Physics Department, University of Minnesota, Minneapolis, Minnesota, USA.
Biophys J. 2012 Oct 17;103(8):1657-65. doi: 10.1016/j.bpj.2012.09.015. Epub 2012 Oct 16.
Mammalian cells express two classes of phosphatidylinositol 4-kinase (PI4K), designated as Types II and III, that phosphorylate phosphatidylinositol to generate PI4P. A number of studies have indicated that these enzymes are important for Golgi trafficking and both early and late stages of endocytosis. In this study, we focus on PI4KIIβ, a protein that is evenly distributed between membrane and soluble fractions, and is believed to participate in stimulus-dependent phosphoinositide signaling. Using molecular brightness analysis, we found that EGFP-tagged PI4KIIβ exists as two distinct species in the cytoplasm: a soluble monomer and a high-order complex enriched with multiple copies of PI4KIIβ. This observation was confirmed by an autocorrelation analysis that identified two species with distinct mobilities. We further demonstrate that the high-order complex enriched with PI4KIIβ is sensitive to inhibition of palmitoylation, indicating that it is associated with membranes, very likely vesicles. Indeed, we show that the high-order PI4KIIβ complex is sensitive to expression of dynamin 2 (K44A), a dominant-negative inhibitor of endocytosis. Using dual-color heterospecies partition analysis, we directly detected that PI4KIIβ comoves with clathrin light chain on vesicles. This analysis allows us to isolate the comobile species in the presence of strong background contribution from the monomeric pool of PI4KIIβ. Our results strongly suggest that PI4KIIβ is involved in an early stage of endocytosis and is associated with clathrin-coated vesicles. Moreover, we establish molecular brightness as a powerful tool for characterizing cellular cytosolic vesicles that are otherwise difficult to characterize by other techniques.
哺乳动物细胞表达两类磷脂酰肌醇 4-激酶（PI4K），分别命名为 II 型和 III 型，它们将磷脂酰肌醇磷酸化为 PI4P。许多研究表明，这些酶对于高尔基体运输以及胞吞作用的早期和晚期阶段都很重要。在本研究中，我们专注于 PI4KIIβ，这是一种均匀分布于膜和可溶性部分的蛋白质，被认为参与刺激依赖性磷酸肌醇信号转导。使用分子亮度分析，我们发现 EGFP 标记的 PI4KIIβ 在细胞质中存在两种不同的形式：可溶性单体和富含多个 PI4KIIβ 拷贝的高级复合物。这一观察结果通过自相关分析得到了证实，该分析确定了两种具有不同迁移率的物种。我们进一步证明，富含 PI4KIIβ 的高级复合物对棕榈酰化抑制敏感，表明它与膜，很可能是囊泡相关。事实上，我们表明，富含 PI4KIIβ 的高级复合物对 dynamin 2（K44A）的表达敏感，dynamin 2 是胞吞作用的一种显性抑制物。通过双色异种分区分析，我们直接检测到 PI4KIIβ 与网格蛋白轻链在囊泡上共迁移。这种分析允许我们在存在来自 PI4KIIβ 单体池的强背景贡献的情况下分离共迁移物种。我们的结果强烈表明 PI4KIIβ 参与胞吞作用的早期阶段，并与网格蛋白包被的囊泡相关。此外，我们建立了分子亮度作为一种强大的工具，用于表征其他技术难以表征的细胞胞质囊泡。
