PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “COP-Coated Vesicles”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1995

COP-coated vesicles in intracellular protein transport.

COP-coated vesicles have originally been implicated in vesicular transport between subcompartments of the Golgi complex in mammals in a cis to trans direction. More recently, a role for COP-coated vesicles in transport between the endoplasmic reticulum (ER) and Golgi in mammalian cells has been proposed. Under certain conditions COP-coats have been localized to special domains of the transitional ER and to the cis side of the Golgi complex. This led to the assumption that COP-coated vesicles are involved in export of proteins from the ER. In addition, new findings point to a function of COP-coated vesicles in back transport of proteins from the Golgi to the ER. At present it is not known whether COP-coated vesicles move only in one or in both directions between ER and Golgi.

Biological Transport↗
PubMed1994

COP-coated vesicles are involved in the mitotic fragmentation of Golgi stacks in a cell-free system.

Rat liver Golgi stacks fragmented when incubated with mitotic but not interphase cytosol in a process dependent on time, temperature, energy (added in the form of ATP) and cdc2 kinase. The cross-sectional length of Golgi stacks fell in the presence of mitotic cytosol by approximately 50% over 30 min without a corresponding decrease in the number of cisternae in the stack. The loss of membrane from stacked and single cisternae occurred with a half-time of approximately 20 min, and was matched by the appearance of both small (50-100 nm in diameter) and large (100-200 nm in diameter) vesicular profiles. Small vesicular profiles constituted more than 50% of the total membrane after 60 min of incubation and they were shown to be vesicles or very short tubules by serial sectioning. In the presence of GTP gamma S all of the small vesicles were COP-coated and both the extent and the rate at which they formed were sufficient to account for the production of small vesicles during mitotic incubation. The involvement of the COP-mediated budding mechanism was confirmed by immunodepletion of one of the subunits of COP coats (the coatomer) from mitotic cytosol. Vesicles were no longer formed but highly fenestrated networks appeared, an effect reversed by the readdition of purified coatomer. Together these experiments provide strong support for our hypothesis that the observed vesiculation of the Golgi apparatus during mitosis in animal cells is caused by continued budding of COP-coated transport vesicles but an inhibition of their fusion with their target membranes.

Animals↗
PubMed1993

zeta-COP, a subunit of coatomer, is required for COP-coated vesicle assembly.

cDNA encoding the 20-kD subunit of coatomer, zeta-COP, predicts a protein of 177-amino acid residues, similar in sequence to AP17 and AP19, subunits of the clathrin adaptor complexes. Polyclonal antibody directed to zeta-COP blocks the binding of coatomer to Golgi membranes and prevents the assembly of COP-coated vesicles on Golgi cisternae. Unlike other coatomer subunits (beta-, beta'-, gamma-, and epsilon-COP), zeta-COP exists in both coatomer bound and free pools.

Amino Acid Sequence↗
PubMed1994

En bloc incorporation of coatomer subunits during the assembly of COP-coated vesicles.

The cDNA encoding epsilon-COP, the 36-kD subunit of coatomer, was cloned from a bovine liver cDNA library and sequenced. Immunoblotting with an anti-epsilon-COP antibody showed that epsilon-COP exists in COP-coated vesicles as well as in the cytosolic coatomer. Using the cloned cDNA, recombinant His6- tagged epsilon-COP was overexpressed in cultured Chinese hamster ovary (CHO) cells, from which metabolically radiolabeled coatomer was purified by taking advantage of the His6 tag. Radiolabeled coatomer was employed to establish that all the subunits of the coatomer enter coated vesicles as an intact unit.

Amino Acid Sequence↗
PubMed1991

ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: a novel role for a GTP-binding protein.

ADP-ribosylation factor (ARF) is an abundant and highly conserved low molecular weight GTP-binding protein that was originally identified as a key element required for the action of cholera toxin in mammalian cells, but whose physiological role is unknown. We report that ARF family proteins are highly concentrated in non-clathrin-coated transport vesicles and are coat proteins. About three copies of ARF are present on the outside of coated vesicles per alpha-COP (and thus per coatomer). ARF is highly enriched in coated vesicles as compared with parental Golgi cisternae, as shown both by biochemical and morphological methods, and ARF is removed from transport vesicles through uncoating during transport. Furthermore, ARF binds to Golgi cisternae in a GTP-dependent manner independently of coated vesicle budding. These observations strongly suggest a new role for GTP-binding proteins: ARF proteins may modulate vesicle budding and uncoating through controlled GTP hydrolysis.

ADP-Ribosylation Factors↗
PubMed1993

Stepwise assembly of functionally active transport vesicles.

Budding of COP-coated vesicles (the likely carriers of newly synthesized proteins from the endoplasmic reticulum through the Golgi stack) from Golgi cisternae requires ADP-ribosylation factor (ARF), coatomer proteins from the cytosol, GTP, and fatty acyl-coenzyme A (CoA). The assembly of coated buds on the membranes requires coatomer, ARF, and GTP. When palmitoyl-CoA is added, membrane fission occurs at the coated bud, releasing coated vesicles. We show that COP-coated vesicles can be generated stepwise in vitro and isolated in a functionally active form, demonstrating that the minimal set of cytosolic components required for their formation as well as principal steps in their assembly have been identified.

ADP-Ribosylation Factors↗
PubMed1995

Mitotic disassembly of the Golgi apparatus in vivo.

Populations enriched in prophase cells were obtained either by using a cell line with a temperature-sensitive mutation in the mitotic kinase, p34cdc2, or by treating cells with olomoucine, an inhibitor of this kinase. Both methods resulted in efficient and reversible block of the cells at the G2/M boundary. After cells were released from the cell cycle block, the morphological changes to the Golgi apparatus were characterised using both quantitative conventional electron microscopy and immuno-gold microscopy. The early mitotic phases were divided into six stages (G2 to pro-metaphase) based on the morphology of the nucleus. During prophase the cross-sectional length of Golgi stacks decreased prior to unstacking. At the same time, small vesicular profiles, typically 50-70 nm in diameter, accumulated in the vicinity of the stacks. The disappearance of Golgi stacks was accompanied by the transient appearance of tubular networks. By the time cells entered prometaphase, the stacks had completely disassembled and only clusters consisting of Golgi vesicles and short tubular elements were left. When cells were released from the G2/M boundary and pulsed briefly with [AlF4]- to prevent uncoating of transport vesicles, vesicular profiles with a morphology reminiscent of COP-coated vesicles appeared. These vesicular profiles were either associated with Golgi stacks or, at later stages, with clusters, but were formed at all stages of disassembly. Together these results provide further support for our model that continued budding of vesicles from the rims of Golgi cisternae is at least partly responsible for the disassembly of the Golgi apparatus.

Animals↗
PubMed1994

Coat proteins in intracellular membrane transport.

Transport of newly synthesized material from the endoplasmic reticulum (ER) towards the Golgi complex, through the Golgi cisternae, and out of the trans-Golgi network (TGN) is thought to be mediated by vesicular carriers. Different types of vesicle are involved in this biosynthetic membrane traffic. All are coated with protein complexes on their cytoplasmic surface. COP-coated vesicles have recently been implicated in transport of cargo from the ER to the TGN, and clathrin-coated vesicles from the TGN to endosomes, but the carriers moving material to the cell surface are still unknown. Sequence homologies between subunits of the COP- and the clathrin-adaptor complexes suggest that coat proteins may belong to a family of proteins with related functions. The precise role of the coat proteins is not fully understood, although they have been implicated in clustering of cargo into buds and in budding of vesicles. In addition, coat proteins may play an essential role in targeting of transport intermediates and may serve to regulate membrane fusion.

Animals↗
PubMed1995

Cytotoxicity of brefeldin A correlates with its inhibitory effect on membrane binding of COP coat proteins.

The fungal metabolite brefeldin A (BFA) causes the inhibition of protein secretion and the disruption of the structure and function of organelles along the exocytic and endocytic pathways including the Golgi complex. Such effects of BFA have been ascribed in large part to its ability to prevent recruitment of cytosolic coat proteins onto organelle membranes. Here we show that mammalian cell lines differ from one another with respect to sensitivity to this drug. The BFA sensitivity of a given cell line appears to be dependent on the species or the order from which the cell line originates, rather than on the cell line itself. In each cell line, the dose of BFA required for inhibition of cell growth and of protein secretion correlates with the dose required for inhibition of binding of beta-COP, a coat protein of COP-coated vesicles, but not that for inhibition of binding of gamma-adaptin, a component of HA-I/AP-1 adaptor of clathrin-coated vesicles. These observations suggest that: (i) there are at least two targets for BFA that differ from each other in sensitivity to this drug, (ii) the difference in the sensitivity to BFA of the beta-COP binding is determined by the difference in the structure of a target protein for this drug, and (iii) the cytotoxicity of BFA is ascribed mainly to its inhibitory effect on the membrane binding of COP-coat proteins.

3T3 Cells↗
PubMed1994

ADP-ribosylation factor and coatomer couple fusion to vesicle budding.

The coat proteins required for budding COP-coated vesicles from Golgi membranes, coatomer and ADP-ribosylation factor (ARF) protein, are shown to be required to reconstitute the orderly process of transport between Golgi cisternae in which fusion of transport vesicles begins only after budding ends. When either coat protein is omitted, fusion is uncoupled from budding-donor and acceptor compartments pair directly without an intervening vesicle. Coupling may therefore results from the sequestration of fusogenic membrane proteins into assembling coated vesicles that are only exposed when the coat is removed after budding is complete. This mechanism of coupling explains the phenomenon of "retrograde transport" triggered by uncouplers such as the drug brefeldin A.

ADP-Ribosylation Factors↗
PubMed1995

Immunocytochemical localization of beta-COP to the ER-Golgi boundary and the TGN.

Recent data strongly suggest that the coatomer (COP) complex is involved in membrane transport between the ER and Golgi complex. This vesicular coat has been implicated in ER to Golgi, in intra Golgi as well as in Golgi to ER traffic. In this study we present a detailed immunocytochemical analysis of the distribution of beta-COP in different tissue culture cells. Our results extend previous studies by showing, using electron microscopy, that beta-COP accumulates on vesicular profiles and buds in the intermediate compartment (IC) under conditions that block ER to Golgi transport (15 degrees C). Importantly, under these conditions beta-COP co-localizes on these structures with a passenger protein, the membrane glycoprotein of vesicular stomatis virus (ts-O45-G). Furthermore, quantitative immunofluorescence microscopy of cells with ts-045-G accumulated in the ER, IC and trans-Golgi network, shifted briefly to the permissive temperature, showed that beta-COP was associated with many of the putative transport intermediates containing the viral glycoprotein which is in transit between the ER/IC and the cis-Golgi. The simplest interpretation of these data is that COP-coated vesicles are involved in anterograde transport of ts-045-G from the IC to the Golgi complex. Since many putative COP vesicle lacked the G protein following release of the 15 degrees C block this pool could be involved in retrograde transport. We also show that beta-COP is present on the membranes of the trans-Golgi network. However, in contrast to the ER-Golgi boundary, we could find no convincing evidence that this pool of beta-COP is associated with buds or trans-Golgi network-derived transport vesicles.

Animals↗
PubMed1995

Yeast SEC16 gene encodes a multidomain vesicle coat protein that interacts with Sec23p.

Temperature-sensitive mutations in the SEC16 gene of Saccharomyces cerevisiae block budding of transport vesicles from the ER. SEC16 was cloned by complementation of the sec16-1 mutation and encodes a 240-kD protein located in the insoluble, particulate component of cell lysates. Sec16p is released from this particulate fraction by high salt, but not by nonionic detergents or urea. Some Sec16p is localized to the ER by immunofluorescence microscopy. Membrane-associated Sec16p is incorporated into transport vesicles derived from the ER that are formed in an in vitro vesicle budding reaction. Sec16p binds to Sec23p, a COPII vesicle coat protein, as shown by the two-hybrid interaction assay and affinity studies in cell extracts. These findings indicate that Sec16p associates with Sec23p as part of the transport vesicle coat structure. Genetic analysis of SEC16 identifies three functionally distinguishable domains. One domain is defined by the five temperature-sensitive mutations clustered in the middle of SEC16. Each of these mutations can be complemented by the central domain of SEC16 expressed alone. The stoichiometry of Sec16p is critical for secretory function since overexpression of Sec16p causes a lethal secretion defect. This lethal function maps to the NH2-terminus of the protein, defining a second functional domain. A separate function for the COOH-terminal domain of Sec16p is shown by its ability to bind Sec23p. Together, these results suggest that Sec16p engages in multiple protein-protein interactions both on the ER membrane and as part of the coat of a completed vesicle.

Alleles↗
PubMed1995

COPII: a membrane coat that forms endoplasmic reticulum-derived vesicles.

Vesicle budding from the endoplasmic reticulum (ER) has been reconstituted with washed membranes and three soluble proteins: Sec13 complex, Sec23 complex and the small GTPase Sar1p. The proteins that drive this cell-free vesicle budding reaction form an approximately 10 nm thick electron dense coat on ER-derived vesicles. Although the overall mechanism of membrane budding driven by various cytoplasmic coats appears similar, the constituents of this new membrane coat are molecularly distinct from the non-clathrin coat (COP) involved in intra-Golgi transport and the clathrin-containing coats. The new vesicle coat has been termed COPII.

Biological Transport↗
PubMed1994

COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum.

In vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex, and Sec23p complex). Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP. However, GMP-PNP vesicles fail to target and fuse with the Golgi complex whereas GTP vesicles are functional. All the cytosolic proteins required for vesicle formation are retained on GMP-PNP vesicles, while Sar1p dissociates from GTP vesicles. Thin section electron microscopy of purified preparations reveals a uniform population of 60-65 nm vesicles with a 10 nm thick electron dense coat. The subunits of this novel coat complex are molecularly distinct from the constituents of the nonclathrin coatomer involved in intra-Golgi transport. Because the overall cycle of budding driven by these two types of coats appears mechanistically similar, we propose that the coat structures be called COPI and COPII.

Base Sequence↗