Regulatory Mechanisms in Membrane Trafficking

描述（由申请人提供）：在真核细胞中，囊泡运输是物质在膜结合隔室之间或向质膜运输的主要机制，并受到严格监管，以确保货物在正确的时间被运送到正确的目的地。我们的努力旨在理解这种调控的分子基础，我们建议研究空间和时间调控的实例，重点关注囊泡到达受体室时的事件。我们采用结构生物学、生物化学和生物物理学方法。在Aim 1中，我们研究了鸟嘌呤核苷酸交换因子（gef）如何识别和激活小gtpase的Rab家族成员，这些成员在定义细胞器身份并因此确保正确的货物递送中发挥关键作用。我们关注的是高等真核生物中主要的gef家族denn结构域蛋白是如何与其Rab伴侣相互作用的。denn结构域/Rab复合物的晶体结构测定正在顺利进行中，结合动力学和热力学研究，将阐明识别和激活机制。在Aim 2中，我们研究了TRAPP家族中的复合物（在受体区室中参与囊泡识别的栓系因子）是如何在不同的区室中定位的，因为不同的亚基被添加到共享核中。这项工作的一个重要方面是确定TRAPPIII或TRAPPIII亚复合物的结构。完整的TRAPPIII及其关键亚复合物已被重建。对完整的配合物进行了低分辨率的电子显微镜重建，并确定了亚配合物的初始结晶条件。最后，在Aim 3中，我们研究了神经细胞中的特殊蛋白质如何调节SNARE复合物的组装，从而驱动囊泡融合和货物递送，从而仅在响应动作电位时释放神经递质。我们将确定突触蛋白络合蛋白和synaptotagmin如何调节SNARE在质膜上的组装。我们已经确定了与预融合SNARE复合体的模拟物结合的络合蛋白的晶体结构，解释了SNARE组装如何被“夹住”，等待动作电位，并提出了进一步的研究，旨在理解夹住和释放夹住。

DESCRIPTION (provided by applicant): In eukaryotic cells, vesicle trafficking is the principle mechanism by which materials are transported between membrane bound compartments or to the plasma membrane, and it is tightly regulated to ensure that cargo is delivered to the correct destination at the correct time. Our effort is directed at understanding the molecular basis for this regulation, and we propose to investigate instances of both spatial and temporal regulation, focusing on events as the vesicle arrives at the acceptor compartment. We bring to bear structural biology, biochemical and biophysical approaches. In Aim 1 we investigate how guanine nucleotide exchange factors (GEFs) recognize and activate members of the Rab family of small GTPases, which play a key role in defining organelle identity and hence in ensuring correct cargo delivery. We focus on how the DENN-domain proteins, a major family of GEFs in higher eukaryotes, interact with their Rab partners. The crystal structure determination of a DENN-domain/Rab complex is well underway and, together with kinetic and thermodynamic studies, will elucidate recognition and activation mechanisms. In Aim 2, we study how complexes in the TRAPP family, tethering factors that act in vesicle recognition at the acceptor compartment, are localized to different compartments as different subunits are added to a shared core. An important aspect of this work is the determination of the structure for TRAPPIII or a TRAPPIII subcomplex. Intact TRAPPIII as well as key subcomplexes have been reconstituted. Low resolution electron microscopy reconstructions have been obtained for the intact complex and initial crystallization conditions identified for a subcomplex. Lastly, in Aim 3 we examine how specialized proteins in nerve cells regulate the assembly of SNARE complexes, which drive vesicle fusion and cargo delivery, so that neurotransmitter is released only in response to an action potential. We will determine how the synaptic proteins complexin and synaptotagmin regulate SNARE assembly at the plasma membrane. We have determined a crystal structure of complexin bound to a mimetic of a pre-fusion SNARE complex that explains how SNARE assembly can be "clamped", pending an action potential, and propose further studies aimed at understanding clamping and clamp release.