YscE/YscG chaperone-dependent secretion of Yersinia pestis YscF

描述（由申请人提供）：鼠疫耶尔森菌，鼠疫病原体，使用III型分泌系统（T3SS）将效应蛋白注射到真核细胞中。耶尔森氏菌T3S装置的组装和功能依赖于21种必需的耶尔森氏菌分泌（Ysc）蛋白。在功能基础结构组装完成后，T3S装置仅分泌早期底物（YscF、YscI和YscP），这些底物的功能是组装棒状（YscI）和针状（YscF）结构；然而，人们对YscF和其他早期底物的独特特征知之甚少，这些特征使它们能够选择性识别和分泌。在本提案中，我们将描述识别YscF作为早期T3S底物的独特分子特征；以及识别这些特征的T3S设备的组件。重要的是，细胞质中的YscF只存在于与其异二聚体YscE/YscG伴侣的1:1复合物中，这种相互作用对于防止YscF的蛋白水解降解至关重要。我们还提供了实验证据，表明YscE/YscG伴侣对YscF的分泌至关重要，而不依赖于其稳定YscF的作用。首先，我们将研究YscF n端分泌信号在YscF分级分泌中的作用。此外，还将发现结合并稳定YscF，但不分泌YscF的YscE和YscG点突变体。总之，这些研究将定义YscF和YscE/YscG伴侣的独特分子特征，这些特征是识别YscEFG复合物和选择性分泌YscF所必需的。最后，我们将使用表征YscKYscQYscL分类平台和YscN atp酶在识别天然和突变的YscEFG复合物中的作用。这些研究将定义允许T3S装置区分早期和晚期T3S底物的识别事件，这是建立分泌层次结构所必需的过程。

DESCRIPTION (provided by applicant): Yersinia pestis, the etiologic agent of plague, uses a type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. The assembly and function of the Yersinia T3S apparatus is dependent upon 21 essential Yersinia secretion (Ysc) proteins. Following assembly of a functional base structure, the T3S apparatus secretes only early substrates (YscF, YscI and YscP) that function to assemble the rod (YscI) and needle (YscF) structures; however, little is known about the unique features of YscF and other early substrates that enable their selective recognition and secretion. In this proposal, we will characterize the unique molecular features that identify YscF as an early T3S substrate; as well as the components of the T3S apparatus that recognize these features. Importantly, cytoplasmic YscF is found exclusively in a 1:1:1 complex with its heterodimeric YscE/YscG chaperone, an interaction that is essential to prevent proteolytic degradation of YscF. We also provide experimental evidence that the YscE/YscG chaperone is essential for YscF secretion independent of its role in stabilizing YscF. Initially, we will investigate the role of the YscF N-terminal secretion signal in the hierarchical secretion of YscF. In addition, YscE and YscG point mutants that bind and stabilize YscF, but do not secrete YscF will be identified. Together, these studies will define the unique molecular features of YscF and the YscE/YscG chaperone that are required for recognition of the YscEFG complex and selective secretion of YscF. Finally, we will use characterize the role of a YscKYscQYscL sorting platform and YscN ATPase in the recognition of native and mutant YscEFG complexes. These studies will define the recognition events that allow the T3S apparatus to differentiate early and late T3S substrates, a process that is essential for establishing a secretion hierarchy.