Department of Biochemistry and Molecular Biology, The University of Chicago, 929 East 57th Street, Chicago, IL 60637, USA.
Department of Molecular and Cellular Biology, Northwest Labs, Harvard University, Cambridge, MA 02138, USA.
Cell Rep. 2017 Sep 5;20(10):2287-2293. doi: 10.1016/j.celrep.2017.08.035.
The Get1/2 transmembrane complex drives the insertion of tail-anchored (TA) proteins from the cytosolic chaperone Get3 into the endoplasmic reticulum membrane. Mechanistic insight into how Get1/2 coordinates this process is confounded by a lack of understanding of the basic architecture of the complex. Here, we define the oligomeric state of full-length Get1/2 in reconstituted lipid bilayers by combining single-molecule and bulk fluorescence measurements with quantitative in vitro insertion analysis. We show that a single Get1/2 heterodimer is sufficient for insertion and demonstrate that the conserved cytosolic regions of Get1 and Get2 bind asymmetrically to opposing subunits of the Get3 homodimer. Altogether, our results define a simplified model for how Get1/2 and Get3 coordinate TA protein insertion.
Get1/2 跨膜复合物驱动尾部锚定（TA）蛋白从细胞质伴侣 Get3 插入内质网膜。由于缺乏对复合物基本结构的理解，Get1/2 如何协调这一过程的机制洞察力受到阻碍。在这里，我们通过将单分子和体相荧光测量与定量体外插入分析相结合，在重建的脂质双层中定义全长 Get1/2 的寡聚状态。我们表明，单个 Get1/2 异二聚体足以进行插入，并证明 Get1 和 Get2 的保守细胞质区域以不对称的方式与 Get3 同源二聚体的相对亚基结合。总之，我们的结果定义了一个简化的模型，说明 Get1/2 和 Get3 如何协调 TA 蛋白插入。
