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Botulinum neurotoxins are known to have seven serotypes (BoNT/A–G). Here we report a new BoNT serotype, tentatively named BoNT/X, which has the lowest sequence identity with other BoNTs and is not recognized by antisera against known BoNTs. Similar to BoNT/B/D/F/G, BoNT/X cleaves vesicle-associated membrane proteins (VAMP) 1, 2 and 3, but at a novel site (Arg66-Ala67 in VAMP2). Remarkably, BoNT/X is the only toxin that also cleaves non-canonical substrates VAMP4, VAMP5 and Ykt6. To validate its activity, a small amount of full-length BoNT/X was assembled by linking two non-toxic fragments using a transpeptidase (sortase). Assembled BoNT/X cleaves VAMP2 and VAMP4 in cultured neurons and causes flaccid paralysis in mice. Thus, BoNT/X is a novel BoNT with a unique substrate profile. Its discovery posts a challenge to develop effective countermeasures, provides a novel tool for studying intracellular membrane trafficking, and presents a new potential therapeutic toxin for modulating secretions in cells. There are seven well-established types of Botulinum neurotoxins (BoNTs). Here the authors report the identification and characterization of a new type of BoNT—BoNT/X—which cleaves a different site on canonical BoNTs substrates and targets SNARE family members not cleaved by known BoNTs.

The trans -Golgi network (TGN) has been known as a key platform to sort and transport proteins to their final destinations in post-Golgi membrane trafficking. However, how the TGN sorts proteins with different destinies still remains elusive. Here, we examined 3D localization and 4D dynamics of TGN-localized proteins of Arabidopsis thaliana that are involved in either secretory or vacuolar trafficking from the TGN, by a multicolor high-speed and high-resolution spinning-disk confocal microscopy approach that we developed. We demonstrate that TGN-localized proteins exhibit spatially and temporally distinct distribution. VAMP721 (R-SNARE), AP (adaptor protein complex)−1, and clathrin which are involved in secretory trafficking compose an exclusive subregion, whereas VAMP727 (R-SNARE) and AP-4 involved in vacuolar trafficking compose another subregion on the same TGN. Based on these findings, we propose that the single TGN has at least two subregions, or “zones”, responsible for distinct cargo sorting: the secretory-trafficking zone and the vacuolar-trafficking zone. The trans -Golgi network (TGN) serves as a platform to sort and transport proteins to their final destinations. Here the authors show that the TGN of Arabidopsis consists of spatially and temporally distinct subregions and propose that these zones may sort cargo to different destinations.

Mutations in lysosomal-associated membrane protein 2 (LAMP-2) gene are associated with Danon disease, which often leads to cardiomyopathy/heart failure through poorly defined mechanisms. Here, we identify the LAMP-2 isoform B (LAMP-2B) as required for autophagosome–lysosome fusion in human cardiomyocytes (CMs). Remarkably, LAMP-2B functions independently of syntaxin 17 (STX17), a protein that is essential for autophagosome–lysosome fusion in non-CMs. Instead, LAMP-2B interacts with autophagy related 14 (ATG14) and vesicle-associated membrane protein 8 (VAMP8) through its C-terminal coiled coil domain (CCD) to promote autophagic fusion. CMs derived from induced pluripotent stem cells (hiPSC-CMs) from Danon patients exhibit decreased colocalization between ATG14 and VAMP8, profound defects in autophagic fusion, as well as mitochondrial and contractile abnormalities. This phenotype was recapitulated by LAMP-2B knockout in non-Danon hiPSC-CMs. Finally, gene correction of LAMP-2 mutation rescues the Danon phenotype. These findings reveal a STX17-independent autophagic fusion mechanism in human CMs, providing an explanation for cardiomyopathy in Danon patients and a foundation for targeting defective LAMP-2B–mediated autophagy to treat this patient population.

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.

The mechanism by which nutrient status regulates the fusion of autophagosomes with endosomes/lysosomes is poorly understood. Here, we report that O -linked β- N -acetylglucosamine ( O -GlcNAc) transferase (OGT) mediates O -GlcNAcylation of the SNARE protein SNAP-29 and regulates autophagy in a nutrient-dependent manner. In mammalian cells, OGT knockdown, or mutating the O -GlcNAc sites in SNAP-29, promotes the formation of a SNAP-29-containing SNARE complex, increases fusion between autophagosomes and endosomes/lysosomes, and promotes autophagic flux. In Caenorhabditis elegans , depletion of ogt-1 has a similar effect on autophagy; moreover, expression of an O -GlcNAc-defective SNAP-29 mutant facilitates autophagic degradation of protein aggregates. O -GlcNAcylated SNAP-29 levels are reduced during starvation in mammalian cells and in C. elegans . Our study reveals a mechanism by which O -GlcNAc-modification integrates nutrient status with autophagosome maturation. Zhang and colleagues report that starvation reduces O -GlcNAcylation of the SNARE protein SNAP-29. This promotes formation of a competent SNARE complex that increases autophagosome–lysosome fusion, increasing autophagosome maturation and flux.

Cells mitigate ER stress through the unfolded protein response (UPR). Here, we report formation of ER whorls as an effector mechanism of the ER stress response. We found that strong ER stress induces formation of ER whorls, which contain ER-resident proteins such as the Sec61 complex and PKR-like ER kinase (PERK). ER whorl formation is dependent on PERK kinase activity and is mediated by COPII machinery, which facilitates ER membrane budding to form tubular-vesicular ER whorl precursors. ER whorl precursors then go through Sec22b-mediated fusion to form ER whorls. We further show that ER whorls contribute to ER stress-induced translational inhibition by possibly modulating PERK activity and by sequestering translocons in a ribosome-free environment. We propose that formation of ER whorls reflects a new type of ER stress response that controls inhibition of protein translation.

The endoplasmic reticulum (ER) is connected to the plasma membrane (PM) through the plant-specific NETWORKED protein, NET3C, and phylogenetically conserved vesicleassociated membrane protein-associated proteins (VAPs). Ten VAP homologues (VAP27-1 to 27-10) can be identified in the Arabidopsis genome and can be divided into three clades. Representative members from each clade were tagged with fluorescent protein and expressed in Nicotiana benthamiana. Proteins from clades I and III localized to the ER as well as to ER/PM contact sites (EPCSs), whereas proteins from clade II were found only at the PM. Some of the VAP27-labelled EPCSs localized to plasmodesmata, and we show that the mobility of VAP27 at EPCSs is influenced by the cell wall. EPCSs closely associate with the cytoskeleton, but their structure is unaffected when the cytoskeleton is removed. VAP27-labelled EPCSs are found in most cell types in Arabidopsis, with the exception of cells in early trichome development. Arabidopsis plants expressing VAP27-GFP fusions exhibit pleiotropic phenotypes, including defects in root hair morphogenesis. A similar effect is also observed in plants expressing VAP27 RNAi. Taken together, these data indicate that VAP27 proteins used at EPCSs are essential for normal ER–cytoskeleton interaction and for plant development.

The SNARE complex is a key regulator of vesicular traffic, executing membrane fusion between transport vesicles or organelles and target membranes. A functional SNARE complex consists of four coiled-coil helical bundles, three of which are supplied by Q-SNAREs and another from an R-SNARE. Arabidopsis thaliana VAMP727 is an R-SNARE, with homologs only in seed plants. We have found that VAMP727 colocalizes with SYP22/ VAM3, a Q-SNARE, on a subpopulation of prevacuolar compartments/endosomes closely associated with the vacuolar membrane. Genetic and biochemical analyses, including examination of a synergistic interaction of vamp727 and syp22 mutations, histological examination of protein localization, and coimmunoprecipitation from Arabidopsis lysates indicate that VAMP727 forms a complex with SYP22, VTI11, and SYP51 and that this complex plays a crucial role in vacuolar transport, seed maturation, and vacuole biogenesis. We suggest that the VAMP727 complex mediates the membrane fusion between the prevacuolar compartment and the vacuole and that this process has evolved as an essential step for seed development.

Transverse (T)-tubules make-up a specialized network of tubulated muscle cell membranes involved in excitation-contraction coupling for power of contraction. Little is known about how T-tubules maintain highly organized structures and contacts throughout the contractile system despite the ongoing muscle remodeling that occurs with muscle atrophy, damage and aging. We uncovered an essential role for autophagy in T-tubule remodeling with genetic screens of a developmentally regulated remodeling program in Drosophila abdominal muscles. Here, we show that autophagy is both upregulated with and required for progression through T-tubule disassembly stages. Along with known mediators of autophagosome-lysosome fusion, our screens uncovered an unexpected shared role for Rab2 with a broadly conserved function in autophagic clearance. Rab2 localizes to autophagosomes and binds to HOPS complex members, suggesting a direct role in autophagosome tethering/fusion. Together, the high membrane flux with muscle remodeling permits unprecedented analysis both of T-tubule dynamics and fundamental trafficking mechanisms.

Autophagy, a conserved catabolic process implicated in a diverse array of human diseases, requires efficient fusion between autophagosomes and lysosomes to function effectively. Recently, SNAP47 has been identified as a key component of the dual-purpose SNARE complex mediating autophagosome-lysosome fusion in both bulk and selective autophagy. However, the spatiotemporal regulatory mechanisms of this SNARE complex remain unknown. In this study, we found that SNAP47 undergoes acetylation followed by deacetylation during bulk autophagy and mitophagy. The acetylation status of SNAP47 is regulated by the acetyltransferase CBP and the deacetylase HDAC2. Notably, the spatiotemporal regulatory dynamics of SNAP47 acetylation differ between bulk autophagy and mitophagy due to distinct regulation on the activity of acetyltransferase and deacetylase. Acetylated SNAP47 inhibits autophagosome-lysosome fusion by indirectly impeding SNARE complex assembly. Mechanistically, deacetylated SNAP47 recruits HOPS components to autophagic vacuoles independently of STX17 and STX17-SNAP47 interaction, while acetylated SNAP47 inhibits this recruitment, consequently leading to the failure of SNARE complex assembly. Taken together, our study uncovers a SNAP47 acetylation-dependent regulatory mechanism governing autophagosome-lysosome fusion by modulating the recruitment of HOPS to autophagic vacuoles without involving STX17, SNAP47-STX17 interaction and ternary SNARE complex formation. Autophagy involves autophagosome-lysosome fusion, regulated by the SNAP47-containing SNARE complex. This study reveals how acetylation and deacetylation of SNAP47 control fusion by modulating HOPS recruitment.