Beclin 1 self‐association is independent of autophagy induction by amino acid deprivation and rapamycin treatment

Autophagy, a process of self‐digestion of cellular constituents, regulates the balance between protein synthesis and protein degradation. Beclin 1 represents an important component of the autophagic machinery. It interacts with proteins that positively regulate autophagy, such as Vps34, UVRAG, and Ambra1, as well as with anti‐apoptotic proteins such as Bcl‐2 via its BH3‐like domain to negatively regulate autophagy. Thus, Beclin 1 interactions with several proteins may regulate autophagy. To identify novel Beclin 1 interacting proteins, we utilized a GST‐Beclin 1 fusion protein. Using mass spectroscopic analysis, we identified Beclin 1 as a protein that interacts with GST‐Beclin 1. Further examination by cross linking and co‐immunoprecipitation experiments confirmed that Beclin 1 self‐interacts and that the coiled coil and the N‐terminal region of Beclin 1 contribute to its oligomerization. Importantly, overexpression of vps34, UVRAG, or Bcl‐x_L, had no effect on Beclin 1 self‐interaction. Moreover, this self‐interaction was independent of autophagy induction by amino acid deprivation or rapamycin treatment. These results suggest that full‐length Beclin 1 is a stable oligomer under various conditions. Such an oligomer may provide a platform for further protein–protein interactions. J. Cell. Biochem. 110: 1262–1271, 2010. Published 2010 Wiley‐Liss, Inc.     

Dynamin Regulates Autophagy by Modulating Lysosomal Function

Autophagy is a central lysosomal degradation pathway required for maintaining cellular homeostasis and its dysfunction is associated with numerous human diseases. To identify players in autophagy, we tested w1200 chemically induced mutations on the X chromosome in Drosophila fat body clones and discovered that shibire(shi) plays an essential role in starvation-induced autophagy. shi encodes a dynamin protein required for fission of clathrin-coated vesicles from the plasma membrane during endocytosis. We showed that Shi is dispensable for autophagy initiation and autophagosomeelysosome fusion, but required for lysosomal/autolysosomal acidification. We also showed that other endocytic core machinery components like clathrin and AP2 play similar but not identical roles in regulating autophagy and lysosomal function as dynamin. Previous studies suggested that dynamin directly regulates autophagosome formation and autophagic lysosome reformation(ALR) through its excision activity. Here, we provide evidence that dynamin also regulates autophagy indirectly by regulating lysosomal function.     

Thapsigargin distinguishes membrane fusion in the late stages of endocytosis and autophagy

A close relationship exists between autophagy and endocytosis with both sharing lysosomes as their common end-point. Autophagy even requires a functional endocytic pathway. The point at which the two pathways merge, i.e., fusion of autophagosomes and endosomes with lysosomes is poorly understood. Early work in yeast and more recent studies in mammalian cells suggested that conventional membrane trafficking pathways control the fusion of autophagosomes with lysosomes; Rab GTPases are required to recruit tethering proteins which in turn coordinate the SNARE family of proteins that directly drive membrane fusion. Some components required for endosomes to fuse with lysosomes are also shared by autophagosomes; both are thought to require the GTPase Rab7 and the homotypic fusion and vacuole protein sorting (HOPS) complex. Essentially, the autophagosome becomes endosome-like, allowing it to recruit the common fusion machinery to deliver its contents to the lysosome. This raises an interesting question of how the cell determines when the autophagosome is ready to fuse with the endocytic system and bestows upon it the properties required to recruit the fusion machinery. Our recent work has highlighted this conundrum and shown that autophagosome fusion with lysosomes has specific distinctions from the parallel endosomal-lysosomal pathway.     

Thapsigargin distinguishes membrane fusion in the late stages of endocytosis and autophagy

A close relationship exists between autophagy and endocytosis with both sharing lysosomes as their common end-point. Autophagy even requires a functional endocytic pathway. The point at which the two pathways merge, i.e., fusion of autophagosomes and endosomes with lysosomes is poorly understood. Early work in yeast and more recent studies in mammalian cells suggested that conventional membrane trafficking pathways control the fusion of autophagosomes with lysosomes; Rab GTPases are required to recruit tethering proteins which in turn coordinate the SNARE family of proteins that directly drive membrane fusion. Some components required for endosomes to fuse with lysosomes are also shared by autophagosomes; both are thought to require the GTPase Rab7 and the homotypic fusion and vacuole protein sorting (HOPS) complex. Essentially, the autophagosome becomes endosome-like, allowing it to recruit the common fusion machinery to deliver its contents to the lysosome. This raises an interesting question of how the cell determines when the autophagosome is ready to fuse with the endocytic system and bestows upon it the properties required to recruit the fusion machinery. Our recent work has highlighted this conundrum and shown that autophagosome fusion with lysosomes has specific distinctions from the parallel endosomal-lysosomal pathway.     

Autophagosome–lysosome fusion in neurons requires INPP5E,a protein associated with Joubert syndrome

Autophagy is a multistep membrane traffic pathway. In contrast to autophagosome formation, the mechanisms underlying autophagosome–lysosome fusion remain largely unknown. Here, we describe a novel autophagy regulator, inositol polyphosphate‐5‐phosphatase E (INPP5E), involved in autophagosome–lysosome fusion process. In neuronal cells, INPP5E knockdown strongly inhibited autophagy by impairing the fusion step. A fraction of INPP5E is localized to lysosomes, and its membrane anchoring and enzymatic activity are necessary for autophagy. INPP5E decreases lysosomal phosphatidylinositol 3,5‐bisphosphate (PI(3,5)P₂), one of the substrates of the phosphatase, that counteracts cortactin‐mediated actin filament stabilization on lysosomes. Lysosomes require actin filaments on their surface for fusing with autophagosomes. INPP5E is one of the genes responsible for Joubert syndrome, a rare brain abnormality, and mutations found in patients with this disease caused defects in autophagy. Taken together, our data reveal a novel role of phosphoinositide on lysosomes and an association between autophagy and neuronal disease.     

TRIM13 regulates caspase-8 ubiquitination,translocation to autophagosomes and activation during ER stress induced cell death

The emerging evidences suggest that endoplasmic (ER) stress is involved in onset of many pathological conditions like cancer and neurodegeneration. The persistent ER stress results in misfolded protein aggregates, which are degraded through the process of autophagy or lead to cell death through activation of caspases. The regulation of crosstalk of autophagy and cell death during ER stress is emerging. Ubiquitination plays regulatory role in crosstalk of autophagy and cell death. In the current study, we describe the role of TRIM13, RING E3 ubiquitin ligase, in regulation of ER stress induced cell death. The expression of TRIM13 sensitizes cells to ER stress induced death. TRIM13 induced autophagy is essential for ER stress induced caspase activation and cell death. TRIM13 induces K63 linked poly-ubiquitination of caspase-8, which results in its stabilization and activation during ER stress. TRIM13 regulates translocation of caspase-8 to autophagosome and its fusion with lysosome during ER stress. This study first time demonstrated the role of TRIM13 as novel regulator of caspase-8 activation and cell death during ER stress.     

The role of ESCRT proteins in fusion events involving lysosomes, endosomes and autophagosomes

ESCRT (endosomal sorting complex required for transport) proteins were originally identified for their role in delivering endocytosed proteins to the intraluminal vesicles of late-endosomal structures termed multivesicular bodies. Multivesicular bodies then fuse with lysosomes, leading to degradation of the internalized proteins. Four ESCRT complexes interact to concentrate cargo on the endosomal membrane, induce membrane curvature to form an intraluminal bud and finally pinch off the bud through a membrane-scission event to produce the intraluminal vesicle. Recent work suggests that ESCRT proteins are also required downstream of these events to enable fusion of multivesicular bodies with lysosomes. Autophagy is a related pathway required for the degradation of organelles, long-lived proteins and protein aggregates which also converges on lysosomes. The proteins or organelle to be degraded are encapsulated by an autophagosome that fuses either directly with a lysosome or with an endosome to form an amphisome, which then fuses with a lysosome. A common machinery is beginning to emerge that regulates fusion events in the multivesicular body and autophagy pathways, and we focus in the present paper on the role of ESCRT proteins. These fusion events have been implicated in diseases including frontotemporal dementia, Alzheimer's disease, lysosomal storage disorders, myopathies and bacterial pathogen invasion, and therefore further examination of the mechanisms involved may lead to new insight into disease pathogenesis and treatments.     

Members of the CIP4 family of proteins participate in the regulation of platelet‐derived growth factor receptor‐β‐dependent actin reorganization and migration

Background information. The F‐BAR {Fes/CIP4 [Cdc42 (cell division cycle 42)‐interacting protein 4] homology and BAR (Bin/amphiphysin/Rvs)} proteins have emerged as important co‐ordinators of signalling pathways that regulate actin assembly and membrane dynamics. The presence of the F‐BAR domain is the hallmark of this family of proteins and the CIP4 (Cdc42‐interacting protein 4) was one of the first identified vertebrate F‐BAR proteins. There are three human CIP4 paralogues, namely CIP4, FBP17 (formin‐binding protein 17) and Toca‐1 (transducer of Cdc42‐dependent actin assembly 1). The CIP4‐like proteins have been implicated in Cdc42‐dependent actin reorganization and in regulation of membrane deformation events visible as tubulation of lipid bilayers. Results. We performed side‐by‐side analyses of the three CIP4 paralogues. We found that the three CIP4‐like proteins vary in their effectiveness to catalyse membrane tubulation and actin reorganization. Moreover, we show that the CIP4‐dependent membrane tubulation is enhanced in the presence of activated Cdc42. Some F‐BAR members have been shown to have a role in the endocytosis of the EGF (epidermal growth factor) receptor and this prompted us to study the involvement of the CIP4‐like proteins in signalling of the PDGFRβ [PDGF (platelet‐derived growth factor) β‐receptor]. We found that knock‐down of CIP4‐like proteins resulted in a prolonged formation of PDGF‐induced dorsal ruffles, as well as an increased PDGF‐dependent cell migration. This was most likely a consequence of a sustained PDGFRβ activation caused by delayed internalization of the receptor in the cells treated with siRNA (small interfering RNA) specific for the CIP4‐like proteins. Conclusions. Our findings show that CIP4‐like proteins induced membrane tubulation downstream of Cdc42 and that they have important roles in PDGF‐dependent actin reorganization and cell migration by regulating internalization and activity of the PDGFRβ. Moreover, the results suggest an important role for the CIP4‐like proteins in the regulation of the activity of the PDGFRβ.     

Influence of hydrophobic and electrostatic residues on SARS‐coronavirus S2 protein stability: Insights into mechanisms of general viral fusion and inhibitor design

Severe acute respiratory syndrome (SARS) is an acute respiratory disease caused by the SARS‐coronavirus (SARS‐CoV). SARS‐CoV entry is facilitated by the spike protein (S), which consists of an N‐terminal domain (S1) responsible for cellular attachment and a C‐terminal domain (S2) that mediates viral and host cell membrane fusion. The SARS‐CoV S2 is a potential drug target, as peptidomimetics against S2 act as potent fusion inhibitors. In this study, site‐directed mutagenesis and thermal stability experiments on electrostatic, hydrophobic, and polar residues to dissect their roles in stabilizing the S2 postfusion conformation was performed. It was shown that unlike the pH‐independent retroviral fusion proteins, SARS‐CoV S2 is stable over a wide pH range, supporting its ability to fuse at both the plasma membrane and endosome. A comprehensive SARS‐CoV S2 analysis showed that specific hydrophobic positions at the C‐terminal end of the HR2, rather than electrostatics are critical for fusion protein stabilization. Disruption of the conserved C‐terminal hydrophobic residues destabilized the fusion core and reduced the melting temperature by 30°C. The importance of the C‐terminal hydrophobic residues led us to identify a 42‐residue substructure on the central core that is structurally conserved in all existing CoV S2 fusion proteins (root mean squared deviation = 0.4 Å). This is the first study to identify such a conserved substructure and likely represents a common foundation to facilitate viral fusion. We have discussed the role of key residues in the design of fusion inhibitors and the potential of the substructure as a general target for the development of novel therapeutics against CoV infections.     

A Targeted siRNA Screen to Identify SNAREs Required for Constitutive Secretion in Mammalian Cells

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry‐based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein‐specific siRNA screen (38 SNAREs, 4 SNARE‐like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post‐Golgi SNAREs SNAP‐29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP‐29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP‐29‐depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19‐interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP‐23, SNAP‐25, SNAP‐29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post‐Golgi‐specific R‐SNARE in this process.     

Hidden Behind Autophagy: The Unconventional Roles of ATG Proteins

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular catabolic transport route that generally allows the lysosomal degradation of cytoplasmic components, including bulk cytosol, protein aggregates, damaged or superfluous organelles and invading microbes. Target structures are sequestered by double‐membrane vesicles called autophagosomes, which are formed through the concerted action of the autophagy (ATG)‐related proteins. Until recently it was assumed that ATG proteins were exclusively involved in autophagy. A growing number of studies, however, have attributed functions to some of them that are distinct from their classical role in autophagosome biogenesis. Autophagy‐independent roles of the ATG proteins include the maintenance of cellular homeostasis and resistance to pathogens. For example, they assist and enhance the turnover of dead cells and microbes upon their phagocytic engulfment, and inhibit murine norovirus replication. Moreover, bone resorption by osteoclasts, innate immune regulation triggered by cytoplasmic DNA and the ER‐associated degradation regulation all have in common the requirement of a subset of ATG proteins. Microorganisms such as coronaviruses, Chlamydia trachomatis or Brucella abortus have even evolved ways to manipulate autophagy‐independent functions of ATG proteins in order to ensure the completion of their intracellular life cycle. Taken together these novel mechanisms add to the repertoire of functions and extend the number of cellular processes involving the ATG proteins.     

Actin remodeling to facilitate membrane fusion

Actin and its associated proteins participate in several intracellular trafficking mechanisms. This review assesses recent work that shows how actin participates in the terminal trafficking event of membrane bilayer fusion. A recent flurry of reports defines a role for Rho proteins in membrane fusion and also demonstrates that this role is distinct from any vesicle transport mechanism. Rho proteins are well known to govern actin remodeling, which implicates this process as a condition of membrane fusion. A small but significant body of work examines actin-regulated events of intracellular membrane fusion, exocytosis and endocytosis. In general, actin has been shown to act as a negative regulator of exocytosis. Cortical actin filaments act as a barrier that requires transient removal to allow vesicles to undergo docking at the plasma membrane. However, once docked, F-actin synthesis may act as a positive regulator to give the final stimulus to drive membrane fusion. F-actin synthesis is clearly needed for endocytosis and intracellular membrane fusion events. What may seem like dissimilar results are perhaps snapshots of a single mechanism of membranous actin remodeling (i.e. dynamic disassembly and reassembly) that is universally needed for all membrane fusion events.     

Protein clearing pathways in ALS

In the present review a large amount of experimental and clinical studies on ALS are discussed in an effort to dissect common pathogenic mechanisms which may provide novel information and potential therapeutic strategies for motor neuron degeneration.Protein clearing systems play a critical role in motor neuron survival during excitotoxic stress, aging and neurodegenerative disorders. Among various mechanisms which clear proteins from the cell recent studies indicate autophagy as the most prominent pathway to promote survival of motor neurons.Autophagy regulates the clearance of damaged mitochondria, endoplasmic reticulum and misfolded proteins in eukaryotic cells. Upon recruitment of the autophagy pathway, an autophagosome is produced and directed towards lysosomal degradation.Here we provide evidence that in both genetic and sporadic amyotrophic lateral sclerosis (ALS, the most common motor neuron disorder) a defect in the autophagy machinery is common. In fact, swollen, disrupted mitochondria and intracellular protein aggregates accumulate within affected motor neurons. These structures localize within double membrane vacuoles, autophagosomes, which typically cluster in perinuclear position. In keeping with this, when using autophagy inhibitors or suppressing autophagy promoting genes, motor symptoms and motor neuron death are accelerated. Conversely stimulation of autophagy alleviates motor neuron degeneration.Therefore, autophagy represents an important target when developing novel treatments in ALS.     

AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.     

Characterization of trypsin-sensitive factor(s) required for endosome-endosome fusion.

Fusion of endosomes appears to be required at early steps of receptor-mediated endocytosis. These fusion events have been reconstituted using a cell-free assay and have been shown to require both cytosolic and membrane-associated proteins. We report here that trypsinization of endosomes completely inhibited fusion. Addition of untreated cytosol cannot restore fusion of trypsinized endosomes. However, fusion activity is restored by the addition of either untreated vesicles or a high salt extract containing peripheral membrane proteins (KE). KE contains both the membrane-associated factor(s) required for the reconstitution of fusion using trypsinized endosomes and the factors that are normally provided by the cytosol. The restorative activity of KE was sensitive to trypsin treatment or incubation at 100 degrees C, but was largely N-ethylmaleimide (NEM)-resistant. This and other criteria demonstrated that the trypsin-sensitive factor is distinct from N-ethylmaleimide-sensitive factor (NSF), an NEM-sensitive protein involved in vesicular fusion, and from other known factors that may participate in membrane fusion events. Preliminary fractionation studies indicate that the restorative activity of KE is associated with one or more high molecular weight proteins. The present study indicates that a novel trypsin-sensitive protein(s) is involved in endosome-endosome fusion. This factor is membrane-associated and is not found in an active form in cytosol as prepared.     

Autophagosome Requires Specific Early Sec Proteins for Its Formation and NSF/SNARE for Vacuolar Fusion

Double membrane structure, autophagosome, is formed de novo in the process of autophagy in the yeast Saccharomyces cerevisiae, and many Apg proteins participate in this process. To further understand autophagy, we analyzed the involvement of factors engaged in the secretory pathway. First, we showed that Sec18p (N-ethylmaleimide-sensitive fusion protein, NSF) and Vti1p (soluble N-ethylmaleimide-sensitive fusion protein attachment protein, SNARE), and soluble N-ethylmaleimide-sensitive fusion protein receptor are required for fusion of the autophagosome to the vacuole but are not involved in autophagosome formation. Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) (pathway, which shares mostly the same machinery with autophagy. Subcellular fractionation and electron microscopic analyses showed that Cvt vesicles, but not autophagosomes, can be formed in sec12 cells. Three other coatmer protein (COPII) mutants, sec16, sec23, and sec24, were also defective in autophagy. The blockage of autophagy in these mutants was not dependent on transport from endoplasmic reticulum-to-Golgi, because mutations in two other COPII genes, SEC13 and SEC31, did not affect autophagy. These results demonstrate the requirement for subgroup of COPII proteins in autophagy. This evidence demonstrating the involvement of Sec proteins in the mechanism of autophagosome formation is crucial for understanding membrane flow during the process.     

Endophilin,Lamellipodin, and Mena cooperate to regulate F‐actin‐dependent EGF‐receptor endocytosis

The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin‐mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F‐actin‐dependent manner, suggesting that F‐actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin‐coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.     

Insights into links between autophagy and the ubiquitin system from the structure of LC3B bound to the LIR motif from the E3 ligase NEDD4

Members of the LC3/GABARAP family of ubiquitin‐like proteins function during autophagy by serving as membrane linked protein‐binding platforms. Their C‐termini are physically attached to membranes through covalent linkage to primary amines on lipids such as phosphatidylethanolamine, while their ubiquitin‐like fold domains bind “LIR” (LC3‐Interacting Region) sequences found within an extraordinarily diverse array of proteins including regulators of autophagy, adaptors that recruit ubiquitinated cargoes to be degraded, and even proteins controlling processes at membranes that are not associated with autophagy. Recently, LC3/GABARAP proteins were found to bind the ubiquitin E3 ligase NEDD4 to influence ubiquitination associated with autophagy in human cell lines. Here, we use purified recombinant proteins to define LC3B interactions with a specific LIR sequence from NEDD4, present a crystal structure showing atomic details of the interaction, and show that LC3B‐binding can steer intrinsic NEDD4 E3 ligase activity. The data provide detailed molecular insights underlying recruitment of an E3 ubiquitin ligase to phagophores during autophagy.     

Ubiquitin and endocytic internalization in yeast and animal cells

Endocytosis is involved in a wide variety of cellular processes, and the internalization step of endocytosis has been extensively studied in both lower and higher eukaryotic cells. Studies in mammalian cells have described several endocytic pathways, with the main emphasis on clathrin-dependent endocytosis. Genetic studies in yeast have underlined the critical role of actin and actin-binding proteins, lipid modification, and the ubiquitin conjugation system. The combined results of studies of endocytosis in higher and lower eukaryotic cells reveal an interesting interplay in the two systems, including a crucial role for ubiquitin-associated events. The ubiquitylation of yeast cell-surface proteins clearly acts as a signal triggering their internalization. Mammalian cells display variations on the common theme of ubiquitin-linked endocytosis, according to the cell-surface protein considered. Many plasma membrane channels, transporters and receptors undergo cell-surface ubiquitylation, required for the internalization or later endocytic steps of some cell-surface proteins, whereas for others, internalization involves interaction with the ubiquitin conjugation system or with ancillary proteins, which are themselves ubiquitylated. Epsins and Eps15 (or Eps15 homologs), are commonly involved in the process of endocytosis in all eukaryotes, their critical role in this process stemming from their capacity to bind ubiquitin, and to undergo ubiquitylation.     

Prefused lysosomes cluster on autophagosomes regulated by VAMP8

Lysosome–autophagosome fusion is critical to autophagosome maturation. Although several proteins that regulate this fusion process have been identified, the prefusion architecture and its regulation remain unclear. Herein, we show that upon stimulation, multiple lysosomes form clusters around individual autophagosomes, setting the stage for membrane fusion. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein on lysosomes—vesicle-associated membrane protein 8 (VAMP8)—plays an important role in forming this prefusion state of lysosomal clusters. To study the potential role of phosphorylation on spontaneous fusion, we investigated the effect of phosphorylation of C-terminal residues of VAMP8. Using a phosphorylation mimic, we observed a decrease of fusion in an ensemble lipid mixing assay and an increase of unfused lysosomes associated with autophagosomes. These results suggest that phosphorylation not only reduces spontaneous fusion for minimizing autophagic flux under normal conditions, but also preassembles multiple lysosomes to increase the fusion probability for resuming autophagy upon stimulation. VAMP8 phosphorylation may thus play an important role in chemotherapy drug resistance by influencing autophagosome maturation.Subject terms: Cancer therapeutic resistance, Membrane fusion