Regulatory Mechanisms in Membrane Trafficking

描述(申请人提供)：在真核细胞中，囊泡运输是物质在膜结合的隔室之间或向质膜运输的主要机制，它受到严格的监管，以确保货物在正确的时间被送到正确的目的地。我们的努力旨在了解这种调节的分子基础，我们建议研究空间和时间调节的实例，重点关注囊泡到达受体隔室时的事件。我们运用了结构生物学、生化和生物物理方法。在目标1中，我们研究了鸟嘌呤核苷酸交换因子(GEF)是如何识别和激活Rab家族小GTP酶的成员的，这些小GTP酶在确定细胞器的身份从而确保正确的货物递送方面发挥了关键作用。我们重点研究Denn结构域蛋白是高等真核生物中GEF的一个主要家族，如何与它们的RAB伙伴相互作用。Denn结构域/Rab络合物的晶体结构测定正在顺利进行，并将与动力学和热力学研究一起阐明识别和激活机制。在目标2中，我们研究了TRapp家族中的复合体是如何随着不同的亚基添加到共享的核心中而定位到不同的隔室的，TRapp家族是在受体区段起作用的拴系因子。这项工作的一个重要方面是确定TRAPPIII或TRAPPIII亚复合体的结构。完整的TRAPPIII以及关键的亚复合体已经被重组。获得了完整络合物的低分辨电子显微镜重建和亚络合物的初始结晶条件。最后，在目标3中，我们研究了神经细胞中的特殊蛋白质如何调节SNARE复合体的组装，SNARE复合体驱动囊泡融合和货物运输，从而使神经递质仅在动作电位的反应下被释放。我们将确定突触蛋白复合素和突触素是如何调节质膜上的SNAR组装的。我们已经确定了与融合前SNAR复合体的模拟物结合的络合物的晶体结构，该晶体结构解释了SNAR组装如何在等待动作电位的情况下被“钳制”，并提出了旨在理解钳制和钳制释放的进一步研究。

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.