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β-Carotene has been widely used in the food and feed industry and has significant commercial value. This study aimed to increase the β-carotene production in engineered Yarrowia lipolytica by optimizing the host metabolic network. The DID2 gene, a subunit of the endosomal sorting complex required for transport (ESCRT), was integrated into a β-carotene producing strain. The β-carotene production was increased by 260%, and the biomass increased by 10% for engineered Y. lipolytica. Meanwhile, DID2 elevated the mRNA level and protein level of the genes in the β-carotene synthesis pathway, then increased precursors (FPP, Lycopene) utilization. DID2 also increased the mRNA level of the genes in the glucose pathway, pentose phosphate pathway, and tricarboxylic acid cycle and promoted glucose utilization and cofactors consumption. The ESCRT protein complex subunit, DID2, improved β-carotene production in engineered Y. lipolytica and beneficial to glucose utilization and cofactors consumption. This study provided new finding of the DID2 gene’s function and it mostly could be used for many other natural product productions.

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.

Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells. A library of ∼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC–MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes.

Eukaryotic cells are compartmentalized into organelles by intracellular membranes. While the organelles are distinct, many of them make intimate contact with one another. These contacts were first observed in the 1950s, but only recently have the functions of these contact sites begun to be understood. In yeast, the endoplasmic reticulum (ER) makes extensive intermembrane contacts with the plasma membrane (PM), covering ∼40% of the PM. Many functions of ER-PM contacts have been proposed, including nonvesicular lipid trafficking, ion transfer, and as signaling hubs. Surprisingly, cells that lack ER-PM contacts grow well, indicating that alternative pathways may be compensating for the loss of ER-PM contact. To better understand the function of ER-PM contact sites we used saturating transposon mutagenesis to identify synthetic lethal mutants in a yeast strain lacking ER-PM contact sites. The strongest hits were components of the ESCRT complexes. The synthetic lethal mutants have low levels of some lipid species but accumulate free fatty acids and lipid droplets. We found that only ESCRT-III components are synthetic lethal, indicating that Vps4 and other ESCRT complexes do not function in this pathway. These data suggest that ESCRT-III proteins and ER-PM contact sites act in independent pathways to maintain lipid homeostasis.

Polyphosphates (polyP) are energy-rich polymers of inorganic phosphates assembled into chains ranging from 3 residues to thousands of residues in length. They are thought to exist in all cells on earth and play roles in an eclectic mix of functions ranging from phosphate homeostasis to cell signaling, infection control, and blood clotting. In the budding yeast Saccharomyces cerevisiae, polyP chains are synthesized by the vacuole-bound vacuolar transporter chaperone (VTC) complex, which synthesizes polyP while simultaneously translocating it into the vacuole lumen, where it is stored at high concentrations. VTC's activity is promoted by an accessory subunit called Vtc5. In this work, we found that the conserved AP-3 complex is required for proper Vtc5 localization to the vacuole membrane. In human cells, previous work has demonstrated that mutation of AP-3 subunits gives rise to Hermansky-Pudlak syndrome, a rare disease with molecular phenotypes that include decreased polyP accumulation in platelet dense granules. In yeast AP-3 mutants, we found that Vtc5 is rerouted to the vacuole lumen by the e ndosomal s orting c omplex r equired for t ransport (ESCRT), where it is degraded by the vacuolar protease Pep4. Cells lacking functional AP-3 have decreased levels of polyP, demonstrating that membrane localization of Vtc5 is required for its VTC stimulatory activity in vivo. Our work provides insight into the molecular trafficking of a critical regulator of polyP metabolism in yeast. We speculate that AP-3 may also be responsible for the delivery of polyP regulatory proteins to platelet dense granules in higher eukaryotes. IMPORTANCE Long polymers of inorganic phosphates called polyphosphates are ubiquitous across biological kingdoms. From bacteria to humans, they have diverse functions related to protein homeostasis, energy metabolism, and cell signaling. In this study, we provide new insights into the intracellular trafficking of the polyphosphate biosynthetic machinery in the budding yeast S. cerevisiae. The critical advances of the work are 2-fold. First, it provides an explanation for decreased polyphosphate levels observed in cells mutated for a conserved intracellular trafficking machine. Second, it defines critical pathways that are highly likely to serve as hubs for polyphosphate regulation in yeast and other species.

The abscission checkpoint regulates the ESCRT membrane fission machinery and thereby delays cytokinetic abscission to protect genomic integrity in response to residual mitotic errors. The checkpoint is maintained by Aurora B kinase, which phosphorylates multiple targets, including CHMP4C, a regulatory ESCRT-III subunit necessary for this checkpoint. We now report the discovery that cytoplasmic abscission checkpoint bodies (ACBs) containing phospho-Aurora B and tri-phospho-CHMP4C develop during an active checkpoint. ACBs are derived from mitotic interchromatin granules, transient mitotic structures whose components are housed in splicing-related nuclear speckles during interphase. ACB formation requires CHMP4C, and the ESCRT factor ALIX also contributes. ACB formation is conserved across cell types and under multiple circumstances that activate the checkpoint. Finally, ACBs retain a population of ALIX, and their presence correlates with delayed abscission and delayed recruitment of ALIX to the midbody where it would normally promote abscission. Thus, a cytoplasmic mechanism helps regulate midbody machinery to delay abscission., eLife digest When a cell divides, it must first carefully duplicate its genetic information and package these copies into compartments housed in the two new cells. Errors in this process lead to genetic mistakes that trigger cancer or other harmful biological events. Quality control checks exist to catch errors before it is too late. This includes a final ‘abscission’ checkpoint right before the end of division, when the two new cells are still connected by a thin membrane bridge. If cells fail to pass this ‘no cut’ checkpoint, they delay severing their connection until the mistake is fixed. A group of proteins called ESCRTs is responsible for splitting the two cells apart if nothing is amiss. The abscission checkpoint blocks this process by altering certain proteins in the ESCRT complex, but exactly how this works is not yet clear. To find out more, Williams et al. imaged ESCRT factors in a new experimental system in which the abscission checkpoint is active in many cells. This showed that, in this context, certain ESCRT components were rerouted from the thread of membrane between the daughter cells to previously unknown structures, which Williams et al. named abscission checkpoint bodies. These entities also sequestered other factors that participate in the abscission checkpoint and factors that contribute to gene expression. These results are key to better understand how cells regulate their division; in particular, they provide a new framework to explore when this process goes wrong and contributes to cancer.

Zhou et al. identify the mechanism of autophagosome (AP) closure. They show that Rab5 GTPase regulates an interaction between the ESCRT subunit Snf7 and Atg17 to bring ESCRT to APs where it catalyzes AP closure. These findings highlight the convergence of the endocytic and autophagic pathways at this step., In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17–Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17–Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.

ESCRT (Endosomal Sorting Complex Required for Transport) machinery drives different cellular processes such as endosomal sorting, organelle biogenesis, vesicular trafficking, maintenance of plasma membrane integrity, membrane fission during cytokinesis and enveloped virus budding. The normal cycle of assembly and disassembly of some ESCRT complexes at the membrane requires the AAA-ATPase vacuolar protein sorting 4 (Vps4p). A number of ESCRT proteins are hijacked by clinically significant enveloped viruses including Ebola, and Human Immunodeficiency Virus (HIV) to enable enveloped virus budding and Vps4p provides energy for the disassembly/recycling of these ESCRT proteins. Several years ago, the failure of the terminal budding process of HIV following Vps4 protein inhibition was published; although at that time a detailed understanding of the molecular players was missing. However, later it was acknowledged that the ESCRT machinery has a role in enveloped virus budding from cells due to its role in the multivesicular body (MVB) sorting pathway. The MVB sorting pathway facilitates several cellular activities in uninfected cells, such as the down-regulation of signaling through cell surface receptors as well as the process of viral budding from infected host cells. In this review, we focus on summarising the functional organisation of ESCRT proteins at the membrane and the role of ESCRT machinery and Vps4p during MVB sorting and enveloped viral budding.

Early endosomes are the sorting hub on the endocytic pathway, wherein sorting nexins (SNXs) play important roles for formation of the distinct membranous microdomains with different sorting functions. Tubular endosomes mediate the recycling of clathrin-independent endocytic (CIE) cargoes back toward the plasma membrane. However, the molecular mechanism underlying the tubule formation is still poorly understood. Here we screened the effect on the ARF-6-associated CIE recycling endosomal tubules for all the SNX members in Caenorhabditis elegans (C. elegans). We identified SNX-3 as an essential factor for generation of the recycling tubules. The loss of SNX-3 abolishes the interconnected tubules in the intestine of C. elegans. Consequently, the surface and total protein levels of the recycling CIE protein hTAC are strongly decreased. Unexpectedly, depletion of the retromer components VPS-26/-29/-35 has no similar effect, implying that the retromer trimer is dispensable in this process. We determined that hTAC is captured by the ESCRT complex and transported into the lysosome for rapid degradation in snx-3 mutants. Interestingly, EEA-1 is increasingly recruited on early endosomes and localized to the hTAC-containing structures in snx-3 mutant intestines. We also showed that SNX3 and EEA1 compete with each other for binding to phosphatidylinositol-3-phosphate enriching early endosomes in Hela cells. Our data demonstrate for the first time that PX domain-only C. elegans SNX-3 organizes the tubular endosomes for efficient recycling and retrieves the CIE cargo away from the maturing sorting endosomes by competing with EEA-1 for binding to the early endosomes. However, our results call into question how SNX-3 couples the cargo capture and membrane remodeling in the absence of the retromer trimer complex., Author summary Trafficking of internalized materials through the endolysosomal system is essential for the maintenance of homeostasis and signaling regulation in all eukaryotic cells. Early endosomes are the sorting hub on the endocytic pathway. After internalization, the plasma membrane lipid, proteins, and invading pathogens are delivered to early endosomes for further degradation in lysosomes or for retrieval to the plasma membrane or the trans-Golgi network for reuse. However, when, where and by what mechanism various cargo proteins are sorted from each other and into the different pathways largely remain to be explored. Here, we identified SNX-3, a PX-domain only sorting nexin family member, as a novel regulator for the tubular endosomes underlying recycling of a subset of CIE cargoes. Compared with EEA-1, the superior recruitment of SNX-3 at the CIE-derived subpopulation of endosomes is critical for preventing these endosomes from converging to the classical sorting endosomes and subsequently into the multivesicular endosomal pathway. We speculate that through a spatio-temporal interplay with the retromer, SNX-3 is involved in different recycling transport carriers. Our finding of SNX-3’s role in modulating the formation of tubular endosomes provides insight into the sorting and trafficking of CIE pathways.

The discovery of Asgard archaea has changed the existing ideas on the origins of eukaryotes. Researchers propose that eukaryotic cells evolved from Asgard archaea. This hypothesis partly stems from the presence of multiple eukaryotic signature proteins in Asgard archaea, including homologs of ESCRT proteins that are essential components of the endomembrane system in eukaryotes. However, structural and functional features of Asgard ESCRT remain unknown. Our study provides evidence that Asgard ESCRT is functionally comparable to the eukaryotic counterparts, suggesting that despite the apparent absence of endomembranes in archaea, eukaryotic ESCRT was inherited from an Asgard archaeal ancestor, alongside the emergence of endomembrane system during eukaryogenesis., The emergence of the endomembrane system is a key step in the evolution of cellular complexity during eukaryogenesis. The endosomal sorting complex required for transport (ESCRT) machinery is essential and required for the endomembrane system functions in eukaryotic cells. Recently, genes encoding eukaryote-like ESCRT protein components have been identified in the genomes of Asgard archaea, a newly proposed archaeal superphylum that is thought to include the closest extant prokaryotic relatives of eukaryotes. However, structural and functional features of Asgard ESCRT remain uncharacterized. Here, we show that Vps4, Vps2/24/46, and Vps20/32/60, the core functional components of the Asgard ESCRT, coevolved eukaryote-like structural and functional features. Phylogenetic analysis shows that Asgard Vps4, Vps2/24/46, and Vps20/32/60 are closely related to their eukaryotic counterparts. Molecular dynamics simulation and biochemical assays indicate that Asgard Vps4 contains a eukaryote-like microtubule-interacting and transport (MIT) domain that binds the distinct type 1 MIT-interacting motif and type 2 MIT-interacting motif in Vps2/24/46 and Vps20/32/60, respectively. The Asgard Vps4 partly, but much more efficiently than homologs from other archaea, complements the vps4 null mutant of Saccharomyces cerevisiae, further supporting the functional similarity between the membrane remodeling machineries of Asgard archaea and eukaryotes. Thus, this work provides evidence that the ESCRT complexes from Asgard archaea and eukaryotes are evolutionarily related and functionally similar. Thus, despite the apparent absence of endomembranes in Asgard archaea, the eukaryotic ESCRT seems to have been directly inherited from an Asgard ancestor, to become a key component of the emerging endomembrane system.

The scission of lipid membranes is a common biological process, often mediated by ESCRT complexes in concert with VPS4 assembling around the separation point. The functions of the ESCRT-I and ESCRT-III complexes are well established in certain of these cellular processes; however, the role of ESCRT-II remains contentious. Here, we devised a SNAP-tag fluorescent labelling strategy to understand the domain requirements of EAP45, the main component of ESCRT-II, in HIV egress, late endosome recruitment, and cytokinesis. We used TIRF microscopy to measure the spatial co-occurrence of the HIV structural polyprotein Gag with full length EAP45 in both fixed and live cells. Gag colocalises with the full length EAP45 comparably to ALIX, but this is lost on deletion of the EAP45 N terminus. Our findings reveal the H0 domain of the EAP45 protein is essential for linking to ESCRT-I during HIV budding and in anchoring at the late endosomal membrane, however in cytokinesis it is the Glue domain that is critical.

Primary microcephaly (PM) is a highly heterogeneous neurodevelopmental disorder with many contributing risk genes and loci identified to date. We report a consanguineous family with PM, intellectual disability and short stature. Using whole exome sequencing, we identified a homozygous frameshift variant in programmed cell death 6 interacting protein (PDCD6IP, c.154_158dup; p.Val54Profs*18). This gene, PDCD6IP, plays an important role in the endosomal sorting complexes required for transport (ESCRT) pathway in the abscission stage of cytokinesis and apoptosis, and is required for normal brain development in mice. The clinical features observed in our patient were similar to the phenotypes observed in mouse and zebrafish models of PDCD6IP mutations in previous studies. This study provides evidence that clinical manifestations of PDCD6IP mutations as seen in our patients with PM and ID may be a novel cause for neurodevelopmental disorders.

The emergence of the endomembrane system is a key step in the evolution of cellular complexity during eukaryogenesis. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery is essential and required for the endomembrane system functions in eukaryotic cells. Recently, genes encoding eukaryote-like ESCRT protein components have been identified in the genomes of Asgard archaea, a newly proposed archaeal superphylum that is thought to include the closest extant prokaryotic relatives of eukaryotes. However, structural and functional features of Asgard ESCRT remain uncharacterized. Here we show that Vps4, Vps2/24/46, and Vps20/32/60, the core functional components of the Asgard ESCRT, co-evolved eukaryote-like structural and functional features. Phylogenetic analysis shows that Asgard Vps4, Vps2/24/46, and Vps20/32/60 are closely related to their eukaryotic counterparts. Molecular dynamic simulation and biochemical assays indicate that Asgard Vps4 contains a eukaryote-like Microtubule Interacting and Transport (MIT) domain that binds the distinct type-1 MIT Interacting Motif and type-2 MIT Interacting Motif in Vps2/24/46, and Vps20/32/60, respectively. The Asgard Vps4 partly, but much more efficiently than homologs from other archaea, complements the vps4 null mutant of Saccharomyces cerevisiae, further supporting the functional similarity between the membrane remodeling machineries of Asgard archaea and eukaryotes. Thus, this work provides evidence that the ESCRT complexes from Asgard archaea and eukaryotes are evolutionarily related and functionally similar. Thus, despite the apparent absence of endomembranes in Asgard archaea, the eukaryotic ESCRT seems to have been directly inherited from an Asgard ancestor, to become a key component of the emerging endomembrane system.IMPORTANCEThe discovery of Asgard archaea has changed the exiting ideas on the origins of Eukaryotes. Researchers propose that eukaryotic cells evolve from Asgard archaea. This hypothesis partly stems from the presence of multiple eukaryotic signature proteins in Asgard archaea, including homologues of ESCRT proteins that are essential components of the endomembrane system in eukaryotes. However, structural and functional features of Asgard ESCRT remain unknown. Our study provides evidence that Asgard ESCRT is functionally comparable to the eukaryotic counterparts suggesting that, despite the apparent absence of endomembranes in archaea, eukaryotic ESCRT was inherited from an Asgard archaeal ancestor, alongside the emergence of endomembrane system during eukaryogenesis.

The current COVID-19 (Coronavirus Disease-2019) pandemic is affecting the health and/or socioeconomic welfare of almost everyone in the world. Finding vaccines and therapeutics is therefore urgent, but elucidation of the molecular mechanisms that allow some viruses to cross host species boundaries, becoming a threat to human health, must also be given close attention. Here, analysis of all proteins of SARS-CoV-2 revealed a unique PPxY Late (L) domain motif, 25PPAY28, in a spike (S) protein inside a predicted hot disordered loop subject to phosphorylation and binding. PPxY motifs in enveloped RNA viruses are known to recruit Nedd4 E3 ubiquitin ligases and ultimately the ESCRT complex to enhance virus budding and release, resulting in higher viral loads, hence facilitating new infections. Interestingly, proteins of SARS-CoV-1 do not feature PPxY motifs, which could explain why SARS-CoV-2 is more contagious than SARS-CoV-1. Should an experimental assessment of this hypothesis show that the PPxY motif plays the same role in SARS-CoV-2 as it does in other enveloped RNA viruses, this motif will become a promising target for the development of novel host-oriented antiviral therapeutics for preventing S proteins from recruiting Nedd4 E3 ubiquitin ligase partners.

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu’s itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23., Author summary Vpu triggers the degradation or mis-localization of proteins important to the host’s immune response. Vpu acts as an adaptor, linking cellular protein targets to the ubiquitination and membrane trafficking machinery. Vpu has been localized to various cellular membrane systems. By fusing wild type Vpu and Vpu-mutants to the enzyme ascorbate peroxidase, we defined the cellular proteome in proximity to Vpu and correlated this with the protein’s location. We found that wild type Vpu is proximal to ESCRT proteins, retromer complexes, and sorting and late endosomal proteins. Functionally, we found that the Vpu-mediated degradation of the innate defense protein BST-2 required PTPN23, an ALIX-like protein, consistent with our observation of Vpu’s presence at the limiting membranes of multi-vesicular bodies.

Assimilation of heme is mediated by the cell surface protein Shu1 in Schizosaccharomyces pombe. Shu1 undergoes internalization from the cell surface to the vacuole in response to high concentrations of hemin. Here, we have identified cellular components that are involved in mediating vacuolar targeting of Shu1. Cells deficient in heme biosynthesis and lacking the polyubiquitin gene ubi4+ exhibit poor growth in the presence of exogenous hemin as a sole source of heme. Microscopic analyses of hem1Δ shu1Δ ubi4Δ cells expressing a functional HA4 -tagged Shu1 show that Shu1 localizes to the cell surface. Ubiquitinated Nbr1 functions as a receptor for the endosomal sorting complexes required for transport (ESCRT) that delivers cargos to the vacuole. Inactivation of nbr1+ , ESCRT-0 hse1+ or ESCRT-I sst6+ results in hem1Δ cells being unable to use exogenous hemin for the growth. Using lysate preparations from hemin-treated cells, Shu1-Nbr1 and Shu1-Hse1 complexes are detected by coimmunoprecipitation experiments. Further analysis by immunofluorescence microscopy shows that Shu1 is unable to reach vacuoles of hemin-treated cells harboring a deletion for one of the following genes: ubi4+ , nbr1+ , hse1+ and sst6+ . Together, these results reveal that hemin-mediated vacuolar targeting of Shu1 requires Ubi4-dependent ubiquitination, the receptor Nbr1 and the ESCRT proteins Hse1 and Sst6.

The ESCRT pathway, comprising the in sequence acting ESCRT-0, -I, -II, -III and Vps4 complexes, conducts the abscission of membranes away from the cytosol. Whereas the components of the central ESCRT-III core complex have been thoroughly investigated, the function of the components of the associated two auxiliary ESCRT sub-complexes are not well-understood in metazoans, especially at the organismal level. We here present the developmental analysis of the Drosophila orthologs of the auxiliary ESCRTs Chmp5 and Ist1, DChmp5 and DIst1, which belong to the two auxiliary sub-complexes. While each single null mutant displayed mild defects in development, the Dist1 Dchmp5 double mutant displayed a severe defect, indicating that the two genes act synergistically, but in separate pathways. Moreover, the presented results indicate that the auxiliary ESCRTs provide robustness against cold during development of diverse poikilothermic organisms, probably by preventing the accumulation of the ESCRT-III core component Shrub on the endosomal membrane., Summary: The analysis of Chmp5 and Ist1, which belong to the two ESCRT auxiliary sub-complexes in Drosophila, suggests that these ESCRT proteins provide robustness against cold in diverse poikilothermic organisms.

The Herpesviridae are structurally complex DNA viruses whose capsids undergo primary envelopment at the inner nuclear membrane and secondary envelopment at organelles in the cytoplasm. In both locations, there is evidence that envelope formation and scission involve the participation of multiple viral proteins and also the cellular ESCRT apparatus. It nevertheless appears that the best-understood viral strategies for ESCRT recruitment, those adopted by the retroviruses and many other families of enveloped RNA viruses, are not utilized by the Herpesviridae, at least during envelopment in the cytoplasm. Thus, although a large number of herpesvirus proteins have been assigned roles in envelopment, there is a dearth of candidates for the acquisition of the ESCRT complex and the control of envelope scission. This review summarizes our current understanding of ESCRT association by enveloped viruses, examines what is known of herpesvirus ESCRT utilization in the nucleus and cytoplasm, and identifies candidate cellular and viral proteins that could link enveloping herpesviruses to cellular ESCRT components.

Multivesicular bodies (MVBs) are critical intermediates in the trafficking of ubiquitinated endocytosed surface proteins to the lysosome/vacuole for destruction. Recognizing and packaging ubiquitin modified cargoes to the MVB pathway require ESCRT (Endosomal sorting complexes required for transport) machinery, which consists of four core subcomplexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. Our previous results showed that ESCRT-0 is essential for fungal development and pathogenicity in Fusarium graminearum. We then, in this study, systemically studied the protein-protein interactions within F. graminearum ESCRT-I, -II or -III complex, as well as between ESCRT-0 and ESCRT-I, ESCRT-I and ESCRT-II, and ESCRT-II and ESCRT-III complexes and found that loss of any ESCRT component resulted in abnormal function in endocytosis. In addition, ESCRT deletion mutants displayed severe defects in growth, deoxynivalenol (DON) production, virulence, sexual, and asexual reproduction. Importantly genetic complementation with corresponding ESCRT genes fully rescued all these defective phenotypes, indicating the essential role of ESCRT machinery in fungal development and plant infection in F. graminearum. Taken together, the protein-protein interactome and biological functions of the ESCRT machinery is first profoundly characterized in F. graminearum, providing a foundation for further exploration of ESCRT machinery in filamentous fungi.

Our fundamental understanding of the roles played by the endosomal sorting complex required for transport (ESCRT) machinery in cells comes from interdisciplinary approaches that combine numerous in vivo and in vitro techniques. Here, we focus on methods used to biochemically characterize Caenorhabditis elegans ESCRT components in vitro, including the production and characterization of recombinant ESCRT complexes and their use in membrane interaction studies. Key methodologies used include gel filtration chromatography, glycerol density gradient analysis, multi-angle light scattering, liposome co-flotation, and single-liposome fluorescence microscopy. Collectively, these studies have enabled us to define subunit stoichiometry of soluble C. elegans ESCRT complexes and to demonstrate that the late-acting ESCRT-III complex facilitates membrane bending and remodeling, at least in part by virtue of its ability to sense the curvature of lipid bilayers.

Ubiquitinated membrane proteins such as epidermal growth factor receptor (EGFR) are delivered to early endosomes and then sorted to lysosomes via multivesicular bodies (MVBs) for degradation. The regulatory mechanism underlying formation of intralumenal vesicles en route to generation of MVBs is not fully understood. In this study, we found that SH3YL1, a phosphoinositide-binding protein, had a vesicular localization pattern overlapping with internalized EGF in endosomes in the degradative pathway. Deficiency of SH3YL1 prevents EGF trafficking from early to late endosomes and inhibits degradation of EGFR. Moreover, we show that SH3YL1 mediates EGFR sorting into MVBs in a manner dependent on its carboxy-terminal SH3 domain, which is necessary for the interaction with an ESCRT-I component, Vps37B. Taken together, our observations reveal an indispensable role of SH3YL1 in MVB-sorting and EGFR degradation mediated by ESCRT complexes.

The progressive deposition of misfolded and aggregated forms of Tau protein in the brain is a pathological hallmark of tauopathies, such as Alzheimer's disease (AD) and frontotemporal degeneration (FTD). The misfolded Tau can be released into the extracellular space and internalized by neighboring cells, acting as seeds to trigger the robust conversion of soluble Tau into insoluble filamentous aggregates in a prion-like manner, ultimately contributing to the progression of the disease. However, molecular mechanisms accountable for the propagation of Tau pathology are poorly defined. We reviewed the Tau processing imbalance in endosomal, lysosomal, and exosomal pathways in AD. Increased exosome release counteracts the endosomal-lysosomal dysfunction of Tau processing but increases the number of aggregates and the propagation of Tau. This review summarizes our current understanding of the underlying tauopathy mechanisms with an emphasis on the emerging role of the endosomal-lysosomal-exosome pathways in this process. The components CHMP6, TSG101, and other components of the ESCRT complex, as well as Rab GTPase such as Rab35 and Rab7A, regulate vesicle cargoes routing from endosome to lysosome and affect Tau traffic, degradation, or secretion. Thus, the significant molecular pathways that should be potential therapeutic targets for treating tauopathies are determined.

Extracellular vesicles are novel mediators of cell-cell communication. They are present in all species and involved in physiological and pathological processes. One class of extracellular vesicles, the exosomes, originate from an endosomal compartment, the MultiVesicular Body (MVB), and are released from the cell upon fusion of the MVB with the plasma membrane. Although different molecular mechanisms have been associated with MVB biogenesis and exosome secretion, how they coordinate remains poorly documented. We recently found that the small GTPase Ral contributes to exosome release in nematodes and mammalian tumor cells. More specifically, we found that C. elegans RAL-1 is required for the biogenesis of MVBs, and later for MVB fusion with the plasma membrane. Here, we discuss our results in relationship with other factors involved in extracellular vesicle production such as the ESCRT complex and Phospholipase 1D. We propose models to explain Ral function in exosome secretion, its conservation in animals, and its possible role in tumor progression.

UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of tegument, the complex protein layer between the capsid and envelope. UL36p performs multiple functions in the HSV life cycle, including a critical but unknown role in capsid cytoplasmic envelopment. We tested whether UL36p is essential for envelopment because it is required to engage capsids with the cellular ESCRT/Vps4 apparatus. A green fluorescent protein (GFP)-fused form of the dominant negative ATPase Vps4-EQ was used to irreversibly tag ESCRT envelopment sites during infection by UL36p-expressing and UL36-null HSV strains. Using fluorescence microscopy and scanning electron microscopy, we quantitated capsid/Vps4-EQ colocalization and examined the ultrastructure of the corresponding viral assembly intermediates. We found that loss of UL36p resulted in a two-thirds reduction in the efficiency of capsid/Vps4-EQ association but that the remaining UL36p-null capsids were still able to engage the ESCRT envelopment apparatus. It appears that although UL36p helps to couple HSV capsids to the ESCRT pathway, this is likely not the sole reason for its absolute requirement for envelopment. IMPORTANCE Envelopment of the HSV capsid is essential for the assembly of an infectious virion and requires the complex interplay of a large number of viral and cellular proteins. Critical to envelope assembly is the virally encoded protein UL36p, whose function is unknown. Here we test the hypothesis that UL36p is essential for the recruitment of cellular ESCRT complexes, which are also known to be required for envelopment.

Exosomes, extracellular vesicles with diameters ranging from 30 to 150 nm, are widely present in various body fluids. Recently, microRNAs (miRNAs) have been identified in exosomes, the biogenesis, release, and uptake of which may involve the endosomal sorting complex required for transport (ESCRT complex) and relevant proteins. After release, exosomes are taken up by neighboring or distant cells, and the miRNAs contained within modulate such processes as interfering with tumor immunity and the microenvironment, possibly facilitating tumor growth, invasion, metastasis, angiogenesis and drug resistance. Therefore, exosomal miRNAs have a significant function in regulating cancer progression. Here, we briefly review recent findings regarding tumor-derived exosomes, including RNA sorting and delivering mechanism. We then describe the intercommunication occurring between different cells via exosomal miRNAs in tumor microenvironmnt, with impacts on tumor proliferation, vascularization, metastasis and other biological characteristics. Finally, we highlight the potential role of these molecules as biomarkers in cancer diagnosis and prognosis and tumor resistance to therapeutics.

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.

Since its discovery more than 50 years ago, the endo-lysosomal system has emerged as a central integrator of different cellular activities. This vesicular trafficking apparatus governs processes as diverse as the transduction of stimuli by growth factor receptors, the recycling and secretion of signaling molecules and the regulation of cellular homeostasis through autophagy. Accordingly, dysfunctions of the vesicular transport machinery have been linked to a growing number of pathologies. In this review we take the "Endosomal Sorting Complex Required for Transport" (ESCRT) as an example to illustrate the multiple functions of an evolutionarily conserved endosomal transport machinery. We describe the major concepts that have emerged from the study of this machinery at the level of the development and the physiology of multi-cellular organisms. In particular, we highlight the essential contributions of ESCRT proteins on the regulation of three biological processes: the endocytic regulation of cell signaling, autophagy and its role in neuronal morphogenesis and finally the biogenesis and function of extracellular vesicles.

The endosomal sorting complex required for transport (ESCRT) machinery is necessary for budding of many enveloped viruses. Recently, it was demonstrated that Vps4, the key regulator for recycling of the ESCRT-III complex, is required for efficient infection by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, ESCRT assembly, regulation, and function are complex, and little is known regarding the details of participation of specific ESCRT complexes in AcMNPV infection. In this study, the core components of ESCRT-I (Tsg101 and Vps28) and ESCRT-III (Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60) were cloned from Spodoptera frugiperda . Using a viral complementation system and RNA interference (RNAi) assays, we found that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. In cells knocking down or overexpressing dominant negative (DN) forms of the components of ESCRT-I and ESCRT-III complexes, entering virions were partially trapped within the cytosol. To examine only egress, cells were transfected with the double-stranded RNA (dsRNA) targeting an individual ESCRT-I or ESCRT-III gene and viral bacmid DNA or viral bacmid DNA that expressed DN forms of ESCRT-I and ESCRT-III components. We found that ESCRT-III components (but not ESCRT-I components) are required for efficient nuclear egress of progeny nucleocapsids. In addition, we found that several baculovirus core or conserved proteins (Ac11, Ac76, Ac78, GP41, Ac93, Ac103, Ac142, and Ac146) interact with Vps4 and components of ESCRT-III. We propose that these viral proteins may form an “egress complex” that is involved in recruiting ESCRT-III components to a virus egress domain on the nuclear membrane. IMPORTANCE The ESCRT system is hijacked by many enveloped viruses to mediate budding and release. Recently, it was found that Vps4, the key regulator of the cellular ESCRT machinery, is necessary for efficient entry and egress of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, little is known about the roles of specific ESCRT complexes in AcMNPV infection. In this study, we demonstrated that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. The components of ESCRT-III (but not ESCRT-I) are also necessary for efficient nuclear egress of progeny nucleocapsids. Several baculovirus core or conserved proteins were found to interact with Vps4 and components of ESCRT-III, and these interactions may suggest the formation of an “egress complex” involved in the nuclear release or transport of viral nucleocapsids.

The endosomal sorting complex required for transport (ESCRT) is made of subcomplexes (ESCRT 0-III), crucial to membrane remodelling at endosomes, nuclear envelope and cell surface. ESCRT-III shapes membranes and in most cases cooperates with the ATPase VPS4 to mediate fission of membrane necks from the inside. The first ESCRT complexes mainly serve to catalyse the formation of ESCRT-III but can be bypassed by accessory proteins like the Alg-2 interacting protein-X (ALIX). In the nervous system, ALIX/ESCRT controls the survival of embryonic neural progenitors and later on the outgrowth and pruning of axons and dendrites, all necessary steps to establish a functional brain. In the adult brain, ESCRTs allow the endosomal turn over of synaptic vesicle proteins while stable ESCRT complexes might serve as scaffolds for the postsynaptic parts. The necessity of ESCRT for the harmonious function of the brain has its pathological counterpart, the mutations in CHMP2B of ESCRT-III giving rise to several neurodegenerative diseases.

The endosomal sorting complex required for transport (ESCRT) machinery is an ancient system that deforms membrane and severs membrane necks from the inside. Extensive evidence has accumulated to demonstrate the conserved functions of plant ESCRTs in multivesicular body (MVB) biogenesis and MVB-mediated membrane protein sorting. In addition, recent exciting findings have uncovered unique plant ESCRT components and point to emerging roles for plant ESCRTs in non-endosomal sorting events such as autophagy, cytokinesis, and viral replication. Plant-specific processes, such as abscisic acid (ABA) signaling and chloroplast turnover, provide further evidence for divergences in the functions of plant ESCRTs during evolution. We summarize the multiple roles and current working models for plant ESCRT machinery and speculate on future ESCRT studies in the plant field.

Long interspersed nuclear element-1s (LINE-1s, or L1s) are an active family of retrotransposable elements that continue to mutate mammalian genomes. Despite the large contribution of L1 to mammalian genome evolution, we do not know where active L1 particles (particles in the process of retrotransposition) are located in the cell, or how they move towards the nucleus, the site of L1 reverse transcription. Using a yeast model of LINE retrotransposition, we identified ESCRT (endosomal sorting complex required for transport) as a critical complex for LINE retrotransposition, and verified that this interaction is conserved for human L1. ESCRT interacts with L1 via a late domain motif, and this interaction facilitates L1 replication. Loss of the L1/ESCRT interaction does not impair RNP formation or enzymatic activity, but leads to loss of retrotransposition and reduced L1 endonuclease activity in the nucleus. This study highlights the importance of the ESCRT complex in the L1 life cycle and suggests an unusual mode for L1 RNP trafficking., Author summary Long interspersed nuclear elements (LINEs) are a class of retrotransposable elements that mutate mammalian genomes. LINEs have been highly successful in the human genome, multiplying to over 800,000 copies. The LINE-encoded replication machinery is also used by other retrotransposons, and in total, has been responsible for the generation of over 1/3 of human DNA sequence. To replicate, a LINE mRNA forms a ribonucleoprotein particle (RNP) with its proteins. This RNP eventually enters the nucleus to integrate a cDNA copy of itself into chromosomes. The events between RNP formation and successful integration are difficult to study and largely unknown. Here we show that the ESCRT complex plays a conserved role in LINE retrotransposition in both yeast and humans. ESCRT is a membrane budding complex involved in cellular trafficking and membrane budding/fusion. Our results imply that membranes play an integral part of LINE replication, and ESCRT may be required for RNP trafficking towards the nucleus.

Endosomal sorting complex required for transport (ESCRT) is involved in membrane protein deg- radation through the recognition and sorting of ubiquity- lated cargo proteins into the multivesicular body before fusion with the lysosome/vacuole. However, recent studies have challenged this canonical cellular function of ESCRT and have implicated a role for this machinery in multiple intracellular pathways. Here, we provide evidence that ESCRT complexes contribute to the regulation of tran- scription elongation in Saccharomyces cerevisiae. Most strains deficient in each subunit of ESCRT-0, -I, -II, and - III showed significant sensitivity to 6-azauracil or myco- phenolic acid, a phenotype associated with transcription elongation defects. Moreover, these deletion strains sig- nificantly reduced transcription activation through Gcn4, a regulator of the general amino acid control. The tran- scription factor Rim101, which is proteolytically activated through the multimerized component Vps32/Snf7 of ESCRT-III and its collaborative proteins, was not associ- ated with transcription elongation or Gcn4 activation. In addition, we observed that ESCRT complexes were crosslinked at the 3 0 region of the coding sequence in the actively transcribed gene. In summary, these results sug- gest that ESCRT complexes promote genes transcription during the late stages of elongation and are required for transcription activation through Gcn4.

Eukaryotic chromosomal ends are protected by telomeres from fusion, degradation, and unwanted double-strand break repair events. Therefore, telomeres preserve genome stability and integrity. Telomere length can be maintained by telomerase, which is expressed in most human primary tumors but is not expressed in the majority of somatic cells. Thus, telomerase may be a highly relevant anticancer drug target. Genome-wide studies in the yeast Saccharomyces cerevisiae identified a set of genes associated with telomere length maintenance (TLM genes). Among the tlm mutants with short telomeres, we found a strong enrichment for those affecting vacuolar and endosomal traffic (particularly the endosomal sorting complex required for transport [ESCRT] pathway). Here, we present our results from investigating the surprising link between telomere shortening and the ESCRT machinery. Our data show that the whole ESCRT system is required to safeguard proper telomere length maintenance. We propose a model of impaired end resection resulting in too little telomeric overhang, such that Cdc13 binding is prevented, precluding either telomerase recruitment or telomeric overhang protection., IMPORTANCE Telomeres are the ends of eukaryotic chromosomes. They are necessary for the proper replication of the genome and protect the chromosomes from degradation. In a large-scale systematic screen for mutants that affect telomere length in yeast, we found that mutations in any of the genes encoding the ESCRT complexes, required for the formation of transport vesicles within the cell, cause telomere shortening. We carried out an analysis of the mechanisms disrupted in these mutants and found that they are defective for the ability to elongate short telomeres, probably due to faulty end processing. We discuss the significance of these findings and how they could be relevant to anticancer therapies.

Exosomes are a particular type of extracellular vesicle, characterized by their endosomal origin as intraluminal vesicles present in large endosomes with a multivesicular structure. After these endosomes fuse with the plasma membrane, exosomes are secreted into the extracellular space. The ability of exosomes to carry and selectively deliver bioactive molecules (e.g., lipids, proteins, and nucleic acids) confers on them the capacity to modulate the activity of receptor cells, even if these cells are located in distant tissues or organs. Since exosomal cargo depends on cell type, a detailed understanding of the mechanisms that regulate the biochemical composition of exosomes is fundamental to a comprehensive view of exosome function. Here, we review the latest advances concerning exosome function and biogenesis in T cells, with particular focus on the mechanism of protein sorting at multivesicular endosomes. Exosomes secreted by specific T-cell subsets can modulate the activity of immune cells, including other T-cell subsets. Ceramide, tetraspanins and MAL have been revealed to be important in exosome biogenesis by T cells. These molecules, therefore, constitute potential molecular targets for artificially modulating exosome production and, hence, the immune response for therapeutic purposes., Research in the laboratory of MA is supported by grants (BFU2012-32532 and BFU2015-67266-R) from the Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional (MINECO/FEDER).

Members of the crenarchaeal kingdom, such as Sulfolobus, divide by binary fission yet lack genes for the otherwise near-ubiquitous tubulin and actin superfamilies of cytoskeletal proteins. Recent work has established that Sulfolobus homologs of the eukaryotic ESCRT-III and Vps4 components of the ESCRT machinery play an important role in Sulfolobus cell division. In eukaryotes, several pathways recruit ESCRT-III proteins to their sites of action. However, the positioning determinants for archaeal ESCRT-III are not known. Here, we identify a protein, CdvA, that is responsible for recruiting Sulfolobus ESCRT-III to membranes. Overexpression of the isolated ESCRT-III domain that interacts with CdvA results in the generation of nucleoid-free cells. Furthermore, CdvA and ESCRT-III synergize to deform archaeal membranes in vitro. The structure of the CdvA/ESCRT-III interface gives insight into the evolution of the more complex and modular eukaryotic ESCRT complex.

EGFR is a key player in cell proliferation and survival signaling, and its sorting into MVBs for eventual lysosomal degradation is controlled by the coordination of multiple ESCRT complexes on the endosomal membrane. HD-PTP is a cytosolic protein tyrosine phosphatase, and is associated with EGFR trafficking by interacting with the ESCRT-0 protein STAM2 and the ESCRT-III protein CHMP4B via its N-terminal Bro1 domain. Intriguingly, the homologous domain of two other human Bro1 domain-containing proteins, Alix and Brox, binds CHMP4B but not STAM2, despite their high structural similarity. To elucidate this binding specificity, we determined the complex structure of the HD-PTP Bro1 domain bound to the STAM2 core region. STAM2 binds to the hydrophobic concave pocket of the HD-PTP Bro1 domain, as CHMP4B does to the pocket of Alix, Brox, or HD-PTP but in the opposite direction. Critically, Thr145 of HD-PTP, corresponding to Lys151 of Alix and Arg145 of Brox, is revealed to be a determinant residue enabling this protein to bind STAM2, as the Alix- or Brox-mimicking mutations of this residue blocks the intermolecular interaction. This work therefore provides the structural basis for how HD-PTP recognizes the ESCRT-0 component to control EGFR sorting.

The sarcoplasmic reticulum is a network of tubules and cisternae localized in close association with the contractile apparatus, and regulates Ca(2+)dynamics within striated muscle cell. The sarcoplasmic reticulum maintains its shape and organization despite repeated muscle cell contractions, through mechanisms which are still under investigation. The ESCRT complexes are essential to organize membrane subdomains and modify membrane topology in multiple cellular processes. Here, we report for the first time that ESCRT-II proteins play a role in the maintenance of sarcoplasmic reticulum integrity inC. elegans ESCRT-II proteins colocalize with the sarcoplasmic reticulum marker ryanodine receptor UNC-68. The localization at the sarcoplasmic reticulum of ESCRT-II and UNC-68 are mutually dependent. Furthermore, the characterization of ESCRT-II mutants revealed a fragmentation of the sarcoplasmic reticulum network, associated with an alteration of Ca(2+)dynamics. Our data provide evidence that ESCRT-II proteins are involved in sarcoplasmic reticulum shaping.

Most membrane-enveloped viruses depend on host proteins of the endosomal sorting complex required for transport (ESCRT) machinery for their release. HIV-1 is the prototypic ESCRT-dependent virus. The direct interactions between HIV-1 and the early ESCRT factors TSG101 and ALIX have been mapped in detail. However, the full pathway of ESCRT recruitment to HIV-1 budding sites, which culminates with the assembly of the late-acting CHMP4, CHMP3, CHMP2, and CHMP1 subunits, is less completely understood. Here, we report the biochemical reconstitution of ESCRT recruitment to viral assembly sites, using purified proteins and giant unilamellar vesicles. The myristylated full-length Gag protein of HIV-1 was purified to monodispersity. Myr-Gag forms clusters on giant unilamellar vesicle membranes containing the plasma membrane lipid PI(4,5)P 2 . These Gag clusters package a fluorescent oligonucleotide, and recruit early ESCRT complexes ESCRT-I or ALIX with the appropriate dependence on the Gag PTAP and LYP(X) n L motifs. ALIX directly recruits the key ESCRT-III subunit CHMP4. ESCRT-I can only recruit CHMP4 when ESCRT-II and CHMP6 are present as intermediary factors. Downstream of CHMP4, CHMP3 and CHMP2 assemble synergistically, with the presence of both subunits required for efficient recruitment. The very late-acting factor CHMP1 is not recruited unless the pathway is completed through CHMP3 and CHMP2. These findings define the minimal sets of components needed to complete ESCRT assembly at HIV-1 budding sites, and provide a starting point for in vitro structural and biophysical dissection of the system.

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.

The biogenesis of multivesicular endosomes and the sorting of activated signaling receptors into multivesicular endosomes depend on soluble protein complexes (ESCRT complexes) which transiently interact with the receptor cargo and the endosomal membrane. Previously it was shown that the transmembrane protein secretory carrier membrane protein (SCAMP) 3 which is present on endosomes interacts with ESCRT components. Here we report that SCAMP3 plays a role in the biogenesis of multivesicular endosomes. We find that SCAMP3 plays a role in EGF receptor sorting into multivesicular endosomes and in the formation of intralumenal vesicles within these endosomes in vitro and thus also controls EGF receptor targeting to lysosomes. We also find that SCAMP3 regulates the EGF dependent biogenesis of multivesicular endosomes. We conclude that the transmembrane protein SCAMP3 has a positive role in sorting into and budding of intralumenal vesicles and thereby controls the process of multivesicular endosome biogenesis. © 2011 John Wiley Sons A/S.

In 1999, mutations in the gene encoding the microtubule severing AAA ATPase spastin were identified as a major cause of a genetic neurodegenerative condition termed hereditary spastic paraplegia (HSP). This finding stimulated intense study of the spastin protein and over the last decade, a combination of cell biological, in vivo, in vitro and structural studies have provided important mechanistic insights into the cellular functions of the protein, as well as elucidating cell biological pathways that might be involved in axonal maintenance and degeneration. Roles for spastin have emerged in shaping the endoplasmic reticulum and the abscission stage of cytokinesis, in which spastin appears to couple membrane modelling to microtubule regulation by severing. This article is part of a Special Issue entitled: AAA ATPases: structure and function.

The terminal stage of cytokinesis – a process termed abscission – is the severing of the thin intercellular bridge that connects the two daughter cells. Recent work provides new insight into the mechanism by which this microtubule-dense membrane bridge is resolved, and highlights important roles for multi-protein assemblies in different facets of abscission. These include the endosomal sorting complex required for transport (ESCRT), which appears to have a decisive role in the final scission event, and vesicle tethering complexes, which potentially act at an earlier stage, and might serve to prepare the abscission site. Here, we review recent studies of the structure, function and regulation of these complexes as related to abscission. We focus largely on studies of cytokinesis in mammalian cells. However, cell division in other systems, such as plants and Archae, is also considered, reflecting the mechanistic conservation of membrane-scission processes during cell division.

Structural remodeling of synapses in response to growth signals leads to long-lasting alterations in neuronal function in many systems. Synaptic growth factor receptors alter their signaling properties during transit through the endocytic pathway, but the mechanisms controlling cargo traffic between endocytic compartments remain unclear. Nwk (Nervous Wreck) is a presynaptic F-BAR/SH3 protein that regulates synaptic growth signaling in Drosophila melanogaster. In this paper, we show that Nwk acts through a physical interaction with sorting nexin 16 (SNX16). SNX16 promotes synaptic growth signaling by activated bone morphogenic protein receptors, and live imaging in neurons reveals that SNX16-positive early endosomes undergo transient interactions with Nwk-containing recycling endosomes. We identify an alternative signal termination pathway in the absence of Snx16 that is controlled by endosomal sorting complex required for transport (ESCRT)–mediated internalization of receptors into the endosomal lumen. Our results define a presynaptic trafficking pathway mediated by SNX16, NWK, and the ESCRT complex that functions to control synaptic growth signaling at the interface between endosomal compartments., Damon Runyon Cancer Research Foundation (Postdoctoral grant), Charles A. King Trust Postdoctoral Fellowship Program, National Institute of Neurological Disorders and Stroke (U.S.) (K99/R00 award, K99NS060947), National Institutes of Health (U.S.) (award NS43244)

ESCRT (endosomal sorting complex required for transport) proteins were originally identified for their role in delivering endocytosed proteins to the intraluminal vesicles of late-endosomal structures termed multivesicular bodies. Multivesicular bodies then fuse with lysosomes, leading to degradation of the internalized proteins. Four ESCRT complexes interact to concentrate cargo on the endosomal membrane, induce membrane curvature to form an intraluminal bud and finally pinch off the bud through a membrane-scission event to produce the intraluminal vesicle. Recent work suggests that ESCRT proteins are also required downstream of these events to enable fusion of multivesicular bodies with lysosomes. Autophagy is a related pathway required for the degradation of organelles, long-lived proteins and protein aggregates which also converges on lysosomes. The proteins or organelle to be degraded are encapsulated by an autophagosome that fuses either directly with a lysosome or with an endosome to form an amphisome, which then fuses with a lysosome. A common machinery is beginning to emerge that regulates fusion events in the multivesicular body and autophagy pathways, and we focus in the present paper on the role of ESCRT proteins. These fusion events have been implicated in diseases including frontotemporal dementia, Alzheimer's disease, lysosomal storage disorders, myopathies and bacterial pathogen invasion, and therefore further examination of the mechanisms involved may lead to new insight into disease pathogenesis and treatments.

The process of budding of many enveloped viruses utilizes the cellular ESCRT (endosomal sorting complex required for transport) machinery, that is normally involved in the formation of luminal vesicles of endosomal multivesiculate bodies (MVB). A late step in the MVB pathway involves the recruitment of VPS4, an AAA+ ATPase, to the ESCRT complexes. Our earlier work had shown that the formation of influenza virus-like particles was not inhibited by dominant negative VPS4A. However, it was not known if there was a role of VPS4 and the ESCRT pathway in influenza virus particle budding and this needed to be investigated. It was found that neither siRNA knockdown of VPS4A and VPS4B expression nor the use of cell lines that inducibly express VPS4A or VPS4B dominant negative mutants, inhibited influenza virus budding. In contrast, and in keeping with more recent data, vesicular stomatitis virus budding was diminished by VPS4 dysfunction.

The endosomal sorting complexes required for transport (ESCRTs) catalyse one of the most unusual membrane remodelling events in cell biology. ESCRT-I and ESCRT-II direct membrane budding away from the cytosol by stabilizing bud necks without coating the buds and without being consumed in the buds. ESCRT-III cleaves the bud necks from their cytosolic faces. ESCRT-III-mediated membrane neck cleavage is crucial for many processes, including the biogenesis of multivesicular bodies, viral budding, cytokinesis and, probably, autophagy. Recent studies of ultrastructures induced by ESCRT-III overexpression in cells and the in vitro reconstitution of the budding and scission reactions have led to breakthroughs in understanding these remarkable membrane reactions.

Ubiquitin (Ub) sorting receptors facilitate the targeting of ubiquitinated membrane proteins into multivesicular bodies (MVBs). Ub-binding domains (UBDs) have been described in several endosomal sorting complexes required for transport (ESCRT). Using available structural information, we have investigated the role of the multiple UBDs within ESCRTs during MVB cargo selection. We found a novel UBD within ESCRT-I and show that it contributes to MVB sorting in concert with the known UBDs within the ESCRT complexes. These experiments reveal an unexpected level of coordination among the ESCRT UBDs, suggesting that they collectively recognize a diverse set of cargo rather than act sequentially at discrete steps.