Protein Trafficking In The Endosomal-Lysosomal System

我们研究跨膜蛋白被分类到细胞内区室的分子机制，例如内体、溶酶体和一组称为溶酶体相关细胞器（例如黑素体和血小板致密体）的细胞类型特异性细胞器。对这些区室的分选是通过作为膜涂层成分的衔接蛋白识别跨膜蛋白的胞质结构域中存在的信号来介导的。这些接头蛋白包括异四聚体 AP-1、AP-2、AP-3 和 AP-4 复合体、单体 GGA1、GGA2 和 GGA3 蛋白 (GGA) 以及异五聚体逆转录体复合体。正确的分选还需要介导囊泡束缚和融合的运输机制的其他组件的功能，例如异四聚体 GARP 复合体。我们实验室目前的工作旨在阐明外壳蛋白和囊泡束缚因子的结构、调节和生理作用，并研究由遗传缺陷（赫曼斯基-普德拉克综合征）或病原体（HIV-1）利用这些蛋白质引起的人类疾病。 AP-4 复合物是 AP 复合物家族中最近发现且了解最少的。在最近的工作中，我们发现AP-4带有阿尔茨海默病（AD）淀粉样前体蛋白（APP）的胞质尾部。 AP-4-APP 相互作用的破坏将 APP 的分布从内体转移到反式高尔基体网络 (TGN)，并增强 APP 经 γ 分泌酶催化加工成致病性淀粉样β 肽。这些发现表明 AP-4 是 APP 加工和贩运的新型调节剂，也是 AD 的潜在危险因素。 逆转录复合体是一种分选装置，介导从内体到 TGN 的逆行转运。该功能对于高等真核生物中许多重要的生理过程至关重要，包括溶酶体酶分选、APP 加工以及发育过程中形态发生素梯度的形成。逆转录体包含由两个分选连接蛋白 (SNX) 亚基组成的膜结合亚复合物和由 Vps26、Vps29 和 Vps35 组成的货物识别亚复合物。在之前的研究中，我们发现了retromer复合物在将甘露糖6-磷酸受体从内体回收到TGN中的作用，并表明这种回收对于酸性水解酶分选到溶酶体是必需的。我们与 James Hurley (NIDDK) 合作，还解析了 Vps26-Vps29-Vps35 复合物的晶体结构。 在过去的一年里，我们发现除了 SNX 子复合物之外，Vps26-Vps29-Vps35 子复合物向膜的募集还涉及小的 GTP 结合蛋白 Rab7。 SNX 子复合物或 Rab7 的扰动会导致 Vps26-Vps29-Vps35 子复合物从膜上解离。反过来，这导致酸性水解酶的错误分选以及随后未降解物质在溶酶体中的积累，这是溶酶体贮积症的表型特征。 由逆转录酶作用形成的内体运输载体必须与 TGN 对接并融合才能运送货物。我们之前表明，GARP（一种最初在酵母中描述的多蛋白复合物）在哺乳动物细胞中发挥着这样的作用。干扰 GARP 会阻碍 6-磷酸甘露糖受体和志贺毒素等货物从内体到 TGN 的递送，表明 GARP 在逆行运输中具有普遍作用。最近在肌萎缩侧索硬化症动物模型 Wobbler 小鼠突变体中发现了 GARP 亚基之一 Vps54 的突变。我们发现，Wobbler 突变并不能阻止 GARP 在逆行运输中的功能，这表明该疾病可能是由于运输中的细微缺陷或 GARP 的某些其他功能所致。 对 GARP 功能分子机制的进一步研究表明，该复合物在逆行转运中发挥两个不同的作用：（1）将囊泡转运中间体与 TGN 束缚，以及（2）在 TGN 处与 SNARE 蛋白、突触蛋白 6、突触蛋白 16 和 Vamp4 相互作用，促进它们组装成 SNARE 复合物。因此，GARP 通过参与两个连续的独立步骤来协调逆行转运中间体的束缚和融合。

We investigate the molecular mechanisms by which transmembrane proteins are sorted to intracellular compartments such as endosomes, lysosomes and a group of cell-type-specific organelles known as lysosome-related organelles (e.g., melanosomes and platelet dense bodies). Sorting to these compartments is mediated by recognition of signals present in the cytosolic domains of the transmembrane proteins by adaptor proteins that are components of membrane coats. Among these adaptor proteins are the heterotetrameric AP-1, AP-2, AP-3 and AP-4 complexes, the monomeric GGA1, GGA2 and GGA3 proteins (GGAs), and the heteropentameric retromer complex. Proper sorting also requires the function of other components of the trafficking machinery that mediate vesicle tethering and fusion, such as the heterotetrameric GARP complex. Current work in our laboratory is aimed at elucidating the structure, regulation and physiological roles of coat proteins and vesicle tethering factors, and investigating human diseases that result from genetic defects (Hermansky-Pudlak syndrome) or pathogens' (HIV-1) exploitation of these proteins. The AP-4 complex is the most-recently discovered and least well-understood of the family of AP complexes. In recent work, we have found that AP-4 with the cytosolic tail of the Alzheimers Disease (AD) amyloid precursor protein (APP). Disruption of the AP-4-APP interaction shifts the distribution of APP from endosomes to the trans-Golgi network (TGN) and enhances gamma-secretase-catalyzed processing of APP to the pathogenic amyloid-beta peptide. These findings establish AP-4 as a novel regulator of APP processing and trafficking, and as a potential risk factor for AD. The retromer complex is a sorting device that mediates retrograde transport from endosomes to the TGN. This function is essential for many important physiological processes in higher eukaryotes, including lysosomal enzyme sorting, processing of APP, and formation of morphogen gradients during development. The retromer comprises a membrane-binding subcomplex made up of two sorting nexin (SNX) subunits and a cargo-recognition subcomplex composed of Vps26, Vps29 and Vps35. In previous studies, we discovered a role for the retromer complex in the retrieval of mannose 6-phosphate receptors from endosomes to the TGN and showed that this retrieval is essential for acid hydrolase sorting to lysosomes. In collaboration with James Hurley (NIDDK), we also solved the crystal structure of the Vps26-Vps29-Vps35 complex. Over the past year, we found that the recruitment of the Vps26-Vps29-Vps35 subcomplex to membranes involves, in addition to the SNX subcomplex, the small GTP-binding protein Rab7. Perturbation of either the SNX subcomplex or Rab7 results in dissociation of the Vps26-Vps29-Vps35 subcomplex from membranes. In turn, this leads to missorting of acid hydrolases and consequent accumulation of undegraded materials in lysosomes, a phenotype characteristic of lysosomal storage disorders. Endosomal transport carriers formed by the action of retromer must dock at and fuse with the TGN in order to deliver their cargo. We previously showed that GARP, a multi-protein complex originally described in yeast, plays such a role in mammalian cells. Interference with GARP blocks the delivery of cargos such as mannose 6-phosphate receptors and Shiga toxin from endosomes to the TGN, indicating that GARP has a general role in retrograde transport. A mutation in one of the GARP subunits, Vps54, was recently identified in the Wobbler mouse mutant, an animal model of amyotrophic lateral sclerosis. We have found that the Wobbler mutation does not prevent the function of GARP in retrograde transport, suggesting that the disease is likely due to a subtle defect in transport or to some other function of GARP. Further investigation of the molecular mechanism of GARP function showed that this complex plays two distinct roles in retrograde transport: (1) tethering of vesicular transport intermediates with the TGN, and (2) interaction with the SNARE proteins, Syntaxin 6, Syntaxin 16 and Vamp4 at the TGN, in a way that promotes their assembly into SNARE complexes. GARP thus orchestrates the tethering and fusion of retrograde transport intermediates by participating in two consecutive, independent steps.