Formation Of Intralumenal Vesicles Research Articles

Lgd regulates ESCRT-III complex accumulation at multivesicular endosomes to control intralumenal vesicle formation.

Membrane remodeling mediated by heteropolymeric filaments composed of ESCRT-III subunits is an essential process that occurs at a variety of organelles to maintain cellular homeostasis. Members of the evolutionarily conserved Lgd/CC2D1 protein family have been suggested to regulate ESCRT-III polymer assembly, although their specific roles, particularly in vivo, remain unclear. Using the Caenorhabditis elegans early embryo as a model system, we show that Lgd/CC2D1 localizes to endosomal membranes, and its loss impairs endolysosomal cargo sorting and degradation. At the ultrastructural level, the absence of Lgd/CC2D1 results in the accumulation of enlarged endosomal compartments that contain a reduced number of intralumenal vesicles (ILVs). However, unlike aberrant endosome morphology caused by depletion of other ESCRT components, ILV size is only modestly altered in embryos lacking Lgd/CC2D1. Instead, loss of Lgd/CC2D1 impairs normal accumulation of ESCRT-III on endosomal membranes, likely slowing the kinetics of ILV formation. Together, our findings suggest a role for Lgd/CC2D1 in the recruitment and/or stable assembly of ESCRT-III subunits on endosomal membranes to facilitate efficient ILV biogenesis.

Bro1 family proteins harmonize cargo sorting with vesicle formation.

The Endosomal Sorting Complexes Required for Transport (ESCRTs) drive membrane remodeling in a variety of cellular processes that include the formation of endosomal intralumenal vesicles (ILVs) during multivesicular body (MVB) biogenesis. During MVB sorting, ESCRTs recognize ubiquitin (Ub) attached to membrane protein cargo and execute ILV formation by controlling the activities of ESCRT-III polymers regulated by the AAA-ATPase Vps4. Exactly how these events are coordinated to ensure proper cargo loading into ILVs remains unclear. Here we discuss recent work documenting the ability of Bro1, an ESCRT-associated Ub-binding protein, to coordinate ESCRT-III and Vps4-dependent ILV biogenesis with upstream events such as cargo recognition.

Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis.

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT-driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III-dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1-Vps4-ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.

Tuning membrane tension

Cell Biology The tension of a cell's plasma membrane influences many processes that require membrane remodeling, including cell migration and endocytosis. However, little is known about how organelle membrane tension may regulate cell function. After endocytosis, endosomal membranes are remodeled in an ESCRT-III–dependent process that leads to the formation of intralumenal vesicles (ILVs). Mercier et al. investigated this phenomenon using a specific fluorescent membrane tension probe. ESCRT-III subunits were recruited to endosomal membranes as membrane tension reduced. Tension-dependent membrane recruitment was associated with ESCRT-III polymerization and membrane deformation and with increased ILV formation. In cultured cells, endosomal membrane tension decreased when ILV formation was triggered by epidermal growth factor. Thus, endosomal membrane tension regulates ILV formation, and reduced tension facilitates intracellular membrane-remodeling processes. Nat. Cell Biol. 22 , 947 (2020).

Endosomal membrane tension regulates ESCRT-III-dependent intra-lumenal vesicle formation.

Plasma membrane tension strongly affects cell surface processes, such as migration, endocytosis and signalling. However, it is not known whether membrane tension of organelles regulates their functions, notably intracellular traffic. The ESCRT-III complex is the major membrane remodelling complex that drives Intra-Lumenal Vesicle (ILV) formation on endosomal membranes. Here, we made use of a fluorescent membrane tension probe to show that ESCRT-III subunits are recruited onto endosomal membranes when membrane tension is reduced. We find that tension-dependent recruitment is associated with ESCRT-III polymerization and membrane deformation in vitro, and correlates with increased ILV formation in ESCRT-III decorated endosomes in vivo. Finally, we find that endosomal membrane tension decreases when ILV formation is triggered by EGF under physiological conditions. These results indicate that membrane tension is a major regulator of ILV formation and of endosome trafficking, leading us to conclude that membrane tension can control organelle functions.

Growth factor stimulation promotes multivesicular endosome biogenesis by prolonging recruitment of the late-acting ESCRT machinery

The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early- and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation.

SH3YL1 cooperates with ESCRT-I in the sorting and degradation of the EGF receptor.

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 C-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.

Endosomal lipids in trafficking and signaling

During endocytosis, cell surface components including housekeeping receptors and activated signaling receptors are delivered to early endosomes, where they are sorted to be either reutilized via recycling pathways or transported towards late endosomes and then lysosomes for degradation. In the latter case, down‐regulated receptors and other molecules destined for the lysosomes are incorporated into lumenal invaginations of the early endosome membrane, which are pinched off as free cargo‐containing vesicles. These multivesicular regions detach and become multivesicular endosomes, which are transported to and fuse with late endosomes. Eventually, intralumenal vesicles (ILVs) and their cargo are delivered to lysosomes and degraded. Alternatively, ILVs can also be released extracellularly as exosomes, upon fusion of multivesicular endosomes with the plasma membrane. Low‐density lipoproteins (LDL) that have been endocytosed are eventually delivered to multivesicular late endosomes. There, cholesteryl esters are deesterified so that free cholesterol can be released and exported to other cellular destinations. The export mechanisms remain ill defined, but it is clear that disruption of the process has profound consequences for cellular sterol homeostasis and can result in the neurodegenerative disease Niemann‐Pick C (NPC). Our previous studies suggest that the fate of LDL‐derived cholesterol is linked to the unconventional phospholipid lysobisphosphatidic acid (LBPA) that is abundant in late endosome intralumenal membranes, since interfering with LBPA functions phenocopies NPC at the cellular level. Our approaches to study the mechanisms of ILV formation and endosomal cholesterol transport will be discussed, including biochemical strategies and unbiased high content image‐based screens.Support or Funding InformationSwiss National Science Foundation; Swiss National Centre of Competence in Research Chemical Biology; LipidX from the Swiss SystemsX.ch initiativeThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Conformational Changes in the Endosomal Sorting Complex Required for the Transport III Subunit Ist1 Lead to Distinct Modes of ATPase Vps4 Regulation

Intralumenal vesicle formation of the multivesicular body is a critical step in the delivery of endocytic cargoes to the lysosome for degradation. Endosomal sorting complex required for transport III (ESCRT-III) subunits polymerize on endosomal membranes to facilitate membrane budding away from the cytoplasm to generate these intralumenal vesicles. The ATPase Vps4 remodels and disassembles ESCRT-III, but the manner in which Vps4 activity is coordinated with ESCRT-III function remains unclear. Ist1 is structurally homologous to ESCRT-III subunits and has been reported to inhibit Vps4 function despite the presence of a microtubule-interacting and trafficking domain-interacting motif (MIM) capable of stimulating Vps4 in the context of other ESCRT-III subunits. Here we report that Ist1 inhibition of Vps4 ATPase activity involves two elements in Ist1: the MIM itself and a surface containing a conserved ELYC sequence. In contrast, the MIM interaction, in concert with a more open conformation of the Ist1 core, resulted in stimulation of Vps4. Addition of the ESCRT-III subunit binding partner of Ist1, Did2, also converted Ist1 from an inhibitor to a stimulator of Vps4 ATPase activity. Finally, distinct regulation of Vps4 by Ist1 corresponded with altered ESCRT-III disassembly in vitro. Together, these data support a model in which Ist1-Did2 interactions during ESCRT-III polymerization coordinate Vps4 activity with the timing of ESCRT-III disassembly.

A non-canonical ESCRT pathway, including histidine domain phosphotyrosine phosphatase (HD-PTP), is used for down-regulation of virally ubiquitinated MHC class I.

The Kaposi's sarcoma-associated herpes virus (KSHV) K3 viral gene product effectively down-regulates cell surface MHC class I. K3 is an E3 ubiquitin ligase that promotes Lys(63)-linked polyubiquitination of MHC class I, providing the signal for clathrin-mediated endocytosis. Endocytosis is followed by sorting into the intralumenal vesicles (ILVs) of multivesicular bodies (MVBs) and eventual delivery to lysosomes. The sorting of MHC class I into MVBs requires many individual proteins of the four endosomal sorting complexes required for transport (ESCRTs). In HeLa cells expressing the KSHV K3 ubiquitin ligase, the effect of RNAi-mediated depletion of individual proteins of the ESCRT-0 and ESCRT-I complexes and three ESCRT-III proteins showed that these are required to down-regulate MHC class I. However, depletion of proteins of the ESCRT-II complex or of the ESCRT-III protein, VPS20 (vacuolar protein sorting 20)/CHMP6 (charged MVB protein 6), failed to prevent the loss of MHC class I from the cell surface. Depletion of histidine domain phosphotyrosine phosphatase (HD-PTP) resulted in an increase in the cell surface concentration of MHC class I in HeLa cells expressing the KSHV K3 ubiquitin ligase. Rescue experiments with wild-type (WT) and mutant HD-PTP supported the conclusion that HD-PTP acts as an alternative to ESCRT-II and VPS20/CHMP6 as a link between the ESCRT-I and those ESCRT-III protein(s) necessary for ILV formation. Thus, the down-regulation of cell surface MHC class I, polyubiquitinated by the KSHV K3 ubiquitin ligase, does not employ the canonical ESCRT pathway, but instead utilizes an alternative pathway in which HD-PTP replaces ESCRT-II and VPS20/CHMP6.

Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation

Endocytic trafficking of signaling receptors is an important mechanism for limiting signal duration. Components of the Endosomal Sorting Complexes Required for Transport (ESCRT), which target ubiquitylated receptors to intra-lumenal vesicles (ILVs) of multivesicular bodies, are thought to terminate signaling by the epidermal growth factor receptor (EGFR) and direct it for lysosomal degradation. In a genetic screen for mutations that affect Drosophila eye development, we identified an allele of Vacuolar protein sorting 4 (Vps4), which encodes an AAA ATPase that interacts with the ESCRT-III complex to drive the final step of ILV formation. Photoreceptors are largely absent from Vps4 mutant clones in the eye disc, and even when cell death is genetically prevented, the mutant R8 photoreceptors that develop fail to recruit surrounding cells to differentiate as R1-R7 photoreceptors. This recruitment requires EGFR signaling, suggesting that loss of Vps4 disrupts the EGFR pathway. In imaginal disc cells mutant for Vps4, EGFR and other receptors accumulate in endosomes and EGFR target genes are not expressed; epistasis experiments place the function of Vps4 at the level of the receptor. Surprisingly, Vps4 is required for EGFR signaling even in the absence of Shibire, the Dynamin that internalizes EGFR from the plasma membrane. In ovarian follicle cells, in contrast, Vps4 does not affect EGFR signaling, although it is still essential for receptor degradation. Taken together, these findings indicate that Vps4 can promote EGFR activity through an endocytosis-independent mechanism.

Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation.

Endocytic trafficking of signaling receptors is an important mechanism for limiting signal duration. Components of the Endosomal Sorting Complexes Required for Transport (ESCRT), which target ubiquitylated receptors to intra-lumenal vesicles (ILVs) of multivesicular bodies, are thought to terminate signaling by the epidermal growth factor receptor (EGFR) and direct it for lysosomal degradation. In a genetic screen for mutations that affect Drosophila eye development, we identified an allele of Vacuolar protein sorting 4 (Vps4), which encodes an AAA ATPase that interacts with the ESCRT-III complex to drive the final step of ILV formation. Photoreceptors are largely absent from Vps4 mutant clones in the eye disc, and even when cell death is genetically prevented, the mutant R8 photoreceptors that develop fail to recruit surrounding cells to differentiate as R1-R7 photoreceptors. This recruitment requires EGFR signaling, suggesting that loss of Vps4 disrupts the EGFR pathway. In imaginal disc cells mutant for Vps4, EGFR and other receptors accumulate in endosomes and EGFR target genes are not expressed; epistasis experiments place the function of Vps4 at the level of the receptor. Surprisingly, Vps4 is required for EGFR signaling even in the absence of Shibire, the Dynamin that internalizes EGFR from the plasma membrane. In ovarian follicle cells, in contrast, Vps4 does not affect EGFR signaling, although it is still essential for receptor degradation. Taken together, these findings indicate that Vps4 can promote EGFR activity through an endocytosis-independent mechanism.

Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry

Rotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the endosomal sorting complex required for transport (ESCRT) machinery were found. Here we provide evidence for a role of the ESCRT complex in the entry of simian and human RV strains in both monkey and human epithelial cells. In addition, the ESCRT-associated ATPase VPS4A and phospholipid lysobisphosphatidic acid, both crucial for the formation of intralumenal vesicles in multivesicular bodies, were also found to be required for cell entry. Interestingly, it seems that regardless of the molecules that rhesus RV and human RV strains use for cell-surface attachment and the distinct endocytic pathway used, all these viruses converge in early endosomes and use multivesicular bodies for cell entry. Furthermore, the small GTPases RHOA and CDC42, which regulate different types of clathrin-independent endocytosis, as well as early endosomal antigen 1 (EEA1), were found to be involved in this process. This work reports the direct involvement of the ESCRT machinery in the life cycle of a nonenveloped virus and highlights the complex mechanism that these viruses use to enter cells. It also illustrates the efficiency of high-throughput RNAi screenings as genetic tools for comprehensively studying the interaction between viruses and their host cells.

Precise and timely delivery of proteins within cells continues to be an exciting area of cell biology

To deliver the right amount of the right protein to the right place at the right time is a fundamental cell biology event underlying diverse cellular, physiological, and pathological processes. As cochairs of the Minisymposium on Intracellular Sorting and Trafficking we reviewed a large number of deserving abstracts, but we could choose only six speakers, ranging from junior to senior investigators. The talks covered major cellular organelles involved in sorting and trafficking, including the endoplasmic reticulum (ER), Golgi, endocytic compartments, the plasma membrane, and the primary cilia. We were thrilled to have Gia Voeltz from the University of Colorado at Boulder in our session, and we congratulated her on her Early Career Life Scientist Award. She described her original work in defining the molecular basis governing the organization and dynamics of the different subdomains and connectivity of the ER using in vitro systems, biochemical approaches, and in vivo cell biological validation. The small GTPase Rab10 was identified as a major player. A part of the story was just published (English and Voeltz, 2012 ). Moving to the Golgi apparatus, Wanjin Hong from the Institute of Molecular and Cell Biology in Singapore talked about an approach of data-browsing the Human Protein Atlas (www.proteinatlas.org) to identify several new potential Golgi membrane proteins and focused on the novel Golgi protein TMEM115. TMEM115 is an evolutionarily conserved protein with four predicted transmembrane domains and a C-terminus facing the cytoplasm. Functionally, TMEM115 may regulate retrograde trafficking from the Golgi back to the ER through interaction with the conserved oligomeric Golgi complex. Moving from the Golgi to the cell's surface, Thierry Galli from the Institut Jacques Monod in France focused on Ti-VAMP/VAMP7 and its interacting network in regulating the migration of vesicles from the cell center to the cell periphery via the microtubules to mediate exocytosis. A multiprotein interacting network for Ti-VAMP was described, including the Rab21 guanine nucleotide exchange factor Varp, MACF1, GolginA4, and the kinesin 1 Kif5A. Part of the work has appeared recently (Burgo et al., 2012 ). Also at the cell surface, Ludger Johannes from the Institut Curie in France talked about his exciting discovery underlying the molecular aspects of clathrin-independent endocytosis. His group has identified an endogenous protein that drives the biogenesis of clathrin-independent carriers for the uptake of transmembrane cargo proteins, such as CD44. In experiments on cell and model membranes, they found that glycosphingolipids were key to the formation of endocytic membrane invaginations. These finding were condensed into the first mechanistic model based on specific protein machinery to describe how the uptake of certain endogenous cargoes is initiated without the help of the cytosolic clathrin machinery. Moving to later stages in endocytosis, Anne Spang from the University of Basel in Switzerland described a systematic study to address coordination of various processes, such as membrane fusion, acidification, and intralumenal vesicle formation underlying the transition from the early to the late endosome, which occurs during endosome maturation (Poteryaev et al., 2010 ). Novel roles and the action of the HOPS (homotypic fusion and vacuole protein sorting) and the CORVET (class C core vacuole/endosome tethering) tethering complexes in conjunction with SAND-1/Mon1 and RABX-5 on endosome maturation in the model organism Caenorhabditis elegans were presented. Projecting from the cell surface are primary cilia, which are receiving increased attention due to the growing number of human diseases found to be related to the biogenesis, trafficking, and maintenance of the cilia. Importantly, primary cilia appear to be key signaling platforms. Peter Jackson from Genentech in San Francisco discussed many molecules and interacting networks important for cilia biogenesis and trafficking. A key aspect is the role of the small GTPase Arl3 in targeting myristoylated and prenylated proteins, such as NPHP3, to the primary cilium via UNC119 and PDE6D effectors. Some of this work has already been published (Wright et al., 2011 ). We thank all the speakers for their participation and contri­butions and the discussion participants for making this Minisymposium such a great event.

Doa4 function in ILV budding is restricted through its interaction with the Vps20 subunit of ESCRT-III

Assembly of the endosomal sorting complex required for transport (ESCRT)-III executes the formation of intralumenal vesicles (ILVs) at endosomes. Repeated cycles of ESCRT-III function requires disassembly of the complex by Vps4, an ATPase with a microtubule interaction and trafficking (MIT) domain that binds MIT-interacting motifs (MIM1 or MIM2) in ESCRT-III subunits. We identified a putative MIT domain at the N-terminus of Doa4, which is the ubiquitin (Ub) hydrolase in Saccharomyces cerevisiae that deubiquitinates ILV cargo proteins. The Doa4 N-terminus is predicted to have the α-helical structure common to MIT domains, and it binds directly to a MIM1-like sequence in the Vps20 subunit of ESCRT-III. Disrupting this interaction does not prevent endosomal localization of Doa4 but enhances the defect in ILV cargo protein deubiquitination observed in cells lacking Bro1, which is an ESCRT-III effector protein that stimulates Doa4 catalytic activity. Deletion of the BRO1 gene (bro1Δ) blocks ILV budding, but ILV budding was rescued upon disrupting the interaction between Vps20 and Doa4. This rescue in ILV biogenesis requires Doa4 expression but is independent of its Ub hydrolase activity. Thus, binding of Vps20 to the Doa4 N-terminus inhibits a non-catalytic function of Doa4 that promotes ILV formation.

SNX12 Role in Endosome Membrane Transport

In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.

Cargo ubiquitination is essential for multivesicular body intralumenal vesicle formation

The efficient formation of a variety of transport vesicles is influenced by the presence of cargo, suggesting that cargo itself might have a defining role in vesicle biogenesis. However, definitive in vivo experiments supporting this concept are lacking, as it is difficult to eliminate endogenous cargo. The Endosomal Sorting Complexes Required for Transport (ESCRT) apparatus sorts ubiquitinated membrane proteins into endosomal intralumenal vesicles (ILVs) that accumulate within multivesicular bodies. Here we show that cargo ubiquitination is required for effective recruitment of the ESCRT machinery onto endosomal membranes and for the subsequent formation of ILVs.

Endosomal Na+ (K+)/H+ Exchanger Nhx1/Vps44 Functions Independently and Downstream of Multivesicular Body Formation

The multivesicular body (MVB) is an endosomal intermediate containing intralumenal vesicles destined for membrane protein degradation in the lysosome. In Saccharomyces cerevisiae, the MVB pathway is composed of 17 evolutionarily conserved ESCRT (endosomal sorting complex required for transport) genes grouped by their vacuole protein sorting Class E mutant phenotypes. Only one integral membrane protein, the endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44, has been assigned to this class, but its role in the MVB pathway has not been directly tested. Herein, we first evaluated the link between Nhx1 and the ESCRT proteins and then used an unbiased phenomics approach to probe the cellular role of Nhx1. Select ESCRT mutants (vps36Δ, vps20Δ, snf7Δ, and bro1Δ) with defects in cargo packaging and intralumenal vesicle formation shared multiple growth phenotypes with nhx1Δ. However, analysis of cellular trafficking and ultrastructural examination by electron microscopy revealed that nhx1Δ cells retain the ability to sort cargo into intralumenal vesicles. In addition, we excluded a role for Nhx1 in Snf7/Bro1-mediated cargo deubiquitylation and Rim101 response to pH stress. Genetic epistasis experiments provided evidence that NHX1 and ESCRT genes function in parallel. A genome-wide screen for single gene deletion mutants that phenocopy nhx1Δ yielded a limited gene set enriched for endosome fusion function, including Rab signaling and actin cytoskeleton reorganization. In light of these findings and the absence of the so-called Class E compartment in nhx1Δ, we eliminated a requirement for Nhx1 in MVB formation and suggest an alternative post-ESCRT role in endosomal membrane fusion.

Regulation of the MVB Pathway by SCAMP3

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.

Association of the Endosomal Sorting Complex ESCRT-II with the Vps20 Subunit of ESCRT-III Generates a Curvature-sensitive Complex Capable of Nucleating ESCRT-III Filaments

The scission of membranes necessary for vesicle biogenesis and cytokinesis is mediated by cytoplasmic proteins, which include members of the ESCRT (endosomal sorting complex required for transport) machinery. During the formation of intralumenal vesicles that bud into multivesicular endosomes, the ESCRT-II complex initiates polymerization of ESCRT-III subunits essential for membrane fission. However, mechanisms underlying the spatial and temporal regulation of this process remain unclear. Here, we show that purified ESCRT-II binds to the ESCRT-III subunit Vps20 on chemically defined membranes in a curvature-dependent manner. Using a combination of liposome co-flotation assays, fluorescence-based liposome interaction studies, and high-resolution atomic force microscopy, we found that the interaction between ESCRT-II and Vps20 decreases the affinity of ESCRT-II for flat lipid bilayers. We additionally demonstrate that ESCRT-II and Vps20 nucleate flexible filaments of Vps32 that polymerize specifically along highly curved membranes as a single string of monomers. Strikingly, Vps32 filaments are shown to modulate membrane dynamics in vitro, a prerequisite for membrane scission events in cells. We propose that a curvature-dependent assembly pathway provides the spatial regulation of ESCRT-III to fuse juxtaposed bilayers of elevated curvature.