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.     

Macrophage interaction of HDL subclasses separated by free flow isotachophoresis

Preparative isotachophoresis (ITP) was used for the fractionation of fasting and postprandial high density lipoproteins (HDL) according to their net charge in the absence of molecular sieve effects. Three major HDL subpopulations with fast, intermediate, and slow mobility have been recognized. Particle size analysis by gradient gel electrophoresis has shown that in the fast-migrating subpopulation particles dominate with a size of HDL3a and HDL2b. The subpopulation with intermediate mobility contains particles with a size between HDL2a and HDL3b, while in the slow migrating subpopulation particles dominate with a size of HDL2b, HDL3a, and HDL3c. The fast-migrating subpopulation is rich in apoA-I and phosphatidylcholine. The particles of this fraction bind at 4 degrees C to HDL receptors on macrophages with high affinity (KD = 7.71 micrograms/ml; Bmax = 245.6 ng). The subpopulations with intermediate mobility is rich in apoA-II, apoE, C apolipoproteins, cholesteryl esters, and sphingomyelin. Its affinity to HDL receptors (KD = 17.7 micrograms/ml; Bmax = 198.4 ng) is lower than that of the HDL particles in the fast-migrating subfraction. The slow-migrating subpopulation consists of particles rich in apoA-IV and is associated with a high LCAT activity. This fraction expresses the highest nonspecific binding to mouse peritoneal macrophages compared to the other HDL fractions and contains only a small amount of particles that interact with HDL receptors by high affinity binding (KD = 7.3 micrograms/ml; Bmax = 95.9 ng). In 37 degrees C binding experiments the fast-migrating subfraction reveals the highest total cell-associated activity. 72% of which is trypsin-resistant. The other subfractions express a lower total cell-associated activity and 45% of the activity of the intermediate- and 43% of the activity of the slow-migrating fraction is trypsin-sensitive. When the HDL fractions are isolated from postprandial sera of the same donor, the fast-migrating particles bind at 4 degrees C with a higher affinity (KD = 4.6 micrograms/ml) while no significant changes are observed in the intermediate- and slow-migrating subpopulations. The slow- and the fast-migrating HDL subpopulations isolated from fasting serum have a high capacity to promote cholesterol removal from macrophages. We hypothesize that the HDL subpopulations rich in apoA-I promote cholesterol removal predominantly via the interaction with HDL receptors, while apoA-IV-rich HDL particles receive their driving force for cholesterol efflux from the concomitant action of LCAT via a predominantly nonspecific interaction of the particles with the cell surface.(ABSTRACT TRUNCATED AT 400 WORDS)     

Clathrin-Coated Vesicle Subtypes in Mammalian Brain Tissue: Detection of Polypeptide Heterogeneity by Immunoprecipitation with Monoclonal Antibodies

A panel of monoclonal antibodies (mAbs) was developed to identify polypeptides sorted in subtypes of brain coated vesicles (CVs) and to separate these by immunoprecipitation. The corresponding antigen of some of the mAbs elicited by CV components was present also in synaptosomal plasma membrane, synaptic vesicles, or microsomes. On immunoblots the mAbs reacted with constitutive brain CV proteins, with cargo molecules, and with a novel CV component that interacts with the actin cytoskeleton. Analysis of radioiodinated brain CVs immunoprecipitated with a tubulin antibody revealed that all brain CVs contained tubulin. The mAb A-7C11 recognized a 40-kilodalton (kDa) polypeptide on the clathrin coat and immunoprecipitated one-quarter of the total brain CVs. The mAb S-11D9 reacted with a 44-kDa antigen and immunoprecipitated 25% of the CVs. This antigen (44 kDa) was present in synaptic vesicles and synaptosomal membrane as well. Moreover, this mAb (S-11D9) reacted with a polypeptide of 56 kDa detected only in synaptosomal membrane. A mAb (C-10B2) that reacted with one of the clathrin light chains (LCb) immunoprecipitated 90% of the brain CVs. One of the mAbs immunoprecipitated a CV subtype that displayed a reversed ratio of the clathrin LCs (LCa greater than LCb). Each of the mAbs yielded different immunofluorescent staining patterns of vesicles in culture cell types that included nerve growth factor-differentiated PC12 cells, neuroblastoma cells, and Madin Darby bovine kidney cells. The data suggest that in brain tissue there is a heterogeneous population of CVs with different polypeptide compositions and subcellular distributions and that each of these subtypes performs a different role in nerve cells.     

Cellular location of rat muscle ferritins and their preferential loss during cell isolation

Heart and other muscles of the rat contain two forms of ferritin separable in polyacrylamide gel electrophoresis. The cellular location of the fast- and slow-migrating ferritins was investigated using primary cultures of hindlimb skeletal muscle, and isolated myocardial cell populations. Muscle and non-muscle cells were isolated in good yield from hearts of adult rats pretreated with large doses of iron to increase their ferritin content. In virtually all cases, the isolated muscle cells contained traces only of the fast-migrating species and the non-muscle cells contained small amounts of the slow-migrating ferritin. During cell isolation, 90-100% of both ferritins was lost and could be recovered in the perfusates and solutions employed, while one third of the total tissue protein, and a larger percentage of creatine phosphokinase, was recovered in the isolated cells. Primary cultures of thigh muscle from adult rats which had differentiated into multi-nucleated myotubes, were incubated for 1-3 days with chelated iron. These cells contained substantial amounts of the electrophoretically fast migrating ferritin, with its characteristic larger Stokes' radius (determined by quantitative polyacrylamide gel electrophoresis). None of the slow-migrating ferritin species was detected, although hindlimb muscle from iron-treated rats contained both forms. It is concluded that the fast-migrating ferritin of muscle, which is much larger and more asymmetric than other ferritins, is confined to the muscle cell population, while the other form is predominantly or exclusively in the non-muscle cells. Both ferritins are lost preferentially over other proteins during procedures which injure muscle tissue.     

Solubilization of proteins from bovine brain coated vesicles by protein perturbants and Triton X-100

To identify integral and peripheral membrane proteins, highly purified coated vesicles from bovine brain were exposed to solutions of various pH, ionic strength, and concentrations of the nonionic detergent Triton X-100. At pH 10.0 or above most major proteins were liberated, but four minor polypeptides sedimented with the vesicles. From quantitative analysis of phospholipids in the pellet and extract, we determined that at a pH of up to 12 all phospholipids could be recovered in the pellet. Electron microscopic examination of coated vesicles at pH 12.0 showed all vesicles devoid of coat structures. Treatment with high ionic strength solutions (0-1.0 M KCl) at pH 6.5-8.5 also liberated all major proteins, except tubulin, which remained sedimentable. The addition of Triton X-100 to coated vesicles or to stripped vesicles from which 90% of the clathrin had been removed resulted in the release of four distinct polypeptides of approximate Mr 38,000, 29,000, 24,000 and 10,000. The 38,000-D polypeptide (pK approximately 5.0), which represents approximately 50% of the protein liberated by Triton X-100, appears to be a glycoprotein on the basis of its reaction with periodic acid-Schiff reagent. Extraction of 90% of the clathrin followed by extraction of 90% of the phospholipids with Triton X-100 produced a protein residue that remained sedimentable and consisted of structures that appeared to be shrunken stripped vesicles. Together our data indicate that most of the major polypeptides of brain coated vesicles behave as peripheral membrane proteins and at least four polypeptides behave as integral membrane proteins. By use of a monoclonal antibody, we have identified one of these polypeptides (38,000 mol wt) as a marker for a subpopulation of calf brain coated vesicles.     

Purification and properties of a new clathrin assembly protein.

A clathrin assembly protein (AP180) has been purified and characterized from coated vesicles of bovine brain. This protein has hitherto escaped detection because in SDS-gel electrophoresis it is obscured by the 180 kd heavy chain of clathrin. Despite the similarity in electrophoretic mobility, AP180 differs from clathrin in both its subunit and native mol. wt, as well as hydrodynamic properties, surface charge and tryptic peptide composition. It also appears immunologically distinct from clathrin, since neither a polyclonal antiserum nor a monoclonal antibody, that have been shown to be specific for AP180, cross-react with the heavy chain of clathrin. AP180 binds to clathrin triskelia and thereby promotes clathrin assembly into regular polyhedral structures of narrow size-distribution (60-90 nm), reminiscent of the surface coat of coated vesicles. In this respect AP180 bears a functional resemblance to the 100-110 kd clathrin assembly polypeptides that have been previously described.     

The subpopulation of brain coated vesicles that carries synaptic vesicle proteins contains two unique polypeptides

Coated vesicles have been purified in the past on the basis of their remarkably homogeneous structure, not their function. We have succeeded in isolating two subpopulations of bovine brain coated vesicles that carry specific "cargoes," in this case two synaptic vesicle membrane polypeptides (Mr = 95,000 and 65,000). Monoclonal antibodies that recognize cytoplasmic domains of these polypeptides can penetrate the clathrin coat and recognize them on the outer surface of the coated vesicle membrane. An immunoadsorption technique could therefore be used to fractionate coated vesicles on the basis of their membrane composition. The subpopulations have the normal complement of conventional coated vesicle proteins. Exclusive, however, to the subpopulations that carry synaptic vesicle polypeptides are two new coated vesicle polypeptides (Mr = 38,000 and 29,000).     

Coated vesicles contain a phosphatidylinositol kinase

When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. We have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of our CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism.     

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking.     

Fragmentation and separation analysis of the photosynthetic membrane from spinach

Membrane vesicles, originating from grana, grana core (appressed grana regions), grana margins and stroma lamellae/end membranes, were analysed by counter current distribution (CCD) using aqueous dextran-polyethylene glycol two-phase systems. Each vesicle population gave rise to distinct peaks in the CCD diagram representing different vesicle subpopulations. The grana vesicles and grana core vesicles each separated into 3 different subpopulations having different chlorophyll a/b ratios and PSI/PSII ratios. Two of the grana core subpopulations had a chlorophyll a/b ratio of 2.0 and PSI/PSII ratio of 0.10 and are among the most PSII enriched thylakoid vesicle preparation obtained so far by a non detergent method. The margin vesicles separated into 3 different populations, with about the same chlorophyll a/b ratios, but different fluorescence emission spectra. The stroma lamellae/end membrane vesicles separated into 4 subpopulations. Plastoglobules, connected to membrane vesicles, were highly enriched in 2 of these subpopulations and it is proposed that these 2 subpopulations originate from stroma lamellae while the 2 others originate from end membranes. Fragmentation and separation analysis shows that the margins of grana constitute a distinct domain of the thylakoid and also allows the estimation of the chlorophyll antenna sizes of PSI and PSII in different thylakoid domains.     

An antibody against 100- to 116-kDa polypeptides in coated vesicles inhibits triskelion binding

There is considerable evidence that the 100- to 116-kDa polypeptides in calf brain coated vesicles are involved in the assembly of clathrin triskelions to form coated vesicles. We have raised polyclonal antibodies against these polypeptides. By Western blot analysis, these antibodies bind to a distinct subset of the six polypeptides in the region 100-116 kDa. Whole cell homogenates from calf brain, calf liver, and rat liver also show immunoreactivity in the 100-kDa region with no other cross reactivity. Isolated coated vesicles from calf liver, rat brain, and soybean roots also cross-react. Stripped coated vesicles, which are depleted of clathrin but which retain the 100- to 116-kDa polypeptides, quantitatively rebind 125I-triskelions. This binding is inhibited in a dose-dependent manner by 100- to 116-kDa antibody but not by nonimmune serum or by anti-clathrin polyclonal antibody. These studies indicate that (1) specific sites on the 100- to 116-kDa polypeptides are required for assembly of coated vesicles, and (2) this antibody will be useful in clarifying more precisely the role of the 100- to 116-kDa polypeptides in coated vesicle recycling.     

Clathrin-coated vesicle assembly polypeptides: physical properties and reconstitution studies with brain membranes

The assembly polypeptides are an integral component of coated vesicles and may mediate the linkage of clathrin to the vesicle membrane. We have purified assembly polypeptides in milligram quantities from bovine brain by an improved procedure. Hydrodynamic and chemical crosslinking studies indicate that the protein is an asymmetric heterotetramer with a molecular weight of 252,000, containing two subunits of Mr 98,000-115,000, one subunit of 52,000, and one subunit of 16,000. Two-dimensional peptide maps of the subunits show that the 16- and 52-kD polypeptides are not derived from the higher molecular weight species, and that the group of bands at 98-115 kD are related. Electron microscopic visualization shows an essentially globular protein with one or two knob-like tails. We demonstrate a specific membrane protein binding site for 125I-labeled assembly polypeptides in 0.1 N sodium hydroxide-extracted bovine brain membranes based on the following criteria: (a) binding is displaceable by unlabeled ligand, (b) the binding site is destroyed by protease treatment of the membranes, and (c) the distribution of binding between vesicle-depleted membranes and coated vesicle membranes parallels the in vivo localization of assembly polypeptides and clathrin. This binding site is likely to be an integral membrane protein because (a) it is enriched in the sodium hydroxide-extracted membranes stripped of most of their peripheral membrane proteins, and (b) the binding site is partially extracted by 0.5% Triton X-100. A similar binding site appears to be present in coated vesicles. Clathrin binds to the hydroxide-stripped membranes in an assembly polypeptides dependent manner, and this binding is diminished by Triton extraction of the membranes. This assay may aid in identification of the membrane receptor for the assembly polypeptides.     

Identification of three coated vesicle components as alpha- and beta- tubulin linked to a phosphorylated 50,000-dalton polypeptide

Coated vesicles are involved in the intracellular transport of membrane proteins between a variety of membrane compartments. The coats of bovine brain coated vesicles contain at least six polypeptides in addition to an 180,000-dalton polypeptide called clathrin. In this report we show that the 54,000- and 56,000-dalton coated vesicle polypeptides are alpha- and beta-tubulin, determined by immunoblotting and two-dimensional gel electrophoresis. An affinity-purified tubulin antiserum can precipitate coated vesicles. The tubulin polypeptides are tightly associated with a 50,000-dalton coated vesicle polypeptide, which is phosphorylated. The phosphorylated 50,000-dalton polypeptide appears to be related to brain microtubule-associated tau proteins since it can be specifically immunoprecipitated by an affinity-purified antiserum directed against these proteins. In addition, gel filtration experiments indicate that at least a fraction of the 50,000-dalton polypeptide may associate with the 100,000-dalton coated vesicle polypeptide. Since brain is a tissue rich in tubulins, liver coated vesicles were analyzed for the presence of alpha- and beta-tubulin. Like brain coated vesicles, liver coated vesicles also contain an endogenous kinase activity, which phosphorylates polypeptides of the same molecular weights and isoelectric points as the brain coated vesicle 50,000-dalton, tau-like polypeptide, and alpha- and beta-tubulin. The phosphorylated 50,000-dalton polypeptide may link the membrane and contents of coated vesicles with components of the cytoskeleton.     

Physicochemical characterization of high-molecular-weight alpha-crystallin subpopulations from the calf lens nucleus

Calf lens nuclear alpha-crystallin was separated into five molecular weight subpopulations by exclusion chromatography on Bio-Gel A-5m. These subpopulations were compared by amino acid analysis, ultraviolet absorption analysis, fluorescence, far- and near-ultraviolet circular dichroism, isoelectric focusing, SDS-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Although only minor differences were detectable in most physicochemical properties, progressive changes were found in the near-ultraviolet circular dichroism spectra and in pellet hardness after centrifugation. Minute amounts of beta-crystallin polypeptides and a 43 kDa component were present in all five subpopulations. In addition, the highest molecular weight aggregates contain some gamma-crystallin polypeptides. A slow re-equilibration of separated subpopulations towards the initial distribution was observed by rechromatography.     

Biochemical and functional characterization of three types of coated vesicles in bovine adrenocortical cells: implication in the intracellular traffic

Three populations of pure coated vesicles from adrenocortical cells, differing in their density, i.e., 1.125-1.155, 1.155-1.175, and 1.175-1.210 g/cm3, are obtained after separation on two successive sucrose-2H2O gradients. They are involved in LDL internalization and in the receptor cycle as confirmed by the presence, in each population, of the LDL receptor. Electron micrographs confirm the existence of three homogeneous populations exhibiting the typical polygonal structure of the clathrin coat. They differ in their size distribution (small, congruent to 70-nm diameter; medium, congruent to 90-nm diameter; large, congruent to 110-nm diameter) and in the organization of clathrin and of the coat proteins as evidenced on electrophoreses carried out under nondenaturing and denaturing conditions. Activity measurements of marker enzymes, phosphodiesterase and galactosyltransferase, suggest that medium coated vesicles might originate from plasma membranes and small ones from the Golgi complex. Large coated vesicles exhibit phosphokinase enzyme and substrate polypeptides different from those of the two other populations, tubulins being the preferred kinase substrates for the small and medium coated vesicles. These kinases are autophosphorylating enzymes and are revealed, by nondenaturing electrophoreses, as different high molecular mass complexes in the three populations. Clathrin and coat proteins are not part of these complexes.     

An approach to understand the complexation of supramolecular dye Congo red with immunoglobulin L chain lambda

Congo red, a dye of high self-assembling tendency, has been found to form complexes with proteins by adhesion of the ribbon-like supramolecular ligand to polypeptide chains of beta-conformation. Complexation is allowed by local or global protein instability, facilitating penetration of the dye to the locus of its binding. At elevated temperatures, L chain lambda of myeloma origin was found to form two distinct complexes with Congo red, easily differentiated in electrophoresis as slow- and fast-migrating fractions, bearing four- and eight-dye-molecule ligands, respectively, in the V domain of each individual chain. The slow-migrating complex is formed after displacement of the N-terminal polypeptide chain fragment (about 20 residues) from its packing locus, thereby exposing the entrance to the binding cavity. In this work the formation and stability of this complex was studied by molecular dynamics (MD) simulations. The effect of three- and five-molecule ligands introduced to the site binding the dye was also analyzed in an attempt to understand the formation of fast-migrating complexes. The wedging of the ligand containing five dye molecules, hence longer than established experimentally as the maximum for the slow-migrating complex, was found to generate significant structural changes. These changes were assumed to represent the crossing of the threshold on the way to forming a fast-migrating complex more capacious for dyes. They led to almost general destabilization of the V domain, making it susceptible to extra dye complexation. Theoretical studies were designed in close reference to experimental findings concerning the number of dye molecules in the ligand inserted to the site binding the dye, the location of the site in the domain, and the conditions of formation of the complexes. The results of the two kinds of studies appeared coherent.     

The association of clathrin fragments with coated vesicle membranes

The association between clathrin triskelions and the clathrin-stripped membranes of coated vesicles has been investigated using a filter assay to separate bound from unbound clathrin. The filter assay is more sensitive and less cumbersome than a sedimentation assay used previously (1). While confirming the high affinity interaction between clathrin and the vesicle membrane, our results yield Scatchard plots that are curvilinear and consistent with a positively cooperative interaction between clathrin and the vesicle membranes. Controlled digestion with trypsin removes the distal portions of the triskelion legs leaving the proximal 31 nm portions that form the hub of the triskelions. These hubs are trimers of large 112,000- and 124,000-dalton fragments of clathrin heavy chains. They competitively inhibit the binding of 125I-labeled intact triskelions to stripped vesicles with a KI identical to the KD for the association of 125I-labeled intact triskelions to stripped vesicles. Furthermore, these large fragment trimers bind to stripped vesicles with approximately the same high affinity as do intact triskelions and also show evidence of a positively cooperative interaction. It is concluded that clathrin binds to coated vesicles by an interaction that is mediated by the proximal 112,000-dalton fragment of the clathrin heavy chains.     

In vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes

We have studied the in vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes. The major 32P-labeled polypeptides of clathrin-coated vesicles isolated from metabolically labeled cells were the the 165-, 100-110-, and 50-kDa polypeptides of the assembly protein, the clathrin beta-light chain, and to a lesser extent the clathrin alpha-light chain. The phosphorylation of the assembled (particulate) and unassembled (soluble) pools of clathrin and assembly protein was compared by immunoprecipitating the respective protein complexes from particulate and soluble cell fractions. Although all the phosphorylated polypeptides were present in both fractions, the extent of labeling was protein and fraction specific: the apparent specific activities of the assembly protein 50-kDa polypeptide and clathrin light chain were higher in the unassembled pool, whereas those of the 100-110-kDa polypeptides were higher in the assembled pool. The amino acids and polypeptide fragments labeled in vivo appeared similar to those labeled in vitro.     

Heterogeneous distribution of synaptophysin and protein 65 in synaptic vesicles isolated from rat cerebral cortex

Rat brain cerebral cortex derived synaptic vesicles sedimenting on a 0.4 M sucrose solution were further fractionated according to size by column chromatography on Sephacryl-1000 and analyzed for their binding activities of antibodies directed against the vesicle-associated proteins synaptophysin, synapsin I, protein 65 and clathrin. Whereas synapsin I and particularly protein 65 and clathrin are associated with a large range of vesicle sizes, synaptophysin elutes with small vesicles only. Using monoclonal antibodies against either synaptophysin or protein 65 and polyacrylamide beads for solid matrix immunoprecipitation, significant differences could be revealed in the protein composition of the resulting vesicle populations. Whereas synapsin I is associated with both synaptophysin and protein 65 immunoprecipitated vesicle populations, synaptophysin appears to be only a minor constituent of vesicles precipitated with anti-protein 65. Vesicles precipitated with anti-synaptophysin antibodies are enriched in acetylcholine. Our results suggest that the vesicle membrane protein synaptophysin and protein 65 may not have a ubiquitous distribution among synaptic vesicles. Protein 65 containing large vesicle populations contain little synaptophysin and synaptophysin is mainly associated with synaptic vesicles of small diameter.     

Electrophoretic analysis of protoplast, vacuole, and tonoplast vesicle proteins in crassulacean Acid metabolism plants

Protoplasts and vacuoles were isolated and purified in large numbers from the CAM plants Ananas comosus (pineapple) and Sedum telephium for protein characterization. Vacuoles were further fractionated to yield a tonoplast vesicle preparation. Polypeptides of protoplasts, vacuoles, and tonoplast vesicles were compared to whole leaf polypeptides from both plants by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis. Approximately 100 vacuole polypeptides could be resolved of which 25 to 30% were enriched in the tonoplast vesicles. The proteins of protoplasts, vacuoles, and tonoplast vesicles from A. comosus were analyzed further by two-dimensional gel electrophoresis. When one-dimensional electrophoretograms of A. comosus polypeptides were stained with a glycoprotein-specific periodic acid Schiff stain, very few polypeptides appeared to be glycosylated, whereas a large number of glycosylated polypeptides were detected with a silver-based glycoprotein stain particularly in tonoplast vesicles. Analysis of the enzymic content of vacuoles from both plants indicated the presence of a variety of hydrolases, including bromelain as a major constituent of A. comosus. No substrate-specific ATPase, however, could be detected in vacuoles or tonoplast vesicles from either plant.