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PubMed1989

The role of coated vesicles in recycling of synaptic vesicle membrane.

The uptake of extracellular tracers into synaptic nerve terminals has been a phenomenon of persistent interest. Uptake is into synaptic vesicles, hence vesicles spend part of their life in continuity with the plasma membrane, as expected if exocytosis underlies the quantal discharge of neurotransmitters. However, exactly how or when synaptic vesicles acquire extracellular tracers has not been unambiguously determined. Two schools of thought have developed, one holding that vesicles acquire tracers directly via a reversible exo/endocytotic sequence in which they consistently maintain their biochemical identity during their transient continuity with the plasma membrane, the other holding that synaptic vesicles acquire tracers indirectly, via the formation of clathrin-coated vesicles which are spatially and temporally separate from exocytosis and reverse a temporary loss of the vesicles' individual identity upon merger with the plasma membrane. Efforts to distinguish between these two alternatives have generated an interesting diversity of electron microscopic experiments, many of which are reviewed here. However, definitive determination of which view is correct may ultimately require direct visualization of synaptic vesicle turnover in living nerve terminals. To this end, we here review the results of visualizing endocytosis in tissue cultured cells, where light microscopy can provide sufficient resolution to reveal membrane dynamics in living cells. This has allowed visual discrimination of two different types of endocytosis, one clathrin-mediated (coated vesicle formation) and the other actin-mediated (macropinocytosis). Current work is also reviewed which aims at determining experimental methods for inhibiting each type of endocytosis selectively. Hypertonicity and severe cytoplasmic acidification turn out to inhibit coated vesicle formation, while cytochalasin D and mild cytoplasmic acidification selectively inhibit macropinocytosis. Applied to nerves, these various treatments affect synaptic vesicle turnover in a manner that supports the notion that synaptic vesicle membrane recycles via the "indirect" route of coated vesicle formation.

Animals↗
PubMed1989

[Morphological study on the role of coated vesicle in the specialization of synaptic membrane in synaptogenesis].

Our object was to characterize the morphological changes of coated vesicles and synaptic membranes during synaptogenesis. Neurons from spinal cords of fetal mice were established as isolated cells in primary culture. After a few days in vitro, the neurons extended their neurites and started their interaction. At timed intervals thereafter, cultures were fixed for electron microscopic observation. Coated vesicles were prominent in the neuronal cytoplasm at the time of synaptogenesis (about 7-10 days in vitro). Similar vesicles were seen in continuity with some cisternae in the Golgi regions and there was an increase in number during the synaptogenic period. Indeed it is not established whether the coated vesicles were exocytotic or pinocytotic in nature, but the cisternae which were in continuity with coated vesicles could be labelled by glucose-6-phosphatase (G6Pase) but not by thiamine pyrophosphatase (TPPase). Such vesicles were also seen in continuity with the neuronal plasmalemma near the closest contact site and contributed their undercoating to pre- and postsynaptic densities. The formation of bilateral membrane specialization was described as being structurally similar to synaptic active zones and appeared to be the first definitive sign of synapseformation. It has been suggested that the synaptic dense material may derive wholly or in part from the exocytic coated vesicles which apparently budding off from endoplasmic reticulum cisternae. This incorporation could provide the mechanism for confining specific characteristics of neuronal membrane to the synaptic region.

Animals↗
PubMed1988

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.

Adrenal Cortex↗
PubMed1993

Coated vesicle assembly in the Golgi requires only coatomer and ARF proteins from the cytosol.

Transport vesicles derived from the Golgi apparatus are thought to mediate biosynthetic transport across the Golgi stack. These vesicles are surrounded by a protein coat whose principal constituents are coatomer (a complex of seven distinct subunits or COPs) and ADP-ribosylation factor (ARF, an N-myristylated small GTP-binding protein). The coat proteins of the COP-coated vesicles were originally defined by ultrastructural criteria, however, and it is possible that important but minor coat proteins or cytoplasmic proteins needed for coat assembly may have been overlooked. Here we show that coatomer and ARF are the only cytoplasmic proteins needed for the assembly and budding of COP-coated vesicles. COP-coated buds may therefore form essentially by self-assembly from Golgi cisternae after an initial step in which GTP is used to allow ARF binding.

ADP-Ribosylation Factors↗
PubMed1983

Model for transformations of the clathrin lattice in the coated vesicle pathway.

Transport of receptors by the coated vesicle pathway entails assembly of clathrin triskelions into a lattice in conjunction with receptors in a membrane. The processes by which the receptors are concentrated, the lattice is assembled, transformed into a cage during vesiculation, and subsequently removed from pinched off vesicles are not understood in regard to mechanism, energetics or control. Tubulin and actin assembly are looked to for analogies applicable to clathrin. The present model supposes that clathrin assembly is energy linked and can be described by kinetic equations of the same general form as those for treadmilling in linear polymers. The coat lattice assembles in a steady state involving the degradation of a high energy form of the clathrin triskelions. Diffuse endocytosis receptors are assumed to be associated with individual triskelions and to be able to trigger clustering and coated pit formation by influencing the assembly kinetics of the bound triskelions. A generalization of the treadmilling scheme is proposed by which the kinetic parameters associated with clathrin polymerization can shift simultaneously for an entire lattice to favor alternatively net assembly or disassembly. This shift is effected by a coordinated conversion of the lattice bound receptors. The conversion of the receptors in turn depends on some global property of the membrane compartments (arguably pH, calcium concentration or transmembrane voltage) which is likely to change as a consequence of vesiculation. Thereby, lattice disassembly can be coordinated with the topological conversion from coated pit to coated vesicle.

Adenosine Diphosphate↗
PubMed1985

Transmembrane orientation of the mannose 6-phosphate receptor in isolated clathrin-coated vesicles.

The mannose 6-phosphate (Man-6-P) receptor is an integral membrane glycoprotein which mediates intracellular transport and receptor-mediated endocytosis of lysosomal proteins. Clathrin-coated vesicles, which have been shown to be significantly involved in these processes, have also been shown to be a major subcellular site of the receptor. In order to define the orientation of the Man-6-P receptor within the coated vesicle membrane, highly purified preparations of coated vesicles were prepared from bovine brain employing D2O/sucrose gradient centrifugation and Sephacryl S-1000 column chromatography. Using [35S]methionine-labeled lysosomal enzymes secreted by Chinese hamster ovary cells as receptor ligand, significant binding activity was detected only upon permeabilization of the coated vesicle membranes with detergent. Prior treatment of intact vesicles with proteinase K resulted in similar binding activity upon permeabilization. However, examination of the receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with rabbit anti-receptor serum revealed that proteinase K treatment of intact vesicles reduced the size of the receptor by 12,000 daltons. A similar decrease in size was obtained when the vesicles were treated with carboxypeptidase Y. These results suggest that the Man-6-P receptor is a transmembrane protein with its lysosomal enzyme binding site oriented toward the lumen of the coated vesicle and its C-terminal end exposed to the exterior or cytoplasmic portion of the vesicle membrane.

Animals↗
PubMed1991

Clathrin-coated vesicles from human placenta contain GTP-binding proteins.

Biochemical and morphological techniques were used to investigate the GTP-binding proteins of clathrin-coated vesicles. Binding of [3H]GTP to clathrin coats was demonstrated by electron microscopic autoradiography. Purified coated vesicles bound 5.2 pmol [35S]GTP gamma S/mg protein. Addition of GTP or GTP gamma S, but not ATP nor GMP, inhibited binding of [35S]GTP gamma S to intact coated vesicles and Triton X-100-extracted coats. A series of 23-24 kDa GTP-binding proteins with isoelectric points between pH 5-8 were detected in coated vesicles. We suggest that the low molecular weight ras-like GTP-binding protein(s) play a role in regulating vesicle-mediated protein transport or signal transduction within intracellular organelles.

Autoradiography↗
PubMed1980

Role of coated vesicles, microfilaments, and calmodulin in receptor-mediated endocytosis by cultured B lymphoblastoid cells.

Cell surface receptor IgM molecules of cultured human lymlphoblastoid cells (WiL2) patch and redistribute into a cap over the Golgi region of the cell after treatment with multivalent anti-IgM antibodies. During and after the redistribution, ligand-receptor clusters are endocytosed into coated pits and coated vesicles. Morphometric analysis of the distribution of ferritin-labeled ligand at EM resolution reveals the following sequence of events in the endocytosis of cell surface IgM: (a) binding of the multivalent ligand in a diffuse cell surface distribution, (b) clustering of the ligand-receptor complexes, (c) recruitment of clathrin coats to the cytoplasmic surface of the cell membrane opposite ligand-receptor clusters, (d) assembly and (e) internalization of coated vesicles, and (f) delivery of label into a large vesicular compartment, presumably partly lysosomal. Most of the labeled ligand enters this pathway. The recruitment of clathrin coats to the membrane opposite ligand-receptor clusters is sensitive to the calmodulin-directed drug Stelazine (trifluoperazine dihydrochloride). In addition, Stelazine inhibits an alternate pathway of endocytosis that does not involve coated vesicle formation. The actin-directed drug dihydrocytochalasin B has no effect on the recruitment of clathrin to the ligand-receptor clusters and the formation of coated pits and little effect on the alternate pathway, but this drug does interfere with subsequent coated vesicle formation and it inhibits capping. Cortical microfilaments that decorate with heavy meromyosin with constant polarity are observed in association with the coated regions of the plasma membrane and with coated vesicles. SDS-polyacrylamide gel electrophoresis analysis of a coated vesicle preparation isolated from WiL2 cells demonstrates that the major polypeptides in the fraction are a 175-kdalton component that comigrates with calf brain clathrin, a 42-kdalton component that comigrates with rabbit muscle actin and a 18.5-kdalton minor component that comigrates with calmodulin as well as 110-, 70-, 55-, 36-, 30-, and 17-kdalton components. These results clarify the pathways of endocytosis in this cell and suggest functional roles for calmodulin, especially in the formation of clathrin-coated pits, and for actin microfilaments in coated vesicle formation and in capping.

B-Lymphocytes↗
PubMed1998

Uptake by COPI-coated vesicles of both anterograde and retrograde cargo is inhibited by GTPgammaS in vitro.

On the basis of the cell surface protein CD8 we have constructed reporter molecules for both anterograde and retrograde transport from the Golgi complex. The cytoplasmic tail of CD8 was exchanged by a construct comprising a hemagglutinin (HA) epitope, the C-terminal sequence of the viral protein E19 (containing a KKXX retrieval signal) followed by a myc epitope (CD8-LT). Due to this masking of the KKXX retrieval signal CD8-LT is transported to the cell surface. Since the KKXX motif is joined to the myc epitope via a thrombin cleavage site, CD8-LT in isolated Golgi membranes can be proteolytically converted into an unmasked reporter molecule for retrograde transport (CD8-ST) in vitro. A CHO cell line stably expressing CD8-LT was generated and used for the isolation of Golgi membranes. These membranes were shown to contain CD8-LT en route to the cell surface. By addition of thrombin, CD8-LT could be efficiently converted into CD8-ST, and this allows us to study the sorting into coat protein COPI-coated vesicles of these different kinds of cargo on a comparative basis. COPI-coated vesicles were generated in vitro from Golgi membranes containing either CD8-LT or CD8-ST. When the incubation was performed in the presence of GTP, both CD8-LT and CD8-ST were packaged into COPI-coated vesicles. However, COPI-coated vesicles generated in the presence of the slowly hydrolyzable analogue of GTP, GTP(&ggr ;)S contained strikingly lower amounts of CD8-LT and CD8-ST. While COPI-coated vesicles accumulated about 12-fold in the presence of GTPgammaS these vesicles together contained only one fifth of cargo compared to the few vesicles generated in the absence of GTPgammaS. These data indicate that cargo packaging into COPI-coated vesicles requires hydrolysis of GTP.

Amino Acid Sequence↗
PubMed2001

Dominant-interfering Hsc70 mutants disrupt multiple stages of the clathrin-coated vesicle cycle in vivo.

Within the clathrin-coated vesicle (CCV) cycle, coat assembly drives the internalization of receptors from the cell surface and disassembly allows for the processing of internalized ligands. The heat shock cognate protein, hsc70, has been implicated in regulating coat disassembly. We find that in cells overexpressing ATPase-deficient hsc70 mutants, uncoating of CCVs is inhibited in vivo, and the majority of unassembled cytosolic clathrin shifts to an assembled pool that cofractionates with AP1 and AP2. Surprisingly, this assembled pool of coat proteins accumulates in the absence of cargo receptors, suggesting that disruption of hsc70 activity may cause misassembly of empty clathrin cages. The strongest effect of overexpression of hsc70 mutants is a block in transferrin receptor (TfnR) recycling, which cannot be accounted for by the degree of inhibition of uncoating of endocytic CCVs. These results suggest that hsc70 participates in multiple transport and/or sorting events between endosomal compartments. Additionally, the mutant-expressing cells are defective at internalizing transferrin. In the most potent case, the initial rate of uptake is inhibited 10-fold, and TfnR levels double at the cell surface. Our findings demonstrate that hsc70 indeed regulates coat disassembly and also suggest that this chaperone broadly modulates clathrin dynamics throughout the CCV cycle.

Adenosine Triphosphatases↗
PubMed1983

Fusion of coated vesicles with lysosomes: measurement with a fluorescence assay.

A fluorescence assay was developed to measure the rate of fusion of highly purified clathrin-coated vesicles isolated from bovine brain with purified lysosomes isolated from bovine kidney. Coated vesicles and stripped vesicles, prepared by removal of clathrin from coated vesicles with dilute alkaline buffer, were labeled with the nonfluorescent dye 6-carboxydiacetylfluorescein. Fusion of the vesicles with lysosomes resulted in mixing of the vesicle contents and exposure of 6-carboxydiacetylfluorescein to lysosomal esterases, which hydrolyze the probe's acetate groups to give the fluorescent 6-carboxyfluorescein. Fusion was therefore measured by recording the increase in fluorescence obtained upon mixing the vesicles with lysosomes. The results of the experiments indicated that the clathrin coat of coated vesicles inhibited the fusion of the vesicle membrane with that of the lysosome. In addition, fusion appears to require free Ca2+ and does not require vesicle-surface protein.

Animals↗
PubMed1985

Gangliosides and sialosylglycoproteins in coated vesicles from bovine brain.

The content of gangliosides and sialosylglycoproteins was investigated in a coated-vesicle-enriched fraction prepared from bovine brain by the method of Pearse [(1975) J. Mol. Biol. 97, 93-98] and further purified by g.p.c. (glass-permeation chromatography) [Pfeffer & Kelly (1981) J. Cell Biol. 91, 385-391]. From morphological criteria and from the analysis of the polypeptide pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the coated-vesicle fraction (CV-fraction) appeared more than 95% pure. The ganglioside-NeuAc (N-acetylneuraminate), glycoprotein-NeuAc, phospholipid and cholesterol contents of CV-fraction were compared with those of bovine brain synaptic plasma membranes (SPM). The cholesterol to phospholipid molar ratio was 0.47 +/- 0.07 in CV-fraction and 1.06 +/- 0.08 in SPM. The ganglioside-NeuAc and glycoprotein-NeuAc to phospholipid molar ratios were 0.047 and 0.020 respectively in CV-fraction and 0.039 and 0.016 respectively in SPM. The (Na+ + K+)-dependent ATPase activity sensitive to ouabain (in mumol of Pi/h per nmol of phospholipid) was 1.04 in CV-fraction and 0.63 in SPM; the ratio between this activity and the activity resistant to ouabain was 2 in CV-fraction and 1.4 in SPM. A t.l.c. analysis of the ganglioside fractions showed that most of the ganglioside species present in SPM were present in CV-fraction. In a rat brain coated-vesicle preparation not subjected to g.p.c., the activities [as sugar-radioactivity (c.p.m.) transferred/h per mumol of phospholipid] of the enzymes CMP-NeuAc:sialosyl-lactosylceramide (GM3) sialosyl-, UDP-Gal:N-acetylgalactosaminyl(sialosyl)lactosylceramide (GM2) galactosyl- and UDP-GalNAc:sialosyl-lactosylceramide (GM3) N-acetylgalactosaminyl-transferases, which were considered Golgi-apparatus markers, were about 19, 16 and 10% respectively of those determined in rat brain neuronal perikaryon-enriched fractions. Taken together, the results indicate that most of the major gangliosides are constituents of coated vesicles.

Adenosine Triphosphatases↗
PubMed1984

Interaction of basic compounds with coated vesicles.

The effect of poly- and dibasic amines, including chloroquine and quinacrine, on the dissociation of coated vesicles at pH 7.4 in 0.01 M 2-(N-morpholino)ethanesulfonic acid has been evaluated by light scattering and sucrose gradient centrifugation. The degree of inhibition of dissociation by the polybases is proportional to the number of amine groups in each compound. However, very little difference in effectiveness was found in a series of dibasic compounds, NH2(CH2)2-5NH2. Chloroquine and quinacrine contain dibasic aliphatic chains as well as aromatic ring systems. These two antimalarials are more effective in inhibiting dissociation of coated vesicles than the dibasic aliphatic amines. The ring systems therefore appear to be contributing, independently, to the free energy of stabilization of the coat structure of coated vesicles. It is suggested that the interaction of poly- or dibasic compounds with clathrin or coated vesicles could influence the turnover of ligands in receptor-mediated endocytosis.

Alkanesulfonates↗
PubMed1988

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.

Adaptor Proteins, Vesicular Transport↗
PubMed1998

Enrichment of presenilin 1 peptides in neuronal large dense-core and somatodendritic clathrin-coated vesicles.

Presenilin 1 is an integral membrane protein specifically cleaved to yield an N-terminal and a C-terminal fragment, both membrane-associated. More than 40 presenilin 1 mutations have been linked to early-onset familial Alzheimer disease, although the mechanism by which these mutations induce the Alzheimer disease neuropathology is not clear. Presenilin 1 is expressed predominantly in neurons, suggesting that the familial Alzheimer disease mutants may compromise or change the neuronal function (s) of the wild-type protein. To elucidate the function of this protein, we studied its expression in neuronal vesicular systems using as models the chromaffin granules of the neuroendocrine chromaffin cells and the major categories of brain neuronal vesicles, including the small clear-core synaptic vesicles, the large dense-core vesicles, and the somatodendritic and nerve terminal clathrin-coated vesicles. Both the N- and C-terminal presenilin 1 proteolytic fragments were greatly enriched in chromaffin granule and neuronal large dense-core vesicle membranes, indicating that these fragments are targeted to these vesicles and may regulate the large dense-core vesicle-mediated secretion of neuropeptides and neurotransmitters at synaptic sites. The presenilin 1 fragments were also enriched in the somatodendritic clathrin-coated vesicle membranes, suggesting that they are targeted to the somatodendritic membrane, where they may regulate constitutive secretion and endocytosis. In contrast, these fragments were not enriched in the small clear-core synaptic vesicle or in the nerve terminal clathrin-coated vesicle membranes. Taken together, our data indicate that presenilin 1 proteolytic fragments are targeted to specific populations of neuronal vesicles where they may regulate vesicular function. Although full-length presenilin 1 was present in crude homogenates, it was not detected in any of the vesicles studied, indicating that, unlike the presenilin fragments, full-length protein may not have a vesicular function.

Animals↗
PubMed1978

Coated vesicles in the rat anterior pituitary gland.

Ultrastructural documentation of the formation of coated vesicles in prolactin cells of the rat anterior pituitary gland is presented. Coated vesicles were observed forming from the secretory granular membrane in the Golgi area and between the Golgi complex and the plasmalemma. They were also found budding off the membrane lining the exocytotic pocket. Following peroxidase reaction product could be seen forming from the membrane of the exocytotic pocket and adjacent to it. These vesicles were found in transit towards the Golgi area or within lysosomes. It is proposed that these coated vesicles are important for membrane conservation and that this is occurring in developing secretory granules near the Golgi complex, in more mature secretory granules and at the level of the plasmalemma during exocytosis.

Animals↗
PubMed1995

Role of clathrin-coated vesicles in glycoprotein transport from the cell surface to the Golgi complex.

Plasma membrane glycoproteins recycle to the Golgi complex, but the route followed by these proteins is not known. To elucidate the pathway of transport, the involvement of clathrin-coated vesicles was tested. This was accomplished by comparing the traffic of wild type low density lipoprotein receptor (LDLR) and FH 683, a mutant receptor whose endocytosis from the cell surface in coated vesicles is reduced by 90-95%. Wild type LDLR traveled from the cell surface to the sialyltransferase compartment of the Golgi with a half-time of 2.5 h in K562 human leukemia cells expressing receptor from a transfected cDNA. In contrast, FH 683 LDLR recycled to the Golgi at 33% of the wild type rate, suggesting that wild type LDLR is largely transported to the Golgi by a pathway that involves clathrin-coated vesicles. Moreover, because clathrin-coated vesicles that bud from the plasma membrane are transported to endosomes, surface-to-Golgi transport probably involves an endosomal intermediate. Finally, because there was substantial transport of mutant LDLR to the Golgi even though its endocytosis in coated vesicles was greatly reduced, there may be a second pathway of surface-to-Golgi traffic. Our results suggest that wild type LDLR may move from plasma membrane to Golgi by two routes. Two-thirds of the traffic proceeds via a coated vesicle-mediated pathway while the remainder may follow a clathrin-independent pathway.

Asialoglycoproteins↗
PubMed1992

100-kD proteins of Golgi- and trans-Golgi network-associated coated vesicles have related but distinct membrane binding properties.

The 100-110-kD proteins (alpha-, beta-, beta'-, and gamma-adaptins) of clathrin-coated vesicles and the 110-kD protein (beta-COP) of the nonclathrin-coated vesicles that mediate constitutive transport through the Golgi have homologous protein sequences. To determine whether homologous processes are involved in assembly of the two types of coated vesicles, the membrane binding properties of their coat proteins were compared. After treatment of MDBK cells with the fungal metabolite Brefeldin A (BFA), beta-COP was redistributed to the cytoplasm within 15 s, gamma-adaptin and clathrin in the trans-Golgi network (TGN) dispersed within 30 s, but the alpha-adaptin and clathrin present on coated pits and vesicles derived from the plasma membrane remained membrane associated even after a 15-min exposure to BFA. In PtK1 cells and MDCK cells, BFA did not affect beta-COP binding or Golgi morphology but still induced redistribution of gamma-adaptin and clathrin from TGN membranes to the cytoplasm. Thus BFA affects the binding of coat proteins to membranes in the Golgi region (Golgi apparatus and TGN) but not plasma membranes. However, the Golgi binding interactions of beta-COP and gamma-adaptin are distinct and differentially sensitive to BFA. BFA treatment did not release gamma-adaptin or clathrin from purified clathrin-coated vesicles, suggesting that their distribution to the cytoplasm after BFA treatment of cells was due to interference with their rebinding to TGN membranes after a normal cycle of disassembly. This was confirmed using an in vitro assay in which gamma-adaptin binding to TGN membranes was blocked by BFA and enhanced by GTP gamma S, similar to the binding of beta-COP to Golgi membranes. These results suggest the involvement of GTP-dependent proteins in the association of the 100-kD coat proteins with membranes in the Golgi region of the cell.

Adaptor Protein Complex alpha Subunits↗