Institute for Research in Biomedicine-IRB, Barcelona, Spain.
PLoS One. 2013 May 1;8(5):e62195. doi: 10.1371/journal.pone.0062195. Print 2013.
Over the last decades there has been an explosion of new methodologies to study protein complexes. However, most of the approaches currently used are based on in vitro assays (e.g. nuclear magnetic resonance, X-ray, electron microscopy, isothermal titration calorimetry etc). The accurate measurement of parameters that define protein complexes in a physiological context has been largely limited due to technical constrains. Here, we present PICT (Protein interactions from Imaging of Complexes after Translocation), a new method that provides a simple fluorescence microscopy readout for the study of protein complexes in living cells. We take advantage of the inducible dimerization of FK506-binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain to translocate protein assemblies to membrane associated anchoring platforms in yeast. In this assay, GFP-tagged prey proteins interacting with the FRB-tagged bait will co-translocate to the FKBP-tagged anchor sites upon addition of rapamycin. The interactions are thus encoded into localization changes and can be detected by fluorescence live-cell imaging under different physiological conditions or upon perturbations. PICT can be automated for high-throughput studies and can be used to quantify dissociation rates of protein complexes in vivo. In this work we have used PICT to analyze protein-protein interactions from three biological pathways in the yeast Saccharomyces cerevisiae: Mitogen-activated protein kinase cascade (Ste5-Ste11-Ste50), exocytosis (exocyst complex) and endocytosis (Ede1-Syp1).
在过去的几十年中，出现了许多研究蛋白质复合物的新方法。然而，目前使用的大多数方法都是基于体外检测（例如核磁共振、X 射线、电子显微镜、等温滴定量热法等）。由于技术限制，在生理环境下准确测量定义蛋白质复合物的参数在很大程度上受到限制。在这里，我们提出了 PICT（复合物在易位后通过成像进行相互作用），这是一种新的方法，可通过荧光显微镜对活细胞中的蛋白质复合物进行研究。我们利用 FK506 结合蛋白（FKBP）和 FKBP-雷帕霉素结合（FRB）结构域的诱导二聚化，将蛋白质组装体易位到酵母中与膜相关的锚定平台上。在该测定中，与 FRB 标记的诱饵相互作用的 GFP 标记的猎物蛋白在加入雷帕霉素后将共同易位到 FKBP 标记的锚定部位。因此，相互作用被编码为定位变化，可以通过不同生理条件下或在受到干扰时的荧光活细胞成像进行检测。PICT 可以自动化进行高通量研究，并可用于量化体内蛋白质复合物的离解速率。在这项工作中，我们使用 PICT 分析了来自酵母 Saccharomyces cerevisiae 中三个生物学途径的蛋白质-蛋白质相互作用：有丝分裂原激活的蛋白激酶级联（Ste5-Ste11-Ste50）、胞吐作用（胞吐复合蛋白）和胞吞作用（Ede1-Syp1）。
