Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
Autophagy. 2013 May;9(5):770-7. doi: 10.4161/auto.23978. Epub 2013 Feb 27.
We characterized phosphoinositide binding of the S. cerevisiae PROPPIN Hsv2 qualitatively with density flotation assays and quantitatively through isothermal titration calorimetry (ITC) measurements using liposomes. We discuss the design of these experiments and show with liposome flotation assays that Hsv2 binds with high specificity to both PtdIns3P and PtdIns(3,5)P 2. We propose liposome flotation assays as a more accurate alternative to the commonly used PIP strips for the characterization of phosphoinositide-binding specificities of proteins. We further quantitatively characterized PtdIns3P binding of Hsv2 with ITC measurements and determined a dissociation constant of 0.67 µM and a stoichiometry of 2:1 for PtdIns3P binding to Hsv2. PtdIns3P is crucial for the biogenesis of autophagosomes and their precursors. Besides the PROPPINs there are other PtdIns3P binding proteins with a link to autophagy, which includes the FYVE-domain containing proteins ZFYVE1/DFCP1 and WDFY3/ALFY and the PX-domain containing proteins Atg20 and Snx4/Atg24. The methods described could be useful tools for the characterization of these and other phosphoinositide-binding proteins.
我们使用密度浮选分析定性地研究了酿酒酵母 PROPPIN Hsv2 与磷脂酰肌醇结合的特性，并用脂质体进行了等温滴定微量热法（ITC）测量来定量研究。我们讨论了这些实验的设计，并通过脂质体浮选分析表明，Hsv2 特异性地结合 PtdIns3P 和 PtdIns(3,5)P2。我们提出脂质体浮选分析作为一种更准确的方法来替代常用的 PIP 条带，用于研究蛋白质与磷脂酰肌醇结合特异性。我们进一步通过 ITC 测量定量研究了 Hsv2 与 PtdIns3P 的结合，并确定了 Hsv2 与 PtdIns3P 结合的解离常数为 0.67µM，结合比为 2:1。PtdIns3P 对于自噬体及其前体的生物发生至关重要。除了 PROPPINs 之外，还有其他与自噬相关的 PtdIns3P 结合蛋白，包括含有 FYVE 结构域的蛋白 ZFYVE1/DFCP1 和 WDFY3/ALFY，以及含有 PX 结构域的蛋白 Atg20 和 Snx4/Atg24。所描述的方法可以成为研究这些和其他磷脂结合蛋白的有用工具。
