内体分拣转运复合体的组成及其功能研究

内体分拣转运复合体(endosomal sorting complex required for transport,ESCRT)是一种能够识别并分拣泛素化蛋白质货物的蛋白复合体,由四个亚复合体(ESCRT-0、ESCRT-Ⅰ、ESCRT-Ⅱ、ESCRT-Ⅲ)和一些辅助成分构成。研究表明,ESCRT途径能参与病毒的出芽过程、调控细胞自噬,并且与包括肿瘤和神经退行性疾病在内的重要疾病有关。因此,ESCRT复合体结构与功能研究对未来新型治疗药物的开发具有重要意义。该文综述了ESCRT的结构、各成员在多种生命活动中的功能、组装因子之间的互作关系以及ESCRT复合体的功能,旨在为日后深入研究ESCRT的作用机制开辟更科学的研究方向。     

病毒劫持ESCRT系统促进自身复制的研究进展

内吞体分选转运复合体（endosomal sorting complex required for transport，ESCRT）系统驱动细胞的不同生命进程，包括内体分选、细胞器生物发生、囊泡运输、维持质膜完整性、细胞质分裂期间的膜裂变、有丝分裂后的核膜重组、自噬过程中吞噬孔的封闭以及包膜病毒出芽等。越来越多的证据表明，ESCRT系统能够被不同家族病毒劫持用于自身增殖。在病毒生命周期的不同阶段，病毒可以通过各种方式干扰或利用ESCRT系统介导的生理过程，最大限度地提高感染宿主的机会。此外，许多逆转录病毒和RNA病毒蛋白具有“晚期结构域”基序，可招募宿主ESCRT亚基蛋白帮助病毒内吞、运输、复制、出芽以及外排。因此，病毒“晚期结构域”基序和ESCRT亚基蛋白可能是病毒感染治疗中具有广泛应用前景的药物靶点。本文重点综述了ESCRT系统的组成及功能，ESCRT亚基和病毒“晚期结构域”基序对病毒复制的影响以及ESCRT介导的抗病毒作用，以期为抗病毒药物的开发和利用提供参考。     

多囊体生物发生和蛋白质分拣——ESCRT、Vps4、Ubiquitination一个都不能少

多囊体(multivesicular body,MVB)是由晚期内吞体的限制膜内陷出芽而形成的动态的亚细胞结构,是真核细胞重要的膜和蛋白质运输与分拣中心,并与信号转导、胞质分裂、基因沉默、自噬、蛋白质的质量控制、病毒出芽等密切相关.多囊体生物发生涉及20多种囊泡分拣蛋白(Vps),最重要的是在内吞体膜上形成的4种内吞体运输分拣复合物(ESCRT 0、Ⅰ、Ⅱ、Ⅲ)和Vps4.ESCRT 0与包涵素在内吞体膜上形成微域并富集泛素化的货物蛋白.ESCRTⅠ和Ⅱ诱导MVB囊泡出芽、促进囊泡形成并分拣货物蛋白到囊泡中.ESCRTⅢ收缩及剪切芽颈,完成最后的膜脱落过程.Vps4解离ESCRT以循环利用.泛素化及泛素化蛋白也能修饰或调控ESCRT的定位及功能.这些研究表明,泛素化蛋白、ESCRT和Vps4在内吞体膜上的相继协同作用是驱动紧密偶联的多囊体生物发生及蛋白质分拣的主要力量.本文以蛋白质-蛋白质相互作用为主,综述了ESCRT复合物及Vps4多聚体的组装机制、相互作用、生理功能以及泛素化蛋白和泛素化对ESCRT的调控,并对下一步研究进行了展望.     

禾谷镰刀菌AP蛋白家族的生物信息学分析

衔接蛋白(adaptor protein, AP)复合物在真核细胞膜运输途径中参与多种囊泡的形成。禾谷镰刀菌是重要的植物病原真菌,囊泡运输及蛋白转运是其生长发育及致病性所必需的。本研究通过同源比对,在禾谷镰刀菌中共鉴定到12个AP蛋白复合物相关基因,其编码的蛋白质组成3个异源四聚体(AP-1, AP-2和AP-3)。氨基酸序列分析表明AP-1、AP-2和AP-3复合体中的相同亚基具有保守的功能结构域以及保守基序。系统进化分析显示,子囊菌门、担子菌门和壶菌门的AP蛋白复合物相应亚基聚为一类,而植物和动物另聚为一类,说明了真菌的AP蛋白复合体进化可能早于真菌与动植物的分化。基因表达分析显示,Fg AP-1σ亚基在营养菌丝、分生孢子和子囊的表达量是其他亚基的2～5倍,AP-1和AP-3复合体中所有亚基的基因在营养菌丝中表达量最高,AP-2复合体中所有亚基的基因则在分生孢子阶段表达量最高;AP-1、AP-2和AP-3复合体中各个亚基的基因在侵染小麦后的不同时间段均有表达,暗示AP蛋白在禾谷镰刀菌生长发育及侵染致病过程中可能起重要作用。     

COP9信号传导体与调节盖子复合体的进化

COP9信号传导体和26S蛋白酶体的调节盖子复合体皆为含有8个亚基的蛋白复合体,在真核生物体中普遍存在,它们的相应亚基在大小和氨基酸序列上具有一一对应关系.从NCBI站点的所有数据库中获得了裂殖酵母、酿酒酵母、线虫、果蝇、哺乳动物和拟南芥等多种生物的复合体的亚基序列共8组.COP9信号传导体与调节盖子复合体相应亚基之间的氨基酸序列一致性大于12%,它们均具有一些保守的区域,而且保守位点分布均匀,表明它们来自于同一祖先.在基于氨基酸序列构建的系统发育树中,各组序列分别形成两个分支:一个分支由COP9信号传导体亚基和相似蛋白组成,另一分支由相应的调节盖子复合体亚基和相似蛋白构成.各个分支中单细胞生物的序列位于动、植物序列的根部,表明COP9信号传导体与调节盖子复合体的基因重复发生在真核单细胞生物和多细胞生物分化以前,并且二者的亚基基因沿各自的方向独立进化.几乎所有编码两个蛋白复合体的基因在基因组中均为单拷贝,第Ⅴ、Ⅵ组的亚基保守程度最高,暗示着它们在复合体中起着关键的作用.对COP9信号传导体和调节盖子复合体的相应亚基基因两两之间进行dN/dS的相关性分析,分别鉴定出21和15对亚基编码序列间具有显著的Pearson相关关系,推测其相应亚基间可能通过承担相互关联的重要的生物学功能而协同进化.     

胞泌复合体在植物中的功能研究进展

囊泡运输是真核生物的一种重要的细胞学活动, 广泛参与多种生物学过程。该过程主要包括囊泡形成、转运、拴系及与目的膜融合4个环节。目前已知9种多蛋白亚基拴系复合体参与不同途径的胞内转运过程, 其中, 胞泌复合体(exocyst complex)介导了运输囊泡与质膜的拴系过程。对胞泌复合体调控机制的认识主要源于酵母(Saccharomyces cerevisiae)和动物细胞的研究。近年来, 植物胞泌复合体的研究也取得了较大进展, 初步结果显示复合体在功能方面具有一些植物特异的调控特点, 广泛参与植物生长发育和逆境响应。该文主要综述胞泌复合体在植物中的研究进展, 旨在为植物胞泌复合体功能研究提供参考。     

胞泌复合体在植物中的功能研究进展

囊泡运输是真核生物的一种重要的细胞学活动, 广泛参与多种生物学过程。该过程主要包括囊泡形成、转运、拴系及与目的膜融合4个环节。目前已知9种多蛋白亚基拴系复合体参与不同途径的胞内转运过程, 其中, 胞泌复合体(exocyst complex)介导了运输囊泡与质膜的拴系过程。对胞泌复合体调控机制的认识主要源于酵母(Saccharomyces cerevisiae)和动物细胞的研究。近年来, 植物胞泌复合体的研究也取得了较大进展, 初步结果显示复合体在功能方面具有一些植物特异的调控特点, 广泛参与植物生长发育和逆境响应。该文主要综述胞泌复合体在植物中的研究进展, 旨在为植物胞泌复合体功能研究提供参考。     

COP9信号传导体与调节盖子复合体的进化

COP9信号传导体和26S蛋白酶体的调节盖子复合体皆为含有8个亚基的蛋白复合体,在真核生物体中普遍存在,它们的相应亚基在大小和氨基酸序列上具有一一对应关系。从NCBI站点的所有数据库中获得了裂殖酵母、酿酒酵母、线虫、果蝇、哺乳动物和拟南芥等多种生物的复合体的亚基序列共8组。COP9信号传导体与调节盖子复合体相应亚基之间的氨基酸序列一致性大于12％,它们均具有一些保守的区域,而且保守位点分布均匀,表明它们来自于同一祖先。在基于氨基酸序列构建的系统发育树中,各组序列分别形成两个分支：一个分支由COP9信号传导体亚基和相似蛋白组成,另一分支由相应的调节盖子复合体亚基和相似蛋白构成。各个分支中单细胞生物的序列位于动、植物序列的根部,表明COP9信号传导体与调节盖子复合体的基因重复发生在真核单细胞生物和多细胞生物分化以前,并且二者的亚基基因沿各自的方向独立进化。几乎所有编码两个蛋白复合体的基因在基因组中均为单拷贝,第Ⅴ、Ⅵ组的亚基保守程度最高,暗示着它们在复合体中起着关键的作用。对COP9信号传导体和调节盖子复合体的相应亚基基因两两之间进行dN／dS的相关性分析,分别鉴定出21和15对亚基编码序列间具有显著的Pearson相关关系,推测其相应亚基间可能通过承担相互关联的重要的生物学功能而协同进化。     

Cohesin结构及功能研究进展

Cohesin是一类在真核生物进化过程中保守的蛋白复合体,由4个重要亚基相互作用形成环状结构,在细胞分裂过程中参与维持染色体的有序排布。在动物中研究发现cohesin还可以作为分子间的连结器介导绝缘子/增强子–启动子间长距离交互,导致基因表达增强或者抑制,但在植物中关于cohesin在调控基因表达和维持染色体构象方面的研究却相对滞后。本文介绍了cohesin的结构特点和主要组成亚基,对调控cohesin在染色质上动态变化的相关因子进行了总结,并结合近年来植物中cohesin的功能研究和动物中cohesin在三维基因组及转录调控中的重要作用,展望了植物中cohesin在转录调控中的潜在功能。     

F-box蛋白质在植物生长发育中的功能

在真核生物中, 泛素介导的蛋白降解途径参与了许多生物学过程。SCF复合体是一种非常重要的E3泛素连接酶, 在植物中研究的最为深入。F-box蛋白包含一个F-box 基序, 是SCF复合体的一个亚基, 它决定了底物识别的特异性。目前, 从各种植物中已鉴定出大量的F-box蛋白质, 它们参与了植物激素(乙烯, 生长素, GA, JA)的信号传导以及自交不亲和、花器官发育等生物学过程, F-box蛋白还参与了植物的胁迫反应。最新研究结果显示, 一个F-box蛋白TIR1是生长素的受体。因此, F-box蛋白质介导的泛素化蛋白质降解途径可能是植物基因表达调控的重要机制。     

Efficient cargo sorting by ESCRT-I and the subsequent release of ESCRT-I from multivesicular bodies requires the subunit Mvb12

The endosomal sorting complex required for transport (ESCRT)-I protein complex functions in recognition and sorting of ubiquitinated transmembrane proteins into multivesicular body (MVB) vesicles. It has been shown that ESCRT-I contains the vacuolar protein sorting (Vps) proteins Vps23, Vps28, and Vps37. We identified an additional subunit of yeast ESCRT-I called Mvb12, which seems to associate with ESCRT-I by binding to Vps37. Transient recruitment of ESCRT-I to MVBs results in the rapid degradation of Mvb12. In contrast to mutations in other ESCRT-I subunits, which result in strong defects in MVB cargo sorting, deletion of MVB12 resulted in only a partial sorting phenotype. This trafficking defect was fully suppressed by overexpression of the ESCRT-II complex. Mutations in MVB12 did not affect recruitment of ESCRT-I to MVBs, but they did result in delivery of ESCRT-I to the vacuolar lumen via the MVB pathway. Together, these observations suggest that Mvb12 may function in regulating the interactions of ESCRT-I with cargo and other proteins of the ESCRT machinery to efficiently coordinate cargo sorting and release of ESCRT-I from the MVB.     

Endosomal and non-endosomal functions of ESCRT proteins

The three endosomal sorting complexes required for transport (ESCRTs) are integral to the degradation of endocytosed membrane proteins and multivesicular body (MVB) biogenesis. Here, we review evidence that ESCRTs have evolved as a specialized machinery for the degradative sorting of ubiquitinated membrane proteins and we highlight recent studies that have shed light on the mechanisms by which these complexes mediate protein sorting, MVB biogenesis, tumour suppression and viral budding. We also discuss evidence that some ESCRT subunits have evolved additional functions that are unrelated to membrane trafficking.     

Ist1 regulates Vps4 localization and assembly

The ESCRT protein complexes are recruited from the cytoplasm and assemble on the endosomal membrane into a protein network that functions in sorting of ubiquitinated transmembrane proteins into the multivesicular body (MVB) pathway. This transport pathway packages cargo proteins into vesicles that bud from the MVB limiting membrane into the lumen of the compartment and delivers these vesicles to the lysosome/vacuole for degradation. The dissociation of ESCRT machinery by the AAA-type ATPase Vps4 is a necessary late step in the formation of MVB vesicles. This ATP-consuming step is regulated by several Vps4-interacting proteins, including the newly identified regulator Ist1. Our data suggest that Ist1 has a dual role in the regulation of Vps4 activity: it localizes to the ESCRT machinery via Did2 where it positively regulates recruitment of Vps4 and it negatively regulates Vps4 by forming an Ist1-Vps4 heterodimer, in which Vps4 cannot bind to the ESCRT machinery. The activity of the MVB pathway might be in part determined by outcome of these two competing activities.     

Binding to Any ESCRT Can Mediate Ubiquitin‐Independent Cargo Sorting

The ESCRT (endosomal sorting complex required for transport) machinery is known to sort ubiquitinated transmembrane proteins into vesicles that bud into the lumen of multivesicular bodies (MVBs). Although the ESCRTs themselves are ubiquitinated they are excluded from the intraluminal vesicles and recycle back to the cytoplasm for further rounds of sorting. To obtain insights into the rules that distinguish ESCRT machinery from cargo we analyzed the trafficking of artificial ESCRT‐like protein fusions. These studies showed that lowering ESCRT‐binding affinity converts a protein from behaving like ESCRT machinery into cargo of the MVB pathway, highlighting the close relationship between machinery and the cargoes they sort. Furthermore, our findings give insights into the targeting of soluble proteins into the MVB pathway and show that binding to any of the ESCRTs can mediate ubiquitin‐independent MVB sorting.     

Structural studies of phosphoinositide 3-kinase-dependent traffic to multivesicular bodies

Three large protein complexes known as ESCRT I, ESCRT II and ESCRT III drive the progression of ubiquitinated membrane cargo from early endosomes to lysosomes. Several steps in this process critically depend on PtdIns3P, the product of the class III phosphoinositide 3-kinase. Our work has provided insights into the architecture, membrane recruitment and functional interactions of the ESCRT machinery. The fan-shaped ESCRT I core and the trilobal ESCRT II core are essential to forming stable, rigid scaffolds that support additional, flexibly-linked domains, which serve as gripping tools for recognizing elements of the MVB (multivesicular body) pathway: cargo protein, membranes and other MVB proteins. With these additional (non-core) domains, ESCRT I grasps monoubiquitinated membrane proteins and the Vps36 subunit of the downstream ESCRT II complex. The GLUE (GRAM-like, ubiquitin-binding on Eap45) domain extending beyond the core of the ESCRT II complex recognizes PtdIns3P-containing membranes, monoubiquitinated cargo and ESCRT I. The structure of this GLUE domain demonstrates that it has a split PH (pleckstrin homology) domain fold, with a non-typical phosphoinositide-binding pocket. Mutations in the lipid-binding pocket of the ESCRT II GLUE domain cause a strong defect in vacuolar protein sorting in yeast.     

Plant endosomal trafficking pathways

Endosomes regulate both the recycling and degradation of plasma membrane (PM) proteins, thereby modulating many cellular responses triggered at the cell surface. Endosomes also play a role in the biosynthetic pathway by taking proteins to the vacuole and recycling vacuolar cargo receptors. In plants, the trans-Golgi network (TGN) acts as an early/recycling endosome whereas prevacuolar compartments/multivesicular bodies (MVBs) take PM proteins to the vacuole for degradation. Recent studies have demonstrated that some of the molecular complexes that mediate endosomal trafficking, such as the retromer, the ADP-ribosylation factor (ARF) machinery, and the Endosomal Sorting Complexes Required for Transport (ESCRTs) have both conserved and specialized functions in plants. Whereas there is disagreement on the subcellular localization of the plant retromer, its function in recycling vacuolar sorting receptors (VSRs) and modulating the trafficking of PM proteins has been well established. Studies on Arabidopsis ESCRT components highlight the essential role of this complex in cytokinesis, plant development, and vacuolar organization. In addition, post-translational modifications of plant PM proteins, such as phosphorylation and ubiquitination, have been demonstrated to act as sorting signals for endosomal trafficking.     

Divergent pathways lead to ESCRT-III-catalyzed membrane fission

Endosomal sorting complexes required for transport (ESCRT) have been implicated in topologically similar but diverse cellular and pathological processes including multivesicular body (MVB) biogenesis, cytokinesis and enveloped virus budding. Although receptor sorting at the endosomal membrane producing MVBs employs the regulated assembly of ESCRT-0 followed by ESCRT-I, -II, -III and the vacuolar protein sorting (VPS)4 complex, other ESCRT-catalyzed processes require only a subset of complexes which commonly includes ESCRT-III and VPS4. Recent progress has shed light on the pathway of ESCRT assembly and highlights the separation of tasks of different ESCRT complexes and associated partners. The emerging picture suggests that among all ESCRT-catalyzed processes, divergent pathways lead to ESCRT-III assembly within the neck of a budding structure catalyzing membrane fission.     

Dictyostelium Tom1 Participates to an Ancestral ESCRT-0 Complex

Sorting of ubiquitinated proteins to multivesicular bodies (MVBs) in mammalian cells relies on proteins with a Vps27/Hrs/STAM (VHS) domain. Here, we show that the amoeba Dictyostelium presents only one protein with a VHS domain: DdTom1. We demonstrate that the VHS domain of DdTom1 is followed by a Golgi-localized, γ-ear-containing, ADP-ribosylation-factor-binding and Tom1 (GAT) domain that binds ubiquitin, and by a non-conserved C-terminal domain that can recruit clathrin, EGFr pathway substrate 15 and tumor susceptibility gene 101, a component of the MVB biogenesis machinery [endosomal complexes required for transport (ESCRT) complexes]. Both VHS and GAT domains interact with phospholipids and therefore could ensure the recruitment of DdTom1 to endosomal membranes. We propose that DdTom1 participates in an ancestral ESCRT-0 complex implicated in the sorting of ubiquitinated proteins into MVBs.     

The emerging shape of the ESCRT machinery

The past two years have seen an explosion in the structural understanding of the endosomal sorting complex required for transport (ESCRT) machinery that facilitates the trafficking of ubiquitylated proteins from endosomes to lysosomes via multivesicular bodies (MVBs). A common organization of all ESCRTs is a rigid core attached to flexibly connected modules that recognize other components of the MVB pathway. Several previously unsuspected key links between multiple ESCRT subunits, phospholipids and ubiquitin have now been elucidated, which, together with the detailed morphological analyses of ESCRT-depletion phenotypes, provide new insights into the mechanism of MVB biogenesis.     

Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

ABSTRACT: BACKGROUND: In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. RESULTS: Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. CONCLUSIONS: Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway.