Molecular mechanism underlying SNARE-mediated membrane fusion enlightened by all-atom molecular dynamics simulations.

The SNAP receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin mediate neurotransmitter release by forming tight SNARE complexes that fuse synaptic vesicles with the plasma membranes in microseconds. Membrane fusion is generally explained by the action of proteins on macroscopic membrane properties such as curvature, elastic modulus, and tension, and a widespread model envisions that the SNARE motifs, juxtamembrane linkers, and C-terminal transmembrane regions of synaptobrevin and syntaxin-1 form continuous helices that act mechanically as semirigid rods, squeezing the membranes together as they assemble ("zipper") from the N to the C termini. However, the mechanism underlying fast SNARE-induced membrane fusion remains unknown. We have used all-atom molecular dynamics simulations to investigate this mechanism. Our results need to be interpreted with caution because of the limited number and length of the simulations, but they suggest a model of membrane fusion that has a natural physicochemical basis, emphasizes local molecular events over general membrane properties, and explains extensive experimental data. In this model, the central event that initiates fast (microsecond scale) membrane fusion occurs when the SNARE helices zipper into the juxtamembrane linkers which, together with the adjacent transmembrane regions, promote encounters of acyl chains from both bilayers at the polar interface. The resulting hydrophobic nucleus rapidly expands into stalk-like structures that gradually progress to form a fusion pore, aided by the SNARE transmembrane regions and without clearly discernible intermediates. The propensity of polyunsaturated lipids to participate in encounters that initiate fusion suggests that these lipids may be important for the high speed of neurotransmitter release.

Complexin has a dual synaptic function as checkpoint protein in vesicle priming and as a promoter of vesicle fusion.

The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity.

Rab GTPases and phosphoinositides fine-tune SNAREs dependent targeting specificity of intracellular vesicle traffic.

In the secretory pathway the destination of trafficking vesicles is determined by specific proteins that, with the notable exception of SNAREs, are recruited from soluble pools. Previously we have shown that microinjected proteoliposomes containing early or late endosomal SNAREs, respectively, are targeted to the corresponding endogenous compartments, with targeting specificity being dependent on the recruitment of tethering factors by some of the SNAREs. Here, we show that targeting of SNARE-containing liposomes is refined upon inclusion of polyphosphoinositides and Rab5. Intriguingly, targeting specificity is dependent on the concentration of PtdIns(3)P, and on the recruitment of PtdIns(3)P binding proteins such as rabenosyn-5 and PIKfyve, with conversion of PtdIns(3)P into PtdIns(3,5)P2 re-routing the liposomes towards late endosomes despite the presence of GTP-Rab5 and early endosomal SNAREs. Our data reveal a complex interplay between permissive and inhibitory targeting signals that sharpen a basic targeting and fusion machinery for conveying selectivity in intracellular membrane traffic.

Tomosyns attenuate SNARE assembly and synaptic depression by binding to VAMP2-containing template complexes.

Tomosyns are widely thought to attenuate membrane fusion by competing with synaptobrevin-2/VAMP2 for SNARE-complex assembly. Here, we present evidence against this scenario. In a novel mouse model, tomosyn-1/2 deficiency lowered the fusion barrier and enhanced the probability that synaptic vesicles fuse, resulting in stronger synapses with faster depression and slower recovery. While wild-type tomosyn-1m rescued these phenotypes, substitution of its SNARE motif with that of synaptobrevin-2/VAMP2 did not. Single-molecule force measurements indeed revealed that tomosyn's SNARE motif cannot substitute synaptobrevin-2/VAMP2 to form template complexes with Munc18-1 and syntaxin-1, an essential intermediate for SNARE assembly. Instead, tomosyns extensively bind synaptobrevin-2/VAMP2-containing template complexes and prevent SNAP-25 association. Structure-function analyses indicate that the C-terminal polybasic region contributes to tomosyn's inhibitory function. These results reveal that tomosyns regulate synaptic transmission by cooperating with synaptobrevin-2/VAMP2 to prevent SNAP-25 binding during SNARE assembly, thereby limiting initial synaptic strength and equalizing it during repetitive stimulation.

Overexpression of the alfalfa (Medicago sativa) gene, MsKMS1, negatively regulates seed germination in transgenic tobacco (Nicotiana tabacum).

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-associated proteins are a class of transmembrane proteins involved in intracellular trafficking pathways. However, the functions of many SNARE domain-containing proteins remain unclear. We have previously identified a SNARE-associated gene in alfalfa (Medicago sativa ) KILLING ME SLOWLY1 (MsKMS1 ), which is involved in various abiotic stresses. In this study, we investigated the function of MsKMS1 in the seed germination of transgenic tobacco (Nicotiana tabacum ). Phylogenetic analysis showed that MsKMS1 was homologous to the SNARE-associated or MAPR component-related proteins of other plants. Germination assays revealed that MsKMS1 negatively regulated seed germination under normal, D-mannitol and abscisic acid-induced stress conditions, yet MsKMS1 -overexpression could confer enhanced heat tolerance in transgenic tobacco. The suppressive effect on germination in MsKMS1 -overexpression lines was associated with higher abscisic acid and salicylic acid contents in seeds. This was accompanied by the upregulation of abscisic acid biosynthetic genes (ZEP and NCED ) and the downregulation of gibberellin biosynthetic genes (GA20ox2 and GA20ox3 ). Taken together, these results suggested that MsKMS1 negatively regulated seed germination by increasing abscisic acid and salicylic acid contents through the expression of genes related to abscisic acid and gibberellin biosynthesis. In addition, MsKMS1 could improve heat tolerance during the germination of transgenic tobacco seeds.

High-speed measurements of SNARE-complexin interactions using magnetic tweezers.

In neuroscience, understanding the mechanics of synapses, especially the function of force-sensitive proteins at the molecular level, is essential. This need emphasizes the importance of precise measurement of synaptic protein interactions. Addressing this, we introduce high-resolution magnetic tweezers (MT) as a novel method to probe the mechanics of synapse-related proteins with high precision. We demonstrate this technique through studying SNARE-complexin interactions, crucial for synaptic transmission, showcasing its capability to apply specific forces to individual molecules. Our results reveal that high-resolution MT provides in-depth insights into the stability and dynamic transitions of synaptic protein complexes. This method is a significant advancement in synapse biology, offering a new tool for researchers to investigate the impact of mechanical forces on synaptic functions and their implications for neurological disorders.

SM protein Sly1 and a SNARE Habc domain promote membrane fusion through multiple mechanisms.

SM proteins including Sly1 are essential cofactors of SNARE-mediated membrane fusion. Using SNARE and Sly1 mutants and chemically defined in vitro assays, we separate and assess proposed mechanisms through which Sly1 augments fusion: (i) opening the closed conformation of the Qa-SNARE Sed5; (ii) close-range tethering of vesicles to target organelles, mediated by the Sly1-specific regulatory loop; and (iii) nucleation of productive trans-SNARE complexes. We show that all three mechanisms are important and operate in parallel, and that close-range tethering promotes trans-complex assembly when cis-SNARE assembly is a competing process. Further, we demonstrate that the autoinhibitory N-terminal Habc domain of Sed5 has at least two positive activities: it is needed for correct Sed5 localization, and it directly promotes Sly1-dependent fusion. "Split Sed5," with Habc presented solely as a soluble fragment, can function both in vitro and in vivo. Habc appears to facilitate events leading to lipid mixing rather than promoting opening or stability of the fusion pore.

SNARE chaperone Sly1 directly mediates close-range vesicle tethering.

The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway.

Sec18 binds the tethering/SM complex HOPS to engage the Qc-SNARE for membrane fusion.

Membrane fusion is regulated by Rab GTPases, their tethering effectors such as HOPS, SNARE proteins on each fusion partner, SM proteins to catalyze SNARE assembly, Sec17 (SNAP), and Sec18 (NSF). Though concentrated HOPS can support fusion without Sec18, we now report that fusion falls off sharply at lower HOPS levels, where direct Sec18 binding to HOPS restores fusion. This Sec18-dependent fusion needs adenine nucleotide but neither ATP hydrolysis nor Sec17. Sec18 enhances HOPS recognition of the Qc-SNARE. With high levels of HOPS, Qc has a Km for fusion of a few nM. Either lower HOPS levels, or substitution of a synthetic tether for HOPS, strikingly increases the Km for Qc to several hundred nM. With dilute HOPS, Sec18 returns the Km for Qc to low nM. In contrast, HOPS concentration and Sec18 have no effect on Qb-SNARE recognition. Just as Qc is required for fusion but not for the initial assembly of SNAREs in trans, impaired Qc recognition by limiting HOPS without Sec18 still allows substantial trans-SNARE assembly. Thus, in addition to the known Sec18 functions of disassembling SNARE complexes, oligomerizing Sec17 for membrane association, and allowing Sec17 to drive fusion without complete SNARE zippering, we report a fourth Sec18 function, the Sec17-independent binding of Sec18 to HOPS to enhance functional Qc-SNARE engagement.

Impaired TFEB-mediated autophagy-lysosome fusion promotes tubular cell cycle G2/M arrest and renal fibrosis by suppressing ATP6V0C expression and interacting with SNAREs.

Increasing evidence suggests that autophagy plays a major role during renal fibrosis. Transcription factor EB (TFEB) is a critical regulator of autophagy- and lysosome-related gene transcription. However, the pathophysiological roles of TFEB in renal fibrosis and fine-tuned mechanisms by which TFEB regulates fibrosis remain largely unknown. Here, we found that TFEB was downregulated in unilateral ureteral obstruction (UUO)-induced human and mouse fibrotic kidneys, and kidney-specific TFEB overexpression using recombinant AAV serotype 9 (rAAV9)-TFEB in UUO mice alleviated renal fibrosis pathogenesis. Mechanically, we found that TFEB's prevention of extracellular matrix (ECM) deposition depended on autophagic flux integrity and its subsequent blockade of G2/M arrest in tubular cells, rather than the autophagosome synthesis. In addition, we together RNA-seq with CUT&Tag analysis to determine the TFEB targeted gene ATP6V0C, and revealed that TFEB was directly bound to the ATP6V0C promoter only at specific site to promote its expression through CUT&Run-qPCR and luciferase reporter assay. Interestingly, TFEB induced autophagic flux integrity, mainly dependent on scaffold protein ATP6V0C-mediated autophagosome-lysosome fusion by bridging with STX17 and VAMP8 (major SNARE complex) by co-immunoprecipitation analysis, rather than its mediated lysosomal acidification and degradation function. Moreover, we further investigated the underlying mechanism behind the low expression of TEFB in UUO-induced renal fibrosis, and clearly revealed that TFEB suppression in fibrotic kidney was due to DNMT3a-associated TFEB promoter hypermethylation by utilizing methylation specific PCR (MSP) and bisulfite-sequencing PCR (BSP), which could be effectively recovered by 5-Aza-2'-deoxycytidine (5A-za) to alleviate renal fibrosis pathogenesis. These findings reveal for the first time that impaired TFEB-mediated autophagosome-lysosome fusion disorder, tubular cell G2/M arrest and renal fibrosis appear to be sequentially linked in UUO-induced renal fibrosis and suggest that DNMT3a/TFEB/ATP6V0C may serve as potential therapeutic targets to prevent renal fibrosis.

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.

The Ykt6-Snap29-Syx13 SNARE complex promotes crinophagy via secretory granule fusion with Lamp1 carrier vesicles.

In the Drosophila larval salivary gland, developmentally programmed fusions between lysosomes and secretory granules (SGs) and their subsequent acidification promote the maturation of SGs that are secreted shortly before puparium formation. Subsequently, ongoing fusions between non-secreted SGs and lysosomes give rise to degradative crinosomes, where the superfluous secretory material is degraded. Lysosomal fusions control both the quality and quantity of SGs, however, its molecular mechanism is incompletely characterized. Here we identify the R-SNARE Ykt6 as a novel regulator of crinosome formation, but not the acidification of maturing SGs. We show that Ykt6 localizes to Lamp1+ carrier vesicles, and forms a SNARE complex with Syntaxin 13 and Snap29 to mediate fusion with SGs. These Lamp1 carriers represent a distinct vesicle population that are functionally different from canonical Arl8+, Cathepsin L+ lysosomes, which also fuse with maturing SGs but are controlled by another SNARE complex composed of Syntaxin 13, Snap29 and Vamp7. Ykt6- and Vamp7-mediated vesicle fusions also determine the fate of SGs, as loss of either of these SNAREs prevents crinosomes from acquiring endosomal PI3P. Our results highlight that fusion events between SGs and different lysosome-related vesicle populations are critical for fine regulation of the maturation and crinophagic degradation of SGs.

Control of Munc13-1 Activity by Autoinhibitory Interactions Involving the Variable N-terminal Region.

Regulation of neurotransmitter release during presynaptic plasticity underlies varied forms of information processing in the brain. Munc13s play essential roles in release via their conserved C-terminal region, which contains a MUN domain involved in SNARE complex assembly, and controls multiple presynaptic plasticity processes. Munc13s also have a variable N-terminal region, which in Munc13-1 includes a calmodulin binding (CaMb) domain involved in short-term plasticity and a C2A domain that forms an inhibitory homodimer. The C2A domain is activated by forming a heterodimer with the zinc-finger domain of αRIMs, providing a link to αRIM-dependent short- and long-term plasticity. However, it is unknown how the functions of the N- and C-terminal regions are integrated, in part because of the difficulty of purifying Munc13-1 fragments containing both regions. We describe for the first time the purification of a Munc13-1 fragment spanning its entire sequence except for a flexible region between the C2A and CaMb domains. We show that this fragment is much less active than the Munc13-1 C-terminal region in liposome fusion assays and that its activity is strongly enhanced by the RIM2α zinc-finger domain together with calmodulin. NMR experiments show that the C2A and CaMb domains bind to the MUN domain and that these interactions are relieved by the RIM2α ZF domain and calmodulin, respectively. These results suggest a model whereby Munc13-1 activity in promoting SNARE complex assembly and neurotransmitter release are inhibited by interactions of the C2A and CaMb domains with the MUN domain that are relieved by αRIMs and calmodulin.

Emerging roles of O-GlcNAcylation in protein trafficking and secretion.

The emerging roles of O-GlcNAcylation, a distinctive post-translational modification, are increasingly recognized for their involvement in the intricate processes of protein trafficking and secretion. This modification exerts its influence on both conventional and unconventional secretory pathways. Under healthy and stress conditions, such as during diseases, it orchestrates the transport of proteins within cells, ensuring timely delivery to their intended destinations. O-GlcNAcylation occurs on key factors, like coat protein complexes (COPI and COPII), clathrin, SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and GRASP55 (Golgi reassembly stacking protein of 55 kDa) that control vesicle budding and fusion in anterograde and retrograde trafficking and unconventional secretion. The understanding of O-GlcNAcylation offers valuable insights into its critical functions in cellular physiology and the progression of diseases, including neurodegeneration, cancer, and metabolic disorders. In this review, we summarize and discuss the latest findings elucidating the involvement of O-GlcNAc in protein trafficking and its significance in various human disorders.

The crustacean DNA virus tegument protein VP26 binds to SNAP29 to inhibit SNARE complex assembly and autophagic degradation.

Autophagy generally functions as a cellular surveillance mechanism to combat invading viruses, but viruses have evolved various strategies to block autophagic degradation and even subvert it to promote viral propagation. White spot syndrome virus (WSSV) is the most highly pathogenic crustacean virus, but little is currently known about whether crustacean viruses such as WSSV can subvert autophagic degradation for escape. Here, we show that even though WSSV proliferation triggers the accumulation of autophagosomes, autophagic degradation is blocked in the crustacean species red claw crayfish. Interestingly, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex including CqSNAP29, CqVAMP7, and the novel autophagosome SNARE protein CqSyx12 is required for autophagic flux to restrict WSSV replication, as revealed by gene silencing experiments. Simultaneously, the expressed WSSV tegument protein VP26, which likely localizes on autophagic membrane mediated by its transmembrane region, binds the Qb-SNARE domain of CqSNAP29 to competitively inhibit the binding of CqSyx12-Qa-SNARE with CqSNAP29-Qb-SNARE; this in turn disrupts the assembly of the CqSyx12-SNAP29-VAMP7 SNARE complex, which is indispensable for the proposed fusion of autophagosomes and lysosomes. Consequently, the autophagic degradation of WSSV is likely suppressed by the expressed VP26 protein in vivo in crayfish, thus probably protecting WSSV components from degradation via the autophagosome-lysosome pathway, resulting in evasion by WSSV. Collectively, these findings highlight how a DNA virus can subvert autophagic degradation by impairing the assembly of the SNARE complex to achieve evasion, paving the way for understanding host-DNA virus interactions from an evolutionary point of view, from crustaceans to mammals.IMPORTANCEWhite spot syndrome virus (WSSV) is one of the largest animal DNA viruses in terms of its genome size and has caused huge economic losses in the farming of crustaceans such as shrimp and crayfish. Detailed knowledge of WSSV-host interactions is still lacking, particularly regarding viral escape from host immune clearance. Intriguingly, we found that the presence of WSSV-VP26 might inhibit the autophagic degradation of WSSV in vivo in the crustacean species red claw crayfish. Importantly, this study is the first to show that viral protein VP26 functions as a core factor to benefit WSSV escape by disrupting the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which is necessary for the proposed fusion of autophagosomes with lysosomes for subsequent degradation. These findings highlight a novel mechanism of DNA virus evasion by blocking SNARE complex assembly and identify viral VP26 as a key candidate for anti-WSSV targeting.

Asymmetric tethering by exocyst in vitro requires a Rab GTPase, an R-SNARE and a Sac1-sensitive phosphoinositide lipid.

Tethering factors play a critical role in deciphering the correct combination of vesicle and target membrane, before SNARE complex formation and membrane fusion. The exocyst plays a central role in tethering post-Golgi vesicles to the plasma membrane, although the mechanism by which this occurs is poorly understood. We recently established an assay for measuring exocyst-mediated vesicle tethering in vitro and we have adapted this assay to examine the ability of exocyst to tether vesicles in an asymmetric manner. We demonstrate that exocyst differs from another post-Golgi vesicle tethering protein, Sro7, in that it is fully capable of tethering vesicles with a functional Rab GTPase, Sec4, to vesicles lacking a functional Rab GTPase. Using this assay, we show that exocyst requires both the Rab and R-SNARE, Snc1, to be present on the same membrane surface. Using Sac1 phosphatase treatment, we demonstrate a likely role for phosphoinositides on the opposing Rab-deficient membrane. This suggests a specific model for exocyst orientation and its points of contact between membranes during heterotypic tethering of post-Golgi vesicles with the plasma membrane.

All SNAP25 molecules in the vesicle-plasma membrane contact zone change conformation during vesicle priming.

In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25-based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild-type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging reveals a rapid FRET increase preceding individual fusion events by 65 ms. The experiments are performed under conditions of a steady-state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle-plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step.

Structure of a membrane tethering complex incorporating multiple SNAREs.

Most membrane fusion reactions in eukaryotic cells are mediated by multisubunit tethering complexes (MTCs) and SNARE proteins. MTCs are much larger than SNAREs and are thought to mediate the initial attachment of two membranes. Complementary SNAREs then form membrane-bridging complexes whose assembly draws the membranes together for fusion. Here we present a cryo-electron microscopy structure of the simplest known MTC, the 255-kDa Dsl1 complex of Saccharomyces cerevisiae, bound to the two SNAREs that anchor it to the endoplasmic reticulum. N-terminal domains of the SNAREs form an integral part of the structure, stabilizing a Dsl1 complex configuration with unexpected similarities to the 850-kDa exocyst MTC. The structure of the SNARE-anchored Dsl1 complex and its comparison with exocyst reveal what are likely to be common principles underlying MTC function. Our structure also implies that tethers and SNAREs can work together as a single integrated machine.

VAMP5 promotes Fcγ receptor-mediated phagocytosis and regulates phagosome maturation in macrophages.

Phagosome formation and maturation reportedly occur via sequential membrane fusion events mediated by synaptosomal-associated protein of 23 kDa (SNAP23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. Vesicle-associated membrane protein 5 (VAMP5), also a plasmalemma SNARE, interacts with SNAP23; however, its precise function in phagocytosis in macrophages remains elusive. To elucidate this aspect, we investigated the characteristics of macrophages in the presence of VAMP5 overexpression or knockdown and found that VAMP5 participates in Fcγ receptor-mediated phagosome formation, although not directly in phagosome maturation. Overexpressed VAMP5 was localized to the early phagosomal membrane but no longer localized to the lysosomal-associated membrane protein 1-positive maturing phagosomal membrane. Analyses using compound-based selective inhibitors demonstrated that VAMP5 dissociation from early phagosomes occurs in a clathrin- and dynamin-dependent manner and is indispensable for SNAP23 function in subsequent membrane fusion during phagosome maturation. Accordingly, to the best of our knowledge, we demonstrate, for the first time, that VAMP5 exerts an immunologically critical function during phagosome formation and maturation via SNARE-based membrane trafficking in macrophages.

RUNDC1 negatively mediates the fusion of autophagosomes with lysosomes via regulating SNARE complex assembly.

Macroautophagy/autophagy is an essential pro-survival mechanism activated in response to nutrient deficiency. The proper fusion between autophagosomes and lysosomes is a critical step for autophagic degradation. We recently reported that RUNDC1 (RUN domain containing 1) inhibits autolysosome formation via clasping the ATG14-STX17-SNAP29 complex to hinder VAMP8 binding. We showed that RUNDC1 colocalizes with LC3 and associates with mature autophagosomes in cell lines and the zebrafish model. We utilized liposome fusion and in vitro autophagosome-lysosome fusion assays to demonstrate that RUNDC1 inhibits autolysosome formation. Moreover, we found that RUNDC1 clasps the ATG14-STX17-SNAP29 complex via stimulating ATG14 homo-oligomerization to inhibit ATG14 dissociation, which in turn prevents VAMP8 from binding to STX17-SNAP29. Our results demonstrate that RUNDC1 is a negative regulator of autophagy that restricts autophagosome fusion with lysosomes and is crucial for zebrafish survival in nutrient-deficient conditions. Here, we summarize our findings and discuss their implications for our understanding of autophagy regulation.