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The autophagosomal SNARE Syntaxin17 (Syx17) forms a complex with Snap29 and Vamp7/8 to promote autophagosome-lysosome fusion via multiple interactions with the tethering complex HOPS. Here we demonstrate that, unexpectedly, one more SNARE (Ykt6) is also required for autophagosome clearance in Drosophila. We find that loss of Ykt6 leads to large-scale accumulation of autophagosomes that are unable to fuse with lysosomes to form autolysosomes. Of note, loss of Syx5, the partner of Ykt6 in ER-Golgi trafficking does not prevent autolysosome formation, pointing to a more direct role of Ykt6 in fusion. Indeed, Ykt6 localizes to lysosomes and autolysosomes, and forms a SNARE complex with Syx17 and Snap29. Interestingly, Ykt6 can be outcompeted from this SNARE complex by Vamp7, and we demonstrate that overexpression of Vamp7 rescues the fusion defect of ykt6 loss of function cells. Finally, a point mutant form with an RQ amino acid change in the zero ionic layer of Ykt6 protein that is thought to be important for fusion-competent SNARE complex assembly retains normal autophagic activity and restores full viability in mutant animals, unlike palmitoylation or farnesylation site mutant Ykt6 forms. As Ykt6 and Vamp7 are both required for autophagosome-lysosome fusion and are mutually exclusive subunits in a Syx17-Snap29 complex, these data suggest that Vamp7 is directly involved in membrane fusion and Ykt6 acts as a non-conventional, regulatory SNARE in this process.

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis, and pathogen defense. A subset of SNAREs, including the Arabidopsis thaliana SNARE SYP121, are known also to coordinate solute uptake via physical interactions with K+ channels and tomoderate their gating at the plasma membrane. Here, we identify a second subset of SNAREs that interact to control these K+ channels, but with opposing actions on gating. We show that VAMPs (vesicle-associated membrane proteins), which target vesicles to the plasma membrane, also interact with and suppress the activities of the inward-rectifying K+ channels KAT1 and KC1. Interactions were evident in yeast split-ubiquitin assays, they were recovered in vivo by ratiometric bimolecular fluorescence complementation, and they were sensitive to mutation of a single residue, Tyr-57, within the longin domain of VAMP721. Interaction was also recovered on exchange of the residue at this site in the homolog VAMP723, which normally localizes to the endoplasmic reticulum and otherwise did not interact. Functional analysis showed reduced channel activity and alterations in voltage sensitivity that are best explained by a physical interaction with the channel gates. These actions complement those of SYP121, a cognate SNARE partner of VAMP721, and lead us to propose that the channel interactions reflect a “hand-off” in channel control between the two SNARE proteins that is woven together with vesicle fusion.

Arabidopsis synaptotagmin 1 (SYT1) is localized on the endoplasmic reticulum–plasma membrane (ER–PM) contact sites in leaf and root cells. The ER–PM localization of Arabidopsis SYT1 resembles that of the extended synaptotagmins (E-SYTs) in animal cells. In mammals, E-SYTs have been shown to regulate calcium signaling, lipid transfer, and endocytosis. Arabidopsis SYT1 was reported to be essential for maintaining cell integrity and virus movement. This study provides detailed insight into the subcellular localization of SYT1 and VAP27-1, another ER–PM-tethering protein. SYT1 and VAP27-1 were shown to be localized on distinct ER–PM contact sites. The VAP27-1-enriched ER–PM contact sites (V-EPCSs) were always in contact with the SYT1-enriched ER–PM contact sites (S-EPCSs). The V-EPCSs still existed in the leaf epidermal cells of the SYT1 null mutant; however, they were less stable than those in the wild type. The polygonal networks of cortical ER disassembled and the mobility of VAP27-1 protein on the ER–PM contact sites increased in leaf cells of the SYT1 null mutant. These results suggest that SYT1 is responsible for stabilizing the ER network and V-EPCSs.

We investigated the roles of components of neuronal synapses for development of the Drosophila air sac primordium (ASP). The ASP, an epithelial tube, extends specialized signaling filopodia called cytonemes that take up signals such as Dpp (Decapentaplegic, a homolog of the vertebrate bone morphogenetic protein) from the wing imaginal disc. Dpp signaling in the ASP was compromised if disc cells lacked Synaptobrevin and Synaptotagmin-1 (which function in vesicle transport at neuronal synapses), the glutamate transporter, and a voltage-gated calcium channel, or if ASP cells lacked Synaptotagmin-4 or the glutamate receptor GluRII.Transient elevations of intracellular calcium in ASP cytonemes correlate with signaling activity. Calcium transients in ASP cells depend on GluRII, are activated by L-glutamate and by stimulation of an optogenetic ion channel expressed in the wing disc, and are inhibited by EGTA and by the GluR inhibitor NASPM (1-naphthylacetyl spermine trihydrochloride). Activation of GluRII is essential but not sufficient for signaling. Cytoneme-mediated signaling is glutamatergic.

To colonize phagocytes, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan and the GP63 metalloprotease. How these virulence glycoconjugates are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. Here, we show that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages. Pharmacological disruption of the trafficking between the ER and the Golgi hindered the exit of these molecules from the PV and dampened the cleavage of host proteins by GP63. Silencing by RNA interference of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and syntaxin-5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediates the spreading of Leishmania effectors within host cells. Our findings unveil a mechanism whereby a vacuolar pathogen takes advantage of the host cell's secretory pathway to promote egress of virulence factors beyond the PV.

Neurotransmission depends on the exo-endocytosis of synaptic vesicles at active zones. Synaptobrevin 2 [also known as vesicle-associated membrane protein 2 (VAMP2)], the most abundant synaptic vesicle protein and a major soluble NSF attachment protein receptor (SNARE) component, is required for fast calcium-triggered synaptic vesicle fusion. In contrast to the extensive knowledge about the mechanism of SNARE-mediated exocytosis, little is known about the endocytic sorting of synaptobrevin 2. Here we show that synaptobrevin 2 sorting involves determinants within its SNARE motif that are recognized by the ANTH domains of the endocytic adaptors AP180 and clathrin assembly lymphoid myeloid leukemia (CALM). Depletion of CALM or AP180 causes selective surface accumulation of synaptobrevin 2 but not vGLUT1 at the neuronal surface. Endocytic sorting of synaptobrevin 2 is mediated by direct interaction of the ANTH domain of the related endocytic adaptors CALM and AP180 with the N-terminal half of the SNARE motif centered around M46, as evidenced by NMR spectroscopy analysis and site-directed mutagenesis. Our data unravel a unique mechanism of SNARE motif-dependent endocytic sorting and identify the ANTH domain proteins AP180 and CALM as cargo-specific adaptors for synaptobrevin endocytosis. Defective SNARE endocytosis may also underlie the association of CALM and AP180 with neurodevelopmental and cognitive defects or neurodegenerative disorders.

The soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex drives the majority of intracellular and exocytic membrane fusion events. Whether and how SNAREs cooperate to mediate fusion has been a subject of intense study, with estimates ranging from a single SNARE complex to 15. Here we show that there is no universally conserved number of SNARE complexes involved as revealed by our observation that this varies greatly depending on membrane curvature. When docking rates of small (∼40 nm) and large (∼100 nm) liposomes reconstituted with different synaptobrevin (the SNARE present in synaptic vesicles) densities are taken into account, the lipid mixing efficiency was maximal with small liposomes with only one synaptobrevin, whereas 23–30 synaptobrevins were necessary for efficient lipid mixing in large liposomes. Our results can be rationalized in terms of strong and weak cooperative coupling of SNARE complex assembly where each mode implicates different intermediate states of fusion that have been recently identified by electron microscopy. We predict that even higher variability in cooperativity is present in different physiological scenarios of fusion, and we further hypothesize that plasticity of SNAREs to engage in different coupling modes is an important feature of the biologically ubiquitous SNARE-mediated fusion reactions.

The homotypic fusion of yeast vacuoles, each with 3Q‐ and 1R‐SNARE, requires SNARE chaperones (Sec17p/Sec18p and HOPS) and regulatory lipids (sterol, diacylglycerol and phosphoinositides). Pairs of liposomes of phosphatidylcholine/phosphatidylserine, bearing three vacuolar Q‐SNAREs on one and the R‐SNARE on the other, undergo slow lipid mixing, but this is unaffected by HOPS and inhibited by Sec17p/Sec18p. To study these essential fusion components, we reconstituted proteoliposomes of a more physiological composition, bearing vacuolar lipids and all four vacuolar SNAREs. Their fusion requires Sec17p/Sec18p and HOPS, and each regulatory lipid is important for rapid fusion. Although SNAREs can cause both fusion and lysis, fusion of these proteoliposomes with Sec17p/Sec18p and HOPS is not accompanied by lysis. Sec17p/Sec18p, which disassemble SNARE complexes, and HOPS, which promotes and proofreads SNARE assembly, act synergistically to form fusion‐competent SNARE complexes, and this synergy requires phosphoinositides. This is the first chemically defined model of the physiological interactions of these conserved fusion catalysts.

SNAREs (soluble N -ethylmaleimide-sensitive factor attachment protein receptors) facilitate intracellular vesicle trafficking and membrane fusion in eukaryotic cells, and play a vital role in growth, development and pathogenicity of phytopathogens. Fusarium head blight (FHB) caused by F. graminearum is one of the most devastating diseases of wheat and barley worldwide. Sec22 is a member of the SNARE family of proteins and its homologues have been shown to have diverse biological roles in different organisms. However, the functions of this protein in the development and pathogenesis of F. graminearum are currently unknown. In this study, we employed integrated biochemical, microbiological and molecular genetic approaches to investigate the roles of FgSec22 in F. graminearum . Our data reveal that this SNARE protein is localized to endoplasmic reticulum (ER) and is indispensable for normal conidiation, conidial morphology and pathogenesis of this phytopathogenic fungus. Our biochemical assay of deoxynivalenol (DON) reveals the active involvement of this protein in the production of this mycotoxin in F. graminearum . This has further been confirmed by qRT-PCR analyses of trichothecene ( TRI ) genes’ expression where the ΔFgsec22 deletion mutant demonstrated a significant down-regulation of these genes in comparison to the wild-type PH-1. Unlike the wild-type and the complemented strain, the mutant strain presents a remarkable defect in colony formation which reflects the critical role it plays in vegetative growth. Collectively, our data support that the SNARE protein FgSec22 is required for vegetative growth, pathogenesis and DON biosynthesis in F. graminearum .

Intracellular membrane fusion requires R-SNAREs and Q-SNAREs to assemble into a four-helical parallel coiled-coil, with their hydrophobic anchors spanning the two apposed membranes. Based on the fusion properties of chemically defined SNARE- proteoliposomes, it has been proposed that the assembly of this helical bundle transduces force through the entire bilayer via the transmembrane SNARE anchor domains to drive fusion. However, an R-SNARE, Nyv1p, with a genetically engineered lipid anchor that spans half of the bilayer suffices for the fusion of isolated vacuoles, although this organelle has other R-SNAREs. To demonstrate unequivocally the fusion activity of lipid-anchored Nyv1p, we reconstituted proteoliposomes with purified lipid-anchored Nyv1p as the only protein. When these proteoliposomes were incubated with those bearing cognate Q-SNAREs, there was trans-SNARE complex assembly but, in accord with prior studies of the neuronal SNAREs, little lipid mixing. However, the addition of physiological fusion accessory proteins (HOPS, Sec17p, and Sec18p) allows lipid-anchored Nyv1p to support fusion, suggesting that trans-SNARE complex function is not limited to force transduction across the bilayers through the transmembrane domains.