A chemical inhibitor of IST1-CHMP1B interaction impairs endosomal recycling and induces noncanonical LC3 lipidation.

The endosomal sorting complex required for transport (ESCRT) machinery constitutes multisubunit protein complexes that play an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, including cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair, and autophagy. Given the versatile functionality of ESCRTs, and the intricate organizational structure of the ESCRT machinery, the targeted modulation of distinct ESCRT complexes is considerably challenging. This study presents a pseudonatural product targeting IST1-CHMP1B within the ESCRT-III complexes. The compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting, or biogenesis of extracellular vesicles, it rapidly inhibits transferrin receptor recycling in cells, resulting in the accumulation of transferrin in stalled sorting endosomes. Stalled endosomes become decorated by lipidated LC3, suggesting a link between noncanonical LC3 lipidation and inhibition of the IST1-CHMP1B complex.

ALIX and TSG101 are essential for cellular entry and replication of two porcine alphacoronaviruses.

Alphacoronaviruses are the primary coronaviruses responsible for causing severe economic losses in the pig industry with the potential to cause human outbreaks. Currently, extensive studies have reported the essential role of endosomal sorting and transport complexes (ESCRT) in the life cycle of enveloped viruses. However, very little information is available about which ESCRT components are crucial for alphacoronaviruses infection. By using RNA interference in combination with Co-immunoprecipitation, as well as fluorescence and electron microscopy approaches, we have dissected the role of ALIX and TSG101 for two porcine alphacoronavirus cellular entry and replication. Results show that infection by two porcine alphacoronaviruses, including porcine epidemic diarrhea virus (PEDV) and porcine enteric alphacoronavirus (PEAV), is dramatically decreased in ALIX- or TSG101-depleted cells. Furthermore, PEDV entry significantly increases the interaction of ALIX with caveolin-1 (CAV1) and RAB7, which are crucial for viral endocytosis and lysosomal transport, however, does not require TSG101. Interestingly, PEAV not only relies on ALIX to regulate viral endocytosis and lysosomal transport, but also requires TSG101 to regulate macropinocytosis. Besides, ALIX and TSG101 are recruited to the replication sites of PEDV and PEAV where they become localized within the endoplasmic reticulum and virus-induced double-membrane vesicles. PEDV and PEAV replication were significantly inhibited by depletion of ALIX and TSG101 in Vero cells or primary jejunal epithelial cells, indicating that ALIX and TSG101 are crucial for PEDV and PEAV replication. Collectively, these data highlight the dual role of ALIX and TSG101 in the entry and replication of two porcine alphacoronaviruses. Thus, ESCRT proteins could serve as therapeutic targets against two porcine alphacoronaviruses infection.

Identification of membrane curvature sensing motifs essential for VPS37A phagophore recruitment and autophagosome closure.

VPS37A, an ESCRT-I complex component, is required for recruiting a subset of ESCRT proteins to the phagophore for autophagosome closure. However, the mechanism by which VPS37A is targeted to the phagophore remains obscure. Here, we demonstrate that the VPS37A N-terminal domain exhibits selective interactions with highly curved membranes, mediated by two membrane-interacting motifs within the disordered regions surrounding its Ubiquitin E2 variant-like (UEVL) domain. Site-directed mutations of residues in these motifs disrupt ESCRT-I localization to the phagophore and result in defective phagophore closure and compromised autophagic flux in vivo, highlighting their essential role during autophagy. In conjunction with the UEVL domain, we postulate that these motifs guide a functional assembly of the ESCRT machinery at the highly curved tip of the phagophore for autophagosome closure. These results advance the notion that the distinctive membrane architecture of the cup-shaped phagophore spatially regulates autophagosome biogenesis.

Lethal Giant Disc is a target of Cdk1 and regulates ESCRT-III localization during germline stem cell abscission.

Abscission is the final step of cytokinesis that allows the physical separation of sister cells through the scission of the cellular membrane. This deformation is driven by ESCRT-III proteins, which can bind membranes and form dynamic helices. A crucial step in abscission is the recruitment of ESCRT-III proteins at the right time and place. Alix is one of the best characterized proteins that recruits ESCRT-III proteins from yeast to mammals. However, recent studies in vivo have revealed that pathways acting independently or redundantly with Alix are also required at abscission sites in different cellular contexts. Here, we show that Lgd acts redundantly with Alix to properly localize ESCRT-III to the abscission site in germline stem cells (GSCs) during Drosophila oogenesis. We further demonstrate that Lgd is phosphorylated at multiple sites by the CycB/Cdk1 kinase. We found that these phosphorylation events potentiate the activity of Shrub, a Drosophila ESCRT-III, during abscission of GSCs. Our study reveals that redundancy between Lgd and Alix, and coordination with the cell cycle kinase Cdk1, confers robust and timely abscission of Drosophila germline stem cells.

The discovery of potent USP2/USP8 dual-target inhibitors for the treatment of breast cancer via structure guided optimization of ML364.

USP2 and USP8 are crucial in the development and progression of breast cancer, primarily through the stabilization of protein substrates such as Her2 and ERα. The dual-target inhibitor ML364, targeting both USP2 and USP8, has garnered significant interest in recent research. In this study, we developed a series of ML364 derivatives using ligand-based drug design strategies. The standout compound, LLK203, demonstrated enhanced inhibitory activity, showing a 4-fold increase against USP2 and a 9-fold increase against USP8, compared to the parent molecule. In MCF-7 breast cancer cells, LLK203 effectively degraded key proteins involved in cancer progression and notably inhibited cell proliferation. Moreover, LLK203 exhibited potent in vivo efficacy in the 4T1 homograft model, while maintaining a low toxicity profile. These results underscore the potential of LLK203 as a promising dual-target inhibitor of USP2/USP8 for breast cancer treatment.

Cytokinetic abscission requires actin-dependent microtubule severing.

Cell division is completed by the abscission of the intercellular bridge connecting the daughter cells. Abscission requires the polymerization of an ESCRT-III cone close to the midbody to both recruit the microtubule severing enzyme spastin and scission the plasma membrane. Here, we found that the microtubule and the membrane cuts are two separate events that are regulated differently. Using HeLa cells, we uncovered that the F-actin disassembling protein Cofilin-1 controls the disappearance of a transient pool of branched F-actin which is precisely assembled at the tip of the ESCRT-III cone shortly before the microtubule cut. Functionally, Cofilin-1 and Arp2/3-mediated branched F-actin favor abscission by promoting local severing of the microtubules but do not participate later in the membrane scission event. Mechanistically, we propose that branched F-actin functions as a physical barrier that limits ESCRT-III cone elongation and thereby favors stable spastin recruitment. Our work thus reveals that F-actin controls the timely and local disassembly of microtubules required for cytokinetic abscission.

Identification of potential novel proteomic markers of Leishmania spp.-derived exosomes.

Introduction: Extracellular vesicles (EVs) are heterogenous cell-derived membrane-bound structures which can be subdivided into three distinct classes according to distinct morphological characteristics, cellular origins, and functions. Small EVs, or exosomes, can be produced by the protozoan parasite Leishmania through the evolutionarily conserved ESCRT pathway, and act as effectors of virulence and drivers of pathogenesis within mammalian hosts. Techniques for the identification of EVs of non-mammalian origin, however, remain inaccurate in comparison to their well-characterized mammalian counterparts. Thus, we still lack reliable and specific markers for Leishmania-derived exosomes, which poses a significant challenge to the field. Methods: Herein, we utilized serial differential ultracentrifugation to separate Leishmania-derived EV populations into three distinct fractions. Nanoparticle tracking analysis and transmission electron microscopy were used to validate their morphological characteristics, and bioinformatic analysis of LC-MS/MS proteomics corroborated cellular origins and function. Discussion: Proteomic data indicated potential novel proteic markers of Leishmania-derived exosomes, including proteins involved in endosomal machinery and the ESCRT pathway, as well as the parasitic phosphatase PRL-1. Further investigation is required to determine the specificity and sensitivity of these markers.

Bi-allelic variants in SNF8 cause a disease spectrum ranging from severe developmental and epileptic encephalopathy to syndromic optic atrophy.

The endosomal sorting complex required for transport (ESCRT) machinery is essential for membrane remodeling and autophagy and it comprises three multi-subunit complexes (ESCRT I-III). We report nine individuals from six families presenting with a spectrum of neurodevelopmental/neurodegenerative features caused by bi-allelic variants in SNF8 (GenBank: NM_007241.4), encoding the ESCRT-II subunit SNF8. The phenotypic spectrum included four individuals with severe developmental and epileptic encephalopathy, massive reduction of white matter, hypo-/aplasia of the corpus callosum, neurodevelopmental arrest, and early death. A second cohort shows a milder phenotype with intellectual disability, childhood-onset optic atrophy, or ataxia. All mildly affected individuals shared the same hypomorphic variant, c.304G>A (p.Val102Ile). In patient-derived fibroblasts, bi-allelic SNF8 variants cause loss of ESCRT-II subunits. Snf8 loss of function in zebrafish results in global developmental delay and altered embryo morphology, impaired optic nerve development, and reduced forebrain size. In vivo experiments corroborated the pathogenicity of the tested SNF8 variants and their variable impact on embryo development, validating the observed clinical heterogeneity. Taken together, we conclude that loss of ESCRT-II due to bi-allelic SNF8 variants is associated with a spectrum of neurodevelopmental/neurodegenerative phenotypes mediated likely via impairment of the autophagic flux.

Spastin regulates anaphase chromosome separation distance and microtubule-containing nuclear tunnels.

Nuclear envelope reassembly during the final stages of each mitosis depends on disassembling spindle microtubules without disrupting chromosome separation. This process involves the transient recruitment of the ESCRT-III complex and spastin, a microtubule-severing AAA (ATPases associated with diverse cellular activities) mechanoenzyme, to late-anaphase chromosomes. However, dissecting mechanisms underlying these rapid processes, which can be completed within minutes, has been difficult. Here, we combine fast-acting chemical inhibitors with live-cell imaging and find that spindle microtubules, along with spastin activity, regulate the number and lifetimes of spastin foci at anaphase chromosomes. Unexpectedly, spastin inhibition impedes chromosome separation, but does not alter the anaphase localization dynamics of CHMP4B, an ESCRT-III protein, or increase γ-H2AX foci, a DNA damage marker. We show spastin inhibition increases the frequency of lamin-lined nuclear microtunnels that can include microtubules penetrating the nucleus. Our findings suggest failure to sever spindle microtubules impedes chromosome separation, yet reforming nuclear envelopes can topologically accommodate persistent microtubules ensuring nuclear DNA is not damaged or exposed to cytoplasm.

High expression of VTA1 is an adverse prognostic factor in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a common pathological type of non-small cell lung cancer; identifying preferable biomarkers has become one of the current challenges. Given that VTA1 has been reported associated with tumor progression in various human solid cancers but rarely reported in LUAD, herein, RNA sequencing data from TCGA and GTEx were obtained for analysis of VTA1 expression and differentially expressed gene (DEG). Furthermore, functional enrichment analysis of VTA1-related DEGs was performed by GO/KEGG, GSEA, immune cell infiltration analysis, and protein-protein interaction (PPI) network. In addition, the clinical significance of VTA1 in LUAD was figured out by Kaplan-Meier Cox regression and prognostic nomogram model. R package was used to analyze incorporated studies. As a result, VTA1 was highly expressed in various malignancies, including LUAD, compared with normal samples. Moreover, high expression of VTA1 was associated with poor prognosis in 533 LUAD samples, as well as T stage T2&T3&T4, N stage N1&N2&N3, M stage M1, pathologic stage II&III&IV, and residual tumor R1&R2, et al. (P < 0.05). High VTA1 was an independent prognostic factor in Cox regression analysis; Age and cytogenetics risk were included in the nomogram prognostic model. Furthermore, a total of 4232 DEGs were identified between the high- and the low-expression group, of which 736 genes were up-regulated and 3496 genes were down-regulated. Collectively, high expression of VTA1 is a potential biomarker for adverse outcomes in LUAD. The DEGs and pathways recognized in the study provide a preliminary grasp of the underlying molecular mechanisms of LUAD carcinogenesis and progression.

Origin of eukaryotic-like Vps23 shapes an ancient functional interplay between ESCRT and ubiquitin system in Asgard archaea.

Functional interplay between the endosomal sorting complexes required for transport (ESCRT) and the ubiquitin system underlies the ubiquitin-dependent cargo-sorting pathway of the eukaryotic endomembrane system, yet its evolutionary origin remains unclear. Here, we show that a UEV-Vps23 protein family, which contains UEV and Vps23 domains, mediates an ancient ESCRT and ubiquitin system interplay in Asgard archaea. The UEV binds ubiquitin with high affinity, making the UEV-Vps23 a sensor for sorting ubiquitinated cargo. A steadiness box in the Vps23 domain undergoes ubiquitination through an Asgard E1, E2, and RING E3 cascade. The UEV-Vps23 switches between autoinhibited and active forms, regulating the ESCRT and ubiquitin system interplay. Furthermore, the shared sequence and structural homology among the UEV-Vps23, eukaryotic Vps23, and archaeal CdvA suggest a common evolutionary origin. Together, this work expands our understanding of the ancient ESCRT and ubiquitin system interplay that likely arose antedating divergent evolution between Asgard archaea and eukaryotes.

A role for Vps13-mediated lipid transfer at the ER-endosome contact site in ESCRT-mediated sorting.

Endosomes are specialized organelles that function in the secretory and endocytic protein sorting pathways. Endocytosed cell surface receptors and transporters destined for lysosomal degradation are sorted into intraluminal vesicles (ILVs) at endosomes by endosomal sorting complexes required for transport (ESCRT) proteins. The endosomes (multivesicular bodies, MVBs) then fuse with the lysosome. During endosomal maturation, the number of ILVs increases, but the size of endosomes does not decrease despite the consumption of the limiting membrane during ILV formation. Vesicle-mediated trafficking is thought to provide lipids to support MVB biogenesis. However, we have uncovered an unexpected contribution of a large bridge-like lipid transfer protein, Vps13, in this process. Here, we reveal that Vps13-mediated lipid transfer at ER-endosome contact sites is required for the ESCRT pathway. We propose that Vps13 may play a critical role in supplying lipids to the endosome, ensuring continuous ESCRT-mediated sorting during MVB biogenesis.

Alternative mechanisms of Notch activation by partitioning into distinct endosomal domains.

Different membrane microdomain compositions provide unique environments that can regulate signaling receptor function. We identify microdomains on the endosome membrane of Drosophila endosomes, enriched in lipid-raft or clathrin/ESCRT-0, which are associated with Notch activation by distinct, ligand-independent mechanisms. Transfer of Notch between microdomains is regulated by Deltex and Suppressor of deltex ubiquitin ligases and is limited by a gate-keeper role for ESCRT complexes. Ubiquitination of Notch by Deltex recruits it to the clathrin/ESCRT-0 microdomain and enhances Notch activation by an ADAM10-independent/TRPML-dependent mechanism. This requirement for Deltex is bypassed by the downregulation of ESCRT-III. In contrast, while ESCRT-I depletion also activates Notch, it does so by an ADAM10-dependent/TRPML-independent mechanism and Notch is retained in the lipid raft-like microdomain. In the absence of such endosomal perturbation, different activating Notch mutations also localize to different microdomains and are activated by different mechanisms. Our findings demonstrate the interplay between Notch regulators, endosomal trafficking components, and Notch genetics, which defines membrane locations and activation mechanisms.

Palmitoylation of vacuole membrane protein 1 promotes small extracellular vesicle secretion via interaction with ALIX and influences intercellular communication.

BACKGROUND: Small extracellular vesicles (EVs), exemplified by exosomes, mediate intercellular communication by transporting proteins, mRNAs, and miRNAs. Post-translational modifications are involved in controlling small EV secretion process. However, whether palmitoylation regulates small EV secretion, remains largely unexplored. METHODS: Vacuole Membrane Protein 1 (VMP1) was testified to be S-palmitoylated by Palmitoylation assays. VMP1 mutant plasmids were constructed to screen out the exact palmitoylation sites. Small EVs were isolated, identified and compared between wild-type VMP1 or mutant VMP1 transfected cells. Electron microscope and immunofluorescence were used to detect multivesicular body (MVB) number and morphology change when VMP1 was mutated. Immunoprecipitation and Mass spectrum were adopted to identify the protein that interacted with palmitoylated VMP1, while knock down experiment was used to explore the function of targeted protein ALIX. Taking human Sertoli cells (SCs) and human spermatogonial stem cell like cells (SSCLCs) as a model of intercellular communication, SSCLC maintenance was detected by flow cytometry and qPCR at 12 days of differentiation. In vivo, mouse model was established by intraperitoneal injection with palmitoylation inhibitor, 2-bromopalmitate (2BP) for 3 months. RESULTS: VMP1 was identified to be palmitoylated at cysteine 263,278 by ZDHHC3. Specifically, palmitoylation of VMP1 regulated its subcellular location and enhanced the amount of small EV secretion. Mutation of VMP1 palmitoylation sites interfered with the morphology and biogenesis of MVBs through suppressing intraluminal vesicle formation. Furthermore, inhibition of VMP1 palmitoylation impeded small EV secretion by affecting the interaction of VMP1 with ALIX, an accessory protein of the ESCRT machinery. Taking SCs and SSCLCs as a model of intercellular communication, we discovered VMP1 palmitoylation in SCs was vital to the growth status of SSCLCs in a co-culture system. Inhibition of VMP1 palmitoylation caused low self-maintenance, increased apoptosis, and decreased proliferation rate of SSCLCs. In vivo, intraperitoneal injection of 2BP inhibited VMP1 palmitoylation and exosomal marker expression in mouse testes, which were closely associated with the level of spermatogenic cell apoptosis and proliferation. CONCLUSIONS: Our study revealed a novel mechanism for small EV secretion regulated by VMP1 palmitoylation in Sertoli cells, and demonstrated its pivotal role in intercellular communication and SSC niche.

ESCRT-I protein UBAP1 controls ventricular expansion and cortical neurogenesis via modulating adherens junctions of radial glial cells.

Intricate cerebral cortex formation is orchestrated by the precise behavior and division dynamics of radial glial cells (RGCs). Endocytosis functions in the recycling and remodeling of adherens junctions (AJs) in response to changes in RGC activity and function. Here, we show that conditional disruption of ubiquitin-associated protein 1 (UBAP1), a component of endosomal sorting complex required for transport (ESCRT), causes severe brain dysplasia and prenatal ventriculomegaly. UBAP1 depletion disrupts the AJs and polarity of RGCs, leading to failure of apically directed interkinetic nuclear migration. Accordingly, UBAP1 knockout or knockdown results in reduced proliferation and precocious differentiation of neural progenitor cells. Mechanistically, UBAP1 regulates the expression and surface localization of cell adhesion molecules, and ß-catenin over-expression significantly rescues the phenotypes of Ubap1 knockdown in vivo. Our study reveals a critical physiological role of the ESCRT machinery in cortical neurogenesis by regulating AJs of RGCs.

Plasma membrane damage limits replicative lifespan in yeast and induces premature senescence in human fibroblasts.

Plasma membrane damage (PMD) occurs in all cell types due to environmental perturbation and cell-autonomous activities. However, cellular outcomes of PMD remain largely unknown except for recovery or death. In this study, using budding yeast and normal human fibroblasts, we found that cellular senescence-stable cell cycle arrest contributing to organismal aging-is the long-term outcome of PMD. Our genetic screening using budding yeast unexpectedly identified a close genetic association between PMD response and replicative lifespan regulations. Furthermore, PMD limits replicative lifespan in budding yeast; upregulation of membrane repair factors ESCRT-III (SNF7) and AAA-ATPase (VPS4) extends it. In normal human fibroblasts, PMD induces premature senescence via the Ca2+-p53 axis but not the major senescence pathway, DNA damage response pathway. Transient upregulation of ESCRT-III (CHMP4B) suppressed PMD-dependent senescence. Together with mRNA sequencing results, our study highlights an underappreciated but ubiquitous senescent cell subtype: PMD-dependent senescent cells.

NEDD4L intramolecular interactions regulate its auto and substrate NaV1.5 ubiquitination.

NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, NaV1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For NaV1.5, this process is critical as alterations in Na+ current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of NaV1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of NaV1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na+ current reduction using the NEDD4L HECT domain as cargo of a NaV1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding NaV1.5 ubiquitination.

Clathrin controls bidirectional communication between T cells and antigen presenting cells.

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

Phosphoglycerate kinase 1 acts as a cargo adaptor to promote EGFR transport to the lysosome.

The epidermal growth factor receptor (EGFR) plays important roles in multiple cellular events, including growth, differentiation, and motility. A major mechanism of downregulating EGFR function involves its endocytic transport to the lysosome. Sorting of proteins into intracellular pathways involves cargo adaptors recognizing sorting signals on cargo proteins. A dileucine-based sorting signal has been identified previously for the sorting of endosomal EGFR to the lysosome, but a cargo adaptor that recognizes this signal remains unknown. Here, we find that phosphoglycerate kinase 1 (PGK1) is recruited to endosomal membrane upon its phosphorylation, where it binds to the dileucine sorting signal in EGFR to promote the lysosomal transport of this receptor. We also elucidate two mechanisms that act in concert to promote PGK1 recruitment to endosomal membrane, a lipid-based mechanism that involves phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and a protein-based mechanism that involves hepatocyte growth factor receptor substrate (Hrs). These findings reveal an unexpected function for a metabolic enzyme and advance the mechanistic understanding of how EGFR is transported to the lysosome.

Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2.

Apoptosis linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining giant unilamellar vesicle-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.