VTC Events Mediated by the Rab2 Complex

描述（由申请人提供）：我们的长期目标是了解从泡状管簇（VTCs）中分类和回收蛋白质的机制。VTCs作为运输综合体，将货物从急诊室运送到高尔基复合体。此外，这些结构是顺行和逆行途径分离的第一个位点，因此发挥了关键作用。小的GTPase Rab2通过促进逆行定向囊泡的形成，将回收货物带回内质网，在这一运输步骤中起着至关重要的作用。Rab2介导的囊泡形成需要非典型PKCi/X和GAPDH。在本应用中，我们建议从机制上描述Rab2，并确定Rab2在与VTCs相关的事件中的作用。具体目标是：1)鉴定与Rab2结合的VTC亚室的蛋白质组分；2)通过绘制Rab2复合物的相互结合域来表征其组分之间的相互作用；3)阐明Rab2依赖性逆行囊泡从VTC出芽的分子机制。Rab2、PKC和GAPDH表达的升高与多种癌症的高转移潜力相关，这一观察结果强调了研究Rab2在分泌中的功能的重要性。由于转移是细胞骨架重组和细胞运动增加的反映，Rab2及其效应物影响微管动力学值得注意。然而，Rab2、aPKCi/A和GAPDH在早期分泌途径中的确切功能以及这些蛋白如何促进疾病尚不清楚。本应用中提出的研究将开发一种新的范式，将Rab2-Apkc lA-GAPDH功能障碍与病理后果结合起来。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the mechanism of protein sorting and recycling from vesicular tubular clusters (VTCs). VTCs function as transport complexes delivering cargo from the ER to the Golgi complex. Moreover, these structures are the first site of segregation of the anterograde and retrograde pathways and thereby perform a critical function. The small GTPase Rab2 plays an essential role in this trafficking step by promoting formation of retrograde-directed vesicles that carry recycling cargo back to the ER. Rab2- mediated vesicle formation requires atypical PKCi/X and GAPDH. In this application we propose to characterize Rab2 mechanistically and to establish the role of Rab2 in events associated with VTCs. The Specific Aims are: 1) to identify the protein components of the VTC subcompartment that binds Rab2, 2) to characterize the interaction between components of the Rab2 complex by mapping their reciprocal binding domains, and 3) elucidate the molecular mechanism of Rab2-dependent-retrograde vesicle budding from VTCs. The importance of investigating Rab2 function in secretion is underscored by the observation that elevated Rab2, PKC, and GAPDH expression correlates with high metastatic potential in various cancers. Since metastasis is a reflection of cytoskeletal reorganization and increased cell motility, it is noteworthy that Rab2 and its effectors influence microtubule dynamics. However, the precise function of Rab2, aPKCi/A, and GAPDH in the early secretory pathway and how these proteins contribute to the disease are unknown. The studies proposed in this application will develop a new and novel paradigm that couples Rab2-Apkc lA-GAPDH dysfunction with pathological consequences.