Structural Mechanisms in Retrograde Protein Traffic to the Golgi

蛋白质逆行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、高尔基体驻留的分选受体、圈套和其他蛋白质的逆行运输是维持高尔基体内环境平衡和实现循环分选途径所必需的。最具特征的逆行分选途径之一，从酵母到人类保守，是通过逆转录复合体对酵母中的Vps10和人类的甘露糖6-磷酸受体进行分选。逆转录聚体由一个货物识别复合体(Vps26、Vps29和VPS35)和一个靶向和管状膜的二聚体分类连接素(SNX)复合体组成。逆转聚体是多聚免疫球蛋白受体的细胞穿梭和Wnt梯度形成等过程所必需的。尽管逆转聚体在多种途径中具有重要作用，但其分子功能仍不十分清楚。为了解决这一知识空白，我们试图1)通过X射线结晶学确定逆转录聚体亚单位的结构；2)使用结晶学、扫描诱变、流体力学、小角X射线散射、建模和电子显微镜来确定逆转录聚体与膜和货物相互作用的方式；以及3)使用结合和结构研究来确定逆转录聚体如何与膜和货物相互作用。 反转录复合体是酸性水解酶分选到溶酶体、多聚体Ig受体的跨细胞作用、Wnt梯度形成、铁转运体循环和淀粉样前体蛋白加工所必需的。人类反转录聚体由两个较小的复合体组成，即货物识别Vps26：Vps29：VPS35杂三聚体，以及SNX1和/或SNX2的膜靶向异二聚体或同源二聚体。逆转录货物识别复合体由38 kDa Vps 26、20 kDa Vps 29和92 kDa VPS35亚基组成。2006年该项目的研究表明，Vps26是拦阻蛋白的结构近亲，拦阻蛋白是一类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，发现Vps26通过C-末端叶中的一个保守环与VPS35结合，并发现这个环是Vps26在体内正确定位所必需的。另外两个实验室证明，Vps29具有金属磷酸酯酶折叠，可以结合两个金属离子。与功能性金属磷酸酯酶相比，作为金属磷酸酯酶活性部位催化碱基的关键组氨酸被Phe 63取代。因此，Vps29对于普通的磷酸酶底物来说是完全无效的。然而，金属依赖的活性在体外对一个主要的逆转录物的磷酸化肽，阳离子非依赖性甘露糖6-磷酸受体(CI-MPR)，已有报道。尽管逆转录病毒在多条贩运途径中处于中心地位，但它的确切作用一直是个谜。各种提案都强调了作为外壳、接头或货物蛋白磷酸酶的潜在作用。目前的研究正在采取结构性的方法来解决这个问题。 在上一个资助期，我们测定了一个人VPS29-VPS35亚基的晶体结构，显示了金属磷酸酯酶折叠亚基VPS29是如何作为VPS35的羧基末端一半的支架的。VPS35形成一个马蹄形的右旋α螺旋螺线管，其凹面完全覆盖VPS29的金属结合部位，而凸面则露出一系列疏水的螺旋间沟槽。电子显微镜显示，完整的VPS26-VPS29-VPS35复合体为棒状柔性结构，长约21 nm。来自晶体结构、电子显微镜(与Alasdair Steven，NIAMS合作)、相互作用研究和生物信息学(与Andrey Kajava，CNRS合作)的混合结构模型表明，α-螺线管折叠延伸了VPS35的全长，而VPS26与VPS29结合在相反的一端。这种延伸的结构为SNX复合体和受体货物提供了多个结合部位，并似乎能够弯曲以符合弯曲的囊泡膜。 最近，实验室成员Adriana Rojas与Juan Bonifacino的实验室合作，证明了VPS26-VPS29-VPS35复合体直接并特异性地与GTP负载的小GTPase Rab7相互作用。

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 approach to resolving this question. In the previous funding period, we determined the crystal structure of a human VPS29-VPS35 subcomplex showing how the metallophosphoesterase-fold subunit VPS29 acts as a scaffold for the carboxy-terminal half of VPS35. VPS35 forms a horseshoe-shaped, right-handed, alpha-helical solenoid, the concave face of which completely covers the metal-binding site of VPS29, whereas the convex face exposes a series of hydrophobic interhelical grooves. Electron microscopy shows that the intact VPS26-VPS29-VPS35 complex is a stick-shaped, flexible structure, approximately 21 nm long. A hybrid structural model derived from crystal structures, electron microscopy (in collaboration with Alasdair Steven, NIAMS), interaction studies and bioinformatics (in collaboration with Andrey Kajava, CNRS) shows that the alpha-solenoid fold extends the full length of VPS35, and that VPS26 is bound at the opposite end from VPS29. This extended structure presents multiple binding sites for the SNX complex and receptor cargo, and appears capable of flexing to conform to curved vesicular membranes. Most recently, laboratory member Adriana Rojas collaborated with the laboratory of Juan Bonifacino to demonstrate that the VPS26-VPS29-VPS35 complex directly and specifically interacts with the GTP-loaded form of the small GTPase Rab7.