Preparation of a coated vesicle-enriched fraction from plant cells

A fraction rich in coated vesicles has been prepared from suspension-cultured cells of tobacco (Nicotiana tabacum L.) by sucrose gradient centrifugation. Isolated, negatively-stained plant coated vesicles are approx. 100 nm in diameter, and show the characteristic basket-like structure of the clathrin coat previously reported for both plant [2–5] and animal [1, 6–9] coated vesicles. Analysis of the various plant subcellular fractions by SDS polyacrylamide gel electrophoresis demonstrates that a polypeptide of 190 000 D is enriched in parallel with the morphologically identifiable coated vesicles. It is postulated that this polypeptide is plant clathrin with a molecular weight about 10 000 D greater than that previously reported for animal clathrin [1, 6].     

The isolation and enrichment of coated vesicles from suspension-cultured carrot cells

Summary A method has been developed to isolate and purify coated vesicles from suspension cultured carrot (Daucus carota L.) cells. It incorporates features of centrifugation methods (sucrose step gradient; Ficoll/D₂O gradient) previously employed in the isolation of coated vesicles from mammalian brain tissue. Most important is the treatment of the crude coated vesicle fraction (postmicrosomal supernatant) with ribonuclease to remove ribosomes which are a serious source of contamination in such fractions. The fraction finally obtained is contaminated to the extent of 30% of total observed particles in negatively stained preparations with naked vesicles whose diameter are smaller than those of the coated vesicles. These vesicles are interpreted as being coated vesicles which have been stripped of their coats. SDS-PAGE of coated vesicle fractions purified by this method reveal significant differences in the polypeptide patterns obtained from plant and animal systems.     

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.     

Serial section analysis of coated pits and coated vesicles in soybean protoplasts

Divergent viewpoints have been expressed regarding the existence of free coated vesicles in animal cells; this question has not been carefully addressed with plant tissues. Soybean suspension culture protoplasts were exposed to cationized ferritin (CF) for short times to label coated pits and coated vesicles. A serial section analysis did not reveal deep coated pits with long necks as reported in animal cells. Serial sections clearly demonstrated CF-labelled coated vesicles to be separate organelles and is consistent with the idea that they transfer CF from coated pits to other cytoplasmic organelles during endocytosis.     

Plant coated vesicles

Abstract. Coated vesicles are organelles frequently encountered in many plant cell types often in association with the plasma membrane, Golgi apparatus, partially coated reticulum and multivesicular bodies. They are readily identified by a characteristic cage or basket composed of interlocking triskelions of the protein clathrin which are bound to the surface of the vesicle membrane. Although their transport function has been well studied and characterized in mammalian systems, the possible importance of coated vesicles as transport organelles in plant cells is only just beginning to be explored. In this review, the authors describe the structure of higher plant coated vesicles and discuss their possible involvement in the endocytosis of marcromolecules, in exocytosis and in the intracellular transport of material between cytoplasmic compartments. Their possible role in maintaining the macromolecular composition of the plasma membrane whilst allowing recycling of excess lipid bilayer and their potential application as vehicles for the introduction of foreign macromolecules into plant cells are discussed.     

Two polypeptides (30 and 38kDa) in plant coated vesicles with clathrin light chain properties

Summary Coated vesicles have been isolated from bovine brain and etiolated zucchini hypocotyls by centrifugal methods. By putting to use two properties of the light chain polypeptides of brain coated vesicles (calcium binding, heat stability) we have been able to demonstrate the presence of two similar polypeptides with apparent molecular masses of 30 and 38 kDa in plant coated vesicles.Abbreviations CV coated vesicle - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - MES 2-(N-Morpholino)-ethanesulfonic acid - Tris tris(hydroxymethyl) aminomethane     

Homology between antigenic determinants of bovine clathrin and clathrin from the brown alga Laminaria digitata

Coated vesicles, essential organelles of intracellular membrane traffic, have been extensively studied in animal and higher plant cells. In the algae, cytological studies only have been performed which demonstrate the presence of such coated vesicles with their surrounding clathrin lattice. The present work has been carried out on coated vesicles isolated for the first time from the brown algae Laminaria digitata. For comparison of the antigenic characteristics of clathrin prepared from the Bovine brain or adrenocortical cells and the clathrin prepared from algae, polyclonal antibodies have been raised to a purified Bovine brain clathrin in Goat and to Bovine adrenocortical clathrin in Rabbit. The positive immunological responses of the coated vesicles and the clathrin from Algae to these antibodies, evidence an homology between antigenic determinants of clathrin from animal and vegetal cells.     

Presence of a partially-coated reticulum in angiosperms

Summary The form and distribution of partially-coated membrane systems and coated vesicles in the leaf glands ofPhaseolus and the root cortical cells ofZea were investigated. Partially-coated membranes exist as a reticulum which is either sparsely branched or extensively anastomosed. Coated vesicles and coated membrane regions may occur at all points that are in the vicinity of such a reticulum. Golgi stack membranes, though associated with the partially-coated reticulum in many cases, show no consistent orientation to it. Occasionally, the reticulum and the associated coated vesicles are located away from any Golgi stack. We examined other plant tissues from such species asAllium cepa, Beta vulgaris andNicotiana tabacum and the partially-coated reticulum was observed in all material. We suggest that membrane flow may be occurring from the partially-coated reticulum to either the Golgi stack or to other intracellular compartments.     

Isolation and partial characterization of clathrin-coated vesicles from pea (Pisum sativum L.) cotyledons

Summary Clathrin-coated vesicles have been isolated from cotyledons of both developing and germinating pea seeds using differential centrifugation, ribonuclease treatment, discontinuous sucrose gradients, and isopycnic centrifugation on a linear D₂O-Ficoll gradient. The yield of coated vesicles from developing pea cotyledons was exceptional, being 1.6 × higher than the yield from hog and bovine brain, 5.3 × higher than the yield from carrot suspension cultures, and 13 × the yield from cotyledons of germinating pea seeds. The pea coated vesicles are similar to other plant coated vesicles in size (approximately 80 nm in diameter) and in having a clathrin heavy chain of 190,000 M_r. The lipid phosphorus to protein ratio, 190–250 nmol P per mg protein, of the coated vesicles from plants is comparable to that reported for highly purified coated vesicles from animals. The nondenatured pea clathrin reacted weakly with an antiserum to bovine brain clathrin, but pea clathrin denatured by sodium dodecyl sulfate did not.Abbreviations CLC Clathrin light chain - CHC clathrin heavy chain - CV coated vesicle - DTT dithiothreitol - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TBS Tris buffered saline     

Improved Coated Vesicle Isolation Allows Better Characterization of Clathrin Polypeptides

Postmicrosomal pellets from plant sources are grossly contaminatedwith nbosomes. Previous purifications of clathrin coated vesicles(CCV) from such subcellular fractions have therefore often involvedan RNase treatment. Performed at 30°C, this step inherentlycarries with it the dangers of proteolysis. We document herea method for CCV isolation which avoids this. Through the inclusionof suitable antiproteases in the homogenizing and subsequentisolation media, we have also been able to improve the qualityof CCV recovered from plant tissues. As a result we have tentativelybeen able to identify clathrin light chains from zucchini hypocotyland pea cotyledon CCV. Similar to light chains from bovine brainthese polypeptides are heat stable, can be solubilized fromneutralized TCA precipitates, bind calcium and clathrin heavychains. However, in contrast to brain CCV the two light chainsof plant CCV are some 10 kDa heavier. Key words: Antiproteases, Ca²⁺-binding, clathrin coated vesicles, clathrin heavy chains, clathrin light chain(s), heat stability, pea cotyledons, RNase, zucchini hypocotyls     

Coated vesicles participate in the receptor-mediated endocytosis of insulin

We have purified coated vesicles from rat liver by differential ultracentrifugation. Electron micrographs of these preparations reveal only the polyhedral structures typical of coated vesicles. SDS PAGE of the coated vesicle preparation followed by Coomassie Blue staining of proteins reveals a protein composition also typical of coated vesicles. We determined that these rat liver coated vesicles possess a latent insulin binding capability. That is, little if any specific binding of 125I-insulin to coated vesicles is observed in the absence of detergent. However, coated vesicles treated with the detergent octyl glucoside exhibit a substantial specific 125I-insulin binding capacity. We visualized the insulin binding structure of coated vesicles by cross-linking 125I-insulin to detergent-solubilized coated vesicles using the bifunctional reagent disuccinimidyl suberate followed by electrophoresis and autoradiography. The receptor structure thus identified is identical to that of the high-affinity insulin receptor present in a variety of tissues. We isolated liver coated vesicles from rats which had received injections of 125I-insulin in the hepatic portal vein. We found that insulin administered in this fashion was rapidly and specifically taken up by liver coated vesicles. Taken together, these data are compatible with a functional role for coated vesicles in the receptor-mediated endocytosis of insulin.     

Identification of Plasmolipin as a Major Constituent of White Matter Clathrin-Coated Vesicles

We have isolated and characterized coated vesicles from bovine white matter and compared them to those isolated from gray matter. The virtual absence of synaptic vesicle antigens in the white matter coated vesicles indicates they are distinct from those found in gray matter and from vesicles derived from synaptic membranes. The white matter coated vesicles also lack compact myelin components, e.g., the myelin proteolipid, galactocerebroside, and sulfatides, as well as the periaxolemmal myelin marker myelin-associated glycoprotein. On the other hand, these vesicles contain 2',3'-cyclic nucleotide phosphohydrolase. The vesicles also contain high levels of plasmolipin, a protein present in myelin and oligodendrocytes. Plasmolipin was found to be four to five times higher in white matter coated vesicles than in gray matter coated vesicles. Based on western blot quantitation, the concentration of plasmolipin in white matter coated vesicles is 3-4% of the vesicle bilayer protein. These studies indicate that a significant proportion of coated vesicles from white matter may be derived from unique membrane domains of the myelin complex or oligodendroglial membrane, which are enriched in plasmolipin.     

Endocytosis of 1,3-β-glucans by broad bean cells at the penetration site of the cowpea rust fungus (haploid stage)

Te penetration hypha of basidiospore-derived infection structures of the cowpea rust fungus (Uromyces vignae Barclay) in epidermal cells of the nonhost, broad bean (Vicia faba L.), was studied with the electron microscope after high-pressure freezing and freeze substitution. After fungal invasion of the epidermis, a plug in the penetration hypha separated the infection structures on the cuticle from the intraepidermal vesicle of the fungus. The plug and the fungal cell wall reacted with a polyclonal 1,3-β-glucan antibody. The plug in the haploid stage seems to have a task similar to the septum formed in the diploid stage of the fungus. Around the penetration hypha, the plant wall stained darkly and a papilla was deposited by the plant. In the papilla, 1,3-β-glucans were labelled by a monoclonal and a polyclonal antibody. In the infected epidermal cell, clathrin-coated pits, coated vesicles, partially coated reticula and multivesicular bodies were found. The contents of the coated pits, coated vesicles, partially coated reticula and multivesicular bodies bound to monoclonal and polyclonal 1,3-β-glucan antibodies. Accumulation and uptake of this paramural material into the plant cell by endocytosis is concentrated at the fungal penetration site. It may influence the host-parasite interaction.     

Isolation and characterization of coated vesicles from filamentous fungi

Coated vesicles have been shown to exist in Neurospora crassa (Ascomycetes) and Uromyces phaseoli (Basidiomycetes) growing germlings. Separation of coated vesicles in both fungi was obtained when the high-speed (100,000g) pellet was fractioned on a Sephacryl S-1000 gel filtration column, according to the procedure of Mueller and Branton. Electron micrographs of negatively stained coated vesicles from fractions of gel filtration show the same striking lattice coated vesicles similar to vertebrate coated vesicles. We observe two major size classes of coated vesicles in both fungi: the larger class (100-180 nm) is similar in size to vertebrate coated vesicles; the smaller class (50-80 nm) is mostly found in both fungi. When examined by SDS-PAGE, the Sephacryl column fractions containing the maximum concentration of electron microscopically visible coated vesicles coincide with the bands of the protein coat reported as clathrin. The protein composition on SDS-PAGE of the coated vesicles indicates a major polypeptide species of 180 kDa and minor 30 to 36 kDa species. Polypeptides of 100 kDa and 64 kDa are also found in the fractions containing coated vesicles.     

Identification of minor components of coated vesicles by use of permeation chromatography

Coated vesicles are thought to be vehicles for the intracellular transport of membranes. Clathrin is the major protein component of coated vesicles. Minor components of these organelles can be identified in highly purified preparations if they can be shown to copurify with clathrin. To show copurification we have made use of the relatively uniform diameter of coated vesicles (50-150 nm) to fractionate conventionally purified coated vesicles according to size in glass bead columns of 200-nm pore size. We have found that bovine brain coated vesicles prepared by the standard procedure of Pearse can be contaminated with large membrane fragments that are removed by permeation chromatography on such glass bead columns. Gel electrophoretic analysis of column fractions shows that only three major polypeptide chains, and a family of polypeptides with molecular weights close to 100,000 are always in constant ratio to clathrin, and are unique to fractions containing coated vesicles. Two other major polypeptides that appear to be components of coated vesicles are also present in other membrane fractions. We have also used permeation chromatography to monitor artifactual membrane trapping during vesicle isolation. Pure radiolabeled synaptic vesicle membranes were added to bovine brain tissue before homogenization. Considerable amounts of the added radioactivity could be recovered in the fractions conventionally pooled in the preparation of coated vesicles. After permeation chromatography, the radioactivity in the coated vesicle peak was reduced essentially to background.     

Cotton fibre differentiation: Occurrence and distribution of coated and smooth vesicles during primary and secondary wall formation

Summary Coated vesicles occur in differentiating cotton fibres during primary and secondary wall formation. The coated vesicles are often associated with the plasmalemma, or with membranes at the secreting face of dictyosomes, corresponding positionally to GERL. During secondary wall formation the number of dictyosome-associated coated vesicles seems to be smaller than during primary wall formation. When sections are stained for periodateoxidizable polysaccharides (Thiéry reaction) the membrane of plasmalemma-associated coated vesicles is intensely stained. The membrane of dictyosome-associated coated vesicles is only weakly stained. On the basis of the present evidence it is not possible to clearly decide, whether the staining in plasmalemma-associated coated vesicles is due to obliquely cut membrane or to vesicle contents. The vesicle coat material is not stained. Possible functions of coated vesicles in differentiating cotton fibres are discussed.Vesicles with contents positively stained with the Thiéry reaction are observed only during primary wall formation. The membrane of these vesicles is smooth and seems to bud from the same cisternae, probably GERL, as do the coated vesicles. During secondary wall formation no vesicles containing periodate-oxidizable polysaccharides could be detected, even under conditions that result in a strong, specific reaction in the cellulosic secondary wall. In some instances polysaccharidic material, resembling secondary wall material, has been seen to adhere to the outside of the plasmalemma. These results are consistent with the hypothesis that, in higher plants, at least part of primary wall material may already be synthesized in dictyosome vesicles, whereas cellulose biosynthesis occurs at the cell surface.     

Purification of coated vesicles by agarose gel electrophoresis

We have applied agarose gel electrophoresis as a novel step in the purification of clathrin-coated vesicles. Preparations of coated vesicles obtained by sedimentation velocity and isopycnic centrifugation are resolved into two distinct fractions upon electrophoresis. The slower migrating fraction contains smooth vesicles, whereas the faster contains only coated vesicles and empty clathrin coats. The faster mobility of the coated vesicles is primarily caused by the acidic nature of clathrin. Coated vesicles from three different cell types have different mobilities. In each case, however, all of the major polypeptides previously attributed to coated vesicles comigrate with the now homogeneous particles, even though a powerful ATPase activity is completely removed.     

Freeze-substitution improves the ultrastructural preservation of legume root hairs

The freeze-substitution method was applied toVicia hirsuta root hairs to test its effectiveness in improving preservation of the cell ultrastruture. Freeze-substitution almost certainly represents more faithfully the structure of the hair cell. A previously unreported ‘pyriformis’ vesicle is described. Also unique to freeze-substituted material are coated secretory vesicles; a smooth plasma membrane profile; mitochondrial ribosomes; long microfilament bundles which are associated with vesicles, mitochondria, coated pits and coated vesicles; microtubule-associated filaments; well-preserved coated vesicles and coated pits with enclosing rings; a pliciform nucleus. The results are discussed in context of previous reports using conventional fixation techniques.     

荞麦水合花粉和花粉管中的被膜小泡

荞麦水合花粉粒和生长中的花粉管中内质网潴泡形成的囊袋状结构较少见,但内质网囊袋中含有丰富的被膜小泡,直径约为100－150nm。刚刚形成的花粉管中,被膜小泡主要来自于花粉粒营养细胞的细胞质。生长中的花粉管的被膜小泡可由高尔基体分泌形成。另外还观察到内质网的碎裂也是荞麦花粉管中产生被膜小泡的一种机制。花粉管的被膜小泡中含有花粉管壁的前体物质,与花粉管的壁融合参与花粉管的生长。被膜小泡可能含有与脂体和造粉质体水解有关的酶,参与此类物质的降解。荞麦花柱和柱头细胞内含物的解体物质参与花粉管的生长。     

Preparation of a homogeneous coated vesicle fraction from bean leaves

Summary Using a procedure previously developed for suspension-cultured carrot cells, we have been able to isolate two different coated vesicle-containing fractions from green bean leaves (Vicia faba). The two fractions differ in their isopycnic densities in D₂O-Ficoll as well as in their diameters. One of the fractions (the less dense of the two) is almost 100% pure as judged by negative staining. Because of this the polypeptide pattern obtained from SDS-PAGE is most clear and has enabled a clear recognition of clathrin light chains, in addition to the 190 kDa heavy chain coat component. Significantly the 100k Da and 50k Da polypeptides typical of brain coated vesicles are absent from bean leaf coated vesicles. Due to a) the high degree of vacuolation b) the presence of large amounts of ribulose bisphosphate carboxylase in the postmicrosomal supernatant, the yield of coated vesicles from bean leaves, as compared to nongreen plant cells, or to bovine brain tissue is extremely low (1 mg coated vesicles from 2.4 kg leaf tissue).Abbreviations D₂O deuterium oxide - EGTA ethylene glycol-bis ( amino ethyl ether) N,N,N,N tetraacetic acid - MES, 2 (N-morpholino)-ethanesulfonic acid - PMSF phenyl methylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TRIS Tris-hydroxy methyl amino methane