Protein Trafficking In The Endosomal-Lysosomal System

我们研究了跨膜蛋白被分选到细胞内隔间的分子机制，如内小体、溶酶体和一组称为溶酶体相关细胞器的特定细胞类型的细胞器(如黑素小体和血小板致密小体)。对这些隔室的分选是通过识别跨膜蛋白胞浆结构域中存在的信号来实现的，适配器蛋白是膜涂层的组成部分。在这些接头蛋白中，有异四聚体AP-1、AP-2、AP-3和AP-4复合体，单体GGA1、GGA2和GGA3蛋白(GGAs)，以及异五聚体逆转录复合体。正确的分选还需要运输机制中其他调节囊泡拴系和融合的组件的功能，如异四聚体GARP复合体。本实验室目前的工作旨在阐明外壳蛋白和囊泡拴系因子的结构、调节和生理作用，并研究由于这些蛋白的遗传缺陷(Hermansky-Pudlak综合征)或病原体(HIV-1)利用而导致的人类疾病。 AP-4复合体是AP复合体家族中最新发现也是最不为人所知的。在最近的工作中，我们发现AP-4与阿尔茨海默病(AD)的胞浆尾巴淀粉样前体蛋白(APP)有关。AP-4-APP相互作用的中断将APP的分布从内小体转移到跨高尔基网络(TGN)，并增强了APP在伽马分泌酶催化下对致病淀粉样β蛋白的加工。这些发现将AP-4确立为APP处理和交易的新调节因子，并成为AD的潜在风险因素。 逆转录复合体是一种分选装置，它介导从内体到TGN的逆行运输。这一功能在高等真核生物的许多重要生理过程中是必不可少的，包括溶酶体酶的分选、APP的加工以及发育过程中形态原梯度的形成。该反转录聚体包括由两个分类连接蛋白(SNX)亚基组成的膜结合亚复合体和由Vps26、Vps29和VPS35组成的货物识别亚复合体。在以前的研究中，我们发现了逆转聚体复合体在将甘露糖6-磷酸受体从内体恢复到TGN中的作用，并表明这种恢复对于酸性水解酶对溶酶体的分选是必不可少的。与James Hurley(NIDDK)合作，我们还解决了Vps26-Vps29-VPS35络合物的晶体结构。 在过去的一年里，我们发现Vps26-Vps29-VPS35亚复合体的膜募集除了SNX亚复合体之外，还包括小的GTP结合蛋白Rab7。SNX亚复合体或Rab7的扰动导致Vps26-Vps29-VPS35亚复合体从膜上解离。反过来，这会导致酸性水解酶的错误分选，从而在溶酶体中积累未降解的物质，这是溶酶体储存障碍的一种表型特征。 通过逆转录作用形成的内体运输载体必须停靠在TGN并与TGN融合才能运送它们的货物。我们之前证明了GARP，一种最初在酵母中描述的多蛋白质复合体，在哺乳动物细胞中发挥着这样的作用。对GARP的干扰阻断了甘露糖6-磷酸受体和志贺毒素等货物从内体到TGN的运输，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的动物模型Wobbler小鼠突变体中发现了GARP亚基之一Vps54的突变。我们发现，Wobbler突变并没有阻止GARP在逆行运输中的功能，这表明该病可能是由于运输中的细微缺陷或GARP的其他一些功能所致。 对GARP功能的分子机制的进一步研究表明，该复合体在逆行转运过程中起着两个不同的作用：(1)囊泡转运中间产物与TGN的连接；(2)与TGN上的SNARE蛋白Synaxin 6、Synaxin 16和VAMP4相互作用，促进它们组装成SNARE复合体。因此，GARP通过参与两个连续的、独立的步骤来协调逆行运输中间体的捆绑和融合。

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.