ESCRTing proteins in the endocytic pathway

The endosomal sorting complex required for transport (ESCRT) machinery is highly conserved and its components have been found in all five major supergroups of eukaryotes. The three ESCRT complexes and associated proteins play critical roles in receptor downregulation, retroviral budding, and other normal and pathological cellular processes. Besides monoubiquitin-dependent protein cargo recognition and sorting, the ESCRT machinery also appears to drive the formation of multivesicular bodies (MVBs). Recent advances in the determination of the function and structure of the ESCRT complexes have improved our understanding of the molecular details underlying the assembly and regulation of the ESCRT machinery.     

The Arabidopsis ESCRT protein�Cprotein interaction network

In yeast, endosomal sorting of monoubiquitylated transmembrane proteins is performed by a subset of the 19 "class E vacuolar protein sorting" proteins. The core machinery consists of 11 proteins that are organised in three complexes termed ESCRT I-III (endosomal sorting complex required for transport I-III) and is conserved in eukaryotic cells. While the pathway is well understood in yeast and animals, the plant ESCRT system is largely unexplored. At least one sequence homolog for each ESCRT component can be found in the Arabidopsis genome. Generally, sequence conservation between yeast/animals and the Arabidopsis proteins is low. To understand details about participating proteins and complex organization we have performed a systematic pairwise yeast two hybrid analysis of all Arabidopsis proteins showing homology to the ESCRT core machinery. Positive interactions were validated using bimolecular fluorescence complementation. In our experiments, most putative ESCRT components exhibited interactions with other ESCRT components that could be shown to occur on endosomes suggesting that despite their low homology to their yeast and animal counterparts they represent functional components of the plant ESCRT pathway.     

ESCRTing in cereals: still a long way to go

The multivesicular body(MVB) sorting pathway provides a mechanism for the delivery of cargo destined for degradation to the vacuole or lysosome. The endosomal sorting complex required for transport(ESCRT) is essential for the MVB sorting pathway by driving the cargo sorting to its destination. Many efforts in plant research have identified the ESCRT machinery and functionally characterised the first plant ESCRT proteins. However, most studies have been performed in the model plant Arabidopsis thaliana that is genetically and physiologically different to crops. Cereal crops are important for animal feed and human nutrition and have further been utilized as promising candidates for recombinant protein production. In this review, I summarize the role of plant ESCRT components in cereals that are involved in efficient adaptation to environmental stress and grain development. A special focus is on barley(Hordeum vulgare L.) ESCRT proteins, where recent studies show their quantitative mapping during grain development, e.g. associating HvSNF7.1 with protein trafficking to protein bodies(PBs) in starchy endosperm. Thus, it is indispensable to identify the molecular key-players within the endomembrane system including ESCRT proteins to optimize and possibly enhance tolerance to environmental stress, grain yield and recombinant protein production in cereal grains.     

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.     

植物ESCRT复合体的功能研究进展

真核细胞中,功能高度保守的内体蛋白分选转运装置ESCRT在胞吞途径和蛋白分泌途径中均扮演重要角色。植物细胞中,该装置包含ESCRT-Ⅰ、ESCRT-Ⅱ、ESCRT-Ⅲ和VPS4/SKD1复合体4个亚基,但缺乏ESCRT-0亚基。ESCRT的每个亚基均由多个蛋白构成。目前,针对ESCRT的研究已经证实,其在泛素化的膜蛋白进入多囊泡体/液泡前体(MVB/PVC)内腔过程中发挥重要调控作用;同时在自噬途径以及应对环境胁迫等方面也具有重要的调节功能。该文首先介绍了植物中ESCRT复合体的组成及生物学功能,然后总结了植物中特有ESCRT复合体组分蛋白的最新研究进展,最后探讨了有关ESCRT复合体研究中尚未解决的重要科学问题。     

The ESCRT machinery: a cellular apparatus for sorting and scission

The ESCRT (endosomal sorting complex required for transport) machinery is a group of multisubunit protein complexes conserved across phyla that are involved in a range of diverse cellular processes. ESCRT proteins regulate the biogenesis of MVBs (multivesicular bodies) and the sorting of ubiquitinated cargos on to ILVs (intraluminal vesicles) within these MVBs. These proteins are also recruited to sites of retroviral particle assembly, where they provide an activity that allows release of these retroviruses. More recently, these proteins have been shown to be recruited to the intracellular bridge linking daughter cells at the end of mitosis, where they act to ensure the separation of these cells through the process of cytokinesis. Although these cellular processes are diverse, they share a requirement for a topologically unique membrane-fission step for their completion. Current models suggest that the ESCRT machinery catalyses this membrane fission.     

Paramyxovirus budding

Our knowledge about envelope virus budding has been dramatically increased, since L-domain motifs were identified within their matrix and retroviral Gag proteins which drive virus budding. These viral proteins have been shown to interact with host cellular proteins involved in endocytosis and/or multi-vesicular body (MVB) sorting via their L-domains. Since budding of many enveloped viruses have been reported to be dependent on the activity of cellular Vps4, which catalyzes the disassembly of ESCRT machinery in the final step of protein sorting, this cellular function is believed to be utilized for efficient virus budding. However, for many enveloped viruses, L-domain motifs have not yet been identified, and the involvement of MVB sorting machinery in virus budding is still unknown. In this review, we will focus on paramyxoviruses among such viruses, and discuss their budding with the latest information.     

ESCRT proteins in physiology and disease

As a mechanism of signal attenuation, receptors for growth factors, peptide hormones and cytokines are internalized in response to ligand binding, followed by degradation in lysosomes. Receptor ubiquitination is a key signal for such downregulation, and four protein complexes known as endosomal sorting complex required for transport (ESCRT)-0, -I, -II and -III have been identified as the machinery required for degradative endosomal sorting of ubiquitinated membrane proteins in yeast and metazoans. Three of these complexes contain ubiquitin-binding domains whereas ESCRT-III instead recruits deubiquitinating enzymes. The concerted action of the ESCRTs not only serves to sort ubiquitinated cargo but is also thought to cause inward vesiculation of endosomal membranes, thereby mediating biogenesis of multivesicular endosomes (MVEs). Because ligand-mediated receptor downregulation plays an important role in signal attenuation, it is not surprising that dysfunction of ESCRT components is associated with disease. In this review we discuss the possible roles of ESCRTs in protection against cancer, neurodegenerative diseases and bacterial infections, and we highlight the fact that many RNA viruses exploit the ESCRT machinery for the final abscission step of their budding from cells. We also review the additional functions of ESCRT proteins in cytokinesis and discuss how these may be related to ESCRT-associated pathologies.     

The ESCRT machinery: From the plasma membrane to endosomes and back again

Abstract

The manipulation and reorganization of lipid bilayers are required for diverse cellular processes, ranging from organelle biogenesis to cytokinetic abscission, and often involves transient membrane disruption. A set of membrane-associated proteins collectively known as the endosomal sorting complex required for transport (ESCRT) machinery has been implicated in membrane scission steps, which transform a single, continuous bilayer into two distinct bilayers, while simultaneously segregating cargo throughout the process. Components of the ESCRT pathway, which include 5 distinct protein complexes and an array of accessory factors, each serve discrete functions. This review focuses on the molecular mechanisms by which the ESCRT proteins facilitate cargo sequestration and membrane remodeling and highlights their unique roles in cellular homeostasis.     

A protein's final ESCRT

In eukaryotic cells, delivery of transmembrane proteins into the lumen of the lysosome for degradation is mediated by the multivesicular body pathway. The function of the ESCRT protein complexes is required for both the formation of multivesicular body lumenal vesicles and the sorting of endosomal cargo proteins into these vesicles. Recent studies have identified additional factors that seem to function as an upstream cargo retention system feeding into the ESCRT machinery, given new insights into the dynamic structure of multivesicular bodies, and identified a potential mechanism for multivesicular body vesicle formation.     

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.     

Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking

A ubiquitin-binding endosomal protein machinery is responsible for sorting endocytosed membrane proteins into intraluminal vesicles of multivesicular endosomes (MVEs) for subsequent degradation in lysosomes. The Hrs-STAM complex and endosomal sorting complex required for transport (ESCRT)-I, -II and -III are central components of this machinery. Here, we have performed a systematic analysis of their importance in four trafficking pathways through endosomes. Neither Hrs, Tsg101 (ESCRT-I), Vps22/EAP30 (ESCRT-II), nor Vps24/CHMP3 (ESCRT-III) was required for ligand-mediated internalization of epidermal growth factor (EGF) receptors (EGFRs) or for recycling of cation-independent mannose 6-phosphate receptors (CI-M6PRs) from endosomes to the trans-Golgi network (TGN). In contrast, both Hrs and ESCRT subunits were equally required for degradation of both endocytosed EGF and EGFR. Whereas depletion of Hrs or Tsg101 caused enhanced recycling of endocytosed EGFRs, this was not the case with depletion of Vps22 or Vps24. Depletion of Vps24 instead caused a strong increase in the levels of CI-M6PRs and a dramatic redistribution of the Golgi and the TGN. These results indicate that, although Hrs-STAM and ESCRT-I, -II and -III have a common function in degradative protein sorting, they play differential roles in other trafficking pathways, probably reflecting their functions at distinct stages of the endocytic pathway.     

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.     

The ESCRT Machinery Is Recruited by the Viral BFRF1 Protein to the Nucleus-Associated Membrane for the Maturation of Epstein-Barr Virus

The cellular endosomal sorting complex required for transport (ESCRT) machinery participates in membrane scission and cytoplasmic budding of many RNA viruses. Here, we found that expression of dominant negative ESCRT proteins caused a blockade of Epstein-Barr virus (EBV) release and retention of viral BFRF1 at the nuclear envelope. The ESCRT adaptor protein Alix was redistributed and partially colocalized with BFRF1 at the nuclear rim of virus replicating cells. Following transient transfection, BFRF1 associated with ESCRT proteins, reorganized the nuclear membrane and induced perinuclear vesicle formation. Multiple domains within BFRF1 mediated vesicle formation and Alix recruitment, whereas both Bro and PRR domains of Alix interacted with BFRF1. Inhibition of ESCRT machinery abolished BFRF1-induced vesicle formation, leading to the accumulation of viral DNA and capsid proteins in the nucleus of EBV-replicating cells. Overall, data here suggest that BFRF1 recruits the ESCRT components to modulate nuclear envelope for the nuclear egress of EBV.     

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.     

The endosomal sorting complex required for transport machinery influences haem uptake and capsule elaboration in Cryptococcus neoformans

Iron availability is a key determinant of virulence in the pathogenic fungus Cryptococcus neoformans. Previous work revealed that the ESCRT (endosomal sorting complex required for transport) protein Vps23 functions in iron acquisition, capsule formation and virulence. Here, we further characterized the ESCRT machinery to demonstrate that defects in the ESCRT‐II and III complexes caused reduced capsule attachment, impaired growth on haem and resistance to non‐iron metalloprotoporphyrins. The ESCRT mutants shared several phenotypes with a mutant lacking the pH‐response regulator Rim101, and in other fungi, the ESCRT machinery is known to activate Rim101 via proteolytic cleavage. We therefore expressed a truncated and activated version of Rim101 in the ESCRT mutants and found that this allele restored capsule formation but not growth on haem, thus suggesting a Rim101‐independent contribution to haem uptake. We also demonstrated that the ESCRT machinery acts downstream of the cAMP/protein kinase A pathway to influence capsule elaboration. Defects in the ESCRT components also attenuated virulence in macrophage survival assays and a mouse model of cryptococcosis to a greater extent than reported for loss of Rim101. Overall, these results indicate that the ESCRT complexes function in capsule elaboration, haem uptake and virulence via Rim101‐dependent and independent mechanisms.     

Functional ESCRT machinery is required for constitutive recycling of claudin-1 and maintenance of polarity in vertebrate epithelial cells

Genetic screens in Drosophila have identified regulators of endocytic trafficking as neoplastic tumor suppressor genes. For example, Drosophila endosomal sorting complex required for transport (ESCRT) mutants lose epithelial polarity and show increased cell proliferation, suggesting that ESCRT proteins could function as tumor suppressors. In this study, we show for the for the first time to our knowledge that ESCRT proteins are required to maintain polarity in mammalian epithelial cells. Inhibition of ESCRT function caused the tight junction protein claudin-1 to accumulate in intracellular vesicles. In contrast E-cadherin and occludin localization was unaffected. We investigated the cause of this accumulation and show that claudin-1 is constitutively recycled in kidney, colon, and lung epithelial cells, identifying claudin-1 recycling as a newly described feature of diverse epithelial cell types. This recycling requires ESCRT function, explaining the accumulation of intracellular claudin-1 when ESCRT function is inhibited. We further demonstrate that small interfering RNA knockdown of the ESCRT protein Tsg101 causes epithelial monolayers to lose their polarized organization and interferes with the establishment of a normal epithelial permeability barrier. ESCRT knockdown also reduces the formation of correctly polarized three-dimensional cysts. Thus, in mammalian epithelial cells, ESCRT function is required for claudin-1 trafficking and for epithelial cell polarity, supporting the hypothesis that ESCRT proteins function as tumor suppressors.     

Proline-rich regions and motifs in trafficking: from ESCRT interaction to viral exploitation

Most membrane-enveloped viruses bud from infected cells by hijacking the host ESCRT machinery. The ESCRTs are recruited to the budding sites by viral proteins that contain short proline (Pro)-rich motifs (PRMs) known as late domains. The late domains probably evolved by co-opting host PRMs involved in the normal functions of ESCRTs in endosomal sorting and cytokinesis. The solution and crystal structures of PRMs bound to their interaction partners explain the conserved roles of Pro and other residues that predominate in these sequences. PRMs are often grouped together in much larger Pro-rich regions (PRRs) of as many as 150 residues. The PRR of the ESCRT-associated protein, ALIX, autoregulates its conformation and activity. The robustness of different viral budding and host pathways to impairments in Pro-based interactions varies considerably. The known biology of PRM recognition in the ESCRT pathway seems, in principle, compatible with antiviral development, given our increasingly nuanced understanding of the relative weakness and robustness of the host and viral processes.