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Phosphatidylinositol phosphate binding domains exhibit complex dissociation properties at the inner leaflet of plasma membrane sheets
bioRxiv - Biophysics Pub Date : 2021-03-01 , DOI: 10.1101/2021.02.17.431012
Madeline R. Sponholtz , Eric N. Senning

The pleckstrin homology (PH) domain is a lipid targeting motif that binds with high specificity to phosphatidylinositol phosphate (PIP) lipids. Using TIRF microscopy, we followed the dissociation of GFP-tagged PH domains from the plasma membranes of rapidly unroofed cells and found that AKT-PH and PLCδ1-PH dissociation kinetics can be distinguished by their effective koff values determined from fitting fluorescence traces to a single exponential equation. Our measurements for the koff of AKT-PH-GFP and PLCδ1-PH-GFP were significantly different (p < 0.05) at 0.39 ± 0.05 s-1 and 0.56 ± 0.17 s-1, respectively. Furthermore, we identified substantial rebinding events in our measurements of PLCδ1-PH-GFP dissociation kinetics. By applying inositol triphosphate (IP3) to samples during the unroofing process, we measured a much faster koff of 1.54 ± 0.42 s-1 for PLCδ1-PH-GFP, indicating that rebinding events are significantly depressed through competitive action by IP3 for the same PH domain binding site as phosphatidylinositol (4,5)-bisphosphate (PIP2). We discuss the complex character of our PLCδ1-PH-GFP fluorescence decays in the context of membrane receptor and ligand theory to address the question of how free PIP2 levels modulate the interaction between membrane associated proteins and the plasma membrane.

中文翻译:

磷酸磷脂酰肌醇磷酸结合域在质膜片内小叶上表现出复杂的解离特性

pleckstrin同源性(PH)域是一种脂质靶向基序,与磷脂酰肌醇磷酸酯(PIP)脂质具有高特异性结合。使用TIRF显微镜,我们遵循GFP标记的PH结构域的解离从快速无顶的细胞的质膜,发现AKT-PH和PLCδ1-PH解离动力学可以通过它们的有效k来区分值从荧光迹线拟合为确定单指数方程式。我们对AKT-PH-GFP和PLCδ1-PH-GFP的k off的测量在0.39±0.05 s -1和0.56±0.17 s -1时有显着差异(p <0.05), 分别。此外,我们在PLCδ1-PH-GFP解离动力学的测量结果中发现了实质性的重结合事件。通过应用三磷酸肌醇(IP 3在去顶工序)到样品中,我们测量快得多ķ关闭的1.54±0.42小号-1为PLCδ1-PH-GFP,表明重新绑定事件通过竞争行为通过IP显著压下3对与磷脂酰肌醇(4,5)-双磷酸酯(PIP2)相同的PH域结合位点。在膜受体和配体理论的背景下,我们讨论了PLCδ1-PH-GFP荧光衰减的复杂特征,以解决游离PIP2水平如何调节膜相关蛋白与质膜之间相互作用的问题。
更新日期:2021-03-02
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