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The amyloid-related cell (ARC) of the neuritic plaques of Alzheimer's disease revealed numerous cytoplasmic projections surrounding extracellular amyloid material. It is proposed that ARC-coated vesicles fuse with the cell membrane, forming coated pits, which may empty their secretory material into the extracellular space where polymerization of amyloid filaments could occur.
Coated vesicles may be an important component of the micropinocytic system of the human placenta. Regions of very dense reaction with glycocalyx stains are restricted to membranes within forming and fully formed coated vesicles. This is interpreted as evidence against permanently grouped specific binding sites having a role in the selective uptake of materials by micropinocytosis, and as support for theories of coated-vesicle formation which take into account the dynamic nature of membrane components. The pyroantimonate precipitation technique which was employed in an attempt to localize cations in placental tissue at term resulted in the deposition of electron-dense material in coated vesicles and basement membrane. Examination of the distribution of coated vesicles in placental tissue explants at 8--12 weeks of gestation revealed a restricted distribution of these organelles. Probably more than 89% of coated vesicles lie within the largest vesicles' diameter from the cell surface. Placental coated vesicles were isolated and examined using negative staining. A polygonally patterened structure was apparent on their surfaces. Analysis of the isolated fraction of coated vesicles using sodium dodecyl sulphate polyacrylamide gel electrophoresis shows the presence of a major protein of molecular weight 180000. This is the same molecular weight that has been given for clathrin, the major protein of the raised polygonally patterned structure on the cytoplasmic surface of coated vesicles from other sources.
Coated vesicles, which are membrane vesicles enclosed by a polyhedral protein lattice, are involved in many cellular events, including intracellular membrane transport and protein secretion, in which they must be able to undergo repeated membrane fusion and fission. The icosahedral lattice of protein surrounding the core of coated vesicles is composed predominantly of clathrin, a 180,000 (180 K) molecular weight protein, and other 30K and 36K polypeptides. In native conditions, the basic subunit of the coat consists of a trimer of clathrin with probably three polypeptides of 30K and/or 36K (refs 9-11). Additional minor proteins of 100K and 55K have been reported in purified coated vesicles. We describe here the presence of cyclic nucleotide- and Ca2+-independent protein kinase activity in coated vesicles. This protein kinase phosphorylates specifically a unique 50K protein which can be co-purified with clathrin and seems to be an integral protein of coated vesicles.
Coated vesicles are involved in the intracellular transport of membrane proteins between a variety of membrane compartments in which they must be able to undergo repeated membrane fusion and fission. We previously described the presence of cyclic nucleotide- and Ca2+-independent protein kinase activity in bovine brain coated vesicles which specifically phosphorylated a unique Mr = 50,000 coated vesicle integral protein (pp50) on a threonine residue. We describe now the presence in bovine brain coated vesicles of the antagonistic enzymatic activity which dephosphorylates pp50. This phosphoprotein phosphatase occurs under two interconvertible active and inactive forms. The activation process needs the simultaneous presence of Mg2+ and ATP or ADP. Unchelated ATP, but not unchelated ADP, inactivates the pp50 phosphatase. The latter is associated with the vesicular core. MgADP activation of the pp50 phosphatase implicates a different mechanism which does not need a phosphorylated intermediate. Thus, the pp50 phosphatase might belong to a new phosphatase type distinct from the four other classes of well known protein phosphatases.
Coat protein (COP)-coated vesicles have been shown to mediate protein transport through early steps of the secretory pathway in yeast and mammalian cells. Here, we attempt to elucidate their role in vesicular trafficking of plant cells, using a combined biochemical and ultrastructural approach. Immunogold labeling of cryosections revealed that COPI proteins are localized to microvesicles surrounding or budding from the Golgi apparatus. COPI-coated buds primarily reside on the cis-face of the Golgi stack. In addition, COPI and Arf1p show predominant labeling of the cis-Golgi stack, gradually diminishing toward the trans-Golgi stack. In vitro COPI-coated vesicle induction experiments demonstrated that Arf1p as well as coatomer could be recruited from cauliflower cytosol onto mixed endoplasmic reticulum (ER)/Golgi membranes. Binding of Arf1p and coatomer is inhibited by brefeldin A, underlining the specificity of the recruitment mechanism. In vitro vesicle budding was confirmed by identification of COPI-coated vesicles through immunogold negative staining in a fraction purified from isopycnic sucrose gradient centrifugation. Similar in vitro induction experiments with tobacco ER/Golgi membranes prepared from transgenic plants overproducing barley alpha-amylase-HDEL yielded a COPI-coated vesicle fraction that contained alpha-amylase as well as calreticulin.
Coated vesicles prepared from bovine brain contained cyclic nucleotides- and Ca2+-calmodulin-independent protein kinases which in the presence of Mg2+ catalyzed the phosphorylation of an endogenous 48,000 Mr protein of coated vesicles (C-48), phosvitin and troponin T. Phosvitin was phosphorylated either in the presence of ATP or GTP. The phosphorylation of C-48, on the other hand, was specific for ATP. Heparin inhibited the phosphorylation of phosvitin but not that of C-48. Mn2+ inhibited the phosphorylation of phosvitin, while Mn2+ substituted for Mg2+ in the phosphorylation of C-48. When the coated vesicles were prepared in the presence of NaF, C-48 contained 2.5-2.8 mol of phosphate/mol. On incubation with Mg2+ and ATP, C-48 incorporated 1.2-1.6 mol of phosphate/mol. With C-48 as a substrate, the value of its apparent Km for ATP was 6 microM. With phosvitin as a substrate, the value of its apparent Km was 20 microM. The phosphorylated amino acid residues in the phosvitin were identified as serine and threonine. Phosphothreonine was detected in C-48. These results suggest that brain coated vesicles possess two different classes of protein kinase, a casein kinase II and C-48 kinase.
Coated vesicles from bovine adrenal medulla contained clathrin and major detergent-insoluble polypeptides of 120-100, 51 and 49 kDa. Intact coated vesicles and vesicles lacking clathrin light chains were bound by immobilized calmodulin in the presence of Ca2+. Clathrin in the form of 700 A cages was not bound. The calmodulin binding components in intact coated vesicles are therefore contributed by the enclosed vesicle or by the 120-100, 50 or 49 kDa polypeptides. The 51 kDa component incorporated 32Pi from labelled ATP by an endogenous kinase activity; no other coat or vesicle membrane protein was phosphorylated in vitro, either by intrinsic or exogenous kinases.
Coated vesicles within dentate gyrus granule cell dendrites were found free in the cytoplasm and also attached to the smooth endoplasmic reticulum and plasmalemma. Coated vesicles began increasing in number 40-48 h following partial dendritic deafferentation. This increase continued up to 15-30 days postlesion, after which the population steadily declined to control values. These results combined with the findings of previous studies suggest an involvement of coated vesicles in synaptic reinnervation.
Coated vesicles, which are found in many eucaryotic cells, seem to play a role in the transfer of membrane and in the uptake and secretion of proteins. They have polyhedral structures whose faces consist of twelve pentagons and a variable number of hexagons and at whose vertices always three edges meet. To study the stability of such structures theoretically I first enumerate all the topologically distinct polyhedra under the conditions that they have at most ten hexagonal faces. Then I estimate their strain energy assuming Hookean elasticity and considering only interactions of short range. The results show that the three structures of coated vesicles which Crowther et al. (1976) reported have the lowest energy among all the polyhedra under certain conditions. Thus, the seemingly complex structures of coated vesicles can be constructed from only one species of structural units according to the principle of lowest strain energy.
Four high-molecular-weight proteins form the main subunits of the coat of Golgi-derived (non-clathrin) coated vesicles. One of these coat proteins, beta-COP, is identical to a Golgi-associated protein of relative mass 110,000 (110K) that shares homology with the adaptin proteins of clathrin-coated vesicles. This connection, and the comparable molecular weights of the coat proteins of Golgi-derived and clathrin-coated vesicles, indicates that they may be structurally related. The identification of beta-COP as the 110K protein explains the blocking of secretion by the drug brefeldin A.
Coated vesicles have been purified from brain, adrenal medulla, and a nonsecreting lymphoma cell line. A single major protein species, clathrin, with an apparent molecular weight of 180,000, forms the coat of all these vesicles. Peptide mapping suggests that the amino acid sequence of clathrin is conserved, irrespective of tissue or species studied. Coated vesicles of different sizes are found. The coats are constructed with variable numbers of clathrin subunits, arranged in closed networks of hexagons and pentagons. The amount of clathrin in lymphoma cells suggests that coated vesicles transfer substantial amounts of membrane within cells, not necessarily in association with a secretory process.
Coated vesicles are involved in transport of membrane proteins between several intracellular membrane-bound compartments. These vesicles possess a specific 50-kDa protein which is phosphorylated and dephosphorylated by a coated-vesicle-specific kinase and phosphatase. We studied this phosphorylation/dephosphorylation cascade system and show that the phosphorylation level of the 50-kDa protein is governed by the ATP/ADP ratio.
Coated vesicles prepared from bovine brains contained a protein kinase activity which catalyzed the phosphorylation of endogenous structural proteins, Mr 150 000, 120 000, 48 000 and 32 000. An endogenous protein, Mr 48 000 was most strongly phosphorylated by this kinase. This protein kinase also phosphorylated exogenous proteins, phosvitin intensely and casein slightly but not histone or protamine. The enzyme activity was independent of cyclic nucleotides or Ca2+/calmodulin. Mg2+ stimulated the kinase activity. Some divalent cations were substituted for Mg2+; the potency decreased in the order Mn2+, Mg2+, Co2+, Ca2+, Zn2+. Two separate subfractions, the outer coat and the inner vesicle (core), were prepared from coated vesicles by a urea treatment followed by sucrose density gradient centrifugation and dialysis. The kinase activity was found predominantly in the coat subfraction.
Coated vesicles were purified from human placenta by sedimentation, isopycnic centrifugation, and gel filtration. Quantitative Western blotting of the endogenous transferrin receptor (tfR) demonstrated the presence, on average, of roughly one receptor per vesicle. TfR appeared undersaturated with transferrin. After solubilizing vesicles in nonionic detergent, we looked for evidence of tfR interactions with other proteins. Solubilized tfR had an unexpectedly high mobility by gel filtration, apparently resulting from its self-association. This property was not seen in purified tfR or in tfR from a different cell fraction. The tfR complexes, though noncovalent, were largely resistant to conditions that disassemble coat proteins, and they did not appear to contain any other protein species.
The plain synaptic vesicle and the ocated vesicle fractions were isolated from rat brains, and the ATPase [EC 3.6.1.3] activities were characterized in terms of ionic effects, drug effects, and protein components. Coated vesicle fraction contained three times as much actomysin-like proteins as plain vesicle fraction, although both fractions had an identical ratio of actin-like protein to myosin-like protein. The ATPases of these two fractions were activated by both Mg2+ and Ca2+, and, in the presence of either of the cations, were inhibited by KCl. Reserpine activated plain vesicle ATPase only in the presence of Cl-. Colchicine and vinblastine inhibited coated vesicle ATPase only. The results are consistent with the view that actomyosin-like proteins are involved in the synaptic retrieval process.
Frog neuromuscular junctions were stimulated by different methods to secrete quanta of ACh, and the attendant changes in the ultrastructure of the nerve terminal were assessed by morphometric analysis of electron micrographs. Secretion was stimulated by electrical stimulation at 2 Hz or by application of the secretagogues, lanthanum, ouabain or black widow spider venom, either in the presence or in the absence of extracellular Ca2+. The numbers of synaptic vesicles, coated vesicles and coated pits, and the length of axolemma and area of axoplasm were measured on the micrographs. There was a significant increase (about threefold) in the total number of coated structures (vesicles plus pits) per micron2 of axoplasm, but the fractional increase in the number of coated pits exceeded the fractional increase in the number of coated vesicles. These increases were positively correlated with the increase in the length of axolemma per unit area and negatively correlated with the changes in concentration of synaptic vesicles, suggesting that they were due to the increases in the surface area of the terminal that accompany a loss of vesicles. However, the increase in the concentration of coated structures was not related to the number of quanta secreted or to the estimated number of vesicles recycled. The lack of correspondence between the fractional increases in the coated pits and coated vesicles and the poor correlation between the numbers of these structures and the overall parameters of the secretory process suggest that, in contrast to the situation in other secretory systems, coated pits and coated vesicles may not play a crucial role in maintaining the functional population of synaptic vesicles at rapidly secreting neuromuscular junctions.
Biochemical and morphological techniques were used to demonstrate the early steps in the endocytosis of transferrin in broken A431 cells. After binding 125I-transferrin, the cells were broken by scraping and then warmed. 125I-transferrin became inaccessible to exogenous anti-transferrin antibody providing a measure of the internalization process. Parallel morphological experiments using transferrin coupled to horseradish peroxidase confirmed internalization in broken cells. The process was characterized and compared with endocytosis in intact cells and showed many similar features. The system was used to show that both the appearance of new coated pits and the scission of coated pits to form coated vesicles were dependent on the addition of cytosol and ATP whereas invagination of pits was dependent on neither.
The transport of the two mannose 6-phosphate receptors (MPRs) from the secretory pathway to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi-specific assembly protein and clathrin. Using an in vitro assay that reconstitutes the ARF-1-dependent translocation of cytosolic AP-1 onto membranes of the TGN, we have previously reported that the MPRs are key components for the efficient recruitment of AP-1 (Le Borgne, R., G. Griffiths, and B. Hoflack. 1996. J. Biol. Chem. 271:2162-2170). Using a polyclonal antibody against the mouse gamma-adaptin, we have now examined the steady state distribution of AP-1 after subcellular fractionation of mouse fibroblasts lacking both MPRs or reexpressing physiological levels of either MPR. We report that the amount of AP-1 bound to membranes and associated with clathrin-coated vesicles depends on the expression level of the MPRs and on the integrity of their cytoplasmic domains. Thus, these results indicate that the concentration of the MPRs, i.e., the major transmembrane proteins sorted toward the endosomes, determines the number of clathrin-coated vesicles formed in the TGN.