Munc18c accelerates SNARE-dependent membrane fusion in the presence of regulatory proteins α-SNAP and NSF.

Intracellular vesicle fusion is driven by the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and their cofactors, including Sec1/Munc18 (SM), α-SNAP, and NSF. α-SNAP and NSF play multiple layers of regulatory roles in the SNARE assembly, disassembling the cis-SNARE complex and the prefusion SNARE complex. How SM proteins coupled with NSF and α-SNAP regulate SNARE-dependent membrane fusion remains incompletely understood. Munc18c, an SM protein involved in the exocytosis of the glucose transporter GLUT4, binds and activates target (t-) SNAREs to accelerate the fusion reaction through a SNARE-like peptide (SLP). Here, using an in vitro reconstituted system, we discovered that α-SNAP blocks the GLUT4 SNAREs-mediated membrane fusion. Munc18c interacts with t-SNAREs to displace α-SNAP, which overcomes the fusion inhibition. Furthermore, Munc18c shields the trans-SNARE complex from NSF/α-SNAP-mediated disassembly and accelerates SNARE-dependent fusion kinetics in the presence of NSF and α-SNAP. The SLP in domain 3a is indispensable in Munc18c-assisted resistance to NSF and α-SNAP. Together, our findings demonstrate that Munc18c protects the prefusion SNARE complex from α-SNAP and NSF, promoting SNARE-dependent membrane fusion through its SLP.

Arabidopsis synaptotagmin 1 mediates lipid transport in a lipid composition-dependent manner.

The endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (EPCSs) are structurally conserved in eukaryotes. The Arabidopsis ER-anchored synaptotagmin 1 (SYT1), enriched in EPCSs, plays a critical role in plant abiotic stress tolerance. It has become clear that SYT1 interacts with PM to mediate ER-PM connectivity. However, whether SYT1 performs additional functions at EPCSs remains unknown. Here, we report that SYT1 efficiently transfers phospholipids between membranes. The lipid transfer activity of SYT1 is highly dependent on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2 ], a signal lipid accumulated at the PM under abiotic stress. Mechanically, while SYT1 transfers lipids fundamentally through the synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain, the efficient lipid transport requires the C2A domain-mediated membrane tethering. Interestingly, we observed that Ca2+ could stimulate SYT1-mediated lipid transport. In addition to PI(4,5)P2 , the Ca2+ activation requires the phosphatidylserine, another negatively charged lipid on the opposed membrane. Together, our studies identified Arabidopsis SYT1 as a lipid transfer protein at EPCSs and demonstrated that it takes conserved as well as divergent mechanisms with other extend-synaptotagmins. The critical role of lipid composition and Ca2+ reveals that SYT1-mediated lipid transport is highly regulated by signals in response to abiotic stresses.

ORP9 and ORP10 form a heterocomplex to transfer phosphatidylinositol 4-phosphate at ER-TGN contact sites.

Oxysterol-binding protein (OSBP) and its related proteins (ORPs) are a family of lipid transfer proteins (LTPs) that mediate non-vesicular lipid transport. ORP9 and ORP10, members of the OSBP/ORPs family, are located at the endoplasmic reticulum (ER)-trans-Golgi network (TGN) membrane contact sites (MCSs). It remained unclear how they mediate lipid transport. In this work, we discovered that ORP9 and ORP10 form a binary complex through intermolecular coiled-coil (CC) domain-CC domain interaction. The PH domains of ORP9 and ORP10 specially interact with phosphatidylinositol 4-phosphate (PI4P), mediating the TGN targeting. The ORP9-ORP10 complex plays a critical role in regulating PI4P levels at the TGN. Using in vitro reconstitution assays, we observed that while full-length ORP9 efficiently transferred PI4P between two apposed membranes, the lipid transfer kinetics was further accelerated by ORP10. Interestingly, our data showed that the PH domains of ORP9 and ORP10 participate in membrane tethering simultaneously, whereas ORDs of both ORP9 and ORP10 are required for lipid transport. Furthermore, our data showed that the depletion of ORP9 and ORP10 led to increased vesicle transport to the plasma membrane (PM). These findings demonstrate that ORP9 and ORP10 form a binary complex through the CC domains, maintaining PI4P homeostasis at ER-TGN MCSs and regulating vesicle trafficking.

Evaluation of an Optimized Workflow for the Radiofrequency Catheter Ablation of Paroxysmal Atrial Fibrillation.

BACKGROUND A significant number of atrial fibrillation (AF) recurrences occur after initial ablation, often due to pulmonary vein reconnections or triggers from non-pulmonary veins. MATERIAL AND METHODS Patients with paroxysmal AF who underwent radiofrequency catheter ablation for the first time were enrolled. Base on propensity score matching (1: 1 matching), 118 patients were selected for an optimized workflow for the radiofrequency catheter ablation of paroxysmal AF (OWCA) group and a conventional group. Comparative analysis of the acute and 12-month clinical outcomes was conducted. Moreover, an artificial intelligence analytics platform was used to evaluate the quality of pulmonary vein isolation (PVI) circles. RESULTS PVI was successfully achieved in all patients. Incidence of first-pass isolation of bilateral PVI circles was higher (P=0.009) and acute pulmonary vein reconnections was lower (P=0.027) in the OWCA group than conventional group. The OWCA group displayed a significant reduction in the number of fractured points (P<0.001), stacked points (P=0.003), and a greater proportion of cases in which the radiofrequency index achieved the target value (P=0.003). Additionally, the contact force consistently met the force over time criteria (P<0.001) for bilateral PVI circles in the OWCA group, accompanied by a shorter operation time (P=0.017). During the 12-month follow-up period, the OWCA group exhibited a higher atrial arrhythmia-free survival rate following the initial ablation procedure than did the conventional group. CONCLUSIONS The optimized workflow for radiofrequency catheter ablation of paroxysmal AF could play a crucial role in creating higher quality PVI circles. This improvement is reflected in a significantly elevated 12-month atrial arrhythmia-free survival rate.

Deep Tumor Penetration of CRISPR-Cas System for Photothermal-Sensitized Immunotherapy via Probiotics.

Since central cells are more malignant and aggressive in solid tumors, improving penetration of therapeutic agents and activating immunity in tumor centers exhibit great potential in cancer therapies. Here, polydopamine-coated Escherichia coli Nissle 1917 (EcN) bearing CRISPR-Cas9 plasmid-loaded liposomes (Lipo-P) are applied for enhanced immunotherapy in deep tumors through activation of innate and adaptive immunity simultaneously. After accumulation in the tumor center through hypoxia targeting, Lipo-P could be detached under the reduction of reactive oxygen species (ROS)-responsive linkers, lowering the thermal resistance of cancer cells via Hsp90α depletion. Owing to that, heating induced by polydopamine upon near-infrared irradiation could achieve effective tumor ablation. Furthermore, mild photothermal therapy induces immunogenic cell death, as bacterial infections in tumor tissues trigger innate immunity. This bacteria-assisted approach provides a promising photothermal-sensitized immunotherapy in deep tumors.

Manganese promotes α-synuclein amyloid aggregation through the induction of protein phase transition.

α-Synuclein (α-Syn) is the major protein component of Lewy bodies, a key pathological feature of Parkinson's disease (PD). The manganese ion Mn2+ has been identified as an environmental risk factor of PD. However, it remains unclear how Mn2+ regulates α-Syn aggregation. Here, we discovered that Mn2+accelerates α-Syn amyloid aggregation through the regulation of protein phase separation. We found that Mn2+ not only promotes α-Syn liquid-to-solid phase transition but also directly induces soluble α-Syn monomers to form solid-like condensates. Interestingly, the lipid membrane is integrated into condensates during Mn2+-induced α-Syn phase transition; however, the preformed Mn2+/α-syn condensates can only recruit lipids to the surface of condensates. In addition, this phase transition can largely facilitate α-Syn amyloid aggregation. Although the Mn2+-induced condensates do not fuse, our results demonstrated that they could recruit soluble α-Syn monomers into the existing condensates. Furthermore, we observed that a manganese chelator reverses Mn2+-induced α-Syn aggregation during the phase transition stage. However, after maturation, α-Syn aggregation becomes irreversible. These findings demonstrate that Mn2+ facilitates α-Syn phase transition to accelerate the formation of α-Syn aggregates and provide new insights for targeting α-Syn phase separation in PD treatment.

Assembly-promoting protein Munc18c stimulates SNARE-dependent membrane fusion through its SNARE-like peptide.

Intracellular vesicle fusion requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and their cognate Sec1/Munc18 (SM) proteins. How SM proteins act in concert with trans-SNARE complexes to promote membrane fusion remains incompletely understood. Munc18c, a broadly distributed SM protein, selectively regulates multiple exocytotic pathways, including GLUT4 exocytosis. Here, using an in vitro reconstituted system, we discovered a SNARE-like peptide (SLP), conserved in Munc18-1 of synaptic exocytosis, is crucial to the stimulatory activity of Munc18c in vesicle fusion. The direct stimulation of the SNARE-mediated fusion reaction by SLP further supported the essential role of this fragment. Interestingly, we found SLP strongly accelerates the membrane fusion rate when anchored to the target membrane but not the vesicle membrane, suggesting it primarily interacts with t-SNAREs in cis to drive fusion. Furthermore, we determined the SLP fragment is competitive with the full-length Munc18c protein and specific to the cognate v-SNARE isoforms, supporting how it could resemble Munc18c's activity in membrane fusion. Together, our findings demonstrate that Munc18c facilitates SNARE-dependent membrane fusion through SLP, revealing that the t-SNARE-SLP binding mode might be a conserved mechanism for the stimulatory function of SM proteins in vesicle fusion.

AAGAB is an assembly chaperone regulating AP1 and AP2 clathrin adaptors.

Multimeric cargo adaptors such as AP2 play central roles in intracellular membrane trafficking. We recently discovered that the assembly of the AP2 adaptor complex, a key player in clathrin-mediated endocytosis, is a highly organized process controlled by alpha- and gamma-adaptin-binding protein (AAGAB, also known as p34). In this study, we demonstrate that besides AP2, AAGAB also regulates the assembly of AP1, a cargo adaptor involved in clathrin-mediated transport between the trans-Golgi network and the endosome. However, AAGAB is not involved in the formation of other adaptor complexes, including AP3. AAGAB promotes AP1 assembly by binding and stabilizing the γ and σ subunits of AP1, and its mutation abolishes AP1 assembly and disrupts AP1-mediated cargo trafficking. Comparative proteomic analyses indicate that AAGAB mutation massively alters surface protein homeostasis, and its loss-of-function phenotypes reflect the synergistic effects of AP1 and AP2 deficiency. Taken together, these findings establish AAGAB as an assembly chaperone for both AP1 and AP2 adaptors and pave the way for understanding the pathogenesis of AAGAB-linked diseases.

NEDD4 ameliorates myocardial reperfusion injury by preventing macrophages pyroptosis.

OBJECTIVES: The inflammatory cascade and cell death post-myocardial ischemia reperfusion (MI/R) are very complex. Despite the understanding that macrophage inflammation has a pivotal role in the pathophysiology of MI/R, the contribution of macrophage inflammatory signals in tailoring the function of vascular endothelium remains unknown. MATERIALS AND METHODS: In the present study, we analyzed the effects of NEDD4 on the NLRP3 inflammasome activation-mediated pyroptosis in vitro after an acute pro-inflammatory stimulus and in vivo in a MI/R mouse model. TTC and Evan's blue dye, Thioflavin S, immunohistochemistry staining, and ELISA were performed in wild-type and NEDD4 deficiency mice. THP-1 cells were transfected with si-NEDD4 or si-SF3A2. HEK293T cells were transfected with NEDD4 or SF3A2 overexpression plasmid. ELISA analyzed the inflammatory cytokines in the cell supernatant. The levels of NEDD4, SF3A2, and NLRP3/GSDMD pathway were determined by Western blot. Protein interactions were evaluated by immunoprecipitation. The protein colocalization in cells was monitored using a fluorescence microscope. RESULTS: NEDD4 inhibited NLRP3 inflammasome activation and pyroptosis in THP-1 cells treated with lipopolysaccharide (LPS) and nigericin (Nig). Mechanistically, NEDD4 maintained the stability of NLRP3 through direct interaction with the SF3A2, whereas the latter association with NLRP3 indirectly interacted with NEDD4 promoting proteasomal degradation of NLRP3. Deletion of NLRP3 expression further inhibited the caspase cascade to induce pyroptosis. Interestingly, inhibiting NLRP3 inflammasome activation in THP-1 cells could prevent cardiac microvascular endothelial cells (CMECs) injury. In addition, NEDD4 deficiency decreased animal survival and increased myocardial infarct size, no-reflow area, and promoted macrophages infiltration post-MI/R. CONCLUSIONS: NEDD4 could be a potential therapeutic target in microvascular injury following myocardial reperfusion. Video Abstract.

A novel stepwise catheter ablation method of the mitral isthmus for persistent atrial fibrillation: efficacy and reproducibility.

BACKGROUND: Ethanol infusion of the vein of Marshall (EI-VOM) has been widely used to facilitate mitral isthmus (MI) ablation. According to the literature, the success rate of achieving a bidirectional conduction block across the MI ranges from 51 to 96%, with no standardized strategy or method available for cardiac electrophysiologists. OBJECTIVES: This study aimed to introduce and evaluate a novel ablation method of MI. METHODS: Consecutive patients with persistent atrial fibrillation (PeAF) that underwent catheter ablation were included. The MI ablation procedure followed a stepwise approach. In step 1, ethanol infusion of the vein of Marshall (EI-VOM) was performed. In step 2, a "V-shape" endocardial linear ablation connecting the left inferior pulmonary vein (LIPV) to mitral annulus (MA) was performed. In step 3, earliest activation sites(EASs) near the ablation line were identified using activation mapping followed by reinforced ablation. In step 4, precise epicardial ablation was performed, with the catheter introduced into the coronary sinus(CS) to target key ablation targets (KATs). RESULTS: 135 patients with PeAF underwent catheter ablation with the stepwise ablation method adopted in 119 cases. Bidirectional conduction blocks were achieved in 117 patients (98.3%). The block rates of every step were 0%, 58.0%, 44.0%, and 92.9%, and the cumulative block rates for the four steps were 0%, 58.0%, 76.5%, and 98.3%, respectively. No patient experienced fatal complications. CONCLUSIONS: Our novel stepwise catheter ablation method for MI yielded a high bidirectional block rate with high reproducibility.

Genetic evidence for an inhibitory role of tomosyn in insulin-stimulated GLUT4 exocytosis.

Exocytosis is a vesicle fusion process driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A classic exocytic pathway is insulin-stimulated translocation of the glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane in adipocytes and skeletal muscles. The GLUT4 exocytic pathway plays a central role in maintaining blood glucose homeostasis and is compromised in insulin resistance and type 2 diabetes. A candidate regulator of GLUT4 exocytosis is tomosyn, a soluble protein expressed in adipocytes. Tomosyn directly binds to GLUT4 exocytic SNAREs in vitro but its role in GLUT4 exocytosis was unknown. In this work, we used CRISPR-Cas9 genome editing to delete the two tomosyn-encoding genes in adipocytes. We observed that both basal and insulin-stimulated GLUT4 exocytosis was markedly elevated in the double knockout (DKO) cells. By contrast, adipocyte differentiation and insulin signaling remained intact in the DKO adipocytes. In a reconstituted liposome fusion assay, tomosyn inhibited all the SNARE complexes underlying GLUT4 exocytosis. The inhibitory activity of tomosyn was relieved by NSF and α-SNAP, which act in concert to remove tomosyn from GLUT4 exocytic SNAREs. Together, these studies revealed an inhibitory role for tomosyn in insulin-stimulated GLUT4 exocytosis in adipocytes. We suggest that tomosyn-arrested SNAREs represent a reservoir of fusion capacity that could be harnessed to treat patients with insulin resistance and type 2 diabetes.

Calcium-dependent and -independent lipid transfer mediated by tricalbins in yeast.

Membrane contact sites (MCSs) formed between the endoplasmic reticulum (ER) and the plasma membrane (PM) provide a platform for nonvesicular lipid exchange. The ER-anchored tricalbins (Tcb1, Tcb2, and Tcb3) are critical tethering factors at ER-PM MCSs in yeast. Tricalbins possess a synaptotagmin-like mitochondrial-lipid-binding protein (SMP) domain and multiple Ca2+-binding C2 domains. Although tricalbins have been suggested to be involved in lipid exchange at the ER-PM MCSs, it remains unclear whether they directly mediate lipid transport. Here, using in vitro lipid transfer assays, we discovered that tricalbins are capable of transferring phospholipids between membranes. Unexpectedly, while its lipid transfer activity was markedly elevated by Ca2+, Tcb3 constitutively transferred lipids even in the absence of Ca2+. The stimulatory activity of Ca2+ on Tcb3 required intact Ca2+-binding sites on both the C2C and C2D domains of Tcb3, while Ca2+-independent lipid transport was mediated by the SMP domain that transferred lipids via direct interactions with phosphatidylserine and other negatively charged lipid molecules. These findings establish tricalbins as lipid transfer proteins, and reveal Ca2+-dependent and -independent lipid transfer activities mediated by these tricalbins, providing new insights into their mechanism in maintaining PM integrity at ER-PM MCSs.

Myricetin Inhibits α-Synuclein Amyloid Aggregation by Delaying the Liquid-to-Solid Phase Transition.

The aggregation of α-synuclein (α-Syn) is a critical pathological hallmark of Parkinson's disease (PD). Prevention of α-Syn aggregation has become a key strategy for treating PD. Recent studies have suggested that α-Syn undergoes liquid-liquid phase separation (LLPS) to facilitate nucleation and amyloid formation. Here, we examined the modulation of α-Syn aggregation by myricetin, a polyhydroxyflavonol compound, under the conditions of LLPS. Unexpectedly, neither the initial morphology nor the phase-separated fraction of α-Syn was altered by myricetin. However, the dynamics of α-Syn condensates decreased upon myricetin binding. Further studies showed that myricetin dose-dependently inhibits amyloid aggregation in the condensates by delaying the liquid-to-solid phase transition. In addition, myricetin could disassemble the preformed α-Syn amyloid aggregates matured from the condensates. Together, our study shows that myricetin inhibits α-Syn amyloid aggregation in the condensates by retarding the liquid-to-solid phase transition and reveals that α-Syn phase transition can be targeted to inhibit amyloid aggregation.

A small molecule that mitigates bacterial infection disrupts Gram-negative cell membranes and is inhibited by cholesterol and neutral lipids.

Infections caused by Gram-negative bacteria are difficult to fight because these pathogens exclude or expel many clinical antibiotics and host defense molecules. However, mammals have evolved a substantial immune arsenal that weakens pathogen defenses, suggesting the feasibility of developing therapies that work in concert with innate immunity to kill Gram-negative bacteria. Using chemical genetics, we recently identified a small molecule, JD1, that kills Salmonella enterica serovar Typhimurium (S. Typhimurium) residing within macrophages. JD1 is not antibacterial in standard microbiological media, but rapidly inhibits growth and curtails bacterial survival under broth conditions that compromise the outer membrane or reduce efflux pump activity. Using a combination of cellular indicators and super resolution microscopy, we found that JD1 damaged bacterial cytoplasmic membranes by increasing fluidity, disrupting barrier function, and causing the formation of membrane distortions. We quantified macrophage cell membrane integrity and mitochondrial membrane potential and found that disruption of eukaryotic cell membranes required approximately 30-fold more JD1 than was needed to kill bacteria in macrophages. Moreover, JD1 preferentially damaged liposomes with compositions similar to E. coli inner membranes versus mammalian cell membranes. Cholesterol, a component of mammalian cell membranes, was protective in the presence of neutral lipids. In mice, intraperitoneal administration of JD1 reduced tissue colonization by S. Typhimurium. These observations indicate that during infection, JD1 gains access to and disrupts the cytoplasmic membrane of Gram-negative bacteria, and that neutral lipids and cholesterol protect mammalian membranes from JD1-mediated damage. Thus, it may be possible to develop therapeutics that exploit host innate immunity to gain access to Gram-negative bacteria and then preferentially damage the bacterial cell membrane over host membranes.

SNARE zippering requires activation by SNARE-like peptides in Sec1/Munc18 proteins.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze membrane fusion by forming coiled-coil bundles between membrane bilayers. The SNARE bundle zippers progressively toward the membranes, pulling the lipid bilayers into close proximity to fuse. In this work, we found that the +1 and +2 layers in the C-terminal domains (CTDs) of SNAREs are dispensable for reconstituted SNARE-mediated fusion reactions. By contrast, all CTD layers are required for fusion reactions activated by the cognate Sec1/Munc18 (SM) protein or a synthetic Vc peptide derived from the vesicular (v-) SNARE, correlating with strong acceleration of fusion kinetics. These results suggest a similar mechanism underlying the stimulatory functions of SM proteins and Vc peptide in SNARE-dependent membrane fusion. Unexpectedly, we identified a conserved SNARE-like peptide (SLP) in SM proteins that structurally and functionally resembles Vc peptide. Like Vc peptide, SLP binds and activates target (t-) SNAREs, accelerating the fusion reaction. Disruption of the t-SNARE-SLP interaction inhibits exocytosis in vivo. Our findings demonstrated that a t-SNARE-SLP intermediate must form before SNAREs can drive efficient vesicle fusion.

Inducible Exoc7/Exo70 knockout reveals a critical role of the exocyst in insulin-regulated GLUT4 exocytosis.

Insulin promotes glucose uptake by triggering the translocation of glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane through exocytosis. GLUT4 exocytosis is a vesicle fusion event involving fusion of GLUT4-containing vesicles with the plasma membrane. For GLUT4 vesicle fusion to occur, GLUT4 vesicles must first be tethered to the plasma membrane. A key tethering factor in exocytosis is a heterooctameric protein complex called the exocyst. The role of the exocyst in GLUT4 exocytosis, however, remains incompletely understood. Here we first systematically analyzed data from a genome-scale CRISPR screen in HeLa cells that targeted virtually all known genes in the human genome, including 12 exocyst genes. The screen recovered only a subset of the exocyst genes, including exocyst complex component 7 (Exoc7/Exo70). Other exocyst genes, however, were not isolated in the screen, likely because of functional redundancy. Our findings suggest that selection of an appropriate exocyst gene is critical for genetic studies of exocyst functions. Next we developed an inducible adipocyte genome editing system that enabled Exoc7 gene deletion in adipocytes without interfering with adipocyte differentiation. We observed that insulin-stimulated GLUT4 exocytosis was markedly inhibited in Exoc7 KO adipocytes. Insulin signaling, however, remained intact in these KO cells. These results indicate that the exocyst plays a critical role in insulin-stimulated GLUT4 exocytosis in adipocytes. We propose that the strategy outlined in this work could be instrumental in genetically dissecting other membrane-trafficking pathways in adipocytes.

RABIF/MSS4 is a Rab-stabilizing holdase chaperone required for GLUT4 exocytosis.

Rab GTPases are switched from their GDP-bound inactive conformation to a GTP-bound active state by guanine nucleotide exchange factors (GEFs). The first putative GEFs isolated for Rabs are RABIF (Rab-interacting factor)/MSS4 (mammalian suppressor of Sec4) and its yeast homolog DSS4 (dominant suppressor of Sec4). However, the biological function and molecular mechanism of these molecules remained unclear. In a genome-wide CRISPR genetic screen, we isolated RABIF as a positive regulator of exocytosis. Knockout of RABIF severely impaired insulin-stimulated GLUT4 exocytosis in adipocytes. Unexpectedly, we discovered that RABIF does not function as a GEF, as previously assumed. Instead, RABIF promotes the stability of Rab10, a key Rab in GLUT4 exocytosis. In the absence of RABIF, Rab10 can be efficiently synthesized but is rapidly degraded by the proteasome, leading to exocytosis defects. Strikingly, restoration of Rab10 expression rescues exocytosis defects, bypassing the requirement for RABIF. These findings reveal a crucial role of RABIF in vesicle transport and establish RABIF as a Rab-stabilizing holdase chaperone, a previously unrecognized mode of Rab regulation independent of its GDP-releasing activity. Besides Rab10, RABIF also regulates the stability of two other Rab GTPases, Rab8 and Rab13, suggesting that the requirement of holdase chaperones is likely a general feature of Rab GTPases.

The N-peptide-binding mode is critical to Munc18-1 function in synaptic exocytosis.

Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A key SNARE-binding mode of SM proteins involves the N-terminal peptide (N-peptide) motif of syntaxin, a SNARE subunit localized to the target membrane. In in vitro membrane fusion assays, inhibition of N-peptide motif binding previously has been shown to abrogate the stimulatory function of Munc18-1, a SM protein involved in synaptic exocytosis in neurons. The physiological role of the N-peptide-binding mode, however, remains unclear. In this work, we addressed this key question using a "clogged" Munc18-1 protein, in which an ectopic copy of the syntaxin N-peptide motif was directly fused to Munc18-1. We found that the ectopic N-peptide motif blocks the N-peptide-binding pocket of Munc18-1, preventing the latter from binding to the native N-peptide motif on syntaxin-1. In a reconstituted system, we observed that clogged Munc18-1 is defective in promoting SNARE zippering. When introduced into induced neuronal cells (iN cells) derived from human pluripotent stem cells, clogged Munc18-1 failed to mediate synaptic exocytosis. As a result, both spontaneous and evoked synaptic transmission was abolished. These genetic findings provide direct evidence for the crucial role of the N-peptide-binding mode of Munc18-1 in synaptic exocytosis. We suggest that clogged SM proteins will also be instrumental in defining the physiological roles of the N-peptide-binding mode in other vesicle-fusion pathways.

Extended synaptotagmins are Ca2+-dependent lipid transfer proteins at membrane contact sites.

Organelles are in constant communication with each other through exchange of proteins (mediated by trafficking vesicles) and lipids [mediated by both trafficking vesicles and lipid transfer proteins (LTPs)]. It has long been known that vesicle trafficking can be tightly regulated by the second messenger Ca(2+), allowing membrane protein transport to be adjusted according to physiological demands. However, it remains unclear whether LTP-mediated lipid transport can also be regulated by Ca(2+) In this work, we show that extended synaptotagmins (E-Syts), poorly understood membrane proteins at endoplasmic reticulum-plasma membrane contact sites, are Ca(2+)-dependent LTPs. Using both recombinant and endogenous mammalian proteins, we discovered that E-Syts transfer glycerophospholipids between membrane bilayers in the presence of Ca(2+) E-Syts use their lipid-accommodating synaptotagmin-like mitochondrial lipid binding protein (SMP) domains to transfer lipids. However, the SMP domains themselves cannot transport lipids unless the two membranes are tightly tethered by Ca(2+)-bound C2 domains. Strikingly, the Ca(2+)-regulated lipid transfer activity of E-Syts was fully recapitulated when the SMP domain was fused to the cytosolic domain of synaptotagmin-1, the Ca(2+)sensor in synaptic vesicle fusion, indicating that a common mechanism of membrane tethering governs the Ca(2+)regulation of lipid transfer and vesicle fusion. Finally, we showed that microsomal vesicles isolated from mammalian cells contained robust Ca(2+)-dependent lipid transfer activities, which were mediated by E-Syts. These findings established E-Syts as a novel class of LTPs and showed that LTP-mediated lipid trafficking, like vesicular transport, can be subject to tight Ca(2+)regulation.