4P at the plasma membrane using a combination of a moderately hydrophobic domain with the known TGN-targeting PI4P-specific binding PH domain.

中文翻译：

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用于检测质膜中磷脂酰肌醇 4-磷酸的新型 GFP 融合蛋白探针

摘要 4-磷酸磷脂酰肌醇 (PI4P) 在细胞功能中起着至关重要的作用，包括蛋白质运输，主要位于细胞内膜的细胞质表面，包括反式高尔基网络 (TGN) 和质膜。然而，膜相关蛋白的许多 PI4P 结合域仅定位于 TGN，因为需要第二个结合蛋白，例如 ADP-核糖基化因子 1 (ARF1)，以便稳定定位于特定膜。在这项研究中，我们开发了能够使用已知的 TGN 靶向 PI4P 结合域检测质膜上的 PI4P 的新探针。包括 CERT、OSBP、OSH1、FAPP1 与海兔磷酸二酯酶 4 (S(N30)) 短形式的 N 端中等疏水结构域结合，有助于质膜结合，但不能单独促进这种结合。结果，我们发现将 S(N30) 添加到 OSBP (S(N30)-GFP-OSBP-PH)、OSH1 (S(N30)-GFP- OSH1-PH) 或 FAPP1 (S(N30)-GFP-FAPP1-PH) 可以诱导质膜定位，并保留 TGN 定位。S(N30)-GFP-FAPP1-PH 的质膜定位仅由 PI4P 结合介导，而 S(N30)-GFP-OSBP-PH 和 S(N30)-GFP-OSH1-PH 的质膜定位由任一介导PI4P 或 PI(4,5)P2 结合。综合起来，