Hughes William E, Larijani Banafshé, Parker Peter J
Protein Phosphorylation Laboratory, Cancer Research United Kingdom London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.
J Biol Chem. 2002 Jun 21;277(25):22974-9. doi: 10.1074/jbc.M201391200. Epub 2002 Apr 11.
Phospholipase D (PLD) proteins have been identified in secretory and endocytic vesicles, consistent with their proposed role in regulating membrane traffic. However, their sites of catalytic action remain obscure. We have developed here a novel, analytical approach to monitor PLD activation in intact cells employing lifetime imaging microscopy to measure fluorescence resonance energy transfer between protein and membrane phospholipid. Verification and application of this technique demonstrates a dispersed endosomal, epidermal growth factor-induced activation of the PLD1b isoform. Application of this approach will facilitate the spatial resolution of many protein-phospholipid interactions that are key events in the regulation of cellular processes.
磷脂酶D（PLD）蛋白已在分泌囊泡和内吞囊泡中被鉴定出来，这与其在调节膜运输中所起的作用相一致。然而，它们的催化作用位点仍不清楚。我们在此开发了一种新颖的分析方法，利用寿命成像显微镜来测量蛋白质与膜磷脂之间的荧光共振能量转移，以监测完整细胞中PLD的激活。该技术的验证和应用表明，表皮生长因子诱导的PLD1b亚型在内体中呈分散激活状态。这种方法的应用将有助于在空间上解析许多蛋白质 - 磷脂相互作用，而这些相互作用是细胞过程调控中的关键事件。
