Topological regulation of transmembrane proteins through Regulated Alternative Translocation

摘要 跨膜蛋白必须采用适当的膜拓扑结构才能发挥其功能。在……里面 在哺乳动物细胞中，跨膜蛋白的拓扑结构是由 在内质网相关的翻译过程中，哪些跨膜螺旋被插入内质网 核糖体。已有研究认为，蛋白质跨内质膜的转运是一种 构成过程使得跨膜蛋白必须采用固定的拓扑结构。这一假设 受到了我们最近观察到的跨膜方向的挑战 螺旋插入内质网在某些生理条件下是可以逆转的。我们报道了 神经酰胺逆转了两种多位跨膜蛋白的拓扑结构，即TM4SF20和TM4SF20 CCR5.因为这种调节机制不会翻转已经具有 被合成，但通过改变方向来逆转新合成的蛋白质的拓扑 跨膜螺旋通过它跨膜转移，我们将其命名为 按照受监管的替代易位(RAT)进行处理。 该项目的发起是为了通过描绘这一机制来进一步表征RAT 拓扑调节。我们将首先检验TRAM2是神经酰胺传感器的假设 它与新生的跨膜螺旋相互作用，受到老鼠的影响。已开发的方法 因为这项研究可以推广到鉴定神经酰胺相互作用体，这一发现可能揭示更多 神经鞘磷脂介导的信号反应。这些方法也可以不带偏见地应用。 鉴定与大鼠新生的跨膜螺旋相互作用的蛋白质，从而 为这一新的易位调控提供了更多机械性的见解。 该项目还将通过一种新的蛋白质组范围的方法来识别受老鼠影响的蛋白质。 能够在全球范围内测量跨膜蛋白的拓扑结构。完成项目的这一部分 不仅揭示了大鼠的广度，而且为蛋白质组学研究提供了必要的实验数据。 跨膜蛋白拓扑的广泛组装。考虑到只有10%的哺乳动物 跨膜蛋白的拓扑结构由实验证据确定，从而实现了这一点。 该项目将极大地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.