Vitrac Heidi, MacLean David M, Karlstaedt Anja, Taegtmeyer Heinrich, Jayaraman Vasanthi, Bogdanov Mikhail, Dowhan William
From the Department of Biochemistry and Molecular Biology and Center for Membrane Biology, University of Texas McGovern Medical School, Houston, Texas 77030.
From the Department of Biochemistry and Molecular Biology and Center for Membrane Biology, University of Texas McGovern Medical School, Houston, Texas 77030.
J Biol Chem. 2017 Feb 3;292(5):1613-1624. doi: 10.1074/jbc.M116.765719. Epub 2016 Dec 14.
Membrane protein topology and folding are governed by structural principles and topogenic signals that are recognized and decoded by the protein insertion and translocation machineries at the time of initial membrane insertion and folding. We previously demonstrated that the lipid environment is also a determinant of initial protein topology, which is dynamically responsive to post-assembly changes in membrane lipid composition. However, the effect on protein topology of post-assembly phosphorylation of amino acids localized within initially cytoplasmically oriented extramembrane domains has never been investigated. Here, we show in a controlled in vitro system that phosphorylation of a membrane protein can trigger a change in topological arrangement. The rate of change occurred on a scale of seconds, comparable with the rates observed upon changes in the protein lipid environment. The rate and extent of topological rearrangement were dependent on the charges of extramembrane domains and the lipid bilayer surface. Using model membranes mimicking the lipid compositions of eukaryotic organelles, we determined that anionic lipids, cholesterol, sphingomyelin, and membrane fluidity play critical roles in these processes. Our results demonstrate how post-translational modifications may influence membrane protein topology in a lipid-dependent manner, both along the organelle trafficking pathway and at their final destination. The results provide further evidence that membrane protein topology is dynamic, integrating for the first time the effect of changes in lipid composition and regulators of cellular processes. The discovery of a new topology regulatory mechanism opens additional avenues for understanding unexplored structure-function relationships and the development of optimized topology prediction tools.
膜蛋白的拓扑结构和折叠由结构原理和拓扑信号所决定，这些信号在膜蛋白最初插入膜并折叠时被蛋白插入和转运机制识别并解码。我们之前证明脂质环境也是初始蛋白拓扑结构的一个决定因素，它对膜脂质组成的组装后变化具有动态响应。然而，位于最初面向细胞质的膜外结构域内的氨基酸在组装后磷酸化对蛋白拓扑结构的影响从未被研究过。在此，我们在一个可控的体外系统中表明，膜蛋白的磷酸化可以引发拓扑排列的变化。变化速率在数秒的时间尺度上发生，与在蛋白脂质环境变化时观察到的速率相当。拓扑重排的速率和程度取决于膜外结构域的电荷以及脂质双层表面。使用模拟真核细胞器脂质组成的模型膜，我们确定阴离子脂质、胆固醇、鞘磷脂和膜流动性在这些过程中起关键作用。我们的结果证明了翻译后修饰如何以脂质依赖的方式影响膜蛋白拓扑结构，这在细胞器运输途径及其最终目的地都是如此。这些结果进一步证明膜蛋白拓扑结构是动态的，首次整合了脂质组成变化和细胞过程调节因子的影响。一种新的拓扑调节机制的发现为理解未探索的结构 - 功能关系以及开发优化的拓扑预测工具开辟了新途径。
