Membrane-Elasticity Model of Coatless Vesicle Budding Induced by ESCRT Complexes

The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT) directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.     

Blocked coated pits in AtT20 cells result from endocytosis of budding retrovirions

AtT20 cells support the replication of two endogenous retroviruses, a murine leukemia virus and a mouse mammary tumor virus. On glass or plastic substrates, AtT20 cells grow in clumps. In this situation, retroviruses budding from the plasma membrane of one cell can, on rare occasions, be invested by coated pits in the plasma membranes of contiguous cells. These pits can invaginate to depths of 2,000-4,000 A within the cytoplasm drawing with them the viral buds which remain connected to their parental cells by tubular stalks, some of which are only 225 +/- 15 A in diameter. These stalks run down the straight necks of the pits from the buds to the parental cell surfaces. Several lines of evidence indicate that these unique structures are blocked such that neither endocytosis nor budding can go to completion, and that they persist for several hours. The properties of these blocked coated pits are relevant to models of both endocytosis and viral budding. First, they indicate that the invagination of a coated pit is not absolutely dependent on its pinching off to form a coated vesicle, but that uncoating appears to be dependent upon the generation of a free vesicle. Secondly, they suggest that the final stages in the maturation of a retroviral core into a mature nucleoid are dependent on the detachment of the bud from its parental cell and that the driving force of budding is the association of viral transmembrane proteins with viral core proteins. An explanation is offered to account for the formation of these structures despite the phenomenon of viral interference.     

Chirality-induced budding: a raft-mediated mechanism for endocytosis and morphology of caveolae?

The formation of transport carriers (spherical vesicles and tubules) involves membrane budding, growth, and ultimately fission. We propose a mechanism of membrane budding, wherein the tilt and chirality of constituent molecules, confined to a patch of area A, induces buds of approximately 50-100 nm that are comparable to vesicles involved in endocytosis. Because such chiral and tilted lipid molecules are likely to exist in "rafts", we suggest the involvement of this mechanism in generating membrane buds in the clathrin and dynamin-independent, raft-component mediated endocytosis of glycosylphosphatidylinositol-anchored proteins. We argue that caveolae, permanent cell surface structures with characteristic morphology and enriched in raft constituents, are also likely to be formed by this mechanism. Thus, molecular chirality and tilt, and its expression over large spatial scales may be a common organizing principle in membrane budding of transport carriers.     

Dynamin-dependent Transferrin Receptor Recycling by Endosome-derived Clathrin-coated Vesicles

Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the trans-Golgi network. Here we show that these clathrin-coated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that is served by endosome-derived clathrin-coated vesicles, we used cells that overexpressed a temperature-sensitive mutant of dynamin-1 (dynamin-1(G273D)) or, as a control, dynamin-1 wild type. In dynamin-1(G273D)-expressing cells, 29-36% of endocytosed transferrin failed to recycle at the nonpermissive temperature and remained associated with tubular recycling endosomes. Sorting of endocytosed transferrin from fluid-phase endocytosed markers in early endosome antigen 1-labeled sorting endosomes was not inhibited. Dynamin-1(G273D) associated with accumulated clathrin-coated buds on extended tubular recycling endosomes. Brefeldin A interfered with the assembly of clathrin coats on endosomes and reduced the extent of transferrin recycling in control cells but did not further affect recycling by dynamin-1(G273D)-expressing cells. Together, these data indicate that the pathway from recycling endosomes to the plasma membrane is mediated, at least in part, by endosome-derived clathrin-coated vesicles in a dynamin-dependent manner.     

Assembly and disassembly of the ESCRT-III membrane scission complex

The ESCRT (endosomal sorting complex required for transport) pathway promotes the final membrane scission step at the end of cytokinesis, assists viral budding and generates multivesicular bodies (MVBs). These seemingly unrelated processes require a topologically similar membrane deformation and scission event that buds membranes/vesicles out of the cytoplasm. The topology of this budding reaction is 'opposite' to reactions that bud endocytic and secretory vesicles into the cytoplasm. Here we summarize recent findings that help to understand how the ESCRT machinery, in particular the ESCRT-III complex, assembles on its target membranes, executes membrane scission and is disassembled by the AAA-ATPase Vps4.     

A statistical-thermodynamic model of viral budding

We present a simple statistical thermodynamic model for budding of viral nucleocapsids at the cell membrane. The membrane is modeled as a flexible lipid bilayer embedding linker (spike) proteins, which serve to anchor and thus wrap the membrane around the viral capsids. The free energy of a single bud is expressed as a sum of the bending energy of its membrane coat, the spike-mediated capsid-membrane adhesion energy, and the line energy associated with the bud's rim, all depending on the extent of wrapping (i.e., bud size), and density of spikes in the curved membrane. This self-energy is incorporated into a simple free energy functional for the many-bud system, allowing for different spike densities, and hence entropy, in the curved (budding) and planar membrane regions, as well as for the configurational entropy of the polydisperse bud population. The equilibrium spike densities in the coexisting, curved and planar, membrane regions are calculated as a function of the membrane bending energy and the spike-mediated adhesion energy, for different spike and nucleocapsid concentrations in the membrane plane, as well as for several values of the bud's rim energy. We show that complete budding (full wrapping of nucleocapsids) can only take place if the adhesion energy exceeds a certain, critical, bending free energy. Whenever budding takes place, the spike density in the mature virions is saturated, i.e., all spike adhesion sites are occupied. The rim energy plays an important role in determining the size distribution of buds. The fraction of fully wrapped buds increases as this energy increases, resulting eventually in an all-or-nothing mechanism, whereby nucleocapsids at the plasma membrane are either fully enveloped or completely naked (just touching the membrane). We also find that at low concentrations all capsids arriving at the membrane get tightly and fully enveloped. Beyond a certain concentration, corresponding approximately to a stoichiometric spike/capsid ratio, newly arriving capsids cannot be fully wrapped; i.e., the budding yield decreases.     

Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins

The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal compartment that also exhibits budding profiles positive for AP-1. AP-3 colocalizes with clathrin, but to a lesser extent than does AP-1. The AP-3- and AP-1-bearing tubular compartments contain endocytosed transferrin, transferrin receptor, asialoglycoprotein receptor, and low amounts of the cation-independent mannose 6-phosphate receptor and the lysosome-associated membrane proteins (LAMPs) 1 and 2. Quantitative analysis revealed that of these distinct cargo proteins, only LAMP-1 and LAMP-2 are concentrated in the AP-3-positive membrane domains. Moreover, recycling of endocytosed LAMP-1 and CD63 back to the cell surface is greatly increased in AP-3-deficient cells. Based on these data, we propose that AP-3 defines a novel pathway by which lysosomal membrane proteins are transported from tubular sorting endosomes to lysosomes.     

A Single Herpesvirus Protein Can Mediate Vesicle Formation in the Nuclear Envelope

Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission.     

Biogenesis of secretory granules

The biogenesis of secretory granules in endocrine, neuroendocrine, and exocrine cells is thought to involve a selective aggregation of the regulated secretory proteins into a dense-cored structure. The dense-core is then enveloped by membrane in the trans-Golgi network and buds, forming an immature secretory granule. The immature secretory granule then undergoes a maturation process which gives rise to the mature secretory granule. The recent data on the processes of aggregation, budding and maturation are summarized here. In addition, the current knowledge about the mature secretory granule is reviewed with emphasis on the biogenesis of the membrane of this organelle.     

Coordination of Golgi functions by phosphatidylinositol 4-kinases

Phosphatidylinositol 4-kinases (PI4Ks) regulate vesicle-mediated export from the Golgi apparatus via phosphatidylinositol 4-phosphate (PtdIns4P) binding effector proteins that control vesicle budding reactions and regulate membrane dynamics. Evidence has emerged from the characterization of Golgi PI4K effectors that vesicle budding and lipid dynamics are tightly coupled via a regulatory network that ensures that the appropriate membrane composition is established before a transport vesicle buds from the Golgi. An important hub of this network is protein kinase D, which regulates the activity of PI4K and several PtdIns4P effectors that control sphingolipid and sterol content of Golgi membranes. Other newly identified PtdIns4P effectors include Vps74/GOLPH3, a phospholipid flippase called Drs2 and Sec2, a Rab guanine nucleotide exchange factor (GEF). These effectors orchestrate membrane transformation events facilitating vesicle formation and targeting. In this review, we discuss how PtdIns4P signaling is integrated with membrane biosynthetic and vesicle budding machineries to potentially coordinate these crucial functions of the Golgi apparatus.     

Why Enveloped Viruses Need Cores—The Contribution of a Nucleocapsid Core to Viral Budding

During the lifecycle of many enveloped viruses, a nucleocapsid core buds through the cell membrane to acquire an outer envelope of lipid membrane and viral glycoproteins. However, the presence of a nucleocapsid core is not required for assembly of infectious particles. To determine the role of the nucleocapsid core, we develop a coarse-grained computational model with which we investigate budding dynamics as a function of glycoprotein and nucleocapsid interactions, as well as budding in the absence of a nucleocapsid. We find that there is a transition between glycoprotein-directed budding and nucleocapsid-directed budding that occurs above a threshold strength of nucleocapsid interactions. The simulations predict that glycoprotein-directed budding leads to significantly increased size polydispersity and particle polymorphism. This polydispersity can be explained by a theoretical model accounting for the competition between bending energy of the membrane and the glycoprotein shell. The simulations also show that the geometry of a budding particle leads to a barrier to subunit diffusion, which can result in a stalled, partially budded state. We present a phase diagram for this and other morphologies of budded particles. Comparison of these structures against experiments could establish bounds on whether budding is directed by glycoprotein or nucleocapsid interactions. Although our model is motivated by alphaviruses, we discuss implications of our results for other enveloped viruses.     

Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step

Mouse hepatitis coronavirus (MHV) buds into pleomorphic membrane structures with features expected of the intermediate compartment between the ER and the Golgi complex. Here, we characterize the MHV budding compartment in more detail in mouse L cells using streptolysin O (SLO) permeabilization which allowed us to better visualize the membrane structures at the ER-Golgi boundary. The MHV budding compartment shares membrane continuities with the rough ER as well as with cisternal elements on one side of the Golgi stack. It also labeled with p58 and rab2, two markers of the intermediate compartment, and with PDI, usually considered to be a marker of the rough ER. The membranes of the budding compartment, as well as the budding virions themselves, but not the rough ER, labeled with the N-acetyl- galactosamine (GalNAc)-specific lectin Helix pomatia. When the SLO- permeabilized cells were treated with guanosine 5'-(3-O- thio)triphosphate (GTP gamma S), the budding compartment accumulated a large number of beta-cop-containing buds and vesicular profiles. Complementary biochemical experiments were carried out to determine whether vesicular transport was required for the newly synthesized M protein, that contains only O-linked oligosaccharides, to acquire first, GalNAc and second, the Golgi modifications galactose and sialic acid. The results from both in vivo studies and from the use of SLO- permeabilized cells showed that, while GalNAc addition occurred under conditions which block vesicular transport, both cytosol and ATP were prerequisites for the M protein oligosaccharides to acquire Golgi modifications. Collectively, our data argue that transport from the rough ER to the Golgi complex requires only one vesicular transport step and that the intermediate compartment is a specialized domain of the endoplasmatic reticulum that extends to the first cisterna on the cis side of the Golgi stack.     

Morphology and Morphogenesis of Sindbis Virus as Seen with Freeze-Etching Techniques

Freeze-etch electron microscope studies of the morphogenesis and morphology of Sindbis virus confirmed results obtained by other workers employing thin-sectioning techniques. The 68-nm virion was found to have a nucleocapsid 36 nm in diameter surrounded by a double-layered, unit membrane. The membranous envelope is acquired as the capsid buds through the plasma membrane of the infected cell. The freeze-etch technique also provided the following new information. (i) At any one time, budding occurs in patches rather than evenly over the cell surface. (ii) The nucleocapsid is composed of capsomers 7 nm in diameter. (iii) The capsid interacts strongly with the membrane, both prior to budding and after maturation. (iv) The 7- to 10-nm particles characteristic of the internal faces of plasma membranes, which presumably represent host membrane proteins, are present in early stages of budding but disappear as morphogenesis progresses. (v) Fusion of the cell membrane at the base of the budding virion is a two-step process; the inner leaflet fuses into a sphere before the outer one. (vi) The outer surface of the viral envelope is covered with 4-nm subunits with a center-to-center spacing of 6 nm.     

Budding and strobilation inAurelia (Scyphozoa,Cnidaria): Functional requirement and spatial patterns of nucleic acid synthesis

Summary Strobilation and polypoid budding occur at different locations in the scyphistoma (polyp). Initiation and completion of both forms of budding are inhibited by hydroxyurea (HU), which blocks 95% of DNA synthesis within 12 h. Gradients of thymidine incorporation into both cell layers of the body column precede and accompany strobilation, and an epidermal gradient precedes polypoid budding. In both, the highest labelling index is in the zone in which initiation will occur. Polypoid buds show high variation in labelling index, which is therefore not significantly different from body column labelling. Initiation and some elongation of polyp buds occurs in a small percentage of animals in HU, indicating that cell recruitment is important for these processes. Strobilation appears to be more highly dependent on localized nucleic acid synthesis than polypoid budding.     

After Hrs with HIV

To efficiently bud off from infected cells, HIV and other enveloped viruses hijack the host cellular machinery that is normally involved in vacuolar protein sorting and multivesicular body (MVB) biogenesis. The HIV Gag protein mimics hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a modular adaptor protein that links membrane cargo recognition to its degradation after delivery to MVBs. In contrast to T cells, where HIV budding occurs at the plasma membrane, virus buds into vacuoles of macrophages, a process that may facilitate its spread within the infected host.     

Effects of 6-benzylamino-purine and 1-naphthaleneacetic acid on the epiphyllous bud formation in Bryophyllum

Summary The effects of the kinin 6-benzylamino-purine and of 1-naphthaleneacetic acid (NAA) on the epiphyllous bud formation in Bryophyllum were studied under controlled environment.In B. daigremontianum which requires long days for epiphyllous budding, buds were formed under continuous short days after application of the kinin. Similarly, such a treatment caused budding in attached non-aging leaves of B. calycinum which normally form buds only after detachment from the plant. This stimulatory effect of the kinin was strictly bound to the treated leaves (or leaf parts), which also showed an increased growth compared with the opposite non-treated leaves. Root formation in the developing buds was inhibited by the kinin.In both species NAA inhibited the epiphyllous budding under inductive conditions. A similar inhibitory effect was exerted by terminal and axillary buds.The results are discussed in the light of other investigations in this and related fields. It is concluded that epiphyllous bud formation is under the control of a correlative inhibition similar to apical dominance. It is further concluded that even though day-length controls both flowering and epiphyllous budding in B. daigremontianum the two processes must be affected through different biochemical systems.     

Semliki forest virus budding: assay, mechanisms, and cholesterol requirement

All enveloped viruses must bud through a cellular membrane in order to acquire their lipid bilayer, but little is known about this important stage in virus biogenesis. We have developed a quantitative biochemical assay to monitor the budding of Semliki Forest virus (SFV), an enveloped alphavirus that buds from the plasma membrane in a reaction requiring both viral spike proteins and nucleocapsid. The assay was based on cell surface biotinylation of newly synthesized virus spike proteins and retrieval of biotinylated virions using streptavidin-conjugated magnetic particles. Budding of biotin-tagged SFV was continuous for at least 2 h, independent of microfilaments and microtubules, strongly temperature dependent, and relatively independent of continued exocytic transport. Studies of cell surface spike proteins at early times of infection showed that these spikes did not efficiently bud into virus particles and were rapidly degraded. In contrast, at later times of infection, spike protein degradation was markedly reduced and efficient budding was then observed. The previously described cholesterol requirement in SFV exit was shown to be due to a block in budding in the absence of cholesterol and correlated with the continued degradation of spike proteins at all times of virus infection in sterol-deficient cells.     

Inheritance of cortical ER in yeast is required for normal septin organization

How cells monitor the distribution of organelles is largely unknown. In budding yeast, the largest subdomain of the endoplasmic reticulum (ER) is a network of cortical ER (cER) that adheres to the plasma membrane. Delivery of cER from mother cells to buds, which is termed cER inheritance, occurs as an orderly process early in budding. We find that cER inheritance is defective in cells lacking Scs2, a yeast homologue of the integral ER membrane protein VAP (vesicle-associated membrane protein-associated protein) conserved in all eukaryotes. Scs2 and human VAP both target yeast bud tips, suggesting a conserved action of VAP in attaching ER to sites of polarized growth. In addition, the loss of either Scs2 or Ice2 (another protein involved in cER inheritance) perturbs septin assembly at the bud neck. This perturbation leads to a delay in the transition through G2, activating the Saccharomyces wee1 kinase (Swe1) and the morphogenesis checkpoint. Thus, we identify a mechanism involved in sensing the distribution of ER.     

Plasma membrane deformation by circular arrays of ESCRT-III protein filaments

Endosomal sorting complex required for transport III (ESCRT-III) proteins function in multivesicular body biogenesis and viral budding. They are recruited from the cytoplasm to the membrane, where they assemble into large complexes. We used "deep-etch" electron microscopy to examine polymers formed by the ESCRT-III proteins hSnf7-1 (CHMP4A) and hSnf7-2 (CHMP4B). When overexpressed, these proteins target to endosomes and the plasma membrane. Both hSnf7 proteins assemble into regular approximately 5-nm filaments that curve and self-associate to create circular arrays. Binding to a coexpressed adenosine triphosphate hydrolysis-deficient mutant of VPS4B draws these filaments together into tight circular scaffolds that bend the membrane away from the cytoplasm to form buds and tubules protruding from the cell surface. Similar buds develop in the absence of mutant VPS4B when hSnf7-1 is expressed without its regulatory C-terminal domain. We demonstrate that hSnf7 proteins form novel membrane-attached filaments that can promote or stabilize negative curvature and outward budding. We suggest that ESCRT-III polymers delineate and help generate the luminal vesicles of multivesicular bodies.     

Lack of invaginations of the plasma membrane during budding and cell division of Saccharomyces cerevisiae and Schizosaccharomyces pombe

Abstract The plasma membrane of Saccharomyces cerevisiae and Schizosaccharomyces pombe in stationary phase had abundant invaginations. A round uninvaginated area emerged before budding when S. cerevisiae cells were given fresh medium. Middle-sized buds had some invaginations, whereas the neck between the bud and mother had very few. S. pombe which has neither the neck nor the predetermined position to divide had no uninvaginated ring area even in long cells during elongation in fresh medium. However, an uninvaginated ring area emerged as the earliest noticeable stage of cytokinesis. The uninvaginated state of the plasma membrane appeared to be correlated with budding and cell division.