Autophagosome biogenesis requires SNAREs

We recently showed that phagophore biogenesis requires SNAREs. Our data indicate that the exocytic Q/t-SNAREs Sso1/2 and Sec9 are required for one of the earliest steps in autophagosome biogenesis, the homotypic fusion of Atg9-containing vesicles. We propose that this step precedes the formation of Atg9-containing tubulovesicular clusters (TVCs) that is a key step in perivacuolar, phagophore assembly. We also found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 trafficking. Thus, autophagosome biogenesis appears to involve multiple SNARE-mediated fusion events. These findings provide novel insights into the mechanism of autophagosome construction.     

Autophagosome biogenesis requires SNAREs

We recently showed that phagophore biogenesis requires SNAREs. Our data indicate that the exocytic Q/t-SNAREs Sso1/2 and Sec9 are required for one of the earliest steps in autophagosome biogenesis, the homotypic fusion of Atg9-containing vesicles. We propose that this step precedes the formation of Atg9-containing tubulovesicular clusters (TVCs) that is a key step in perivacuolar, phagophore assembly. We also found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 trafficking. Thus, autophagosome biogenesis appears to involve multiple SNARE-mediated fusion events. These findings provide novel insights into the mechanism of autophagosome construction.     

Autophagosome closure requires membrane scission

During the intracellular process of macroautophagy (hereafter autophagy), a membrane-bound organelle, the autophagosome, is generated de novo. The remodeling of the autophagic membrane during the life cycle of the organelle is a complex multistep process and involves several changes in the topology of the autophagic membrane. Here, we focus on the final step of autophagosome formation, the closure of the phagophore, during which the inner and outer autophagic membranes become separate entities. We argue that this topological membrane transformation is a membrane scission event. Surprisingly, not a single recent review describes this substep as membrane scission (or membrane fission). In contrast, a number of publications imply that membrane fusion is involved. We discuss the potential sources for misinterpretation and recommend to consistent use of the unambiguous term “membrane scission.”     

Genomic analysis of homotypic vacuole fusion

Yeast vacuoles undergo fission and homotypic fusion, yielding one to three vacuoles per cell at steady state. Defects in vacuole fusion result in vacuole fragmentation. We have screened 4828 yeast strains, each with a deletion of a nonessential gene, for vacuole morphology defects. Fragmented vacuoles were found in strains deleted for genes encoding known fusion catalysts as well as 19 enzymes of lipid metabolism, 4 SNAREs, 12 GTPases and GTPase effectors, 9 additional known vacuole protein-sorting genes, 16 protein kinases, 2 phosphatases, 11 cytoskeletal proteins, and 28 genes of unknown function. Vacuole fusion and vacuole protein sorting are catalyzed by distinct, but overlapping, sets of proteins. Novel pathways of vacuole priming and docking emerged from this deletion screen. These include ergosterol biosynthesis, phosphatidylinositol (4,5)-bisphosphate turnover, and signaling from Rho GTPases to actin remodeling. These pathways are supported by the sensitivity of the late stages of vacuole fusion to inhibitors of phospholipase C, calcium channels, and actin remodeling. Using databases of yeast protein interactions, we found that many nonessential genes identified in our deletion screen interact with essential genes that are directly involved in vacuole fusion. Our screen reveals regulatory pathways of vacuole docking and provides a genomic basis for studies of this reaction.     

Yeast homotypic vacuole fusion requires the Ccz1-Mon1 complex during the tethering/docking stage

The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle. Accordingly, highly regulated processes control vacuolar fission and fusion events. Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse. Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage. Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane. In the absence of the Ccz1-Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired. The Ccz1-Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1-Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins. Accordingly, we propose that the Ccz1-Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion.     

Autophagosome maturation is impaired in Fabry disease

Fabry disease is a lysosomal storage disorder (LSD) caused by a deficiency in α-galactosidase A. The disease is characterized by severe major organ involvement, but the pathologic mechanisms responsible have not been elucidated. Disruptions of autophagic processes have been reported for other LSDs, but have not yet been investigated in Fabry disease. Renal biopsies were obtained from 5 adult male Fabry disease patients before and after 3 years of enzyme replacement therapy (ERT) with agalsidase alfa. Vacuole accumulation was seen in renal biopsies from all patients compared with control biopsies. Decreases in the number of vacuoles were seen after 3 years of ERT primarily in renal endothelial cells and mesangial cells. Measurement of the levels of LC3, a specific autophagy marker, in cultured cells from Fabry patients revealed increased basal levels compared to cells from non-Fabry subjects and a larger increase in response to starvation than seen in non-Fabry cells. Starvation in the presence of protease inhibitors did not result in a significant increase in LC3 in Fabry cells, whereas a further increase in LC3 was observed in non-Fabry cells, an observation that is consistent with impaired autophagic flux in Fabry disease. Overexpression of LC3 mRNA in Fabry fibroblasts compared to control cells is consistent with an upregulation of autophagy. Furthermore, LC3 and p62/SQSTM1 (that binds to LC3) staining in renal tissues and in cultured fibroblasts from Fabry patients supports impairment of autophagic flux. These findings suggest that Fabry disease is linked to a deregulation of autophagy.     

SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

Dynamin-like GTPases of the atlastin family are thought to mediate homotypic endoplasmic reticulum (ER) membrane fusion; however, the underlying mechanism remains largely unclear. Here, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p-dependent ER fusion. Using this assay, we found that the ER SNAREs Sec22p and Sec20p were required for Sey1p-mediated ER fusion. Consistently, ER fusion was significantly reduced by inhibition of Sec18p and Sec17p, which regulate SNARE-mediated membrane fusion. The involvement of SNAREs in Sey1p-dependent ER fusion was further supported by the physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25-fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion.     

Ion regulation of homotypic vacuole fusion in Saccharomyces cerevisiae

Biological membrane fusion employs divalent cations as protein cofactors or as signaling ligands. For example, Mg2+ is a cofactor for the N-ethylmaleimide-sensitive factor (NSF) ATPase, and the Ca2+ signal from neuronal membrane depolarization is required for synaptotagmin activation. Divalent cations also regulate liposome fusion, but the role of such ion interactions with lipid bilayers in Rab- and soluble NSF attachment protein receptor (SNARE)-dependent biological membrane fusion is less clear. Yeast vacuole fusion requires Mg2+ for Sec18p ATPase activity, and vacuole docking triggers an efflux of luminal Ca2+. We now report distinct reaction conditions where divalent or monovalent ions interchangeably regulate Rab- and SNARE-dependent vacuole fusion. In reactions with 5 mm Mg2+, other free divalent ions are not needed. Reactions containing low Mg2+ concentrations are strongly inhibited by the rapid Ca2+ chelator BAPTA. However, addition of the soluble SNARE Vam7p relieves BAPTA inhibition as effectively as Ca2+ or Mg2+, suggesting that Ca2+ does not perform a unique signaling function. When the need for Mg2+, ATP, and Sec18p for fusion is bypassed through the addition of Vam7p, vacuole fusion does not require any appreciable free divalent cations and can even be stimulated by their chelators. The similarity of these findings to those with liposomes, and the higher ion specificity of the regulation of proteins, suggests a working model in which ion interactions with bilayer lipids permit Rab- and SNARE-dependent membrane fusion.     

Differentiation of endothelial barrier function during normal angiogenesis requires homotypic VE-cadherin adhesion

Endothelial cells express two principal cadherins: VE-cadherin and N-cadherin. We established previously that only VE-cadherin expression was increased during differentiation of barrier function by angiogenic endothelium of the chick chorioallantoic membrane (CAM). Presently anti-VE-cadherin mAb, applied to the CAM at day 4.5 of gestation, served to inhibit the abrupt reduction of macromolecular extravasation that occurs normally at day 5.0. Neither anti-N-cadherin nor nonimmune IgG, on the other hand, prevented this temporal decrease of endothelial permeability. Despite the differential permeability responses, morphometric evaluations defined a reduction of mean paracellular cleft width after the application of either anti-VE-cadherin or anti-N-cadherin. Hence, alteration of molecular sieving characteristics within the junctional clefts, rather than modification of cleft dimensions; likely served as the principal modulator of macromolecular extravasation after inhibition of homotypic VE-cadherin adhesion. These results provide support to the concept that VE-cadherin contributes to the normal differentiation of endothelial barrier function during CAM angiogenesis in vivo.     

Single-phagosome imaging reveals that homotypic fusion impairs phagosome degradative function

Immune cells degrade internalized pathogens in vesicle compartments called phagosomes. Many intracellular bacteria induce homotypic phagosome fusion to survive in host cells, but the fusion interaction between phagosomes and its consequence for phagosome function have scarcely been studied. Here, we characterize homotypic fusion between phagosomes in macrophages and identify how such interactions impact the degradative capacity of phagosomes. By developing a series of particle sensors for measuring biochemical changes of single phagosomes, we show that phagosomes undergo stable fusion, transient “kiss-and-run” fusion, or both in succession. Super-resolution three-dimensional fluorescence microscopy revealed that stably fused phagosomes are connected by membrane “necks” with submicron–sized fusion pores. Furthermore, we demonstrate that, after stable fusion, phagosomes have leaky membranes and thereby impaired degradative functions. Our findings, based on phagosomes that contain synthetic particles, illustrate that homotypic fusion is not exclusive to phagosomes that encapsulate pathogens, as previously believed. The physical process of homotypic fusion is alone sufficient to perturb the degradative functions of phagosomes.     

Normal myoblast fusion requires myoferlin

Muscle growth occurs during embryonic development and continues in adult life as regeneration. During embryonic muscle growth and regeneration in mature muscle, singly nucleated myoblasts fuse to each other to form myotubes. In muscle growth, singly nucleated myoblasts can also fuse to existing large, syncytial myofibers as a mechanism of increasing muscle mass without increasing myofiber number. Myoblast fusion requires the alignment and fusion of two apposed lipid bilayers. The repair of muscle plasma membrane disruptions also relies on the fusion of two apposed lipid bilayers. The protein dysferlin, the product of the Limb Girdle Muscular Dystrophy type 2 locus, has been shown to be necessary for efficient, calcium-sensitive, membrane resealing. We now show that the related protein myoferlin is highly expressed in myoblasts undergoing fusion, and is expressed at the site of myoblasts fusing to myotubes. Like dysferlin, we found that myoferlin binds phospholipids in a calcium-sensitive manner that requires the first C2A domain. We generated mice with a null allele of myoferlin. Myoferlin null myoblasts undergo initial fusion events, but they form large myotubes less efficiently in vitro, consistent with a defect in a later stage of myogenesis. In vivo, myoferlin null mice have smaller muscles than controls do, and myoferlin null muscle lacks large diameter myofibers. Additionally, myoferlin null muscle does not regenerate as well as wild-type muscle does, and instead displays a dystrophic phenotype. These data support a role for myoferlin in the maturation of myotubes and the formation of large myotubes that arise from the fusion of myoblasts to multinucleate myotubes.     

Phosphatidylinositol 4,5-bisphosphate regulates two steps of homotypic vacuole fusion

Yeast vacuoles undergo cycles of fragmentation and fusion as part of their transmission to the daughter cell and in response to changes of nutrients and the environment. Vacuole fusion can be reconstituted in a cell free system. We now show that the vacuoles synthesize phosphoinositides during in vitro fusion. Of these phosphoinositides, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) are important for fusion. Monoclonal antibodies to PI(4,5)P(2), neomycin (a phosphoinositide ligand), and phosphatidylinositol-specific phospholipase C interfere with the reaction. Readdition of PI(4, 5)P(2) restores fusion in each case. Phosphatidylinositol 3-phosphate and PI(3,5)P(2) synthesis are not required. PI(4,5)P(2) is necessary for priming, i.e., for the Sec18p (NSF)-driven release of Sec17p (alpha-SNAP), which activates the vacuoles for subsequent tethering and docking. Therefore, it represents the kinetically earliest requirement identified for vacuole fusion so far. Furthermore, PI(4,5)P(2) is required at a step that can only occur after docking but before the BAPTA sensitive step in the latest stage of the reaction. We hence propose that PI(4,5)P(2) controls two steps of vacuole fusion.     

The Tlg SNARE complex is required for TGN homotypic fusion.

Using a new assay for membrane fusion between late Golgi/endosomal compartments, we have reconstituted a rapid, robust homotypic fusion reaction between membranes containing Kex2p and Ste13p, two enzymes resident in the yeast trans-Golgi network (TGN). Fusion was temperature, ATP, and cytosol dependent. It was inhibited by dilution, Ca+2 chelation, N-ethylmaleimide, and detergent. Coimmunoisolation confirmed that the reaction resulted in cointegration of the two enzymes into the same bilayer. Antibody inhibition experiments coupled with antigen competition indicated a requirement for soluble NSF attachment protein receptor (SNARE) proteins Tlg1p, Tlg2p, and Vti1p in this reaction. Membrane fusion also required the rab protein Vps21p. Vps21p was sufficient if present on either the Kex2p or Ste13p membranes alone, indicative of an inherent symmetry in the reaction. These results identify roles for a Tlg SNARE complex composed of Tlg1p, Tlg2p, Vti1p, and the rab Vps21p in this previously uncharacterized homotypic TGN fusion reaction.     

The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion

Chlamydiae replicate within an intracellular vacuole, termed an inclusion, that is non-fusogenic with vesicles of the endosomal or lysosomal compartments. Instead, the inclusion appears to intersect an exocytic pathway from which chlamydiae intercept sphingomyelin en route from the Golgi apparatus to the plasma membrane. Chlamydial protein synthesis is required to establish this interaction. In an effort to identify those chlamydial proteins controlling vesicle fusion, we have prepared polyclonal antibodies against several Chlamydia trachomatis inclusion membrane proteins. Microinjection of polyclonal antibodies against three C. trachomatis inclusion membrane proteins, IncA, F and G, into the cytosol of cells infected with C. trachomatis demonstrates reactivity with antigens on the cytoplasmic face of the inclusion membrane, without apparent inhibition of chlamydial multiplication. Microinjection of antibodies against the C. trachomatis IncA protein, however, results in the development of an aberrant multilobed inclusion structure remarkably similar to that of C. psittaci GPIC. These results suggest that the C. trachomatis IncA protein is involved in homotypic vesicle fusion and/or septation of the inclusion membrane that is believed to accompany bacterial cell division in C. psittaci . This proposal is corroborated by the expression of C. trachomatis and C. psittaci IncA in a yeast two-hybrid system to demonstrate C. trachomatis , but not C. psittaci , IncA interactions. Despite the inhibition of homotypic fusion of C. trachomatis inclusions, fusion of sphingomyelin-containing vesicles with the inclusion was not suppressed.     

Rat myoblast fusion requires metalloendoprotease activity

The calcium-dependent fusion of cultured rat myoblasts to multinucleate myotubes appears to require the activity of a neutral metalloendoprotease at the time of fusion. Metalloendoprotease inhibitors and synthetic dipeptide substrates prevent myoblast fusion when added to fusion-competent myoblasts with the addition of calcium. Metalloendoprotease activity has been identified and partially characterized in myoblast membranes with a fluorogenic protease substrate, and is inhibited by the same compounds that prevent fusion.     

SNARE status regulates tether recruitment and function in homotypic COPII vesicle fusion

In mammals, coat complex II (COPII)-coated transport vesicles deliver secretory cargo to vesicular tubular clusters (VTCs) that facilitate cargo sorting and transport to the Golgi. We documented in vitro tethering and SNARE-dependent homotypic fusion of endoplasmic reticulum-derived COPII transport vesicles to form larger cargo containers characteristic of VTCs ( Xu, D., and Hay, J. C. (2004) J. Cell Biol. 167, 997-1003). COPII vesicles thus appear to contain all necessary components for homotypic tethering and fusion, providing a pathway for de novo VTC biogenesis. Here we demonstrate that antibodies against the endoplasmic reticulum/Golgi SNARE Syntaxin 5 inhibit COPII vesicle homotypic tethering as well as fusion, implying an unanticipated role for SNAREs upstream of fusion. Inhibition of SNARE complex access and/or disassembly with dominant-negative alpha-soluble NSF attachment protein (SNAP) also inhibited tethering, implicating SNARE status as a critical determinant in COPII vesicle tethering. The tethering-defective vesicles generated in the presence of dominant-negative alpha-SNAP specifically lacked the Rab1 effectors p115 and GM130 but not other peripheral membrane proteins. Furthermore, Rab effectors, including p115, were shown to be required for homotypic COPII vesicle tethering. Thus, our results demonstrate a requirement for SNARE-dependent tether recruitment and function in COPII vesicle fusion. We anticipate that recruitment of tether molecules by an upstream SNARE signal ensures that tethering events are initiated only at focal sites containing appropriately poised fusion machinery.     

A novel membrane-anchored Rab5 interacting protein required for homotypic endosome fusion

The ras-related GTPase rab5 is rate-limiting for homotypic early endosome fusion. We used a yeast two-hybrid screen to identify a rab5 interacting protein, rab5ip. The cDNA sequence encodes a ubiquitous 75-kDa protein with an N-terminal transmembrane domain (TM), a central coiled-coil structure, and a C-terminal region homologous to several centrosome-associated proteins. rab5ip lacking the transmembrane domain (rab5ipTM(-)) had a greater affinity in vitro for rab5-guanosine 5'-O-2-(thio)diphosphate than for rab5-guanosine 5'-3-O-(thio)triphosphate. In transfected HeLa cells, rab5ipTM(-) was partly cytosolic and localized (by immunofluorescence) with a rab5 mutant believed to be in a GDP conformation (GFP-rab5(G78A)) but not with GFP-rab5(Q79L), a GTPase-deficient mutant. rab5ip with the transmembrane domain (rab5ipTM(+)) was completely associated with the particulate fraction and localized extensively with GFP-rab5(wt) in punctate endosome-like structures. Overexpression of rab5ipTM(+) using Sindbis virus stimulated the accumulation of fluid-phase horseradish peroxidase by BHK-21 cells, and homotypic endosome fusion in vitro was inhibited by antibody against rab5ip. rab5ipTM(-) inhibited rab5(wt)-stimulated endosome fusion but did not inhibit fusion stimulated by rab5(Q79L). rab5ip represents a novel rab5 interacting protein that may function on endocytic vesicles as a receptor for rab5-GDP and participate in the activation of rab5.     

Neuronal fusion pore assembly requires membrane cholesterol

Cholesterol has been proposed to play a critical role in regulating neurotransmitter release and synaptic plasticity. The neuronal porosome/fusion pore, the secretory machinery at the nerve terminal, is a 12-17 nm cup-shaped lipoprotein structure composed of cholesterol and a number of proteins, among them calcium channels, and the t-SNARE proteins Syntaxin-1 and SNAP-25. During neurotransmission, synaptic vesicles dock and fuse at the porosome via interaction of their v-SNARE protein with t-SNAREs at the porosome base. Membrane-associated neuronal t-SNAREs interact in a circular array with liposome-associated neuronal v-SNARE to form the t-/v-SNARE ring complex. The SNARE complex along with calcium is required for the establishment of continuity between opposing bilayers. Here we show that although cholesterol is an integral component of the neuronal porosome and is required for maintaining its physical integrity and function, it has no influence on the conformation of the SNARE ring complex.     

自噬体膜的来源

细胞自噬是指细胞通过自噬-溶酶体(autolysosome)降解变性蛋白聚集物和受损细胞器的过程. 自噬对于细胞内环境的稳态、物质的平衡、胚胎发育以及疾病的发生发挥重要作用. 在电镜下观察,自噬体膜是一个双层脂质膜结构. 细胞中因缺乏除了自噬相关蛋白9 (autophagy-related protein 9,ATG9)以外的自噬体膜相关蛋白,故难以确定自噬体膜的来源. 自噬体膜的来源也因此成为目前自噬研究领域的热点问题. 关于自噬体膜的来源,学术界存在两种观点：一种认为自噬体膜是细胞在自噬体组装位点(pre-autophagosomal structure, PAS)重新合成的;另一种观点则认为自噬体膜来源于细胞已有的某些细胞器(如内质网、高尔基体、内吞体、质膜和线粒体). 该文综述了近年有关自噬体膜来源于细胞已有的某些细胞器的研究进展,旨在为相关领域的研究提供参考.