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PubMed1992

Gamma-COP, a coat subunit of non-clathrin-coated vesicles with homology to Sec21p.

Constitutive secretory transport in eukaryotes is likely to be mediated by non-clathrin-coated vesicles, which have been isolated and characterized [(1989) Cell 58, 329-336; (1991) Nature 349, 215-220]. They contain a set of coat proteins (COPs) which are also likely to exist in a preformed cytosolic complex named coatomer [(1991) Nature 349, 248-250]. From peptide sequence and cDNA structure comparisons evidence is presented that one of the subunits of coatomer, gamma-COP, is a true constituent of non-clathrin-coated vesicles, and that gamma-COP is related to sec 21, a secretory mutant of the yeast Saccharomyces cervisiae.

Amino Acid Sequence↗
PubMed1999

Arabidopsis Sec21p and Sec23p homologs. Probable coat proteins of plant COP-coated vesicles.

Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cells has not yet been demonstrated, although the GTP-binding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membrane-bound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-density-gradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.

Arabidopsis↗
PubMed1983

Tubulin as a molecular component of coated vesicles.

Two proteins of 53,000 and 56,000 mol wt have been found to be associated with coated vesicles (CV) purified from bovine brain and chicken liver. These proteins share molecular weights, isoelectric points, and antigenic determinants with alpha- and beta-tubulins purified from bovine brain. Based on SDS PAGE and electron microscopic analysis of controlled pore glass bead exclusion column fractions, both the tubulins and the major CV polypeptide clathrin were found to chromatograph as components of a single kinetic particle. In addition, tubulin and CV antigens assayed by a sensitive enzyme-linked-immunoadsorbent method eluted from the columns with constant stoichiometry. These data provide evidence that tubulin is a molecular component of coated vesicles.

Animals↗
PubMed1990

Coupling of adenosine A1 receptors to a G-protein in coated vesicles isolated from bovine brain: presence of pertussis and cholera toxin substrates.

Adenosine A1 receptors have been described in coated vesicles isolated from bovine brain (Gonzalez-Calero et al., J. Neurochem. 1990, 55, 106-113). Addition of non hydrolyzable GTP analogue (guanyl-5-yl-imidodiphosphate) caused a transition of the receptor from the high- to the low-affinity state, without any significant change in the total binding sites. The presence of G-proteins has been investigated by pertussis and cholera toxins catalyzed ADP-ribosylation. A band of Mr = 41,000 D, similar to the alpha Gi subunit, was specifically labeled in the presence of preactivated pertussis toxin. Bands of Mr = 42,000 D and Mr = 47,000 D were specifically labeled in the presence of preactivated cholera toxin. These results confirm the presence of GTP binding proteins (alpha Gi and alpha Gs) in coated vesicles isolated from bovine brain.

Animals↗
PubMed1998

Interaction of the clathrin-coated vesicle V-ATPase with ADP and sodium azide.

The kinetics of adenosine triphosphate (ATP)-dependent proton transport into clathrin-coated vesicles from bovine brain have been studied. We observe that the vacuolar proton-translocating ATPase (V-ATPase) from clathrin-coated vesicles is subject to two different types of inhibition by ADP. The first is competitive inhibition with respect to ATP, with a Ki for ADP of 11 microM. The second type of inhibition occurs after preincubation of the V-ATPase in the presence of ADP and Mg2+, which results in inhibition of the initial rate of proton transport followed by reactivation over the course of several minutes. The second effect is observed at ADP concentrations as low as 0.1-0.2 microM, indicating that a high affinity inhibitory complex is formed between ADP and the V-ATPase and is only slowly dissociated after the addition of ATP. We have further investigated the effect of sodium azide, an inhibitor of the F-ATPases that has been shown to stabilize an inactive complex between ADP and the F1-F0-ATP synthase (F-ATPase). We observed that azide inhibited ATP-dependent proton transport by the purified, reconstituted V-ATPase with a K0.5 of 0.2-0.4 mM but had no effect on ATP hydrolysis. Azide was shown not to increase the passive proton permeability of reconstituted vesicles and did not stimulate ATP hydrolysis by the reconstituted enzyme, in contrast with CCCP, which both abolished the proton gradient and stimulated hydrolysis. Thus, azide does not appear to act as a simple uncoupler of proton transport and ATP hydrolysis. Rather, azide may have some more direct effect on V-ATPase activity. Possible mechanisms by which azide could exert this effect on the V-ATPase and the contrasting effects of azide on the F- and V-ATPases are discussed.

Adenosine Diphosphate↗
PubMed1991

Stabilization of clathrin coated vesicles by amantadine, tromantadine and other hydrophobic amines.

Amantadine and related compounds stabilized the structure of purified pig brain clathrin coated vesicles (CCV) at biologically relevant concentrations. Incubation of purified CCV for 30 min at 25 degrees C or 37 degrees C caused the release of clathrin, as determined by a centrifugation assay, and a reduction in the number of coated vesicles, by electron microscopy. Amantadine (10 mM), tromantadine (1 mM), amidine D295 (cyclohexylcarboximidamide-(N-benzyl)hydrochloride (10 mM), chloroquine (0.1 mM) and monodansylcadaverine (10 mM) significantly reduced the extent of dissociation.

Amantadine↗
PubMed1997

COPII subunit interactions in the assembly of the vesicle coat.

In vitro analysis of COPII vesicle formation in the yeast Saccharomyces cerevisiae has demonstrated the requirement for three cytosolic factors: Sec31p-Sec13p, Sec23p-Sec24p, and Sar1p. Convergent evidence suggests that the peripheral endoplasmic reticulum (ER) membrane protein Sec16p also represents an important component of the vesicle assembly apparatus: SEC16 interacts genetically with all five COPII genes; Sec16p binds to Sec23p and Sec24p, is found on ER-derived transport vesicles, and is required in vitro for the efficient release of ER-derived vesicle cargo. In this report, we demonstrate an important functional interaction between Sec16p and Sec31p. First, we map onto Sec31p binding regions for Sec16p, Sec23p, Sec24p, and Sec13p. Second, we show that a truncation mutant of Sec31p specifically defective for Sec16p binding is unable to complement a sec31Delta mutant and cannot rescue the secretion defect of a temperature-sensitive sec31 mutant at nonpermissive temperatures. We propose that Sec16p organizes the assembly of a coat that is stabilized both by the interaction of Sec31p with Sec23p and Sec24p and by the interaction of these three components with Sec16p.

Binding Sites↗
PubMed2002

Drs2p-dependent formation of exocytic clathrin-coated vesicles in vivo.

The small GTP binding protein ARF has been implicated in budding clathrin-coated vesicles (CCVs) from Golgi and endosomal membranes. An arf1 synthetic lethal screen identified DRS2/SWA3 along with a clathrin heavy-chain conditional allele (chc1-5/swa5-1) and SWA2, encoding the yeast auxilin-like protein involved in uncoating CCVs. Drs2p/Swa3p is a P-type ATPase and a potential aminophospholipid translocase that localizes to the trans-Golgi network (TGN) in yeast. Genetic and phenotypic analyses of drs2Delta mutants suggested that Drs2p was required for clathrin function. To address a potential role for Drs2p in CCV formation from the TGN in vivo, we have performed epistasis analyses between drs2 and mutations that cause accumulation of distinct populations of post-Golgi vesicles. We find that Drs2p is required to form a specific class of secretory vesicles that accumulate when the actin cytoskeleton is disrupted. Accumulation of these vesicles also requires clathrin and is perturbed by mutation of AP-1, but not AP-2, AP-3, or GGA adaptins. Most of the accumulated vesicles are uncoated; however, clathrin coats can be partially stabilized on these vesicles by deletion of SWA2. These data provide the first in vivo evidence for an integral membrane protein requirement in forming CCVs.

Adaptor Protein Complex 1↗
PubMed1994

Activity and in vitro reassembly of the coated vesicle (H+)-ATPase requires the 50-kDa subunit of the clathrin assembly complex AP-2.

We have previously shown that the 50-kDa subunit of the clathrin assembly complex AP-2 (AP50) stoichiometrically binds to and is immunoprecipitated with the vacuolar (H+)-ATPase (V-ATPase) from clathrin-coated vesicles (Myers, M., and Forgac, M. (1993) J. Biol. Chem. 268, 9184-9186). We now report that treatment of stripped coated vesicles with cystine results in a purified V-ATPase complex lacking the AP50 polypeptide. Removal of AP50 can be reversed upon treatment of the vesicles with dithiothreitol. Removal of AP50 reduces the ATPase activity of the purified V-ATPase by 90% relative to the enzyme containing AP50. This inhibition is not reversed upon treatment of the AP50-depleted enzyme with dithiothreitol in the absence of AP50. The reconstituted V-ATPase depleted of AP50 is devoid of ATP-dependent proton transport activity. We observe further that the peripheral V1 subunits are unable to reassemble onto the integral V0 domain in the absence of AP50. The addition of purified AP-2 containing the AP50 polypeptide restores the ability of the V1 subunits to assemble with the V0 sector to give a V-ATPase complex that is functional in ATP-dependent proton transport. These results indicate that the AP50 polypeptide is necessary for both activity and in vitro reassembly of the V-ATPase complex.

Adaptor Protein Complex 2↗
PubMed2000

Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases.

The vacuolar H(+)-ATPases (or V-ATPases) are a family of ATP-dependent proton pumps that carry out acidification of intracellular compartments in eukaryotic cells. This review is focused on our work on the V-ATPases of clathrin-coated vesicles and yeast vacuoles. The coated-vesicle V-ATPase undergoes trafficking to endosomes and synaptic vesicles, where it functions in receptor recycling and neurotransmitter uptake, respectively. The yeast V-ATPase functions to acidify the central vacuole and is necessary both for protein degradation and for coupled transport processes across the vacuolar membrane. The V-ATPases are multisubunit complexes composed of two functional domains. The V(1) domain is a 570 kDa peripheral complex composed of eight subunits of molecular mass 73-14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V(o) domain is a 260 kDa integral complex composed of five subunits of molecular mass 100-17 kDa (subunits a, d, c, c' and c") that is responsible for proton translocation. To explore the function of individual subunits in the V-ATPase complex as well as to identify residues important in proton transport and ATP hydrolysis, we have employed a combination of chemical modification, site-directed mutagenesis and in vitro reassembly. A central question concerns the mechanism by which vacuolar acidification is controlled in eukaryotic cells. We have proposed that disulfide bond formation between conserved cysteine residues at the catalytic site of the V-ATPase plays an important role in regulating V-ATPase activity in vivo. Other regulatory mechanisms that are discussed include reversible dissociation and reassembly of the V-ATPase complex, changes in the tightness of coupling between proton transport and ATP hydrolysis, differential targeting of V-ATPases within the cell and control of the Cl(-) conductance that is necessary for vacuolar acidification.

Adenosine Triphosphate↗
PubMed1991

Beta-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Golgi complex, shows homology to beta-adaptin.

We have cloned and sequenced beta-COP, a peripheral 110 kd Golgi membrane protein. beta-COP shows significant homology to beta-adaptin. It is present in a membrane-bound form and in a cytosolic complex of 13-14S, with a Stokes radius of approximately 10 nm and an estimated Mr of approximately 550,000. By immunofluorescence labeling, beta-COP is associated with the structures of the Golgi complex. Immunoelectron microscopy has localized beta-COP to non-clathrin-coated vesicles and cisternae of the Golgi complex. These coated vesicles accumulate in rat liver Golgi fractions treated with GTP gamma S and strongly label for beta-COP. Our data suggest that beta-COP is a component of a coat associated with vesicles and cisternae of the Golgi complex.

Amino Acid Sequence↗
PubMed2004

Structure of the functional fragment of auxilin required for catalytic uncoating of clathrin-coated vesicles.

The three-dimensional structure of the C-terminal 20 kDa portion of auxilin, which consists of the clathrin binding region and the C-terminal J-domain, has been determined by NMR. Auxilin is an Hsp40 family protein that catalytically supports the uncoating of clathrin-coated vesicles through recruitment of Hsc70 in an ATP hydrolysis-driven process. This 20 kDa auxilin construct contains the minimal sequential region required to uncoat clathrin-coated vesicles catalytically. The tertiary structure consists of six helices, where the first three are unique to auxilin and believed to be important in the catalytic uncoating of clathrin. The last three helices correspond to the canonical J-domain of Hsp40 proteins. The first helix, helix 1, which contains a conserved FEDLL motif believed to be necessary for clathrin binding, is transient and not packed against the rest of the structure. Helix 1 is joined to helix 2 by a flexible linker. Helix 2 packs loosely against the J-domain surface, whereas helix 3 packs tightly and makes critical contributions to the J-domain core. A long insert loop, also unique to the auxilin J-domain, is seen between helix 4 and helix 5. Comparison with a previously reported structure of auxilin containing only helices 3-6 shows a significant difference in the invariant HPD segment of the J-domain. The region where helix 1 is located corresponds to the expected region of the unstructured G/F-rich domain seen in DnaJ, i.e., the canonical N-terminal J-domain protein. In contrast, the location of helix 1 differs from the substrate binding regions of two other Hsp40 proteins, Escherichia coli Hsc20 and viral large T antigen. The variety of biological functions performed by Hsp40 proteins such as auxilin, as well as the observed differences in the structure and function of their substrate binding regions, supports the notion that Hsp40 proteins act as target-specific adaptors that recruit their more general Hsp70 partners to specific biological roles.

Adenosine Triphosphatases↗
PubMed2004

Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling.

Tandem MS has identified 209 proteins of clathrin-coated vesicles (CCVs) isolated from rat brain. An overwhelming abundance of peptides were assigned to the clathrin coat with a 1:1 stoichiometry observed for clathrin heavy and light chains and a 2:1 stoichiometry of clathrin heavy chain with clathrin adaptor protein heterotetramers. Thirty-two proteins representing many of the known components of synaptic vesicles (SVs) were identified, supporting that a main function for brain CCVs is to recapture SVs after exocytosis. A ratio of vesicle-N-ethylmaleimide-sensitive factor attachment protein receptors to target-N-ethylmaleimide-sensitive factor attachment protein receptors, similar to that previously detected on SVs, supports a single-step model for SV sorting during CCV-mediated recycling of SVs. The uncovering of eight previously undescribed proteins, four of which have to date been linked to clathrin-mediated trafficking, further attests to the value of the current organelle-based proteomics strategy.

Animals↗
PubMed1982

Coated pits and coated vesicles in the endothelial cells of trabecular meshwork.

Coated pits (CPs) and coated vesicles (CVs) were investigated in the trabecular meshwork of man and rabbit. In both man and rabbit CPs and CVs appear to be randomly distributed throughout the trabecular tissue. CVs are located in the peripheral cytoplasm of the trabecular endothelial cells and show an average diameter of 0 . 16 micron. The ratio between the number of CPs and CVs ranges from 0 . 53 (rabbit) to 0 . 6 (man). The number of CPs and CVs is higher in rabbit than in man. Consequently, the overall CPs surface and CVs volume density estimates are higher in rabbits, where CPs represent about 1% of the cell surface and CVs account for about 0 . 4% of the cell cytoplasm. In man these percentages are 0 . 46 and 0 . 17%, respectively. No significant changes of CPs and CVs were found in rabbits with corticosteroid-induced ocular hypertension. However, changes in length of boundary membrane of CPs, though not statistically significant, were accompanied by proportional changes in CVs diameter.

Animals↗
PubMed1991

Bovine brain coated vesicles contain guanine nucleotide regulatory proteins.

Binding of [3H]guanosine triphosphate (GTP) with a high affinity was found to be present in the coated vesicle fraction prepared from bovine cerebral cortex. The binding was saturable and displaced by 1 microM of GTP, guanosine diphosphate and guanosine 5'-(3-O-thio)triphosphate. Incubation of the vesicles with islet-activating protein and [32P]NAD resulted in ADP-ribosylation of a 39,000-41,000-dalton polypeptide. Antibodies to the alpha-subunit of stimulatory guanine nucleotide regulatory proteins (G-proteins) immunoblotted 52,000- and 45,000-dalton polypeptides. The results indicated that stimulatory and inhibitory G-proteins are contained in a fraction of the bovine brain coated vesicles.

Adenosine Diphosphate Ribose↗
PubMed1994

Hydrolysis of amyloid precursor protein-derived peptides by cysteine proteinases and extracts of rat brain clathrin-coated vesicles.

Amyloid precursor proteins (APPs) and C-terminal fragments were colocalized with cysteine proteinase-like enzymes in purified rat brain clathrin-coated vesicles. Vesicular extracts degraded beta A4(12-28), yielding a product profile similar to that of purified rat brain cathepsin B. Cathepsin B degraded this peptide sequentially, with initial cleavage occurring at Val18-Phe19 and Phe19-Phe20 followed by release of dipeptides. Enzyme also hydrolyzed beta A4(1-40) at Phe19-Phe20 bond but at lower rates, likely due to aggregate formation. An octapeptide analogue of the domain adjacent to beta A4 (N-Ac-Val-Lys-Met-Asp-Ala-Glu-Phe-NH2) was also hydrolyzed by brain cathepsins B and L, and metalloendopeptidase 24.11. Enzymes acted at multiple sites, but only 24.11 cleaved the Met-Asp bond, thus resembling a proposed beta-secretase. Data imply that clathrin-coated vesicles contain cysteine-like proteinases capable of initiating the processing of APP or its fragments.

Amino Acid Sequence↗
PubMed1983

Immunocytochemical characterization of clathrin-associated proteins (CAPs). I. Neuronal distribution of CAPs, a component of clathrin-coated vesicles.

Clathrin-associated proteins (CAPs) elicited antibodies in rabbits that were affinity-purified using CAPs-conjugated CNBr-Sepharose 4B. Anti-CAPs IgG formed immunoprecipitates with CAPs and with the clathrin-CAPs complex. Indirect immunoperoxidase-labeling in sections of rat brain cortex using anti-CAPs and anti-clathrin IgG yielded similar staining patterns. Coated perinuclear cisternae and coated vesicles became stained and easily distinguished. Intense staining also was found in synaptic boutons with label between most synaptic vesicles and as a thick crust surrounding coated vesicles. The data demonstrate that clathrin and CAPs polypeptides are in identical subcellular locations.

Animals↗
PubMed1990

Equilibrium kinetics model for the cGMP-stimulated phosphodiesterase of brain coated vesicles.

An equilibrium kinetics model is proposed to described some of the enzymatic properties of the cyclic GMP-stimulated phosphodiesterase activity associated with brain clathrin coated vesicles. The model assumes the presence of pharmacologically distinct regulatory and catalytic domains in the enzyme. The model contemplates that random fashion occupancy of the regulatory site by the substrate, cyclic GMP, induces a conformational change which leads to the generation of an actived catalytic state. Therefore, cyclic GMP is a positive allosteric modulator of the coated vesicle enzyme. Experimental data revealed that occupancy or activation of the regulatory site was not essential for catalysis to occur since hydrolysis occurred after loss (200%) of the activation by cyclic GMP. This constitutes an example of non-essential substrate activation. Analysis of this PDE following activation by cGMP and after loss of the regulation, activation capacity of the enzyme allows the calculation of the various kinetic parameters inherent in the model.

3',5'-Cyclic-GMP Phosphodiesterases↗