Topological regulation of transmembrane proteins through Regulated Alternative Translocation

总结 跨膜蛋白必须采用适当的膜拓扑结构才能发挥其功能。在 在哺乳动物细胞中，跨膜蛋白的拓扑结构是由通过 其中跨膜螺旋在ER相关蛋白上的翻译期间插入ER中， 核糖体已经假定蛋白质跨内质网膜的转运是一种免疫反应。 因此，跨膜蛋白必须采用固定的拓扑结构。这种假设 我们最近观察到，跨膜细胞的方向 插入ER的螺旋在某些生理条件下可以逆转。我们报道 神经酰胺逆转了两个多位跨膜蛋白的拓扑结构，即TM4SF20和 CCR 5。由于这种调节机制不翻转已经发生作用的跨膜蛋白， 但通过改变方向来反转新合成蛋白质的拓扑结构 通过它跨膜螺旋可以跨膜转位，我们将其命名为 调节性替代易位（Regulated Alternative Translocation，RAT） 本项目的启动是为了进一步描述RAT的机制， 拓扑调节我们将开始通过测试TRAM 2是神经酰胺传感器的假设 与经受RAT的新生跨膜螺旋相互作用。制定的方法 这项研究可以概括为确定神经酰胺相互作用组，这一发现可能揭示更多 由鞘脂介导的信号传导反应。这些方法也可以无偏见地应用 鉴定与经受RAT的新生跨膜螺旋相互作用的蛋白质，从而 为这种新的易位调节提供了更多的机制性见解。 该项目还将通过一种新的蛋白质组方法鉴定受RAT影响的蛋白质 能够全面测量跨膜蛋白的拓扑结构。实现项目的这一部分 将不仅揭示RAT的广度，而且为蛋白质组学提供必要的实验数据， 跨膜蛋白拓扑结构的广泛组装。考虑到只有10%的哺乳动物 跨膜蛋白具有由实验证据定义的拓扑结构， 该项目将大大提高我们对跨膜蛋白的理解。

Summary Transmembrane proteins must adopt proper membrane topology to perform their function. In mammalian cells, the topology of transmembrane proteins is determined by the direction through which transmembrane helices are inserted into ER during their translation on ER-associated ribosomes. It has been assumed that protein translocation across the ER membranes is a constitutive process so that transmembrane proteins must adopt a fixed topology. This assumption has been challenged by our recent observation that the direction through which transmembrane helices are inserted into the ER can be reversed under certain physiological conditions. We reported that ceramide inverted the topology of two polytopic transmembrane proteins, namely TM4SF20 and CCR5. Since this regulatory mechanism does not flip transmembrane proteins that have already been synthesized but inverts the topology of newly synthesized proteins by changing the direction through which transmembrane helices are translocated across membranes, we designated this process as Regulated Alternative Translocation (RAT). This project is initiated to further characterize RAT by delineating the mechanism of this topological regulation. We will begin by testing the hypothesis that TRAM2 is the ceramide sensor that interacts with the nascent transmembrane helices subjected to RAT. The approaches developed for this study can be generalized to identify ceramide interactome, a finding that might reveal more signaling reactions mediated by the sphingolipid. These approaches may also be applied unbiasedly to identify proteins interacting with the nascent transmembrane helices subjected to RAT, thereby providing more mechanistic insights into this novel translocation regulation. This project will also identify proteins subject to RAT by a novel proteome-wide approach capable of measuring topology of transmembrane proteins globally. Achieving this part of the project will not only reveal the breadth of RAT but also provide essential experimental data for proteome- wide assembly of topology of transmembrane proteins. Considering that only 10% of mammalian transmembrane proteins have their topology defined by experimental evidence, accomplishing this project should greatly improve our understanding of transmembrane proteins.