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The ubiquitin E2 variant domain of TSG101 (TSG101‐UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disruption of TSG101‐UEV/PTAP interactions has emerged as a promising strategy for the development of host‐oriented broad‐spectrum antivirals with low susceptibility to resistance. TSG101 is a challenging target characterized by an extended and flat binding interface, low affinity for PTAP ligands, and complex binding energetics. Here, we assess the druggability of the TSG101‐UEV/PTAP binding interface by searching for drug‐like inhibitors and evaluating their ability to block PTAP recognition, impair budding, and inhibit viral proliferation. A discovery workflow was established by combining in vitro miniaturized HTS assays and a set of cell‐based activity assays including high‐content bimolecular complementation, virus‐like particle release measurement, and antiviral testing in live virus infection. This approach has allowed us to identify a set of chemically diverse molecules that block TSG101‐UEV/PTAP binding with IC50s in the low μM range and are able to disrupt the interaction between full‐length TSG101 and viral proteins in human cells and inhibit viral replication. State‐of‐the‐art molecular docking studies reveal that the active compounds exploit binding hotspots at the PTAP binding site, unlocking the full binding potential of the TSG101‐UEV binding pockets. These inhibitors represent promising hits for the development of novel broad‐spectrum antivirals through targeted optimization and are also valuable tools for investigating the involvement of ESCRT in the proliferation of different virus families and study the secondary effects induced by the disruption of ESCRT/virus interactions. [ABSTRACT FROM AUTHOR]

Summary: The phytohormone abscisic acid (ABA) functions in the control of plant stress responses, particularly in drought stress. A significant mechanism in attenuating and terminating ABA signals involves regulated protein turnover, with certain ABA receptors, despite their main presence in the cytosol and nucleus, subjected to vacuolar degradation via the Endosomal Sorting Complex Required for Transport (ESCRT) machinery.Collectively our findings show that discrete TOM1‐LIKE (TOL) proteins, which are functional ESCRT‐0 complex substitutes in plants, affect the trafficking for degradation of core components of the ABA signaling and transport machinery.TOL2,3,5 and 6 modulate ABA signaling where they function additively in degradation of ubiquitinated ABA receptors and transporters.TOLs colocalize with their cargo in different endocytic compartments in the root epidermis and in guard cells of stomata, where they potentially function in ABA‐controlled stomatal aperture.Although the tol2/3/5/6 quadruple mutant plant line is significantly more drought‐tolerant and has a higher ABA sensitivity than control plant lines, it has no obvious growth or development phenotype under standard conditions, making the TOL genes ideal candidates for engineering to improved plant performance. [ABSTRACT FROM AUTHOR]

In circulation, T cells are spherical with selectin enriched dynamic microvilli protruding from the surface. Following extravasation, these microvilli serve another role, continuously surveying their environment for antigen in the form of peptide‐MHC (pMHC) expressed on the surface of antigen presenting cells (APCs). Upon recognition of their cognate pMHC, the microvilli are initially stabilized and then flatten into F‐actin dependent microclusters as the T cell spreads over the APC. Within 1–5 min, clathrin is recruited by the ESCRT‐0 component Hrs to mediate release of T cell receptor (TCR) loaded vesicles directly from the plasma membrane by clathrin and ESCRT‐mediated ectocytosis (CEME). After 5‐10 min, Hrs is displaced by the endocytic clathrin adaptor epsin‐1 to induce clathrin‐mediated trans‐endocytosis (CMTE) of TCR‐pMHC conjugates. Here we discuss some of the functional properties of the clathrin machinery which enables it to control these topologically opposite modes of membrane transfer at the immunological synapse, and how this might be regulated during T cell activation. [ABSTRACT FROM AUTHOR]

Cytosolic Mycobacterium marinum are ejected from host cells such as macrophages or the amoeba Dictyostelium discoideum in a non‐lytic fashion. As described previously, the autophagic machinery is recruited to ejecting bacteria and supports host cell integrity during egress. Here, we show that the ESCRT machinery is also recruited to ejecting bacteria, partially dependent on an intact autophagic pathway. As such, the AAA‐ATPase Vps4 shows a distinct localization at the ejectosome structure in comparison to fluorescently tagged Vps32, Tsg101 and Alix. Along the bacterium engaged in ejection, ESCRT and the autophagic component Atg8 show partial colocalization. We hypothesize that both, the ESCRT and autophagic machinery localize to the bacterium as part of a membrane damage response, as well as part of a "frustrated autophagosome" that is unable to engulf the ejecting bacterium. [ABSTRACT FROM AUTHOR]

The discovery of microautophagy, the direct engulfment of cytoplasmic material by the lysosome, dates back to 1966 in a morphological study of mammalian cells by Christian de Duve. Since then, studies on microautophagy have shifted toward the elucidation of the physiological significance of the process. However, in contrast to macroautophagy, studies on the molecular mechanisms of microautophagy have been limited. Only recent studies revealed that ATG proteins involved in macroautophagy are also operative in several types of microautophagy and that ESCRT proteins, responsible for the multivesicular body pathway, play a central role in most microautophagy processes. In this review, we summarize our current knowledge on the function of ATG and ESCRT proteins in microautophagy. [ABSTRACT FROM AUTHOR]

The integrity of the nuclear envelope depends on the function of nuclear pore complexes (NPCs), transport channels that control macromolecular traffic between the nucleus and cytosol. The central importance of NPCs suggests the existence of quality control (QC) mechanisms that oversee their assembly and function. In this perspective, we emphasize the challenges associated with NPC assembly and the need for QC mechanisms that operate at various stages of an NPC's life. This includes cytosolic preassembly QC that helps enforce key nucleoporin–nucleoporin interactions and their ultimate stoichiometry in the NPC in addition to mechanisms that monitor aberrant fusion of the inner and outer nuclear membranes. Furthermore, we discuss whether and how these QC mechanisms may operate to sense faulty mature NPCs to facilitate their repair or removal. The so far uncovered mechanisms for NPC QC provide fertile ground for future research that not only benefits a better understanding of the vital role that NPCs play in cellular physiology but also how loss of NPC function and/or these QC mechanisms might be an input to aging and disease. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) play a critical role in maintaining the equilibrium between the nucleus and cytoplasm, enabling bidirectional transport across the nuclear envelope, and are essential for proper nuclear organization and gene regulation. Perturbations in the regulatory mechanisms governing NPCs and nuclear envelope homeostasis have been implicated in the pathogenesis of several neurodegenerative diseases. The ESCRT‐III pathway emerges as a critical player in the surveillance and preservation of well‐assembled, functional NPCs, as well as nuclear envelope sealing. Recent studies have provided insights into the involvement of nuclear ESCRT‐III in the selective reduction of specific nucleoporins associated with neurodegenerative pathologies. Thus, maintaining quality control of the nuclear envelope and NPCs represents a pivotal element in the pathological cascade leading to neurodegenerative diseases. This review describes the constituents of the nuclear‐cytoplasmic transport machinery, encompassing the nuclear envelope, NPC, and ESCRT proteins, and how their structural and functional alterations contribute to the development of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

In every bacterial cell, the plasma membrane plays a key role in viability as it forms a selective barrier between the inside of the cell and its environment. This barrier function depends on the physical state of the lipid bilayer and the proteins embedded or associated with the bilayer. Over the past decade or so, it has become apparent that many membrane‐organizing proteins and principles, which were described in eukaryote systems, are ubiquitous and play important roles in bacterial cells. In this minireview, we focus on the enigmatic roles of bacterial flotillins in membrane compartmentalization and bacterial dynamins and ESCRT‐like systems in membrane repair and remodeling. [ABSTRACT FROM AUTHOR]

The amyloid‐β (Aβ) peptide, produced from amyloid precursor protein (APP) by β and γ‐secretases, has been implicated in the etiology of Alzheimer's disease (AD). However, the precise intracellular trafficking pathway of APP and its subcellular locations to produce Aβ have remained unclear. To address these issues, we established fly AD models that recapitulated multiple AD‐like symptoms by expressing human APP in the Drosophila nerve system. The ESCRT (endosomal sorting complexes required for transport) machinery regulates the sorting and trafficking of endocytosed proteins, yet its role in AD pathogenesis has not been explored in vivo. We found that knockdown of distinct ESCRT components ameliorated APP‐induced morphological and behavioral defects, including impaired wing expansion, eye degeneration, dopamine neuron loss, locomotor disability, lifespan shortening, and cognitive deficits. Mechanistically, we showed that impaired ESCRT impeded APP's intracellular transportation from early endosomes to late endosomes, resulting in reduced Aβ production and amyloid deposit load. These data suggest that APP undergoes ESCRT‐mediated endocytic trafficking, and Aβ is generated mainly in late endosomes. Our data provide the first in vivo evidence to support a physiological role of ESCRT in AD pathogenesis, suggesting that interfering with ESCRT machinery might be an alternative therapeutic strategy for AD. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus type 1 (HIV‐1) is an intracellular pathogen whose replication cycle strictly depends on the host cell molecular machinery. HIV‐1 crosses twice the plasma membrane, to get in and to get out of the cell. Therefore, the first and the last line of intracellular component encountered by the virus is the cortical actin network. Here, we review the role of actin and actin‐related proteins in HIV‐1 entry, assembly, budding, and release. We first highlight the mechanisms controlling actin polymerization at the entry site that promote the clustering of HIV‐1 receptors, a crucial step for the virus to fuse with the plasma membrane. Then, we describe how actin is transiently depolymerized locally to allow the capsid to cross the actin cortex, before migrating towards the nucleus. Finally, we review the role of several actin‐binding proteins in actin remodeling events required for membrane deformation and curvature at the viral assembly site as well as for virus release. Strikingly, it appears that common actin‐regulating pathways are involved in viral entry and exit. However, while the role of actin remodeling during entry is well understood, this is not the case during exit. We discuss remaining challenges regarding the actin‐dependent mechanisms involved in HIV‐1 entry and exit, and how they could be overcome. [ABSTRACT FROM AUTHOR]

Exosomes are secreted nanovesicles with potent signalling activity that are initially formed as intraluminal vesicles (ILVs) in late Rab7‐positive multivesicular endosomes, and also in recycling Rab11a‐positive endosomes, particularly under some forms of nutrient stress. The core proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) participate in exosome biogenesis and ILV‐mediated destruction of ubiquitinylated cargos. Accessory ESCRT‐III components have reported roles in ESCRT‐III‐mediated vesicle scission, but their precise functions are poorly defined. They frequently only appear essential under stress. Comparative proteomics analysis of human small extracellular vesicles revealed that accessory ESCRT‐III proteins, CHMP1A, CHMP1B, CHMP5 and IST1, are increased in Rab11a‐enriched exosome preparations. We show that these proteins are required to form ILVs in Drosophila secondary cell recycling endosomes, but unlike core ESCRTs, they are not involved in degradation of ubiquitinylated proteins in late endosomes. Furthermore, CHMP5 knockdown in human HCT116 colorectal cancer cells selectively inhibits Rab11a‐exosome production. Accessory ESCRT‐III knockdown suppresses seminal fluid‐mediated reproductive signalling by secondary cells and the growth‐promoting activity of Rab11a‐exosome‐containing EVs from HCT116 cells. We conclude that accessory ESCRT‐III components have a specific, ubiquitin‐independent role in Rab11a‐exosome generation, a mechanism that might be targeted to selectively block pro‐tumorigenic activities of these vesicles in cancer. [ABSTRACT FROM AUTHOR]

Viruses are obligate intracellular pathogens that utilize cellular machinery for many aspects of their propagation and effective egress of virus particles from host cells is one important determinant of virus infectivity. Hijacking host cell processes applies in particular to the hepatitis B virus (HBV), as its DNA genome with about 3 kb in size is one of the smallest viral genomes known. HBV is a leading cause of liver disease and still displays one of the most successful pathogens in human populations worldwide. The extremely successful spread of this virus is explained by its efficient transmission strategies and its versatile particle types, including virions, empty envelopes, naked capsids, and others. HBV exploits distinct host trafficking machineries to assemble and release its particle types including nucleocytoplasmic shuttling transport, secretory, and exocytic pathways, the Endosomal Sorting Complexes Required for Transport pathway, and the autophagy pathway. Understanding how HBV uses and subverts host membrane trafficking systems offers the chance of obtaining new mechanistic insights into the regulation and function of this essential cellular processes. It can also help to identify potential targets for antiviral interventions. Here, I will provide an overview of HBV maturation, assembly, and budding, with a focus on recent advances, and will point out areas where questions remain that can benefit from future studies. Unless otherwise indicated, almost all presented knowledge was gained from cell culture‐based, HBV in vitro‐replication and in vitro‐infection systems. [ABSTRACT FROM AUTHOR]

Endocytosis, secretion, and endosomal trafficking are key cellular processes that control the composition of the plasma membrane. Through the coordination of these trafficking pathways, cells can adjust the composition, localization, and turnover of proteins and lipids in response to developmental or environmental cues. Upon being incorporated into vesicles and internalized through endocytosis, plant plasma membrane proteins are delivered to the trans‐Golgi network (TGN). At the TGN, plasma membrane proteins are recycled back to the plasma membrane or transferred to multivesicular endosomes (MVEs), where they are further sorted into intralumenal vesicles for degradation in the vacuole. Both types of plant endosomes, TGN and MVEs, act as sorting organelles for multiple endocytic, recycling, and secretory pathways. Molecular assemblies such as retromer, ESCRT (endosomal sorting complex required for transport) machinery, small GTPases, adaptor proteins, and SNAREs associate with specific domains of endosomal membranes to mediate different sorting and membrane‐budding events. In this review, we discuss the mechanisms underlying the recognition and sorting of proteins at endosomes, membrane remodeling and budding, and their implications for cellular trafficking and physiological responses in plants. [ABSTRACT FROM AUTHOR]

Damaged lysosomes can be repaired by calcium release-dependent recruitment of the ESCRT machinery. However, the involvement of annexins, another group of calcium-responding membrane repair proteins, has not been fully addressed. Here, we show that although all ubiquitously expressed annexins (ANXA1, A2, A4, A5, A6, A7, and A11) localize to damaged lysosomes, only ANXA1 and ANXA2 are important for repair. Their recruitment is calcium-dependent, ESCRT-independent, and selective towards lysosomes with large injuries. Lysosomal leakage was more severe when ANXA1 or ANXA2 was depleted compared to that of ESCRT components. These findings suggest that ANXA1 and ANXA2 constitute an additional repair mechanism that serves to minimize leakage from damaged lysosomes. [ABSTRACT FROM AUTHOR]

Animal models have played an essential role in understanding the host–pathogen interactions of pathogenic mycobacteria, including the Mycobacterium tuberculosis and emerging nontuberculous mycobacteria (NTM) species such as M. avium and M. abscessus. Drosophila melanogaster has become a well‐established model for the study of innate immunity and is increasingly being used as a tool to study host–pathogen interactions, in part due to its genetic tractability. The use of D. melanogaster has led to greater understanding of the role of the innate immune system in response to mycobacterial infection, including in vitro RNAi screens and in vivo studies. These studies have identified processes and host factors involved in mycobacterial infection, such as those required for cellular entry, those required to control or resist non‐pathogenic mycobacteria, or factors that become dysregulated as a result of mycobacterial infection. Developments in genetic tools for manipulating mycobacterial genomes will allow for more detailed studies into how specific host and pathogen factors interact with one another by using D. melanogaster; however, the full potential of this model has not yet been reached. Here we provide an overview of how D. melanogaster has been used to study mycobacterial infection and discuss the current gaps in our understanding. [ABSTRACT FROM AUTHOR]

Mutations in the ESCRT‐III subunit CHMP2B cause frontotemporal dementia (FTD) and lead to impaired endolysosomal trafficking and lysosomal storage pathology in neurons. We investigated the effect of mutant CHMP2B on synaptic pathology, as ESCRT function was recently implicated in the degradation of synaptic vesicle (SV) proteins. We report here that expression of C‐terminally truncated mutant CHMP2B results in a novel synaptopathy. This unique synaptic pathology is characterised by selective retention of presynaptic SV trafficking proteins in aged mutant CHMP2B transgenic mice, despite significant loss of postsynaptic proteins. Furthermore, ultrastructural analysis of primary cortical cultures from transgenic CHMP2B mice revealed a significant increase in the number of presynaptic endosomes, while neurons expressing mutant CHMP2B display defective SV recycling and alterations to functional SV pools. Therefore, we reveal how mutations in CHMP2B affect specific presynaptic proteins and SV recycling, identifying CHMP2B FTD as a novel synaptopathy. This novel synaptopathic mechanism of impaired SV physiology may be a key early event in multiple forms of FTD, since proteins that mediate the most common genetic forms of FTD all localise at the presynapse. [ABSTRACT FROM AUTHOR]

A number of viruses including HIV use the ESCRT system to bud from the infected cell. We have previously confirmed biochemically that ESCRT‐II is involved in this process in HIV‐1 and have defined the molecular domains that are important for this. Here, using SNAP‐tag fluorescent labelling and both fixed and live cell imaging we show that the ESCRT‐II component EAP45 colocalises with the HIV protein Gag at the plasma membrane in a temporal and quantitative manner, similar to that previously shown for ALIX and Gag. We show evidence that a proportion of EAP45 may be packaged within virions, and we confirm the importance of the N terminus of EAP45 and specifically the H0 domain in this process. By contrast, the Glue domain of EAP45 is more critical for recruitment during cytokinesis, emphasising that viruses have ways of recruiting cellular components that may be distinct from those used by some cellular processes. This raises the prospect of selective interference with the pathway to inhibit viral function while leaving cellular functions relatively unperturbed. [ABSTRACT FROM AUTHOR]

Mitosis and endocytosis are two fundamental cellular processes essential for maintaining a eukaryotic life. Mitosis partitions duplicated chromatin enveloped in the nuclear membrane into two new cells, whereas endocytosis takes in extracellular substances through membrane invagination. These two processes are spatiotemporally separated and seemingly unrelated. However, recent studies have uncovered that endocytic proteins have moonlighting functions in mitosis, and mitotic complexes manifest additional roles in endocytosis. In this review, we summarize important proteins or protein complexes that participate in both processes, compare their mechanism of action, and discuss the rationale behind this multifunctionality. We also speculate on the possible origin of the functional reciprocity from an evolutionary perspective. [ABSTRACT FROM AUTHOR]

Mitochondria play important roles in energy generation and homeostasis maintenance in eukaryotic cells. The damaged or superfluous mitochondria can be nonselectively or selectively removed through the autophagy/lysosome pathway, which was referred as mitophagy. According to the molecular machinery for degrading mitochondria, the selectively removed mitochondria can occur through macromitophagy or micromitophagy. In this study, we show that the endosomal sorting complex required for transport III (ESCRT‐III) in budding yeast regulates macromitophagy induced by nitrogen starvation, but not by the post‐logarithmic phase growth in lactate medium by monitoring a mitochondrial marker, Om45. Firstly, loss of ESCRT‐III subunit Snf7 or Vps4‐Vta1 complex subunit Vps4, two representative subunits of the ESCRT complex, suppresses the delivery and degradation of Om45‐GFP to vacuoles. Secondly, we show that the mitochondrial marker Om45 and mitophagy receptor Atg32 accumulate on autophagosomes marked with Atg8 (mitophagosomes, MPs) in ESCRT mutants. Moreover, the protease‐protection assay indicates that Snf7 and Vps4 are involved in MP closure. Finally, Snf7 interacts with Atg11, which was detected by two ways, glutathione‐S‐transferase (GST) pulldown and bimolecular fluorescence complementation (BiFC) assay, and this BiFC interaction happens on mitochondrial reticulum. Therefore, we proposed that the ESCRT‐III machinery mediates nitrogen starvation‐induced macromitophagy by the interaction between Snf7 and Atg11 so that Snf7 is recruited to Atg32‐marked MPs by the known Atg11–Atg32 interaction to seal them. These results reveal that the ESCRT‐III complex plays a new role in yeast on macromitophagy. [ABSTRACT FROM AUTHOR]

Summary: During evolution, land plants generated unique proteins that participate in endosomal sorting and multivesicular endosome (MVE) biogenesis, many of them with specific phosphoinositide‐binding capabilities. Nonetheless, the function of most plant phosphoinositide‐binding proteins in endosomal trafficking remains elusive.Here, we analysed several Arabidopsis mutants lacking predicted phosphoinositide‐binding proteins and first identified fyve4‐1 as a mutant with a hypersensitive response to high‐boron conditions and defects in degradative vacuolar sorting of membrane proteins such as the borate exporter BOR1‐GFP.FYVE4 encodes a plant‐unique, FYVE domain‐containing protein that interacts with SNF7, a core component of ESCRT‐III (Endosomal Sorting Complex Required for Transport III). FYVE4 affects the membrane association of the late‐acting ESCRT components SNF7 and VPS4, and modulates the formation of intraluminal vesicles (ILVs) inside MVEs. The critical function of FYVE4 in the ESCRT pathway was further demonstrated by the strong genetic interactions with SNF7B and LIP5. Although the fyve4‐1, snf7b and lip5 single mutants were viable, the fyve4‐1 snf7b and fyve4‐1 lip5 double mutants were seedling lethal, with strong defects in MVE biogenesis and vacuolar sorting of ubiquitinated membrane proteins.Taken together, we identified FYVE4 as a novel plant endosomal regulator, which functions in ESCRTing pathway to regulate MVE biogenesis. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are cytosolic fat storage organelles that play roles in lipid metabolism, trafficking and signaling. Breakdown of LDs in Saccharomyces cerevisiae is mainly achieved by lipolysis and lipophagy. In this study, we found that the endosomal sorting complex required for transport (ESCRT) in S. cerevisiae negatively regulated the turnover of a LD marker, Erg6, under both simplified glucose restriction (GR) and acute glucose restriction (AGR) conditions by monitoring the localization and degradation of Erg6. Loss of Vps27, Snf7 or Vps4, representative subunits of the ESCRT machinery, facilitated the delivery of Erg6‐GFP to vacuoles and its degradation depending on the lipophagy protein Atg15 under simplified GR. Additionally, the lipolysis proteins Tgl3 and Tgl4 were also involved in the enhanced vacuolar localization and degradation of Erg6‐GFP in vps4Δ cells. Furthermore, we found that Atg14, which is required for the formation of putatively liquid‐ordered (Lo) membrane domains on the vacuole that act as preferential internalization sites for LDs, abundantly localized to vacuolar membranes in ESCRT mutants. Most importantly, the depletion or overexpression of Atg14 correspondingly abolished or promoted the observed Erg6 degradation in ESCRT mutant cells. We propose that Atg14 together with other proteins promotes Erg6 degradation in ESCRT mutant cells under specific glucose restriction conditions. These results shed new light on the regulation of ESCRT on LD turnover. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) uses the ESCRT (endosomal sorting complexes required for transport) protein pathway to bud from infected cells. Despite the roles of ESCRT‐I and ‐III in HIV budding being firmly established, participation of ESCRT‐II in this process has been controversial. EAP45 is a critical component of ESCRT‐II. Previously, we utilised a CRISPR‐Cas9 EAP45 knockout cell line to assess the involvement of ESCRT‐II in HIV replication. We demonstrated that the absence of ESCRT‐II impairs HIV budding. Here, we show that virus spread is also defective in physiologically relevant CRISPR/Cas9 EAP45 knockout T cells. We further show reappearance of efficient budding by re‐introduction of EAP45 expression into EAP45 knockout cells. Using expression of selected mutants of EAP45, we dissect the domain requirement responsible for this function. Our data show at the steady state that rescue of budding is only observed in the context of a Gag/Pol, but not a Gag expressor, indicating that the size of cargo determines the usage of ESCRT‐II. EAP45 acts through the YPXL‐ALIX pathway as partial rescue is achieved in a PTAP but not a YPXL mutant virus. Our study clarifies the role of ESCRT‐II in the late stages of HIV replication and reinforces the notion that ESCRT‐II plays an integral part during this process as it does in sorting ubiquitinated cargos and in cytokinesis. [ABSTRACT FROM AUTHOR]

Mutations in CHMP2B, an ESCRT‐III (endosomal sorting complexes required for transport) component, are associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Neurodegenerative disorders including FTD are also associated with a disruption in circadian rhythms, but the mechanism underlying this defect is not well understood. Here, we ectopically expressed the human CHMP2B variant associated with FTD (CHMP2BIntron5) in flies using the GMR‐GAL4 driver (GMR>CHMP2BIntron5) and analyzed their circadian rhythms at behavioral, cellular, and biochemical level. In GMR>CHMP2BIntron5 flies, we observed disrupted eclosion rhythms, shortened free‐running circadian locomotor period, and reduced levels of timeless (tim) mRNA—a circadian pacemaker gene. We also observed that the GMR‐GAL4 driver, primarily known for its expression in the retina, drives expression in a subset of tim expressing neurons in the optic lobe of the brain. The patterning of these GMR‐ and tim‐positive neurons in the optic lobe, which appears distinct from the putative clusters of circadian pacemaker neurons in the fly brain, was disrupted in GMR>CHMP2BIntron5 flies. These results demonstrate that CHMP2BIntron5 can disrupt the normal function of the circadian clock in Drosophila. [ABSTRACT FROM AUTHOR]

Ubiquitin is covalently attached to substrate proteins in the form of a single ubiquitin moiety or polyubiquitin chains and has been generally linked to protein degradation, however, distinct types of ubiquitin linkages are also used to control other critical cellular processes like cell signaling. Over forty mammalian G protein‐coupled receptors (GPCRs) have been reported to be ubiquitinated, but despite the diverse and rich complexity of GPCR signaling, ubiquitin has been largely ascribed to receptor degradation. Indeed, GPCR ubiquitination targets the receptors for degradation by lysosome, which is mediated by the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery, and the proteasome. This has led to the view that ubiquitin and ESCRTs primarily function as the signal to target GPCRs for destruction. Contrary to this conventional view, studies indicate that ubiquitination of certain GPCRs and canonical ubiquitin‐binding ESCRTs are not required for receptor degradation and revealed that diverse and complex pathways exist to regulate endo‐lysosomal sorting of GPCRs. In other studies, GPCR ubiquitination has been shown to drive signaling and not receptor degradation and further revealed novel insight into the mechanisms by which GPCRs trigger the activity of the ubiquitination machinery. Here, we discuss the diverse pathways by which ubiquitin controls GPCR endo‐lysosomal sorting and beyond. Ubiquitination of GPCRs and endocytic adaptor proteins facilitates GPCR trafficking to lysosomes and. In addition, ubiquitination of GPCRs and GPCR effectors can mediate the activation of specific signaling pathways. GPCR ubiquitination is also critical for targeting some GPCRs for degradation by proteasomes. This review highlights the multiple pathways by which ubiquitin controls GPCR endo‐lysosomal sorting and signaling, which expand the function of ubiquitin in regulating GPCR biology. [ABSTRACT FROM AUTHOR]

When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB‐lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell‐free MVB‐lysosome fusion assay using Saccharomyces cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS ( homotypic fusion and vacuole protein sorting complex) are required, we found that the Qa‐SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab‐cascade mechanism harmonizes MVB maturation with lysosome fusion. [ABSTRACT FROM AUTHOR]

Endosomes are transportation nodes, mediating selective transport of soluble and transmembrane cargos to and from the Golgi apparatus, plasma membrane and lysosomes. As endosomes mature to become multivesicular bodies (MVBs), Endosomal Sorting Complexes Required for Transport (ESCRTs) selectively incorporate transmembrane cargos into vesicles that bud into the endosome lumen. Luminal vesicles and their cargoes are targeted for destruction when MVBs fuse with lysosomes Common assays of endosomal luminal targeting, including fluorescence microscopy and monitoring of proteolytic cargo maturation, possess significant limitations. We present a quantitative assay system called LUCID (LUCiferase reporter of Intraluminal Deposition) that monitors exposure of chimeric luciferase-cargo reporters to cytosol. Luciferase-chimera signal increases when sorting to the endosome lumen is disrupted, and silencing of signal from the chimera depends upon luminal delivery of the reporter rather than proteolytic degradation. The system presents several advantages, including rapidity, microscale operation and a high degree of reproducibility that enables detection of subtle phenotypic differences. Luciferase reporters provide linear signal over an extremely broad dynamic range, allowing analysis of reporter traffic even at anemic levels of expression. Furthermore, LUCID reports transport kinetics when applied to inducible trafficking reporters. [ABSTRACT FROM AUTHOR]

The endosomal deubiquitylase USP8 has profound effects on endosomal morphology and organisation. Previous reports have proposed both positive (EGFR, MET) and negative roles in the down-regulation of receptors (Frizzled, Smoothened). Here we report an additional influence of USP8 on the retromer-dependent shuttling of ci-M6PR between the sorting endosome and biosynthetic pathway. Depletion of USP8 leads to a steady state redistribution of ci-M6PR from the Trans-Golgi Network (TGN) to endosomal compartments. Consequently we observe a defect in sorting of lysosomal enzymes, evidenced by increased levels of unprocessed Cathepsin D, which is secreted into the medium. The normal distribution of receptor can be restored by expression of siRNA-resistant USP8 but not by a catalytically inactive mutant or a truncated form, lacking a MIT domain required for endosomal localisation. We suggest that effects of USP8 depletion may reflect the loss of ESCRT-0 components which associate with retromer components Vps35 and SNX1, whilst failure to efficiently deliver lysosomal enzymes may also contribute to the observed block in receptor tyrosine kinase degradation. [ABSTRACT FROM AUTHOR]

BACKGROUND: Receptor down-regulation by the multivesicular body (MVB) pathway is critical for many cellular signaling events. MVB generation is mediated by the highly conserved ESCRT (0, I, II, and III) protein complexes. Chmp1 is an ESCRT-III component and a putative tumor suppressor in humans. However, published data on Chmp1 activity are conflicting and its role during tissue development is not well defined. RESULTS: We investigated the function of Drosophila Chmp1 and found that it is an essential gene. In the wing, loss of Chmp1 activity causes a cell fate change from intervein to vein, and interactions between Chmp1 and Drosophila Epidermal Growth Factor Receptor (DER) regulators suggest that Chmp1 negatively regulates DER signaling. Chmp1 knockdown also decreases Blistered expression, which is repressed by DER signaling. We find that Chmp1 protein localizes to the late endosome in Drosophila embryos, which is consistent with its effects on DER signaling resulting from its function in the ESCRT-III complex. CONCLUSIONS: Drosophila Chmp1 negatively regulates DER signaling, likely through its role in MVB formation. Loss of Chmp1 activity in the Drosophila wing induces a cell fate change from intervein to vein that should provide a useful tool for future studies of ESCRT protein activity. Developmental Dynamics 243:977-987, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The multivesicular body ( MVB) is a specialized Rab7+ late endosome ( LE) containing multiple intralumenal vesicles that function in targeting ubiquitinylated cell surface proteins to the lysosome for degradation. African trypanosomes lack a morphologically well-defined MVB, but contain orthologs of the ESCRT ( Endosomal Sorting Complex Required for Transport) machinery that mediates MVB formation. We investigate the role of TbVps23, an early ESCRT component, and TbVps4, the terminal ESCRT ATPase, in lysosomal trafficking in bloodstream form trypanosomes. Both localize to the TbRab7+ LE and RNAi silencing of each rapidly blocks growth. TbVps4 silencing results in approximately threefold accumulation of TbVps23 at the LE, consistent with blocking terminal ESCRT disassembly. Trafficking of endocytic and biosynthetic cargo, but not default lysosomal reporters, is also negatively affected. Others reported that TbVps23 mediates ubiquitin-dependent lysosomal degradation of invariant surface glycoproteins ( ISG65) (Leung et al., Traffic 2008;9:1698-1716). In contrast, we find that TbVps23 ablation does not affect ISG65 turnover, while TbVps4 silencing markedly enhances lysosomal degradation. We propose several models to accommodate these results, including that the ESCRT machinery actually retrieves ISG65 from the LE to earlier endocytic compartments, and in its absence ISG65 traffics more efficiently to the lysosome. Overall, these results confirm that the ESCRT machinery is essential in Trypanosoma brucei and plays important and novel role(s) in LE function in trypanosomes. [ABSTRACT FROM AUTHOR]

Endocytosis and subsequent membrane traffic through endosomes are cellular processes that are integral to eukaryotic evolution, and numerous human diseases are associated with their dysfunction. Consequently, it is important to untangle the molecular machineries that regulate membrane dynamics and protein flow in the endocytic pathway. Central in this context is class III phosphatidylinositol 3-kinase, an evolutionarily conserved enzyme complex that phosphorylates phosphatidylinositol into phosphatidylinositol 3-phosphate. Phosphatidylinositol 3-phosphate recruits specific effector proteins, most of which contain FYVE or PX domains, to promote endocytosis, endosome fusion, endosome motility and endosome maturation, as well as cargo sorting to lysosomes, the biosynthetic pathway or the plasma membrane. Here we review the functions of key phosphatidylinositol 3-phosphate effectors in regulation of endocytic membrane dynamics and protein sorting. [ABSTRACT FROM AUTHOR]

The exit of low-density lipoprotein derived cholesterol (LDL-C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann-Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol-binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N-terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well-established role in disassembling the ESCRT (endosomal sorting complex required for transport)-III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL-C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT-III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT-III. [ABSTRACT FROM AUTHOR]

Enveloped viruses acquire their host-derived membrane at a variety of intracellular locations. Herpesviruses are complex entities that undergo several budding and fusion events during an infection. All members of this large family are believed to share a similar life cycle. However, they seemingly differ in terms of acquisition of their mature envelope. Herpes simplex virus is often believed to bud into an existing intracellular compartment, while the related cytomegalovirus may acquire its final envelope from a novel virus-induced assembly compartment. This review focuses on recent advances in the characterization of cellular compartment(s) potentially contributing to herpes virion final envelopment. It also examines the common points between seemingly distinct envelopment pathways and highlights the dynamic nature of intracellular compartments in the context of herpesvirus infections. [ABSTRACT FROM AUTHOR]

Traffic through endosomes and lysosomes is controlled by small G-proteins of the Rab5 and Rab7 families. Like humans, Saccharomyces cerevisiae has three Rab5s (Vps21, Ypt52 and Ypt53) and one Rab7 (Ypt7). Here, we elucidate the functional roles and regulation of the yeast Rab5s. Using GFP-tagged cargoes, a novel quantitative multivesicular body ( MVB) sorting assay, and electron microscopy, we show that MVB biogenesis and thus MVB cargo sorting is severely impaired in vps21Δ ypt52Δ double mutants. Ypt53, the third Rab5 paralog, is hardly expressed during normal growth but its transcription is strongly induced by cellular stress through the calcineurin-Crz1 pathway. The requirement for Rab5 activity in stress tolerance facilitated identification of Msb3/Gyp3 as the principal Rab5 GAP ( GTPase accelerating protein). In vitro GAP assays verified that Vps21 is a preferred Gyp3 target. Moreover, we demonstrate that Gyp3 spatially restricts active Vps21 to intermediate endosomal compartments by preventing Vps21 accumulation on lysosomal vacuoles. Gyp3, therefore, operates as a compartmental insulator that helps to define the spatial domain of Vps21 signaling in the endolysosomal pathway. [ABSTRACT FROM AUTHOR]

The four protein complexes termed endosomal sorting complexes required for transport (ESCRT) are key mediators of multivesicular body sorting/formation, retroviral budding and cell abscission, which share a membrane deformation process with the same topological change: vesicles budding away from the cytoplasm. Independent studies of the signal transduction pathways that mediate ambient pH sensing and adaptation in yeast and fungi revealed that these pathways share a conserved signaling mechanism that utilizes ESCRT complexes for its activation. This pathway in Saccharomyces cerevisiae, termed the Rim101 pathway, consists of both a sensing complex, which senses ambient alkaline pH, and a proteolytic complex, which proteolyzes and thereby activates the key transcription factor Rim101. Since the proteolytic complex is thought to be formed and activated on a platform of a multimerized ESCRT-III component Snf7, the organization, regulation and function of this pathway are dependent on the function of ESCRT complexes. [ABSTRACT FROM AUTHOR]

The name calpain was historically given to a protease that is activated by Ca2+ and whose primary structure contains a Ca2+-binding penta-EF-hand (PEF) as well as a calpain cysteine protease (CysPc) domain and a C2-domain-like (C2L) domain. In the human genome, CysPc domains are found in 15 genes, but only nine of them encode PEF domains. Fungi and budding yeasts have calpain-like sequences that lack the PEF domain, and each protein (designated PalB and Rim13, respectively) is orthologous to human calpain-7, indicating that the calpain-7 orthologs are evolutionarily more conserved than classical calpains possessing PEF domains. An N-terminal region of calpain-7 has a tandem repeat of microtubule-interacting and transport domains that interact with a subset of endosomal sorting complex required for transport (ESCRT) III proteins. In addition to calpains, PEF domains are found in other Ca2+-binding proteins including ALG-2 that associates with ALIX (an ESCRT-III accessory protein) and TSG101 (an ESCRT-I subunit). Phylogenetic comparison of dissected domain structures of calpains and experimentally confirmed protein-protein interaction networks imply that there is an evolutionary and physical linkage between mammalian calpains and PEF proteins involving the ESCRT system. [ABSTRACT FROM AUTHOR]

The endosomal sorting complexes required for transport (ESCRTs) mediate membrane fission from the cytoplasmic face of the bud neck. ESCRTs were originally identified as factors involved in multivesicular body vesicle biogenesis in yeast but have since been shown to function in other membrane fission events in mammalian cells, including enveloped virus budding and the abscission step of cytokinesis. Several recent studies have revealed that not all ESCRT factors are required for each of these biological processes, and this review summarizes our current understanding of the different requirements for ESCRT factors in these three different ESCRT-mediated mammalian membrane fission processes. [ABSTRACT FROM AUTHOR]

Abstract: The ESCRT (endosomal sorting complex required for transport) pathway promotes the final membrane scission step at the end of cytokinesis, assists viral budding and generates multivesicular bodies (MVBs). These seemingly unrelated processes require a topologically similar membrane deformation and scission event that buds membranes/vesicles out of the cytoplasm. The topology of this budding reaction is ‘opposite’ to reactions that bud endocytic and secretory vesicles into the cytoplasm. Here we summarize recent findings that help to understand how the ESCRT machinery, in particular the ESCRT-III complex, assembles on its target membranes, executes membrane scission and is disassembled by the AAA-ATPase Vps4. [Copyright &y& Elsevier]

The number of surface membrane proteins and their residence time on the plasma membrane are critical determinants of cellular responses to cues that can control plasticity, growth and differentiation. After internalization, the ultimate fate of many plasma membrane proteins is dependent on whether they are sorted for internalization into the lumenal vesicles of multivesicular bodies (MVBs), an obligate step prior to lysosomal degradation. To help to elucidate the mechanisms underlying MVB sorting, we have developed a novel cell-free assay that reconstitutes the sorting of a prototypical membrane protein, the epidermal growth factor receptor, with which we have probed some of its molecular requirements. The sorting event measured is dependent on cytosol, ATP, time, temperature and an intact proton gradient. Depletion of Hrs inhibited biochemical and morphological measures of sorting that were rescued by inclusion of recombinant Hrs in the assay. Moreover, depletion of signal-transducing adaptor molecule (STAM), or addition of mutated ATPase-deficient Vps4, also inhibited sorting. This assay reconstitutes the maturation of late endosomes, including the formation of internal vesicles and the sorting of a membrane protein, and allows biochemical investigation of this process. [ABSTRACT FROM AUTHOR]

Melanosomes are lysosome-related organelles that coexist with lysosomes within melanocytes. The pathways by which melanosomal proteins are diverted from endocytic organelles toward melanosomes are incompletely defined. In melanocytes from mouse models of Hermansky-Pudlak syndrome that lack BLOC-1, melanosomal proteins such as tyrosinase-related protein 1 (Tyrp1) accumulate in early endosomes. Whether this accumulation represents an anomalous pathway or an arrested normal intermediate in melanosome protein trafficking is not clear. Here, we show that early endosomes are requisite intermediates in the trafficking of Tyrp1 from the Golgi to late stage melanosomes in normal melanocytic cells. Kinetic analyses show that very little newly synthesized Tyrp1 traverses the cell surface and that internalized Tyrp1 is inefficiently sorted to melanosomes. Nevertheless, nearly all Tyrp1 traverse early endosomes since it becomes trapped within enlarged, modified endosomes upon overexpression of Hrs. Although Tyrp1 localization is not affected by Hrs depletion, depletion of the ESCRT-I component, Tsg101, or inhibition of ESCRT function by dominant-negative approaches results in a dramatic redistribution of Tyrp1 to aberrant endosomal membranes that are largely distinct from those harboring traditional ESCRT-dependent, ubiquitylated cargoes such as MART-1. The lysosomal protein content of some of these membranes and the lack of Tyrp1 recycling to the plasma membrane in Tsg101-depleted cells suggests that ESCRT-I functions downstream of BLOC-1. Our data delineate a novel pathway for Tyrp1 trafficking and illustrate a requirement for ESCRT-I function in controlling protein sorting from vacuolar endosomes to the limiting membrane of a lysosome-related organelle. [ABSTRACT FROM AUTHOR]

Abstract: Endocytosis is an important regulator of cell–cell signaling and endocytic trafficking has been increasingly implicated in control of tumor suppression. Recent insights from Drosophila indicate that impairment of multiple trafficking steps which lead to receptor degradation can cause tumor formation in epithelial organs. These tumors are characterized by sustained activation of a number of mitogenic signaling pathways, and by subversion of epithelial polarity and the apoptotic response. Cooperation between such alterations, as well as tumor–host interactions, is also observed. The recapitulation of several hallmarks of human cancers in fly tumors provides a framework to understand the role of defective endocytosis in cancer. [Copyright &y& Elsevier]

The endosomal sorting complex required for transport (ESCRT) protein machinery comprises four complexes, ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III, that facilitate receptor sorting into the lumen of multivesicular endosomes (MVEs) in order to terminate signalling receptors for final degradation within the lysosomes. Even though ESCRT proteins appear to be essential for the biogenesis of MVEs in Saccharomyces cerevisae, it is not clear whether ESCRT-independent pathways for MVE biogenesis exist in higher organisms. In this study we maximized inhibition of ESCRT-dependent pathway by depleting cells of key subunits of all four ESCRTs and followed MVE formation and epidermal growth factor (EGF) receptor (EGFR) traffic using electron and confocal microscopy. There was a dramatic alteration in the morphology of components of the endocytic pathway in ESCRT-depleted cells, but early and late endosomes stayed clearly differentiated. Importantly, although EGF-induced formation of MVEs was highly sensitive to ESCRT depletion, EGF-independent formation of MVEs could still occur. The MVEs remaining in ESCRT-depleted cells contained enlarged intralumenal vesicles into which EGFRs were not sorted. Our observations suggest that both ESCRT-dependent and ESCRT-independent mechanisms of MVE biogenesis exist in mammalian cells. [ABSTRACT FROM AUTHOR]

SKD1/VPS4B belongs to the adenosine triphosphatases associated with diverse cellular activities (AAA) family and regulates multivesicular body (MVB) biogenesis. SKD1 changes its oligomeric state during the ATPase cycle and subsequently releases endosomal sorting complex required for transport (ESCRT) complexes from endosomes during the formation of MVBs. In this study, we describe domain motions in monomeric SKD1 on ATP and ADP binding. Nucleotides bind between the α/β and the α-helical domains of SKD1, inducing a ∼20° domain rotation and closure of the binding site, which are similar to the changes observed in the AAA+ ATPase, HslU. Gel filtration and small-angle X-ray scattering experiments showed that the ATP-bound form of SKD1 oligomerizes in solution, whereas ADP-bound and apo forms of SKD1 exist as monomers, even though the conformations of the ADP- and ATP-bound forms are nearly identical. Nucleotide-bound SKD1 structures are compatible with a hexameric ring arrangement reminiscent of the AAA ATPase p97 D1 ring. In the hexameric ring model of SKD1, Arg290 from a neighboring molecule binds to the γ-phosphate of ATP, which promotes oligomerization of the ATP-bound form. ATP hydrolysis would eliminate this interaction and subsequent nucleotide release causes the domains to rotate, which together lead to the disassembly of the SKD1 oligomer. [ABSTRACT FROM AUTHOR]

Plant endosomes are highly dynamic organelles that are involved in the constitutive recycling of plasma membrane cargo and the trafficking of polarized plasma membrane proteins such as auxin carriers. In addition, recent studies have shown that surface receptors such as the plant defense-related FLS2 receptor and the brassinosteroid receptor BRI1 appear to signal from endosomes upon ligand binding and internalization. In yeast and mammals, endosomes are also known to recycle vacuolar cargo receptors back to the trans Golgi network and sort membrane proteins for degradation in the vacuole/lysosome. Some of these sorting mechanisms are mediated by the retromer and endosomal sorting complex required for transport (ESCRT) complexes. Plants contain orthologs of all major retromer and ESCRT complex subunits, but they have also evolved variations in endosomal functions connected to plant-specific features such as the diversity of vacuolar transport pathways. This review focuses on recent studies in plants dealing with the regulation of endosomal recycling functions, architecture and formation of multivesicular bodies, ligand-mediated endocytosis and receptor signaling from endosomes as well as novel endosomal markers and the function of endosomes in the transport and processing of soluble vacuolar proteins. [ABSTRACT FROM AUTHOR]

Lysosomal targeting of ubiquitylated endocytic cargo is mediated in part by the endosomal sorting complex required for transport (ESCRT) complexes, a system conserved between animals and fungi (Opisthokonta). Extensive comparative genomic analysis demonstrates that ESCRT factors are well conserved across the eukaryotic lineage and complexes I, II, III and III-associated are almost completely retained, indicating an early evolutionary origin. The conspicuous exception is ESCRT 0, which functions in recognition of ubiquitylated cargo, and is restricted to the Opisthokonta, suggesting that a distinct mechanism likely operates in the vast majority of eukaryotic organisms. Additional analysis suggests that ESCRT III and ESCRT III-associated components evolved through a concerted model. Functional conservation of the ESCRT system is confirmed by direct study in trypanosomes. Despite extreme sequence divergence, epitope-tagged ESCRT factors TbVps23 and TbVps28 localize to the endosomal pathway, placing the trypanosome multivesicular body (MVB) in juxtaposition to the early endosome and lysosome. Knockdown of TbVps23 partially prevents degradation of an ubiquitylated endocytosed transmembrane domain protein. Therefore, despite the absence of an ESCRT 0 complex, the trypanosome ESCRT/MVB system functions similarly to that of opisthokonts. Thus the ESCRT system is an ancient and well-conserved feature of eukaryotic cells but with key differences between diverse lineages. [ABSTRACT FROM AUTHOR]

Endosomal sorting complex required for transport-III (ESCRT-III) is a large complex built from related ESCRT-III proteins involved in multivesicular body biogenesis. Little is known about the structure and function of this complex. Here, we compare four human ESCRT-III proteins – hVps2-1/CHMP2a, hVps24/CHMP3, hVps20/CHMP6, and hSnf7-1/CHMP4a – to each other, studying the effects of deleting predicted α-helical domains on their behavior in transfected cells. Surprisingly, removing ∼40 amino acids from the C-terminus of each protein unmasks a common ability to associate with endosomal membranes and assemble into large polymeric complexes. Expressing these truncated ESCRT-III proteins in cultured cells causes ubiquitinated cargo to accumulate on enlarged endosomes and inhibits viral budding, while expressing full-length proteins does not. hVps2-1/CHMP2a lacking its C-terminal 42 amino acids further fails to bind to the AAA+ adenosine triphosphatase VPS4B/SKD1, indicating that C-terminal sequences are important for interaction of ESCRT-III proteins with VPS4. Overall, our study supports a model in which ESCRT-III proteins cycle between a default ‘closed’ state and an activated ‘open’ state under control of sequences at their C-terminus and associated factors. [ABSTRACT FROM AUTHOR]

Abstract: Enveloped viruses exit their host cell by budding from a cellular membrane and thereby spread from one cell to another. Virus budding in general involves the distortion of a cellular membrane away from the cytoplasm, envelopment of the viral capsid by one or more lipid bilayers that are enriched in viral membrane glycoproteins, and a fission event that separates the enveloped virion from the cellular membrane. While it was initially thought that virus budding is always driven by viral transmembrane proteins interacting with the inner structural proteins, it is now clear that the driving force may be different depending on the virus. Research over the past years has shown that viral components specifically interact with host cell lipids and proteins, thereby adopting cellular functions and pathways to facilitate virus release. This review summarizes the current knowledge of the cellular membrane systems that serve as viral budding sites and of the viral and cellular factors involved in budding. One of the best studied cellular machineries required for virus egress is the ESCRT complex, which will be described in more detail. [Copyright &y& Elsevier]

The endosomal sorting complex required for transport (ESCRT) is thought to support the formation of intralumenal vesicles of multivesicular bodies (MVBs). The ESCRT is also required for the budding of HIV and has been proposed to be recruited to the HIV-budding site, the plasma membrane of T cells and MVBs in macrophages. Despite increasing data on the function of ESCRT, the ultrastructural localization of its components has not been determined. We therefore localized four proteins of the ESCRT machinery in human T cells and macrophages by quantitative electron microscopy. All the proteins were found throughout the endocytic pathway, including the plasma membrane, with only around 10 and 3% of the total labeling in the cytoplasm and on the MVBs, respectively. The majority of the labeling (45%) was unexpectedly found on tubular–vesicular endosomal membranes rather than on endosomes themselves. The ESCRT labeling was twice as concentrated on early and late endosomes/lysosomes in macrophages compared with that in T cells, where it was twice more abundant at the plasma membrane. The ESCRT proteins were not redistributed on HIV infection, suggesting that the amount of ESCRT proteins located at the budding site suffices for HIV release. These results represent the first systematic ultrastructural localization of ESCRT and provide insights into its role in uninfected and HIV-infected cells. [ABSTRACT FROM AUTHOR]

Class E vacuolar protein-sorting (Vps) proteins were first described in yeast as being involved in receptor-mediated endocytosis and multivesicular body formation. Inactivation by RNA interference of the class E VPS genes of the nematode Caenorhabditis elegans revealed heterogeneous phenotypes. We have further characterized the role of the essential gene Cevps-27, ortholog of human hepatocyte growth factor-regulated tyrosine kinase substrate, during the development of C. elegans. Use of green fluorescent protein fusion constructs and antibody staining revealed that Cevps-27 localizes to endosomal membranes. It is widely expressed but enriched in epithelial cells. Cevps-27 mutants presented enlarged endosomal structures and an accumulation of autophagic vesicles as revealed by electron microscopy and the analysis of the autophagic marker LGG-1. Cevps-27 animals arrested at L2-L3 molt with an inability to degrade their old cuticle. This molting phenotype was more severe when Cevps-27 worms were grown on suboptimal concentrations of cholesterol. Furthermore, defective endocytic trafficking of the low-density lipoprotein receptor-related protein 1 (LRP-1) was also observed in Cevps-27 mutants. These results indicate that CeVPS-27 is required for endosomal and autophagic pathways in C. elegans and plays a crucial role in the control of molting through LRP-1 internalization and cholesterol traffic. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]