Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) is generated through phosphorylation of phosphatidylinositol 4-phosphate (PtdIns(4)P) by Mss4p, the only PtdIns phosphate 5-kinase in yeast cells. PtdIns(4,5)P₂ is involved in various kinds of yeast functions. PtdIns(4)P is not only the immediate precursor of PtdIns(4,5)P₂, but also an essential signaling molecule in the plasma membrane, Golgi, and endosomal system. To analyze the distribution of PtdIns(4,5)P₂ and PtdIns(4)P in the yeast plasma membrane at a nanoscale level, we employed a freeze–fracture electron microscopy (EM) method that physically immobilizes lipid molecules in situ. It has been reported that the plasma membrane of budding yeast can be divided into three distinct areas: furrowed, hexagonal, and undifferentiated flat. Previously, using the freeze–fracture EM method, we determined that PtdIns(4)P is localized in the undifferentiated flat area, avoiding the furrowed and hexagonal areas of the plasma membrane. In the present study, we found that PtdIns(4,5)P₂ was localized in the cytoplasmic leaflet of the plasma membrane, and concentrated in the furrowed area. There are three types of PtdIns 4-kinases which are encoded by stt4, pik1, and lsb6. The labeling density of PtdIns(4)P in the plasma membrane significantly decreased in both pik1^ts and stt4^ts mutants. However, the labeling densities of PtdIns(4,5)P₂ in the plasma membrane of both the pik1^ts and stt4^ts mutants were comparable to that of the wild type yeast. These results suggest that PtdIns(4)P produced by either Pik1p or Stt4p is immediately phosphorylated by Mss4p and converted to PtdIns(4,5)P₂ at the plasma membrane.

4,5-二磷酸磷脂酰肌醇 (PtdIns(4,5)P ₂ ) 是通过 Mss4p（酵母细胞中唯一的 PtdIns 磷酸 5-激酶）磷酸化 4-磷酸磷脂酰肌醇 (PtdIns(4)P) 产生的。PtdIns(4,5)P ₂参与各种酵母功能。PtdIns(4)P 不仅是 PtdIns(4,5)P ₂的直接前体，而且是质膜、高尔基体和内体系统中必不可少的信号分子。分析PtdIns(4,5)P _2的分布和 PtdIns(4)P 在酵母质膜中的纳米级水平，我们采用冷冻断裂电子显微镜 (EM) 方法将脂质分子物理固定在原位。据报道，芽殖酵母的质膜可分为三个不同的区域：皱褶的、六角形的和未分化的平面。以前，使用冷冻断裂 EM 方法，我们确定 PtdIns(4)P 位于未分化的平坦区域，避免了质膜的沟槽和六边形区域。在本研究中，我们发现PtdIns(4,5)P ₂定位于质膜的细胞质小叶中，并集中在皱褶区域。共有三种类型的 PtdIns 4-激酶，分别由stt4、pik1和lsb6。PtdIns(4)P 在质膜中的标记密度在pik1 ^ts和stt4 ^ts突变体中均显着降低。然而，PtdIns(4,5)P ₂在pik1 ^ts和stt4 ^ts突变体的质膜中的标记密度与野生型酵母相当。这些结果表明Pik1p 或Stt4p 产生的PtdIns(4)P 立即被Mss4p 磷酸化并在质膜上转化为PtdIns(4,5)P ₂。