Coated vesicles in plant cells

Coated vesicles represent vital transport intermediates in all eukaryotic cells. While the basic mechanisms of membrane exchange are conserved through the kingdoms, the unique topology of the plant endomembrane system is mirrored by several differences in the genesis, function and regulation of coated vesicles. Efforts to unravel the complex network of proteins underlying the behaviour of these vesicles have recently benefited from the application in planta of several molecular tools used in mammalian systems, as well as from advances in imaging technology and the ongoing analysis of the Arabidopsis genome. In this review, we provide an overview of the roles of coated vesicles in plant cells and highlight salient new developments in the field.     

Low molecular weight GTP-binding proteins are associated with neuronal organelles involved in rapid axonal transport and exocytosis

Recent evidence suggests that low molecular weight GTP-binding proteins may play important roles in a variety of membrane transport processes. In order to address the question of whether these proteins are involved in transport processes in the nerve axon, we have assessed their presence in rapid transport membranes from rabbit optic nerve. We report the characterization of a group of low molecular weight GTP-binding proteins which are constituents of rapid transport vesicles. Although these proteins are components of rapid transport vesicles, they are apparently not major rapidly transported species. They are localized in cytosolic as well as in membrane fractions of axons, and the membrane-associated form behaves as an integral membrane protein(s). These proteins are also found in association with a variety of vesicular and organellar components of neurons including coated vesicles, synaptic vesicles, synaptic plasma membranes, and mitochondria. We discuss the possible roles of these proteins in rapid axonal transport and exocytosis.     

Characterization and regulation of constitutive transport intermediates involved in trafficking from the trans-Golgi network

Transport vesicles or containers (TCs) mediate constitutive protein transport between the trans-Golgi network (TGN) and the plasma membrane. A key question is the nature and regulation of these transport containers or intermediates. We have used a trans-Golgi network resident, TGN38, to investigate TC formation. TGN38 is a recycling membrane glycoprotein that moves to the cell surface via constitutive membrane traffic and returns via the endosomal pathway. An in vitro assay to measure TC formation was devised using rat liver Golgi membranes, cytosolic factors and ATP. Transport intermediates containing TGN38 were produced and found to be smooth vesicles and tubules of up to 200 nm in length. These membrane-enclosed structures contain different constitutively secreted membrane glycoproteins, including molecules involved in immune functions such as MHC Class I and the polymeric Ig receptor, showing that these intermediates correspond to TCs that have been previously identified in vivo. Importantly, TC formation can be stimulated or inhibited by protein kinase and phosphatase inhibitors, showing regulation by intracellular signalling pathways.     

Evidence for segregation of sphingomyelin and cholesterol during formation of COPI-coated vesicles

In higher eukaryotes, phospholipid and cholesterol synthesis occurs mainly in the endoplasmic reticulum, whereas sphingomyelin and higher glycosphingolipids are synthesized in the Golgi apparatus. Lipids like cholesterol and sphingomyelin are gradually enriched along the secretory pathway, with their highest concentration at the plasma membrane. How a cell succeeds in maintaining organelle-specific lipid compositions, despite a steady flow of incoming and outgoing transport carriers along the secretory pathway, is not yet clear. Transport and sorting along the secretory pathway of both proteins and most lipids are thought to be mediated by vesicular transport, with coat protein I (COPI) vesicles operating in the early secretory pathway. Although the protein constituents of these transport intermediates are characterized in great detail, much less is known about their lipid content. Using nano-electrospray ionization tandem mass spectrometry for quantitative lipid analysis of COPI-coated vesicles and their parental Golgi membranes, we find only low amounts of sphingomyelin and cholesterol in COPI-coated vesicles compared with their donor Golgi membranes, providing evidence for a significant segregation from COPI vesicles of these lipids. In addition, our data indicate a sorting of individual sphingomyelin molecular species. The possible molecular mechanisms underlying this segregation, as well as implications on COPI function, are discussed.     

Biogenesis of transport intermediates in the endocytic pathway.

Evidence is accumulating that membrane traffic between organelles can be achieved by different types of intermediates. Small (< 100 nm) and short-lived vesicles mediate transport from the plasma membrane or the trans-Golgi network to endosomes, and formation of these vesicles depends on specific adapter complexes. In contrast, transport from early to late endosomes is achieved by relatively large (approximately 0.5 microm), long-lived and multivesicular intermediates, and their biogenesis depends on endosomal COP-I proteins. Here, we review recent work on the formation of these different transport intermediates, and we discuss, in particular, coat proteins, sorting signals contained in cargo molecules and the emerging role of lipid in vesicle biogenesis.     

A budding and fusing journey through the secretory pathway. Genetic and In Vitro Analysis of Cell Compartmentalization: A UCLA symposium, Taos, NM, USA, February 3-9, 1990

Reconstitution of vesicular transport events and the molecular and genetic analysis of the secretory pathway have taken the field of membrane traffic into a new era. Already, proteins have been discovered that facilitate multiple transport steps, and studies of the identities and modes of action of additional transport components, such as those that specify the targets of transport vesicles, will soon follow. Even after we understand how transport vesicles form, find their targets, and then fuse, other fundamental questions will still remain. How are proteins sorted into distinct transport vesicles? How is the directionality of protein transport achieved? How do organelles maintain their identities in the face of large volumes of membrane traffic? Finally, how is membrane traffic regulated? Answers to each of these fundamental questions are likely to be available in the not-too-distant future.     

A novel fluorescence microscopic approach to quantitatively analyse protein-induced membrane remodelling

Membrane remodelling or the bending and rupture of the lipid bilayer occurs during diverse cellular processes such as cell division, synaptic transmission, vesicular transport, organelle biogenesis and sporulation. These activities are brought about by the localized change in membrane curvature, which in turn causes lipid-packing stress, of a planar lipid bilayer by proteins. For instance, vesicular transport processes are typically characterized by the cooperative recruitment of proteins that induce budding of a planar membrane and catalyse fission of the necks of membrane buds to release vesicles. The analysis of such membrane remodelling reactions has traditionally been restricted to electron microscopy–based approaches or force spectroscopic analysis of membrane tethers pulled from liposome-based model membrane systems. Our recent work has demonstrated the facile creation of tubular model membrane systems of supported membrane tubes (SMrTs), which mimic late-stage intermediates of typical vesicular transport reactions. This review addresses the nature of such an assay system and a fluorescence-intensity-based analysis of changes in tube dimensions that is indicative of the membrane remodelling capacity of proteins.     

The role of microtubules in transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells

The organization of intracellular compartments and the transfer of components between them are central to the correct functioning of mammalian cells. Proteins and lipids are transferred between compartments by the formation, movement and subsequent specific fusion of transport intermediates. These vesicles and membrane clusters must be coupled to the cytoskeleton and to motor proteins that drive motility. Anterograde ER (endoplasmic reticulum)-to-Golgi transport, and the converse step of retrograde traffic from the Golgi to the ER, are now known to involve coupling of membranes to the microtubule cytoskeleton. Here we shall discuss our current understanding of the mechanisms that link membrane traffic in the early secretory pathway to the microtubule cytoskeleton in mammalian cells. Recent data have also provided molecular detail of functional co-ordination of motor proteins to specify directionality, as well as mechanisms for regulating motor activity by protein phosphorylation.     

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.     

Molecular mechanisms of COPII vesicle formation

The first step in protein secretion from eukaryotic cells is mediated by COPII vesicles, known for the cytoplasmic coat proteins that are the minimal machinery required to generate these small transport carriers. The five COPII coat components coordinate to create a vesicle by locally generating membrane curvature and populating the incipient bud with the appropriate cargo. This review describes the molecular details of how the COPII coat sculpts vesicles from the endoplasmic reticulum and highlights some unresolved questions regarding the regulation of this process in the complex environment of the eukaryotic cell.     

Identification, purification, and characterization of the rat liver golgi membrane ATP transporter

Phosphorylation of secretory and integral membrane proteins and of proteoglycans also occurs in the lumen of the Golgi apparatus. ATP, the phosphate donor in these reactions, must first cross the Golgi membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golgi membrane has been previously demonstrated in vitro using intact Golgi membrane vesicles from rat liver and mammary gland. We have now identified and purified the rat liver Golgi membrane ATP transporter. The transporter was purified to apparent homogeneity by a combination of conventional ion exchange, dye color, and affinity chromatography. An approximately 70,000-fold purification (2% yield) was achieved starting from crude rat liver Golgi membranes. A protein with an apparent molecular mass of 60 kDa was identified as the putative transporter by a combination of column chromatography, photoaffinity labeling with an analog of ATP, and native functional size determination on a glycerol gradient. The purified transporter appears to exist as a homodimer within the Golgi membrane, and when reconstituted into phosphatidylcholine liposomes, was active in ATP but not nucleotide sugar or adenosine 3'-phosphate 5'-phosphosulfate transport. The transport activity was saturable with an apparent Km very similar to that of intact Golgi vesicles.     

Biogenesis of tubular ER-to-Golgi transport intermediates

Tubular transport intermediates (TTIs) have been described as one class of transport carriers in endoplasmic reticulum (ER)-to-Golgi transport. In contrast to vesicle budding and fusion, little is known about the molecular regulation of TTI synthesis, transport and fusion with target membranes. Here we have used in vivo imaging of various kinds of GFP-tagged proteins to start to address these questions. We demonstrate that under steady-state conditions TTIs represent approximately 20% of all moving transport carriers. They increase in number and length when more transport cargo becomes available at the donor membrane, which we induced by either temperature-related transport blocks or increased expression of the respective GFP-tagged transport markers. The formation and motility of TTIs is strongly dependent on the presence of intact microtubules. Microinjection of GTPgammaS increases the frequency of TTI synthesis and the length of these carriers. When Rab proteins are removed from membranes by microinjection of recombinant Rab-GDI, the synthesis of TTIs is completely blocked. Microinjection of the cytoplasmic tails of the p23 and p24 membrane proteins also abolishes formation of p24-containing TTIs. Our data suggest that TTIs are ER-to-Golgi transport intermediates that form preferentially when transport-competent cargo exists in excess at the donor membrane. We propose a model where the interaction of the cytoplasmic tails of membrane proteins with microtubules are key determinants for TTI synthesis and may also serve as a so far unappreciated model for aspects of transport carrier formation.     

Na-dependent transport of tricarboxylic acid cycle intermediates by renal brush border membranes. Effects on fluorescence of a potential-sensitive cyanine dye

The effect of the transport of tricarboxylic acid cycle intermediates on the membrane potential of renal brush border vesicles was studied using fluorescence of the cyanine dye, 3,3′-dipropylthiadicarbocyanine iodide. The behavior of the dye in the preparation was established with valinomycin-induced K⁺-diffusion potentials; increases in fluorescence were associated with depolarizing conditions. Addition of 1 mM succinate or citrate to membrane/dye suspensions produced transient increases in fluorescence, indicative of a depolarizing event(s) associated with the transport of these substrates. The transient response in fluorescence was Na⁺ dependent, of greater magnitude under Na⁺-gradient as compared to Na⁺-equilibrium conditions, and was a saturable function of substrate concentration. The specificity of the fluorescence response was identical to that obtained from studies of the competitive inhibition of succinate transport by tricarboxylic acid cycle intermediates and analogs. We conclude that the major tricarboxylic acid cycle intermediates are transported via a common Na⁺-dependent transport system in renal brush border membranes.     

The pre-vacuolar t-SNARE AtPEP12p forms a 20S complex that dissociates in the presence of ATP

Many proteins are transported to the plant vacuole through the secretory pathway in small transport vesicles by a series of vesicle budding and fusion reactions. Vesicles carrying vacuolar cargo bud from the trans-Golgi network are thought to fuse with a pre-vacuolar compartment before being finally transported to the vacuole. In mammals and yeast, the fusion of a vesicle with its target organelle is mediated by a 20S protein complex containing membrane and soluble proteins that appear to be conserved between different species. A number of membrane proteins have been identified in plants that show sequence similarity with a family of integral membrane proteins (t-SNAREs) on target organelles that are required for the fusion of transport vesicles with that organelle. However, the biochemical function of these proteins has remained elusive. Here, we demonstrate for the first time the formation of a 20S complex in plants that has characteristics of complexes involved in vesicle fusion. This complex contains AtPEP12p, an Arabidopsis protein thought to be involved in protein transport to the prevacuolar compartment. In addition, we have shown that AtPEP12p can bind to alpha-SNAP, indicating that AtPEP12p does indeed function as a SNAP receptor or SNARE. These preliminary data suggest that AtPEP12p may function jointly with alpha-SNAP and NSF in the fusion of transport vesicles containing vacuolar cargo proteins with the pre-vacuolar compartment.     

Transport of phosphate in membrane vesicles from mouse fibroblasts transformed by simian virus 40.

Membrane vesicles were prepared from mouse fibroblasts transformed by SV40 virus (SV3T3). Following disruption of the cells by nitrogen cavitation, the membrane vesicles were obtained by differential centrifugation. As measured by enzyme markers, they consist mainly of membrane from the plasma membrane and smooth and rough endoplasmic reticulum. The vesicles transport Pi by two separate, mediated systems: one is independent of Na+, and the other is secondary active transport driven by a Na+ gradient. Electrical and chemical energy can be provided by a Na+ gradient to drive the concentrative uptake of Pi by the vesicles, one or both forces being used to energize transport. Evidence is provided that both the electrical and chemical potentials produced by the asymmetric distribution of Na+ across the membrane of SV3T3 membrane vesicles are utilized to concentrate phosphate in the vesicles. Phosphate transport by the vesicles cannot be accounted for by a small contamination of this fraction with mitochondria (1 to 4%). The Pi transport properties of the membrane vesicles differ from those of the fraction enriched in mitochondria in the following respects: their kinetic properties, and their responses to a Na+ gradient, N-ethylmaleimide, mersalyl, and succinate/acetate. However, the membrane vesicles share some properties of Pi transport with mitochondria. Cyanide, azide, oligomycin, 2,4-dinitrophenol, and carbonyl cyanide m-cholophenylhydrazone, inhibitors of Pi transport by mitochondria, also inhibit membrane vesicle, Pi transport. The vesicles retain all the features of Pi transport by SV3T3 cells that have been examined. They provide a simplified system for a determination of the details of the mechanism of Pi transport under conditions where transport is dissociated from intracellular reactions and in the presence of a defined electrochemical driving force.     

Cardiolipin Regulates the Activity of the Reconstituted Mitochondrial Calcium Uniporter by Modifying the Structure of the Liposome Bilayer

Reconstitution of mitochondrial calcium transport activity requires the incorporation of membrane proteins into a lipidic ambient. Calcium uptake has been measured previously using Cytochrome oxidase vesicles. The enrichment of these vesicles with cardiolipin, an acidic phospholipid that is found only in the inner mitochondrial membrane of eukaryotic cells, strongly inhibits calcium transport, in remarkable contrast with the activation effect that cardiolipin exerts upon other mitochondrial transporters and enzymes. The relation of the inactivation of calcium transport to the physical state of the bilayer was studied by following the polarization changes of 1,6-diphenyl-1,3,5-hexatriene (DPH) and by flow cytometry in the cardiolipin-enriched liposomes with incorporated mitochondrial solubilized proteins. Non-bilayer molecular arrangements in the cardiolipin-supplemented liposomes, detected by flow cytometry, may produce the fluidity changes observed by fluorescence polarization of DPH. Fluidity changes correlate with the abolition of calcium uptake, but have no effect on the establishment of a membrane potential in the vesicles required for calcium transport activity. Changes in the membrane structure and uniporter function are observed in the combined presence of cardiolipin and calcium leading to a modified lipid configuration.     

VIP21, a 21-kD membrane protein is an integral component of trans-Golgi- network-derived transport vesicles

In simple epithelial cells, apical and basolateral proteins are sorted into separate vesicular carriers before delivery to the appropriate plasma membrane domains. To dissect the putative sorting machinery, we have solubilized Golgi-derived transport vesicles with the detergent CHAPS and shown that an apical marker, influenza haemagglutinin (HA), formed a large complex together with several integral membrane proteins. Remarkably, a similar set of CHAPS-insoluble proteins was found after solubilization of a total cellular membrane fraction. This allowed the cloning of a cDNA encoding one protein of this complex, VIP21 (Vesicular Integral-membrane Protein of 21 kD). The transiently expressed protein appeared on the Golgi-apparatus, the plasma membrane and vesicular structures. We propose that VIP21 is a component of the molecular machinery of vesicular transport.     

Organelle biogenesis and intracellular lipid transport in eukaryotes.

The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface. The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane. The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly.     

Lipid cosorting mediated by shiga toxin induced tubulation

To maintain cell membrane homeostasis, lipids must be dynamically redistributed during the formation of transport intermediates, but the mechanisms driving lipid sorting are not yet fully understood. Lowering sphingolipid concentration can reduce the bending energy of a membrane, and this effect could account for sphingolipid depletion along the retrograde pathway. However, sphingolipids and cholesterol are enriched along the anterograde pathway, implying that other lipid sorting mechanisms, such as protein-mediated sorting, can dominate. To characterize the influence of protein binding on the lipid composition of highly curved membranes, we studied the interactions of the B-subunit of Shiga toxin (STxB) with giant unilamellar vesicles containing its glycosphingolipid receptor [globotriaosylceramide (Gb3)]. STxB binding induced the formation of tubular membrane invaginations, and fluorescence microscopy images of these highly curved membranes were consistent with co-enrichment of Gb3 and sphingolipids. In agreement with theory, sorting was stronger for membrane compositions close to demixing. These results strongly support the hypothesis that proteins can indirectly mediate the sorting of lipids into highly curved transport intermediates via interactions between lipids and the membrane receptor of the protein.     

Molecular packing parameters of bipolar lipids.

The bipolar lipid fractions extracted from the thermophilic archaeobacterium Sulfolobus solfataricus have different chemical structures and geometrical shapes. The conditions which lead to the formation of vesicles were investigated in order to study the self-assembly of these molecules. Such conditions are fulfilled when an appropriate mixture of two different molecular species (both bipolar or bipolar and monopolar) is used. According to the theory introduced by Israelachvili and co-workers, lipid self-assembly results from the balance of interaction free energy, entropy and molecular geometry. We have shown that this theory can be extended to bipolar lipids, in spite of their more complex nature, and the experimental results obtained combining 1H-NMR, light scattering and entrapped volume techniques closely match theoretical expectations. To carry out calculations, it was necessary to introduce hypotheses about the disposition of bipolar molecules in the vesicle membrane. These hypotheses have been tested indirectly by measuring the transport properties mediated by carriers or channels, whose transport mechanism can be considered to be a probe of the membrane structure.