Mechanism Of Vesicle Formation Research Articles

Atg27 Is Required for Autophagy-dependent Cycling of Atg9

Autophagy is a catabolic pathway for the degradation of cytosolic proteins or organelles and is conserved among all eukaryotic cells. The hallmark of autophagy is the formation of double-membrane cytosolic vesicles, termed autophagosomes, which sequester cytoplasm; however, the mechanism of vesicle formation and the membrane source remain unclear. In the yeast Saccharomyces cerevisiae, selective autophagy mediates the delivery of specific cargos to the vacuole, the analog of the mammalian lysosome. The transmembrane protein Atg9 cycles between the mitochondria and the pre-autophagosomal structure, which is the site of autophagosome biogenesis. Atg9 is thought to mediate the delivery of membrane to the forming autophagosome. Here, we characterize a second transmembrane protein Atg27 that is required for specific autophagy in yeast. Atg27 is required for Atg9 cycling and shuttles between the pre-autophagosomal structure, mitochondria, and the Golgi complex. These data support a hypothesis that multiple membrane sources supply the lipids needed for autophagosome formation.

A role for BARS at the fission step of COPI vesicle formation from Golgi membrane

The core complex of Coat Protein I (COPI), known as coatomer, is sufficient to induce coated vesicular-like structures from liposomal membrane. In the context of biological Golgi membrane, both palmitoyl-coenzyme A (p-coA) and ARFGAP1, a GTPase-activating protein (GAP) for ADP-Ribosylation Factor 1, also participate in vesicle formation, but how their roles may be linked remains unknown. Moreover, whether COPI vesicle formation from Golgi membrane requires additional factors also remains unclear. We now show that Brefeldin-A ADP-Ribosylated Substrate (BARS) plays a critical role in the fission step of COPI vesicle formation from Golgi membrane. This role of BARS requires its interaction with ARFGAP1, which is in turn regulated oppositely by p-coA and nicotinamide adenine dinucleotide, which act as cofactors of BARS. Our findings not only identify a new factor needed for COPI vesicle formation from Golgi membrane but also reveal a surprising mechanism by which the roles of p-coA and GAP are linked in this process.

Influence of salt and polymer on the critical vesicle concentration in aqueous mixture of zwitterionic/anionic surfactants

Vesicles can be formed spontaneously in aqueous solution of surfactant mixture of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) and lauryl sulfonate betaine (LSB). Different from the catanionic vesicles, the formation is dependent on surfactant concentration, mixing ratio and salinity due to the weak attraction between the two kinds of surfactant molecules, which provides possibility to determine the critical vesicle concentration (CVC), just as the critical micelle concentration (CMC). In this paper, a simple but sensitive conductivity titration was employed to determine the critical concentration since it is concentration-continuous and the structural transition of the ionic surfactant relates directly to their conductivity. Especially, after vesicles are formed, some of the surfactant molecules form the inner layer of the vesicle and do not conduct any more, which will lead to a much evident change in conductivity. As a result, the turning points appear in both the conductivity and molar conductivity titration curve at the transition from monomers to micelles and micelles to vesicles with increasing concentration and/or salinity, with assistant proof from surface tension measurement and TEM. The CVC drops markedly after the addition of salt due to the compression of salt on the double electric layer of the surfactant polar head. But the addition of nonionic polymer PVP (K30) delays the vesicle formation due to the adsorption of surfactant molecules to the polymer. The results may make much contribution to the understanding of the mechanism of vesicle formation.

The molecular machinery of autophagy: unanswered questions

Autophagy is a process in which cytosol and organelles are sequestered within double-membrane vesicles that deliver the contents to the lysosome/vacuole for degradation and recycling of the resulting macromolecules. It plays an important role in the cellular response to stress, is involved in various developmental pathways and functions in tumor suppression, resistance to pathogens and extension of lifespan. Conversely, autophagy may be associated with certain myopathies and neurodegenerative conditions. Substantial progress has been made in identifying the proteins required for autophagy and in understanding its molecular basis; however, many questions remain. For example, Tor is one of the key regulatory proteins at the induction step that controls the function of a complex including Atg1 kinase, but the target of Atg1 is not known. Although autophagy is generally considered to be nonspecific, there are specific types of autophagy that utilize receptor and adaptor proteins such as Atg11; however, the means by which Atg11 connects the cargo with the sequestering vesicle, the autophagosome, is not understood. Formation of the autophagosome is a complex process and neither the mechanism of vesicle formation nor the donor membrane origin is known. The final breakdown of the sequestered cargo relies on well-characterized lysosomal/vacuolar proteases; the roles of lipases, by contrast, have not been elucidated, and we do not know how the integrity of the lysosome/vacuole membrane is maintained during degradation.

Cell-free Reconstitution of Transport from the trans-Golgi Network to the Late Endosome/Prevacuolar Compartment

Vesicle-mediated transport between the trans-Golgi network (TGN) and the late endosome/prevacuolar compartment (PVC) is an essential step in lysosomal/vacuolar biogenesis. In addition, localization of integral membrane proteins to the TGN requires continual cycles of vesicular transport between the TGN and endosomal compartments. Genetic and biochemical analyses in yeast have identified a variety of proteins required for TGN-to-PVC transport. However, the precise mechanisms of vesicle formation, transport, and fusion have not been fully elucidated. To study the steps of TGN-to-PVC transport in mechanistic detail, we have developed a cell-free assay to monitor delivery of the processing protease Kex2p from the TGN to PVC compartments containing a Kex2p substrate. Transport is time-, temperature-, and ATP-dependent and requires the t-SNARE Pep12p. Moreover, cell-free delivery of Kex2p to the PVC results in the co-integration of Kex2p into PVC membranes containing the Kex2p substrate as determined by co-immunoisolation of Kex2p and the substrate using antibody against the Kex2p cytosolic tail. This work represents the first cell-free reconstitution and biochemical analysis of the essential vacuolar/lysosomal sorting step TGN to late endosome transport.

GTP/GDP exchange by Sec12p enables COPII vesicle bud formation on synthetic liposomes.

The generation of COPII vesicles from synthetic liposome membranes requires the minimum coat components Sar1p, Sec23/24p, Sec13/31p, and a nonhydrolyzable GTP analog such as GMP-PNP. However, in the presence of GTP and the full complement of coat subunits, nucleotide hydrolysis by Sar1p renders the coat insufficiently stable to sustain vesicle budding. In order to recapitulate a more authentic, GTP-dependent budding event, we introduced the Sar1p nucleotide exchange catalyst, Sec12p, and evaluated the dynamics of coat assembly and disassembly by light scattering and tryptophan fluorescence measurements. The catalytic, cytoplasmic domain of Sec12p (Sec12DeltaCp) activated Sar1p with a turnover 10-fold higher than the GAP activity of Sec23p stimulated by the full coat. COPII assembly was stabilized on liposomes incubated with Sec12DeltaCp and GTP. Numerous COPII budding profiles were visualized on membranes, whereas a parallel reaction conducted in the absence of Sec12DeltaCp produced no such profiles. We suggest that Sec12p participates actively in the growth of COPII vesicles by charging new Sar1p-GTP molecules that insert at the boundary between a bud and the surrounding endoplasmic reticulum membrane.

Atg21 is a phosphoinositide binding protein required for efficient lipidation and localization of Atg8 during uptake of aminopeptidase I by selective autophagy.

Delivery of proteins and organelles to the vacuole by autophagy and the cytoplasm to vacuole targeting (Cvt) pathway involves novel rearrangements of membrane resulting in the formation of vesicles that fuse with the vacuole. The mechanism of vesicle formation and the origin of the membrane are complex issues still to be resolved. Atg18 and Atg21 are proteins essential to vesicle formation and together with Ygr223c form a novel family of phosphoinositide binding proteins that are associated with the vacuole and perivacuolar structures. Their localization requires the activity of Vps34, suggesting that phosphatidylinositol(3)phosphate may be essential for their function. The activity of Atg18 is vital for all forms of autophagy, whereas Atg21 is required for the Cvt pathway but not for nitrogen starvation-induced autophagy. The loss of Atg21 results in the absence of Atg8 from the pre-autophagosomal structure (PAS), which may be ascribed to a reduced rate of conjugation of Atg8 to phosphatidylethanolamine. A similar defect in localization of a second ubiquitin-like conjugate, Atg12-Atg5, suggests that Atg21 may be involved in the recruitment of membrane to the PAS.

Spontaneous vesicle formation and vesicle-tubular microstructure transition in aqueous solution of a poly-tailed cationic and anionic surfactants mixture.

Spontaneous vesicle formation has been observed in aqueous mixtures of tri-(N-dodecyldimethylhydroxypropylammonium chloride) phosphate (PTA) and bis-(2-ethylhexyl) sulfosuccinate (Aerosol OT), which is supported by negative-staining TEM and dynamic light scattering. The range of vesicle formation in the PTA/AOT mixtures is wide and monodisperse vesicles are obtained. The vesicle diameter increases with the total surfactant concentration. Tubular microstructures, vesicle fusion, and vesicle-tubular microstructure transition have been also observed by negative-staining TEM. The vesicle formation mechanism is discussed from the viewpoint of molecular geometry, conformation, and the interaction between surfactant molecules.

Secretory bulk flow of soluble proteins is efficient and COPII dependent.

COPII-coated vesicles, first identified in yeast and later characterized in mammalian cells, mediate protein export from the endoplasmic reticulum (ER) to the Golgi apparatus within the secretory pathway. In these organisms, the mechanism of vesicle formation is well understood, but the process of soluble cargo sorting has yet to be resolved. In plants, functional complements of the COPII-dependent protein traffic machinery were identified almost a decade ago, but the selectivity of the ER export process has been subject to considerable debate. To study the selectivity of COPII-dependent protein traffic in plants, we have developed an in vivo assay in which COPII vesicle transport is disrupted at two distinct steps in the pathway. First, overexpression of the Sar1p-specific guanosine nucleotide exchange factor Sec12p was shown to result in the titration of the GTPase Sar1p, which is essential for COPII-coated vesicle formation. A second method to disrupt COPII transport at a later step in the pathway was based on coexpression of a dominant negative mutant of Sar1p (H74L), which is thought to interfere with the uncoating and subsequent membrane fusion of the vesicles because of the lack of GTPase activity. A quantitative assay to measure ER export under these conditions was achieved using the natural secretory protein barley alpha-amylase and a modified version carrying an ER retention motif. Most importantly, the manipulation of COPII transport in vivo using either of the two approaches allowed us to demonstrate that export of the ER resident protein calreticulin or the bulk flow marker phosphinothricin acetyl transferase is COPII dependent and occurs at a much higher rate than estimated previously. We also show that the instability of these proteins in post-ER compartments prevents the detection of the true rate of bulk flow using a standard secretion assay. The differences between the data on COPII transport obtained from these in vivo experiments and in vitro experiments conducted previously using yeast components are discussed.

The Itinerary of a Vesicle Component, Aut7p/Cvt5p, Terminates in the Yeast Vacuole via the Autophagy/Cvt Pathways

Aminopeptidase I (API) is delivered to the yeast vacuole by one of two alternative pathways, cytoplasm to vacuole targeting (Cvt) or autophagy, depending on nutrient conditions. Genetic, morphological, and biochemical studies indicate that the two pathways share many of the same molecular components. The Cvt pathway functions during vegetative growth, while autophagy is induced during starvation. Both pathways involve the formation of cytosolic vesicles that fuse with the vacuole. In either case, the mechanism of vesicle formation is not known. Autophagic uptake displays a greater capacity for cytosolic protein sequestration. This suggests the involvement of an inducible protein(s) that allows the vesicle-forming machinery to adapt to the increased degradative needs of the cell. We have analyzed the biosynthesis of Aut7p, a protein required for both pathways. We find Aut7p expression is induced by nitrogen starvation. Aut7p is degraded by a process dependent on both proteinase A and Cvt/autophagy components. Protease accessibility assays demonstrate that Aut7p is located within vesicles in strains defective in vesicle delivery or breakdown. Finally, the aut7/cvt5 mutant accumulates precursor API at a stage prior to vesicle completion. These data suggest that Aut7p is induced during autophagy and delivered to the vacuole together with precursor API by Cvt/autophagic vesicles.

Mechanisms of vesicle formation: Insights from the COP system

The major cytosolic and membrane proteins that represent machinery of coat protein (COP)-coated transport vesicles within the secretory pathway are characterized to date. This has allowed investigation of the molecular mechanisms that underlie the formation of these vesicles. In vitro binding studies and reconstitution experiments have provided insights at the molecular level into the biogenesis of COPII- and COPI-coated vesicles.

Light scattering characterization of extruded lipid vesicles.

By modeling extruded unilamellar lipid vesicles as thin-walled ellipsoidal shells, mathematical analysis provides simple equations which relate the mean elongation and other morphological characteristics of a vesicle population to quantities readily obtained from combined static and dynamic light scattering measurements. For SOPC vesicles extruded through a 100 nm pore-size filter into a 72.9 mM NaCl solution, the inferred elongation ratio (vesicle long axis to short axis) is approximately 3.7 +/- 0.6. When these vesicles were dialyzed into hypertonic or hypotonic solutions, this elongation ratio varied from 1 (for spherical liposomes) in strongly hypotonic solutions to greater than 6 in increasingly hypertonic solutions, beyond which abrupt morphological transformations appear. These results are quantitatively consistent with a mechanism of vesicle formation by extrusion and with the expectation that vesicle volumes change to equalize internal and external osmolarity via water flow, subject to the constraint of constant bilayer area. Our analysis also provides simplified equations to assess the effects of vesicle elongation and polydispersity on liposome parameters that are commonly required to characterize vesicle preparations for diverse applications. The implications of this study for routine light scattering characterization of extruded vesicles are discussed.

Rotational dynamics of lipid–detergent mixtures probed by a cyanine dye: a mechanism for vesicle formation

Time-resolved fluorescence anisotropy and lifetime measurements on the dye 3,3′-diethyloxadicarbocyanine iodide in phospholipid–deoxycholate and phospholipid–octyl glucoside mixtures have been carried out. Earlier we showed that the different populations of the dye could be resolved by virtue of the difference in their microenvironmental emission behaviour. Rotational dynamics of the dye in detergent–phospholipid mixtures show that gradual removal of detergent from the medium leads to a large increase in rotational correlation time corresponding to formation of large vesicles. The lamellar–micellar transition takes place near the critical micellar concentration (c.m.c.) of the detergents. The present results strongly support the mechanism of stepwise formation of lipid vesicles on removal of detergent that was proposed recently by us.

Mechanisms of Liposome Formation

The exact mechanism of liposome formation is still not well understood. When starting from lipid-in-water suspensions there are predominantly two different mechanisms of vesicle formation: fragmentation of bilayers with subsequent self-closure of bilayered fragments and budding off daughter vesicles from mother liposomes. The two mechanisms are qualitatively discussed and related to particular vesicle preparation methods. The article is concluded with a speculative unifying mechanism and discussion on liposome stability.

Na,K-ATPase and carboxyfluorescein distinctly alter vesicle formation in vitro

The mechanism of vesicle formation as well as the precise reasons for their stability are not known. Thus, it is necessary to simulate the process in vitro for studying its mechanism. If phospholipids are suspended in physiological solution by means of cholate and the detergent is then removed by dialysis, the phospholipids self-assemble to form unilamellar vesicles. We report here that the addition of Na,K-ATPase (an integral membrane protein) to the phospholipids changes the vesicle structure, they become larger and a multilamellar population appears. By contrast, carboxyfluorescein, a compound commonly used for labelling the aqueous vesicle compartment, produces an unexpected effect on vesicle structure by inducing complex, tore-like intravesicular multilayer formations associated with a 5-fold increase in diameter. Thus, the presence of a protein in the membrane phase or of a compound in the water phase can influence and direct vesicle formation in vitro; these model systems might give some clues to possible physicochemical or biological factors governing the formation of natural membrane structures.

Rapid recovery of structure and function of the cholinergic synapses in the cat superior cervical ganglionin vivo following stimulation-induced exhaustion

Cat superior cervical ganglia (SCG) were tetanically stimulated in vivo at 30-100 Hz until neural transmission was exhausted, and then were allowed to rest and recover. Changes in their cholinergic synapses were examined electrophysiologically and morphologically during the time of tetanic stimulation and during recovery. For morphometric analysis the presynaptic terminal was subdivided into two areas: an area directly over the active zone, termed zone-I, (bounded by a hemicircle with a diameter equivalent to the active zone length), and the remaining preterminal area, termed zone-II. In control ganglia before stimulation synaptic vesicle density in zone-I (SVD-I) averaged 90 microns-2 and the number of vesicles actually attached to the active zone (SVA) averaged about 2.5 per single profile of nerve terminal. Upon stimulation, the postganglionic potential immediately began to decline in amplitude and disappeared after 1 min of stimulation. Simultaneously, SVD-I declined to less than 35 microns-2 and SVA declined to less than 1 per section. Thereafter, stimulation was terminated and the ganglion was allowed to rest. Recovery of the postganglionic potential was monitored by stimulation at 1 Hz. The postganglionic potential reached control levels after only 1 min of rest. Likewise, the structural parameters, SVD-I and SVA, also rapidly recovered, reaching control levels after only 30 sec of rest, slightly faster than the postganglionic potential. This illustrates that stimulation-induced fatigue of transmitter output and depletion of synaptic vesicles recover to the control level at a high rate in synapses of the cat SCG with a normal supply of blood. In fact, morphological recovery may be slightly faster than electrophysiological recovery. Mechanisms of vesicle formation and migration to the presynaptic area are discussed in light of these observations.

On the mechanism of vesicle formation

A mechanism of vesicle formation is discussed. A recently proposed model in which the transition structures are hexagonal assemblies of rod-like mixed micelles is compared to the classical model in which disk-like mixed micelles are postulated as intermediate structures in the vesicle formation process. The kinetic effect, i.e., the influence of the rate of detergent removal (vesicle formation) on the size distribution of vesicles formed, is qualitatively explained: systems are driven far from equilibrium conditions and the intermediate structures simply do not have time to achieve thermodynamic equilibria.

The mechanism of vesicle formation

The mechanism of vesicle formation

The mechanism of vesicle formation.

Phospholipid molecules exhibit amphiphilic properties and therefore they aggregate either in their crystalline state or in polar solvents into ordered structures with typical lyotropic liquid crystalline symmetries (de Gennes, 1974). At high lipid concentrations in water these are predominantly lamellar phases (Luzzati, 1968), while in aqueous solutions phospholipid molecules normally form self-closed spherical or oval structures where one or several phospholipid bilayers entrap(s) part of the solvent in its/their interior (Bangham & Home, 1964; Papahadjopoulos, 1978). In the case of one bilayer separating the internal and external solvent these structures are called, with respect to their size, small or large unilamellar vesicles (SUV and LUV, respectively) while the term multilamellar vesicles (MLV) is used in the case of many bilayers entrapping some of the solvent. The terms giant vesicle (GV) and large or small oligolamellar vesicles (LOV, SOV) are also used for very large vesicles and structures where several bilayers surround the entrapped solvent. Sometimes, especially in the case of technological applications, the term liposome is a homonym for SUV, LUV and MLV while in the older literature terms liposome and MLV are often synonyms. Vesicles are very important in many different areas of science and technology. In basic research they serve as models for cell membranes and their fusion, transport studies and investigations of membrane proteins that can be reconstituted in vesicles (i.e. 'proteoliposomes'). They also serve as delivery vehicles for drugs, genetic material, enzymes and other (macro)molecules into living cells and through other hydrophobic barriers in pharmacology, medicine, genetic engineering, cosmetic industry and food industry (Gregoriadis, 1984). They are also used as

Short-chain lecithins: How to form unilamellar vesicles from micelles and phospholipid multibilayers

Short-chain lecithins (6–8 carbon fatty acyl chains) form micelles in aqueous solutions. NMR and SANS studies show that a short-chain lecithin molecule in these micelles has the same conformation as a long-chain lecithin in bilayer aggregates. When small amounts of these short-chain lecithins (typically 20 mol% of total phospholipid) are added to aqueous dispersions of long-chain phospholipids, unilamellar vesicles form spontaneously. Induction of unilamellar vesicles from multibilayers does not occur with comparable concentrations of single-chain detergents. Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs) are extremely stable and form with a wide variety of long-chain phospholipids (with different head groups, T m, unsaturation, etc.). We have characterized them in terms of (1) size distribution and coexistence of multilamellar or mixed micellar structures, (2) phase behavior of the long-chain phospholipid, (3) distribution of the short-chain lecithin across the bilayer, (4) dynamics and interactions of the two phospholipids, and (5) susceptibility to water-soluble phospholipases. The results suggest a mechanism for vesicle formation and are critical in understanding the action of phospholipase-A 2 and phospholipase-C on bilayer surfces.