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区室之间运输或运输至质膜的主要机制，并且其受到严格调控以确保货物在正确的时间递送至正确的目的地。我们的努力是针对理解这种调节的分子基础，我们建议调查的情况下，空间和时间的调节，专注于事件的囊泡到达受体室。我们采用结构生物学、生物化学和生物物理学方法。在目的1中，我们研究鸟嘌呤核苷酸交换因子（GEFs）如何识别和激活小GTP酶的Rab家族成员，这在确定细胞器身份，从而确保正确的货物交付中发挥了关键作用。我们专注于DENN结构域蛋白，在高等真核生物的GEF的一个主要家庭，如何与他们的拉布合作伙伴。DENN-结构域/Rab复合物的晶体结构测定正在进行中，连同动力学和热力学研究，将阐明识别和激活机制。在目的2中，我们研究了TRAPP家族中的复合物，在受体室的囊泡识别中起作用的拴系因子，如何定位于不同的室，因为不同的亚基被添加到共享的核心。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以及关键的亚复合物已被重建。低分辨率的电子显微镜重建已获得完整的复杂和初始结晶条件确定的subcomplex。最后，在目标3中，我们研究了神经细胞中的专门蛋白质如何调节SNARE复合物的组装，SNARE复合物驱动囊泡融合和货物递送，使得神经递质仅在响应动作电位时释放。我们将确定突触蛋白复合蛋白和突触结合蛋白如何调节SNARE在质膜上的组装。我们已经确定了一个晶体结构的复合物绑定到一个模拟的融合前陷阱复合物，解释了陷阱组件可以被“钳”，等待动作电位，并提出进一步的研究，旨在了解钳和钳释放。

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.