Brach Thorsten, Soyk Sebastian, Müller Christopher, Hinz Giselbert, Hell Rüdiger, Brandizzi Federica, Meyer Andreas J
Heidelberg Institute for Plant Science, University of Heidelberg, Im Neuenheimer Feld 360, D-69120 Heidelberg, Germany.
Plant J. 2009 Feb;57(3):534-41. doi: 10.1111/j.1365-313X.2008.03704.x. Epub 2008 Oct 2.
We present a novel method to experimentally visualize in vivo the topology of transmembrane proteins residing in the endoplasmic reticulum (ER) membrane or passing through the secretory pathway on their way to their final destination. This approach, so-called redox-based topology analysis (ReTA), is based on fusion of transmembrane proteins with redox-sensitive GFP (roGFP) and ratiometric imaging. The ratio images provide direct information on the orientation of roGFP relative to the membrane as the roGFP fluorescence alters with changes in the glutathione redox potential across the ER membrane. As proof of concept, we produced binary read-outs using oxidized roGFP inside the ER lumen and reduced roGFP on the cytosolic side of the membrane for both N- and C-terminal fusions of single and multi-spanning membrane proteins. Further, successive deletion of hydrophobic domains from the C-terminus of the K/HDEL receptor ERD2 resulted in alternating localization of roGFP and a topology model for AtERD2 with six transmembrane domains.
我们提出了一种新方法，可在体内实验性地可视化内质网（ER）膜中跨膜蛋白的拓扑结构，或在其前往最终目的地的途中穿过分泌途径的跨膜蛋白的拓扑结构。这种方法，即所谓的基于氧化还原的拓扑分析（ReTA），是基于跨膜蛋白与氧化还原敏感型绿色荧光蛋白（roGFP）的融合以及比率成像。当roGFP荧光随内质网膜上谷胱甘肽氧化还原电位的变化而改变时，比率图像提供了关于roGFP相对于膜的方向的直接信息。作为概念验证，我们针对单跨膜和多跨膜蛋白的N端和C端融合，在内质网腔内部使用氧化型roGFP以及在膜的胞质侧使用还原型roGFP产生了二元读数。此外，从K/HDEL受体ERD2的C端连续缺失疏水结构域，导致roGFP的定位交替变化，并得到了具有六个跨膜结构域的AtERD2的拓扑模型。
