Structural Mechanisms in Retrograde Protein Traffic to the Golgi

蛋白质逆向运输到高尔基体的结构机制 高尔基体是真核细胞的中央分选库。脂质、高尔基体驻留分选受体、SNARE和其他蛋白质的逆行运输是维持高尔基体稳态和实现循环分选途径所必需的。从酵母到人类，最具特征的逆行分选途径之一是通过逆转录酶复合物对酵母中的Vps 10和人类中的甘露糖6-磷酸受体进行分选。Retromer由货物识别复合物（Vps 26、Vps 29和Vps 35）和靶向和微管化膜的二聚体分选连接蛋白（SNX）复合物组成。逆转录聚合物是聚合物IG受体转胞吞作用和Wnt梯度形成等过程所必需的。尽管它在多种途径中的重要性，逆转录酶的分子功能仍然有些模糊。为了解决这一知识缺口，我们寻求1）通过X射线晶体学确定retromer亚基的结构; 2）使用晶体学、扫描诱变、流体力学、小角X射线散射、建模和电子显微镜确定亚基如何彼此相互作用以形成完整的复合物;以及3）使用结合和结构研究确定retromer如何与膜和货物相互作用。 逆转录酶复合物是将酸性水解酶分选到溶酶体、聚合物IG受体的转胞吞作用、Wnt梯度形成、铁转运蛋白再循环和淀粉样前体蛋白加工所必需的。人逆转录聚合物由两个较小的复合物组成，即货物识别Vps 26：Vps 29：Vps 35异源三聚体和SNX 1和/或SNX 2的膜靶向异源二聚体或同源二聚体。逆转录分子货物识别复合物由38-kDa Vps 26、20-kDa Vps 29和92-kDa Vps 35亚基组成。2006年在该项目下进行的研究表明，Vps 26是抑制蛋白的结构表亲，抑制蛋白是一种直接与细胞表面受体结合并指导其内化的运输蛋白家族。Juan Bonifacinos实验室与我们的团队合作，表明Vps 26通过C-末端叶中的保守环与Vps 35结合，并发现该环是Vps 26在体内正确定位所必需的。另外两个实验室发现vps 29有一个可以结合两个金属离子的金属磷酸酯酶折叠。与功能性金属磷酸酯酶相比，金属磷酸酯酶活性位点中作为催化碱基的关键His被Phe 63取代。因此，Vps 29相对于通用磷酸酶底物完全无活性。然而，金属依赖性活性在体外对磷酸化肽从一个主要的逆转录货物，阳离子非依赖性甘露糖6-磷酸受体（CI-MPR），已被报道。尽管逆转录酶对多种贩运途径至关重要，但其确切功能一直是个谜。各种提议强调了作为外壳、衔接子或货物蛋白磷酸酶的潜在作用。目前的研究正在采取结构和重建的方法来解决这个问题。

Structural Mechanisms in Retrograde Protein Traffic to the Golgi The Golgi apparatus is the eukaryotic cells central sorting depot. Retrograde traffic of lipids, Golgi-resident sorting receptors, SNAREs, and other proteins is required to maintain Golgi homeostasis and enable cyclical sorting pathways. One of the best characterized retrograde sorting pathways, conserved from yeast to humans, is the sorting of Vps10 in yeast and the mannose 6-phosphate receptors in humans by the retromer complex. Retromer consists of a cargo recognition complex (Vps26, Vps29, and Vps35), and a dimeric sorting nexin (SNX) complex that targets and tubulates membranes. Retromer is required for transcytosis of the polymeric Ig receptor and for Wnt gradient formation, among other processes. Despite its importance in multiple pathways, the molecular function of retromer remains somewhat obscure. To address this gap in knowledge, we seek to 1) determine the structures of retromer subunits by x-ray crystallography; 2) determine how the subunits interact with each other to form the intact complex using crystallography, scanning mutagenesis, hydrodynamics, small angle x-ray scattering, modeling, and electron microscopy; and 3) determine how retromer interacts with membranes and cargo using binding and structural studies. The retromer complex is required for the sorting of acid hydrolases to lysosomes, transcytosis of the polymeric Ig receptor, Wnt gradient formation, iron transporter recycling, and processing of the amyloid precursor protein. Human retromer consists of two smaller complexes, the cargo recognition Vps26:Vps29:Vps35 heterotrimer, and a membrane-targeting heterodimer or homodimer of SNX1 and/or SNX2. The retromer cargo recognition complex consists of the 38-kDa Vps26, 20-kDa Vps29, and 92-kDa Vps35 subunits. Studies under this project in 2006 showed that Vps26 is a structural cousin of the arrestins, a family of trafficking proteins that directly bind to cell surface receptors and direct their internalization. Juan Bonifacinos laboratory, working in collaboration with our group, showed that Vps26 binds to Vps35 through a conserved loop in the C-terminal lobe, and found that this loop is required for the correct localization of Vps26 in vivo. Vps29 was shown by two other labs to have a metallophosphoesterase fold that can bind two metal ions. Compared to functional metallophosphoesterases, a key His that serves as a catalytic base in the metallophosphoesterase active site is replaced by Phe 63. Thus Vps29 is completely inactive with respect to generic phosphatase substrates. However, metal-dependent activity in vitro against a phosphorylated peptide from a major retromer cargo, the cation-independent mannose 6-phosphate receptor (CI-MPR), has been reported. Despite its centrality to multiple trafficking pathways, the precise function of retromer has been enigmatic. Various proposals have emphasized potential roles as a coat, adaptor, or cargo protein phosphatase. Current studies are taking a structural and reconstituion approach to resolving this question.