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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.

Upon starvation, cells rewire their metabolism, switching from glucose-based metabolism to mitochondrial oxidation of fatty acids, which require the transfer of FAs from lipid droplets (LDs) to mitochondria at mitochondria−LD membrane contact sites (MCSs). However, factors responsible for FA transfer at these MCSs remain uncharacterized. Here, we demonstrate that vacuolar protein sorting-associated protein 13D (VPS13D), loss-of-function mutations of which cause spastic ataxia, coordinates FA trafficking in conjunction with the endosomal sorting complex required for transport (ESCRT) protein tumor susceptibility 101 (TSG101). The VPS13 adaptor-binding domain of VPS13D and TSG101 directly remodels LD membranes in a cooperative manner. The lipid transfer domain of human VPS13D binds glycerophospholipids and FAs in vitro. Depletion of VPS13D, TSG101, or ESCRT-III proteins inhibits FA trafficking from LDs to mitochondria. Our findings suggest that VPS13D mediates the ESCRT-dependent remodeling of LD membranes to facilitate FA transfer at mitochondria-LD contacts., Metabolic rewiring requires the mobilization of fatty acids (FA) from lipid droplets (LDs) at membrane contact sites (MCSs), although the details of FA transfer remain unclear. Here, the authors show that VPS13D and the ESCRT complex remodel LD membranes to promote FA trafficking to mitochondria.

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.

Multivesicular body (MVB)-mediated endosomal sorting and macroautophagy are the main pathways mediating the transport of cellular components to the vacuole and are essential for maintaining cellular homeostasis. The interplay of these two pathways remains poorly understood in plants. In this study, we show that FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1 (FREE1), which was previously identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT), essential for MVB biogenesis and plant growth, can be transported to the vacuole for degradation in response to iron deficiency. The vacuolar transport of ubiquitinated FREE1 protein is mediated by the autophagy pathway. As a consequence, the autophagy deficient mutants, atg5-1 and atg7-2, accumulate more endogenous FREE1 protein and display hypersensitivity to iron deficiency. Furthermore, under iron-deficient growth condition autophagy related genes are upregulated to promote the autophagic degradation of FREE1, thereby possibly relieving the repressive effect of FREE1 on iron absorption. Collectively, our findings demonstrate a unique regulatory mode of protein turnover of the ESCRT machinery through the autophagy pathway to respond to iron deficiency in plants.

Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of proteins residing in endosomal membranes. Recent studies revealed that yeast ESCRT machinery also sorts ubiquitinated proteins from the vacuolar membrane for degradation in the vacuole lumen. However, whether mammalian ESCRTs perform a similar function at lysosomes remained unknown. Here, we show that ESCRT-I restricts the size of lysosomes and promotes degradation of proteins from lysosomal membranes, including MCOLN1, a Ca2+ channel protein. Upon ESCRT-I depletion, the lysosomal accumulation of non-degraded proteins coincided with elevated expression of genes annotated to cholesterol biosynthesis and biogenesis of lysosomes, indicative of response to lysosomal stress. Accordingly, the lack of ESCRT-I promoted abnormal cholesterol accumulation in lysosomes and activated TFEB/TFE3 transcription factors. Finally, we discovered that in contrast to basal TFEB/TFE3 signaling that depended on the availability of exogenous lipids, the stress-induced activation of this pathway was Ca2+-MCOLN1-dependent. Hence, we provide evidence that ESCRT-I is crucial for maintaining lysosomal homeostasis and we elucidate mechanisms distinguishing basal from lysosomal stress-induced TFEB/TFE3 signaling.

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.

Macroautophagic clearance of cytosolic materials entails the initiation, growth and closure of autophagosomes. Cargo triggers the assembly of a web of cargo receptors and core machinery. Autophagy-related protein 9 (ATG9) vesicles seed the growing autophagosomal membrane, which is supplied by de novo phospholipid synthesis, phospholipid transport via ATG2 proteins and lipid flipping by ATG9. Autophagosomes close via ESCRT complexes. Here, we review recent discoveries that illuminate the molecular mechanisms of autophagosome formation and discuss emerging questions in this rapidly developing field. In this Review, Hurley and colleagues cover the most recent discoveries and the emerging molecular understanding of the mechanisms of autophagosome formation.

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.

Exosomes are secreted nanovesicles with potent signalling activity that are initially formed as intraluminal vesicles (ILVs) in multivesicular endosomes, which subsequently fuse with the plasma membrane. These ILVs are made in both late endosomes and recycling endosomes, the latter marked by the small GTPase Rab11 and generating exosomes with different cargos and functions. Core proteins within four Endosomal Sorting Complex Required for Transport (ESCRT) assemblies (0-III) play key sequential roles in late endosomal exosome biogenesis and ILV-mediated destruction of ubiquitinylated cargos through the endolysosomal system. They also control additional cellular processes, such as cytokinesis and other vesicle budding. By contrast, the functions of several accessory ESCRTs are not well defined. Here we assess the ESCRT-dependency of Rab11-exosomes, using RNA knockdown inDrosophilasecondary cells (SCs) of the male accessory gland, which have unusually enlarged Rab11-positive compartments. Unexpectedly, not only are core proteins in all four ESCRT complexes required for Rab11-exosome formation, but also accessory ESCRT-III proteins, CHMP1, CHMP5 and IST1. Suppressing expression of these accessory proteins does not affect other aspects of cell morphology, unlike most core ESCRT knockdowns, and does not lead to accumulation of ubiquitinylated cargos. We conclude that accessory ESCRT-III components have a specific and potentially ubiquitin-independent role in Rab11-exosome generation, which might provide a target for blocking the pro-tumorigenic activities of these vesicles in cancer.

The ESCRT complexes drive membrane scission in HIV-1 release, autophagosome closure, MVB biogenesis, cytokinesis, and other cell processes. ESCRT-I is the most upstream complex and bridges the system to HIV-1 Gag in virus release. The crystal structure of the headpiece of human ESCRT-I comprising TSG101–VPS28–VPS37B–MVB12A was determined, revealing an ESCRT-I helical assembly with a 12 molecule repeat. Electron microscopy confirmed that ESCRT-I subcomplexes form helical filaments in solution. Mutation of VPS28 helical interface residues blocks filament formation in vitro and autophagosome closure and HIV-1 release in human cells. Coarse grained simulations of ESCRT assembly at HIV-1 budding sites suggest that formation of a 12-membered ring of ESCRT-I molecules is a geometry-dependent checkpoint during late stages of Gag assembly and HIV-1 budding, and templates ESCRT-III assembly for membrane scission. These data show that ESCRT-I is not merely a bridging adaptor, but has an essential scaffolding and mechanical role in its own right., Reporting Summary Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.

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 endosomal sorting complex required for transport (ESCRT) organises in supramolecular structures on the surface of lipid bilayers to drive membrane invagination and scission of intraluminal vesicles (ILVs), a process also controlled by membrane mechanics. However, ESCRT association with the membrane is also mediated by electrostatic interactions with anionic phospholipids. Phospholipid distribution within natural biomembranes is inhomogeneous due to, for example, the formation of lipid rafts and curvature-driven lipid sorting. Here, we have used phase-separated giant unilamellar vesicles (GUVs) to investigate the link between phosphatidylserine (PS)-rich lipid domains and ESCRT activity. We employ GUVs composed of phase separating lipid mixtures, where unsaturated DOPS and saturated DPPS lipids are incorporated individually or simultaneously to enhance PS localisation in liquid disordered (Ld) and/or liquid ordered (Lo) domains, respectively. PS partitioning between the coexisting phases is confirmed by a fluorescent Annexin V probe. Ultimately, we find that ILV generation promoted by ESCRTs is significantly enhanced when PS lipids localise within Ld domains. However, the ILVs that form are rich in Lo lipids. We interpret this surprising observation as preferential recruitment of the Lo phase beneath the ESCRT complex due to its increased rigidity, where the Ld phase is favoured in the neck of the resultant buds to facilitate the high membrane curvature in these regions of the membrane during the ILV formation process. Ld domains offer lower resistance to membrane bending, demonstrating a mechanism by which the composition and mechanics of membranes can be coupled to regulate the location and efficiency of ESCRT activity.

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.

To complete mitosis, the intercellular bridge that links daughter cells needs to be cleaved. This abscission step is carried out by the sequential recruitment of ESCRT proteins at the midbody. We report here that a new factor, named AKTIP, works in association with ESCRTs. We find that AKTIP binds to the ESCRT I subunit VPS28, and show by high resolution microscopy that AKTIP forms a ring in the dark zone of the intercellular bridge. This ring is positioned in between the circular structures formed by ESCRTs type III. Functionally, we observe that the reduction of AKTIP impinges on the recruitment of the ESCRT III member IST1 at the midbody and causes abscission defects. Taken together, these data indicate that AKTIP is a new factor that contributes to the formation of the ESCRT complex at the midbody and is implicated in the performance of the ESCRT machinery during cytokinetic abscission.

During HIV-1 assembly, the Pr55Gag polyprotein precursor (Gag) interacts with the genomic RNA, with lipids of the plasma membrane, with host proteins (ALIX, TSG101) through the ESCRT complex, with the viral protein Vpr and are involved in intermolecular interactions with other Pr55Gag proteins. This network of interactions is responsible for the formation of the viral particle, the selection of genomic RNA and the packaging of Vpr. The C-terminal domain of Gag encompassed in NCp15 is involved in the majority of these interactions, either by its nucleocapsid or its p6 domains. We study the NCp15 protein as a model of the C-terminal domain of Gag to better understand the role of this domain in the assembly and budding of HIV-1. Here, we report the 1H, 13C and 15N chemical shift assignments of NCp15 obtained by heteronuclear multidimensional NMR spectroscopy as well as the analysis of its secondary structure in solution. These assignments of NCp15 pave the way for interaction studies with its numerous 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.

Extracellular vesicles (EVs) facilitate intercellular communication and are considered a promising therapeutic tool for the treatment of infectious diseases. These vesicles involve microvesicles (MVs) and exosomes and selectively transfer proteins, lipids, mRNAs, and microRNAs from one cell to another. While MVs are formed by extrusion of the plasma membrane, exosomes are a population of vesicles of endosomal origin that are stored inside the multivesicular bodies (MVBs) as intraluminal vesicles (ILVs) and are released when the MVBs fuse with the plasma membrane. Biogenesis of exosomes may be driven by the endosomal sorting complex required for transport (ESCRT) machinery or may be ESCRT independent, and it is still debated whether these are entirely separate pathways. In this manuscript, we report that the protozoan parasite, Giardia lamblia, although lacking a classical endo-lysosomal pathway, is able to produce and release exosome-like vesicles (ElV). By using a combination of biochemical and cell biology analyses, we found that the ElVs have the same size, shape, and protein and lipid composition as exosomes described for other eukaryotic cells. Moreover, we established that some endosome/lysosome peripheral vacuoles (PVs) contain ILV during the stationary phase. Our results indicate that ILV formation and ElV release depend on the ESCRT-associated AAA+-ATPase Vps4a, Rab11, and ceramide in this parasite. Interestingly, EIV biogenesis and release seems to occur in Giardia despite the fact that this parasite has lost most of the ESCRT machinery components during evolution and is unable to produce ceramide de novo. The differences in protozoa parasite EV composition, origin, and release may reveal functional and structural properties of EVs and, thus, may provide information on cell-to-cell communication and on survival mechanisms. Fil: Moyano, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Musso, Juliana Rita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Feliziani, Constanza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Zamponi, Nahuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Frontera, Lorena Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Ropolo, Andrea Silvana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Lanfredi-Rangel, Adriana. Centro de Pesquizas Gonzalo Monis. Fiocruz; Brasil Fil: Lalle, Marco. Department Of Infectious Diseases, Foodborne And Neglec; Italia Fil: Touz, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina

Extracellular vesicles (EVs) facilitate intercellular communication and are considered a promising therapeutic tool for the treatment of infectious diseases. These vesicles involve microvesicles (MVs) and exosomes and selectively transfer proteins, lipids, mRNAs, and microRNAs from one cell to another. While MVs are formed by extrusion of the plasma membrane, exosomes are a population of vesicles of endosomal origin that are stored inside the multivesicular bodies (MVBs) as intraluminal vesicles (ILVs) and are released when the MVBs fuse with the plasma membrane. Biogenesis of exosomes may be driven by the endosomal sorting complex required for transport (ESCRT) machinery or may be ESCRT independent, and it is still debated whether these are entirely separate pathways. In this manuscript, we report that the protozoan parasite, Giardia lamblia, although lacking a classical endo-lysosomal pathway, is able to produce and release exosome-like vesicles (ElV). By using a combination of biochemical and cell biology analyses, we found that the ElVs have the same size, shape, and protein and lipid composition as exosomes described for other eukaryotic cells. Moreover, we established that some endosome/lysosome peripheral vacuoles (PVs) contain ILV during the stationary phase. Our results indicate that ILV formation and ElV release depend on the ESCRT-associated AAA+-ATPase Vps4a, Rab11, and ceramide in this parasite. Interestingly, EIV biogenesis and release seems to occur in Giardia despite the fact that this parasite has lost most of the ESCRT machinery components during evolution and is unable to produce ceramide de novo. The differences in protozoa parasite EV composition, origin, and release may reveal functional and structural properties of EVs and, thus, may provide information on cell-to-cell communication and on survival mechanisms.

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.

The endosomal sorting complexes required for transport (ESCRT) comprise a major trafficking pathway for plasma membrane proteins and are fundamental for ubiquitin-dependent cargo endocytosis. Here, we describe a method for studying the effect of ESCRT complexes on endo-lysosomal membrane trafficking and their role in receptor integrin α5β1 downregulation. Single cell fluorescence ratio image analysis (FRIA), using appropriate fluorescence probes, enables the measurement of dynamics of integrin α5β1 containing vesicles and represents a live cell-based method for studying the role of ESCRTs.

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The AAA ATPase Vps4 disassembles the ESCRT complex from the endosomal membrane. Vps4 contains an N-terminal MIT (microtubule interacting and transport) domain and a C-terminal catalytic domain. The MIT domain binds to MIMs (MIT-interacting motifs), which exist at the C-terminus of ESCRT-III proteins, with a dissociation constant in the micromolar range. Five MIMs have been identified by structural and biophysical methods to date, and the recognition motifs have been refined. Among biophysical approaches used to analyze protein interactions, surface plasmon resonance (SPR) analysis is often suitable for weak interactions, and fluorescence-binding assay has an advantage in terms of sensitivity. We have introduced protein modification tags into the N-terminus of proteins with bacterial expression vectors for biotinylation and FlAsH (fluorescein arsenical hairpin binder) fluorescent labeling. Here, we describe how to purify the MIT domain of Vps4 and the MIMs of ESCRT-III proteins and how to conduct crystallography, SPR, and fluorescence-binding assays.