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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.

The Unc-51 like autophagy activating kinase 1 (ULK1) complex plays a central role in the initiation stage of autophagy. However, the function of ULK1 in the late stage of autophagy is unknown. Here, we report that ULK1, a central kinase of the ULK1 complex involved in autophagy initiation, promotes autophagosome–lysosome fusion. PKCα phosphorylates ULK1 and prevents autolysosome formation. PKCα phosphorylation of ULK1 does not change its kinase activity; however, it decreases autophagosome–lysosome fusion by reducing the affinity of ULK1 for syntaxin 17 (STX17). Unphosphorylated ULK1 recruited STX17 and increased STX17′s affinity towards synaptosomal-associated protein 29 (SNAP29). Additionally, phosphorylation of ULK1 enhances its interaction with heat shock cognate 70 kDa protein (HSC70) and increases its degradation through chaperone-mediated autophagy (CMA). Our study unearths a key mechanism underlying autolysosome formation, a process in which the kinase activity of PKCα plays an instrumental role, and reveals the significance of the mutual regulation of macroautophagy and CMA in maintaining the balance of autophagy. The ULK complex plays a well-known role in initiating autophagy, to recycle cellular components in response to nutritional stress. Here, the authors demonstrate a late role for ULK in auotophagosome–lysosome fusion and provide a direct link between macroautophagy and chaperone mediated autophagy.

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 Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual \"handshaking\" mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.

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.

Autophagy serves as a pro-survival mechanism for a cell or a whole organism to cope with nutrient stress. Our understanding of the molecular regulation of this fusion event remains incomplete. Here, we identified RUNDC1 as a novel ATG14-interacting protein, which is highly conserved across vertebrates, including zebrafish and humans. By gain and loss of function studies, we demonstrate that RUNDC1 negatively modulates autophagy by blocking fusion between autophagosomes and lysosomes via inhibiting the assembly of the STX17-SNAP29-VAMP8 complex both in human cells and the zebrafish model. Moreover, RUNDC1 clasps the ATG14-STX17-SNAP29 complex via stimulating ATG14 homo-oligomerization to inhibit ATG14 dissociation. This also prevents VAMP8 from binding to STX17-SNAP29. We further identified that phosphorylation of RUNDC1 Ser379 is crucial to inhibit the assembly of the STX17-SNAP29-VAMP8 complex via promoting ATG14 homo-oligomerization. In line with our findings, RunDC1 is crucial for zebrafish in their response to nutrient-deficient conditions. Taken together, our findings demonstrate that RUNDC1 is a negative regulator of autophagy that restricts autophagosome fusion with lysosomes by clasping the ATG14-STX17-SNAP29 complex to hinder VAMP8 binding.

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.

Human melanoma cells express various tumour antigens that are recognized by CD8 + cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo . However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that, on conjugation with CTL, human melanoma cells undergo an active late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance, we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients. Cytotoxic T lymphocytes recognise and eliminate tumour cells. Here, the authors show that on contact with these immune cells melanoma cells can resist T cell cytotoxicity by modulating the trafficking of their lysosomal compartment, this results in the degradation of the T cell protein perforin by the protease cathepsin B.

The O-linked β-N-acetylglucosamine (O-GlcNAc) modification and autophagy are associated with diabetic myocardial injury, however, the molecular mechanisms between the two processes remain to be fully elucidated. The purpose of the present study was to elucidate the molecular regulation of autophagy by O-GlcNAc-modified synaptosomal-associated protein 29 (SNAP29) in diabetic myocardial injury. A rat model of type I diabetes was established via intraperitoneal injection of streptozotocin (STZ; 55 mg/kg). Significant increases in the O-GlcNAc modification and accumulation of the autophagy markers microtubule-associated protein 1 light chain 3α II/I and P62, which suggest that autophagic flux is inhibited, were observed in rats 8 weeks following STZ induction. Subsequently, the selective O-GlcNAcase inhibitor, thiamet G, increased the level of O-GlcNAc modification, which further disrupted autophagic flux; deteriorated cardiac diastolic function, as indicated by an increased left ventricular filling peak velocity/atrial contraction flow peak velocity ratio shown by echocardiography; and exacerbated myocardial abnormalities, as characterized by cardiomyocyte disorganization and fat and interstitial fibrosis accumulation. By contrast, 6-diazo-5-oxo-L-norleucine, an inhibitor of glucosamine fructose-6-phosphate aminotransferase isomerizing 1, acted as an O-GlcNAc antagonist and reduced the level of O-GlcNAc modification, which maintained autophagic flux and improved cardiac diastolic function. In vitro, high glucose (25 mM) was used to stimulate primary neonatal rat cardiomyocytes (NRCMs). Consistent with the myocardium of diabetic rats, it was also shown in the NRCMs that O-GlcNAc modification of SNAP29 negatively regulated autophagic flux. The application of the short hairpin RNA interference lysosome-associated membrane protein (LAMP2) and the autophagy inhibitor 3-methyladenine demonstrated that high glucose inhibited autophagy-mediated degradation rather than affected the initial stage of autophagy. Finally, co-immunoprecipitation was used to determine the role of the O-GlcNAc-modified substrate protein SNAP29, which acted as an SNAP29-syntaxin-17 (STX17)-vesicle-associated membrane protein 8 (VAMP8) complex during disease progression. The present study is the first, to the best of our knowledge, to demonstrate that SNAP29 is an O-GlcNAc substrate and that an increase in O-GlcNAc-modified SNAP29 inhibits SNAP29-STX17-VAMP8 complex formation, thereby inhibiting the degradation of autophagy and exacerbating myocardial injury in type I diabetic rats.

Increasing evidences reveal that autophagy inhibitor could enhance the effect of chemotherapy to cancer. However, few autophagy inhibitors are currently approved for clinical application in humans. Berbamine (BBM) is a natural compound extracted from traditional Chinese medicine that is widely used for treatment of a variety of diseases without any obvious side effects. Here we found that BBM is a novel auophagy inhibitor, which potently induced the accumulation of autophagosomes by inhibiting autophagosome-lysosome fusion in human breast cancer cells. Mechanistically, we found that BBM blocked autophagosome-lysosome fusion by inhibiting the interaction of SNAP29 and VAMP8. Furthermore, BBM induced upregulation of BNIP3 and the interaction between SNAP29 and BNIP3. BNIP3 depletion or SNAP29 overexpression abrogated BBM-mediated blockade of autophagosome-lysosome fusion through the interaction between SNAP29 and VAMP8, whereas BNIP3 overexpression blocked autophagosome-lysosome fusion through inhibition of the interaction between SNAP29 and VAMP8. These findings suggest that upregulation of BNIP3 and interaction between BNIP3 and SNAP29 could be involved in BBM-mediated blockade of autophagosome-lysosome fusion through inhibition of the interaction between SNAP29 and VAMP8. Our findings identify the critical role of BNIP3 in blockade of autophagosome-lysosome fusion mediated by BBM, and suggest that BBM could potentially be further developed as a novel autophagy inhibitor, which could enhance the effect of chemotherapy to cancer.