Novel Pathways of Membrane Protein Insertion

最近，我们发现了一个细胞溶质TMD识别复合物（TRC），选择性地与TA蛋白的ER膜的目的相互作用。TRC的一个中心组分被鉴定为高度保守的40 kD ATP酶，并且代表了这种广泛使用的膜蛋白插入途径中的第一个分子因子。与TRC 40合作介导TA蛋白选择性识别、靶向和插入ER的其他成分尚不清楚。我们现在正在采取各种方法来鉴定这些额外的因子，并用确定的组分重建TA蛋白的靶向反应。实现这些目标将定义基本蛋白质运输途径的核心机制和功能，并为未来的机制和结构分析铺平道路。过去一年，我们在两个方面朝着这些目标取得了重大进展。首先，与Robert Keenan合作，解决了TRC40同系物在多种构象中的高分辨率晶体结构。当与生物化学分析相结合时，这项工作有助于提供一种机制，了解TRC40如何识别底物，以及它如何以ATP依赖的方式结合和释放底物。其次，我们确定了这一途径中的其他因素。最值得注意的是，我们确定了一个保守的伴侣复合物，似乎有利于底物捕获TRC40。这种伴侣复合物是新颖的，可能具有更广泛的作用，目前正在研究。我们正在进行的努力旨在确定其他因素，以及了解目前已知因素的功能机制。

Recently, we discovered a cytosolic TMD recognition complex (TRC) that selectively interacts with TA proteins destined for the ER membrane. A central component of TRC was identified as a highly conserved 40 kD ATPase and represents the first molecular factor in this widely used membrane protein insertion pathway. Other components that collaborate with TRC40 to mediate selective recognition, targeting, and insertion of TA proteins into the ER are unknown. We are now taking various approaches to identify these additional factor(s) and reconstitute the targeting reaction for TA proteins with defined components. Achieving these goals will define the core machinery and functions for a fundamental protein trafficking pathway and pave the way for future mechanistic and structural analyses. In the past year, we have made substantial progress towards these goals on two fronts. First, in collaboration with Robert Keenan, the high resolution crystal structure of TRC40 homologs were solved in multiple conformations. When combined with biochemical analysis, this work helped provide a mechanistic understanding how how TRC40 recognizes substrates and how it might function to bind and release them in an ATP-dependent manner. Second, we identified additional factors in this pathway. Most notably, we identified a conserved chaperone complex that seems to facilitate substrate capture by TRC40. This chaperone complex is novel and may have broader roles that are currently being studied. Our ongoing efforts are aimed toward identifying additional factors, as well as understanding the mechanism of function of the currently known factors.