Coatomer Complex Research Articles

Afadin- and α-actinin-binding protein ADIP directly binds β′-COP, a subunit of the coatomer complex

Afadin DIL domain-interacting protein (ADIP) is a novel protein that binds both afadin and α-actinin and localizes at adherens junctions, which are formed by nectins and cadherins, cell–cell adhesion molecules. Afadin is an actin filament (F-actin)-binding protein which connects nectins to the actin cytoskeleton. α-Actinin is another F-actin-binding protein that is indirectly associated with cadherins through the catenin complex. ADIP is at least partly involved in the physical association of nectins and cadherins. We show here that ADIP furthermore binds β′-COP, a subunit of the coatomer complex. ADIP co-localizes with β′-COP at the Golgi complex in Madin Darby canine kidney and normal rat kidney cells. These results suggest that ADIP is involved in vesicle trafficking from the Golgi to the endoplasmic reticulum and through the Golgi complex by interacting with the coatomer complex.

Sorting signals in the cytosolic tail of membrane proteins involved in the interaction with plant ARF1 and coatomer.

In mammals and yeast, a cytosolic dilysine motif is critical for endoplasmic reticulum (ER) localization of type I membrane proteins. Retrograde transport of type I membrane proteins containing dilysine motifs at their cytoplasmic carboxy (C)-terminal tail involves the interaction of these motifs with the COPI coat. The C-terminal dilysine motif has also been shown to confer ER localization to type I membrane proteins in plant cells. Using in vitro binding assays, we have analyzed sorting motifs in the cytosolic tail of membrane proteins, which may be involved in the interaction with components of the COPI coat in plant cells. We show that a dilysine motif in the -3,-4 position (relative to the cytosolic C-terminus) recruits in a very specific manner all the subunits of the plant coatomer complex. Lysines cannot be replaced by arginines or histidines to bind plant coatomer. A diphenylalanine motif in the -7,-8 position, which by itself has a low ability to bind plant coatomer, shows a clear cooperativity with the dilysine motif. Both dilysine and diphenylalanine motifs are present in the cytosolic tail of several proteins of the p24 family of putative cargo receptors, which has several members in plant cells. The cytosolic tail of a plant p24 protein is shown to recruit not only coatomer but also ADP ribosylation factor 1 (ARF1), a process which depends on both dilysine and diphenylalanine motifs. ARF1 binding increases twofold upon treatment with brefeldin A (BFA) and is completely abolished upon treatment with GTPgammaS, suggesting that ARF1 can only interact with the cytosolic tail of p24 proteins in its GDP-bound form.

Novel isotypic gamma/zeta subunits reveal three coatomer complexes in mammals.

In early secretory transport, coat recruitment for the formation of coat protein I (COPI) vesicles involves binding to donor Golgi membranes of the small GTPase ADP-ribosylation factor 1 and subsequent attachment of the cytoplasmic heptameric complex coatomer. Various hypotheses exist as to the precise role of and possible routes taken by COPI vesicles in the mammalian cell. Here we report the ubiquitous expression of two novel isotypes of coatomer subunits gamma- and zeta-COP that are incorporated into coatomer, and show that three isotypes exist of the complex defined by the subunit combinations gamma 1/zeta 1, gamma 1/zeta 2, and gamma 2/zeta 1. In a liver cytosol, these forms make up the total coatomer in a ratio of about 2:1:2, respectively. The coatomer isotypes are located differentially within the early secretory pathway, and the gamma 2/zeta 1 isotype is preferentially incorporated into COPI vesicles. A population of COPI vesicles was characterized that almost exclusively contains gamma 2/zeta 1 coatomer. This existence of three structurally different forms of coatomer will need to be considered in future models of COPI-mediated transport.

Gamma-COP appendage domain - structure and function.

COPI-coated vesicles mediate retrograde transport from the Golgi back to the ER and intra-Golgi transport. The cytosolic precursor of the COPI coat, the heptameric coatomer complex, can be thought of as composed of two subcomplexes. The first consists of the beta-, gamma-, delta- and zeta-COP subunits which are distantly homologous to AP clathrin adaptor subunits. The second consists of the alpha-, beta'- and epsilon-COP subunits. Here, we present the structure of the appendage domain of gamma-COP and show that it has a similar overall fold as the alpha-appendage of AP2. Again, like the alpha-appendage the gamma-COP appendage possesses a single protein/protein interaction site on its platform subdomain. We show that in yeast this site binds to the ARFGAP Glo3p, and in mammalian gamma-COP this site binds to a Glo3p orthologue, ARFGAP2. On the basis of mutations in the yeast homologue of gamma-COP, Sec21p, a second binding site is proposed to exist on the gamma-COP appendage that interacts with the alpha,beta',epsilon COPI subcomplex.

The COPI Complex Functions in Nuclear Envelope Breakdown and Is Recruited by the Nucleoporin Nup153

Nuclear envelope breakdown is a critical step in the cell cycle of higher eukaryotes. Although integral membrane proteins associated with the nuclear membrane have been observed to disperse into the endoplasmic reticulum at mitosis, the mechanisms involved in this reorganization remain to be fully elucidated. Here, using Xenopus extracts, we report a role for the COPI coatomer complex in nuclear envelope breakdown, implicating vesiculation as an important step. We have found that a nuclear pore protein, Nup153, plays a critical role in directing COPI to the nuclear membrane at mitosis and that this event provides feedback to other aspects of nuclear disassembly. These results provide insight into how key steps in nuclear division are orchestrated.

Bicaudal-D regulates COPI-independent Golgi–ER transport by recruiting the dynein–dynactin motor complex

The small GTPase Rab6a is involved in the regulation of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER) in a coat complex coatomer protein I (COPI)-independent pathway. Here, we used a yeast two-hybrid approach to identify binding partners of Rab6a. In particular, we identified the dynein-dynactin-binding protein Bicaudal-D1 (BICD1), one of the two mammalian homologues of Drosophila Bicaudal-D. BICD1 and BICD2 colocalize with Rab6a on the trans-Golgi network (TGN) and on cytoplasmic vesicles, and associate with Golgi membranes in a Rab6-dependent manner. Overexpression of BICD1 enhances the recruitment of dynein-dynactin to Rab6a-containing vesicles. Conversely, overexpression of the carboxy-terminal domain of BICD, which can interact with Rab6a but not with cytoplasmic dynein, inhibits microtubule minus-end-directed movement of green fluorescent protein (GFP)-Rab6a vesicles and induces an accumulation of Rab6a and COPI-independent ER cargo in peripheral structures. These data suggest that coordinated action between Rab6a, BICD and the dynein-dynactin complex controls COPI-independent Golgi-ER transport.

Subunit H of the V-ATPase involved in endocytosis shows homology to beta-adaptins.

The vacuolar ATPase (V-ATPase) is a multisubunit enzyme that facilitates the acidification of intracellular compartments in eukaryotic cells and plays an important role in receptor-mediated endocytosis, intracellular trafficking processes, and protein degradation. In this study we show that the C-terminal fragment of 350 residues of the regulatory subunit H (V1H) of the V-ATPase shares structural and functional homologies with the beta-chains of adaptor protein complexes. Moreover, the fragment is similar to a region in the beta-subunit of COPI coatomer complexes, which suggests the existence of a shared domain in these three different families of proteins. For beta-adaptins, this fragment binds to cytoplasmic di-leucine-based sorting motifs such as in HIV-1 Nef that mediate endocytic trafficking. Expression of this fragment in cells blocks the internalization of transmembrane proteins, which depend on di-leucine-based motifs, whereas mutation of the consensus sequence GEY only partly diminishes the recognition of the sorting motif. Based on recent structural analysis, our results suggest that the di-leucine-binding domain consists of a HEAT or ARM repeat protein fold.

Interaction of γ-COP with a transport motif in the D1 receptor C-terminus

Truncations at the carboxyl termini of G protein-coupled receptors result in defective receptor biogenesis and comprise a number of inherited disorders. In order to evaluate the structural role of the C-terminus in G protein-coupled receptor biogenesis, we generated a series of deletion and substitution mutations in the dopamine D1 receptor and visualized receptor subcellular localization by fusion to a green fluorescent protein. Alanine substitutions of several hydrophobic residues within the proximal C-terminus resulted in receptor transport arrest in the ER. Agonist binding and coupling to adenylyl cyclase was also abolished. In contrast, substitutions conserving C-terminal hydrophobicity produced normal cell surface receptor expression, binding, and stimulatory function. A mechanism for the role of the C-terminus in D1 receptor transport was investigated by searching for candidate protein interactions. The D1 receptor was found to co-precipitate and associate in vitro directly with the gamma-subunit of the COPI coatomer complex. In vitro pull-down assays confirmed that only the D1 C-terminus is required for COPI association, and that identical mutations causing disruption of receptor transport to the cell surface also disrupted binding to COPI. Furthermore, conservative mutations in the D1 C-terminus restored COPI association just as they restored cell surface transport. These results suggest that association between the coatomer complex and hydrophobic residues within the proximal C-terminus of the D1 receptor may serve an important role in receptor transport.

Regulation of GTP Hydrolysis on ADP-ribosylation Factor-1 at the Golgi Membrane

The interaction of the coatomer coat complex with the Golgi membrane is initiated by the active, GTP-bound state of the small GTPase ADP-ribosylation factor 1 (ARF1), whereas GTP hydrolysis triggers coatomer dissociation. The hydrolysis of GTP on ARF1 depends on the action of members of a family of ARF1-directed GTPase-activating proteins (GAPs). Previous studies in well defined systems indicated that the activity of a mammalian Golgi membrane-localized ARF GAP (GAP1) might be subjected to regulation by membrane lipids as well as by the coatomer complex. Coatomer was found to strongly stimulate GAP-dependent GTP hydrolysis on a membrane-independent mutant of ARF1, whereas we reported that GTP hydrolysis on wild type, myristoylated ARF1 loaded with GTP in the presence of phospholipid vesicles was coatomer-independent. To investigate the regulation of ARF1 GAPs under more physiological conditions, we studied GTP hydrolysis on Golgi membrane-associated ARF1. The activities at the Golgi of recombinant GAP1 as well as coatomer-depleted fractions from rat brain cytosol resembled those observed in the presence of liposomes; however, unlike in liposomes, GAP activities on Golgi membranes were approximately doubled upon addition of coatomer. By contrast, endogenous GAP activity in Golgi membrane preparations was unaffected by coatomer. Cytosolic GAP activity was partially reduced following immunodepletion of GAP1, indicating that GAP1 plays a significant although not exclusive role in the regulation of GTP hydrolysis at the Golgi. Unlike the activities of the mammalian proteins, the Saccharomyces cerevisiae Glo3 ARF GAP displayed activity at the Golgi that was highly dependent on coatomer. We conclude that ARF GAPs in themselves can efficiently stimulate GTP hydrolysis on ARF1 at the Golgi, and that coatomer may play an auxiliary role in this reaction, which would lead to an increased cycling rate of ARF1 in COPI-coated regions of the Golgi membrane.

The coatomer of Trypanosoma brucei

Coatomer is a multisubunit complex involved in trafficking of vesicles between the endoplasmatic reticulum and the Golgi apparatus. From sequence homologies, all seven subunits, α-, β-, β′-, γ-, δ-, ε-, and ζ-COP, are encoded in the genome of Trypanosoma brucei. The complete predicted amino-acid sequences of β-, β′-, and ζ-COP show only 20–30% identity with higher eucaryotic homologues. The trypanosome coatomer complex was partially purified using a procedure similar to that used for bovine coatomer.

Overexpression of rat neuronal calcium sensor-1 in rodent NG108-15 cells enhances synapse formation and transmission.

The role of rat neuronal calcium sensor-1 (NCS-1), a Ca2+-binding protein, in synapse formation and transmitter release was examined in mouse neuroblastoma x rat glioma hybrid NG108-15 cells in culture. Wild-type NG108-15 cells expressed rodent NCS-1. Endogenous NCS-1 was partially co-localized with the synaptic protein SNAP-25 at the plasma membrane in both cell bodies and processes, but not with the Golgi marker [beta]-COP, an individual coat subunit of the coatomer complex present on Golgi-derived vesicles. In NG108-15 cells co-cultured with rat myotubes, partial co-localization of SNAP-25 and NCS-1 was observed at the plasma membrane of neurites and growth cones, some of which had synaptic contacts to muscle cells. Transient co-transfection of the rat NCS-1 cDNA and green fluorescent protein (GFP) resulted in NCS-1 overexpression in about 30 % of the cells as determined by fluorescence microscopy. The rate of functional synapse formation with co-cultured rat myotubes increased 2-fold as determined by the presence of miniature endplate potentials (MEPPs) in NCS-1-overexpressing NG108-15 cells compared to non- and mock-transfected cells. The number of neurites per cell, branches per neurite and length of neurites was slightly less in cells that were either transiently transfected (GFP-NCS-1-fluorescence positive) or stably transformed with NCS-1 compared to GFP-NCS-1-negative, non-transfected or mock-transfected NG108-15 cells. The number of action potentials that elicited endplate potentials increased in NG108-15 cells stably transformed with rat NCS-1. The mean number of quanta per impulse (m) increased 5-fold. These results show that NCS-1 functions to facilitate synapse formation, probably because of the increased quantal content of evoked acetylcholine release.

Rer1p, a Retrieval Receptor for Endoplasmic Reticulum Membrane Proteins, Is Dynamically Localized to the Golgi Apparatus by Coatomer

Rer1p, a yeast Golgi membrane protein, is required for the retrieval of a set of endoplasmic reticulum (ER) membrane proteins. We present the first evidence that Rer1p directly interacts with the transmembrane domain (TMD) of Sec12p which contains a retrieval signal. A green fluorescent protein (GFP) fusion of Rer1p rapidly cycles between the Golgi and the ER. Either a lesion of coatomer or deletion of the COOH-terminal tail of Rer1p causes its mislocalization to the vacuole. The COOH-terminal Rer1p tail interacts in vitro with a coatomer complex containing alpha and gamma subunits. These findings not only give the proof that Rer1p is a novel type of retrieval receptor recognizing the TMD in the Golgi but also indicate that coatomer actively regulates the function and localization of Rer1p.

Dsl1p, an essential protein required for membrane traffic at the endoplasmic reticulum/Golgi interface in yeast.

To identify novel factors required for ER to Golgi transport in yeast we performed a screen for genes that, when mutated, confer a dependence on a dominant mutant form of the ER to Golgi vesicle docking factor Sly1p, termed Sly1-20p. DSL1, a novel gene isolated in the screen, encodes an essential protein with a predicted molecular mass of 88 kDa. DSL1 is required for transport between the ER and the Golgi because strains bearing mutant alleles of this gene accumulate the pre-Golgi form of transported proteins at the restrictive temperature. Two strains bearing temperature-sensitive alleles of DSL1 display distinct phenotypes as observed by electron microscopy at the restrictive temperature; although both strains accumulate ER membrane, one also accumulates vesicles. Interestingly, the inviability of strains bearing several mutant alleles of DSL1 can be suppressed by expression of either Erv14p (a protein required for the movement of a specific protein from the ER to the Golgi), Sec21p (the gamma-subunit of the COPI coat protein complex coatomer), or Sly1-20p. Because the strongest suppressor is SEC21, we proposed that Dsl1p functions primarily in retrograde Golgi to ER traffic, although it is possible that Dsl1p functions in anterograde traffic as well, perhaps at the docking stage, as suggested by the suppression by SLY1-20.

The γ-subunit of the coatomer complex binds Cdc42 to mediate transformation

The Ras-related GTP-binding protein Cdc42 is implicated in a variety of biological activities including the establishment of cell polarity in yeast, the regulation of cell morphology, motility and cell-cycle progression in mammalian cells and the induction of malignant transformation. We identified a Cdc42 mutant (Cdc42F28L) which binds GTP in the absence of a guanine nucleotide exchange factor, but still hydrolyses GTP with a turnover number identical to that for wild-type Cdc42. Expression of this mutant in NIH 3T3 fibroblasts causes cellular transformation, mimicking many of the characteristics of cells transformed by the Dbl oncoprotein, a known guanine nucleotide exchange factor for Cdc42. Here we searched for new Cdc42 targets in an effort to understand how Cdc42 mediates cellular transformation. We identified the gamma-subunit of the coatomer complex (gammaCOP) as a specific binding partner for activated Cdc42. The binding of Cdc42 to gammaCOP is essential for a transforming signal distinct from those elicited by Ras.

Selecting sorting signals

During the creation of a COPI transport vesicle, GTP–ARF1, a member of the RAS superfamily, binds to the donor membrane and recruits the coatomer complex to it. The coatomer complex binds to the donor membrane, inducing the formation of the vesicle. Once the vesicle has been formed, there is a stimulation of the GTPase activity on the ARF1 molecule both by coatomer and the ARF1 GTPase-activating protein (GAP), resulting in the dissociation of ARF1 and coatomer from the vesicle. This exposes the targeting proteins that are responsible for the recognition of the acceptor compartment by the transport vesicle. Increasing evidence has accumulated for a model in which the cargo to be transported interacts specifically with the coat proteins involved in creating the vesicle, thereby incorporating that cargo into the vesicle. However, many transported cargo molecules as well as resident nontransported molecules, despite having different sorting signals, are able to compete for the same binding site on the coatomer complex. How then can two proteins competing for the same binding site on the coat protein result in the selective incorporation of only one of the molecules and the exclusion of the molecule to remain in the donor organelle? Jonathan Goldberg presents data in this paper that suggest a very provocative model for how two competing signals can be deciphered by the coat proteins and result in the selective incorporation of one protein over another 1 Goldberg J. Decoding of sorting signals by coatomer through a GTPase switch in the COPI coat complex. Cell. 2000; 100: 671-679 Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar .

Role of COPI in Phagosome Maturation

Phagosomes mature by sequentially fusing with endosomes and lysosomes. Vesicle budding is presumed to occur concomitantly, mediating the retrieval of plasmalemmal components and the regulation of phagosomal size. We analyzed whether fission of vesicles from phagosomes requires COPI, a multimeric complex known to be involved in budding from the Golgi and endosomes. The role of COPI was studied using ldlF cells, that harbor a temperature-sensitive mutation in epsilon-COP, a subunit of the coatomer complex. These cells were made phagocytic toward IgG-opsonized particles by heterologous expression of human FcgammaRIIA receptors. Following incubation at the restrictive temperature, epsilon-COP was degraded in these cells and their Golgi complex dispersed. Nevertheless, phagocytosis persisted for hours in cells devoid of epsilon-COP. Retrieval of transferrin receptors from phagosomes became inefficient in the absence of epsilon-COP, while clearance of the FcgammaRIIA receptors was unaffected. This indicates that fission of vesicles from the phagosomal membrane involves at least two mechanisms, one of which requires intact COPI. Traffic of fluid-phase markers and aggregated IgG-receptor complexes along the endocytic pathway was abnormal in epsilon-COP-deficient cells. In contrast, phagosome fusion with endosomes and lysosomes was unimpaired. Moreover, the resulting phagolysosomes were highly acidic. Similar results were obtained in RAW264.7 macrophages treated with brefeldin A, which precludes COPI assembly by interfering with the activation of adenosine ribosylation factor. These data indicate that neither phagosome formation nor maturation are absolutely dependent on COPI. Our findings imply that phagosomal maturation differs from endosomal progression, which appears to be more dependent on COPI-mediated formation of carrier vesicles.

Gamma2-COP, a novel imprinted gene on chromosome 7q32, defines a new imprinting cluster in the human genome.

We describe a novel imprinted gene, gamma 2-COP (nonclathrincoatprotein), identified in a search for expressed sequences in human chromosome 7q32 where the paternally expressed MEST gene is located. gamma 2-COP contains 24 exons and spans >50 kb of genomic DNA. Like MEST, gamma 2-COP is ubiquitously transcribed in fetal and adult tissues. In fetal tissues, including skeletal muscle, skin, kidney, adrenal, placenta, intestine, lung, chorionic plate and amnion, gamma 2-COP is imprinted and expressed from the paternal allele. In contrast to the monoallelic expression observed in these fetal tissues, biallelic expression was evident in fetal brain and liver and in adult peripheral blood. Biallelic expression in blood is supported by the demonstration of gamma 2-COP transcripts in lymphoblastoid cell lines with maternal uniparental disomy 7. Absence of paternal gamma 2-COP transcripts during embryonic development may contribute to Silver-Russell syndrome. However, on mutation scanning the only gamma 2-COP mutation detected was maternally derived. Amino acid comparison of gamma2-COP protein revealed close relation to gamma-COP, a subunit of the coatomer complex COPI, suggesting a role of gamma2-COP in cellular vesicle traffic. The existence of distinct coatomer complexes could be the basis for the functional heterogeneity of COPI vesicles in retrograde and anterograde transport and/or in cargo selection. Together, gamma 2-COP and MEST constitute a novel imprinting cluster in the human genome that may contain other, as yet unknown, imprinted genes.

Defective expression of the mu3 subunit of the AP-3 adaptor complex in the Drosophila pigmentation mutant carmine.

The adaptor protein (AP) complexes AP-1, AP-2, and AP-3 mediate coated vesicle formation and sorting of integral membrane proteins in the endocytic and late exocytic pathways in mammalian cells. A search of the Drosophila melanogaster expressed sequence tag (EST) database identified orthologs of family members mammalian medium (mu) chain families mu1, mu2, and mu3, of the corresponding AP complexes, and delta-COP, the analogous component of the coatomer (COPI) complex. The Drosophila orthologs exhibit a high degree of sequence identity to mammalian medium chain and delta-COP proteins. Northern analysis demonstrated that medium chain and delta-COP mRNAs are expressed uniformly throughout fly development. Medium chain and delta-COP genes were cytologically mapped and the mu3 gene was found to localize to a region containing the pigmentation locus carmine (cm). Analysis of genomic DNA of the cm1 mutant allele indicated the presence of a large insertion in the coding region of the mu3 gene and Northern analysis revealed no detectable mu3 mRNA. Light microscopy of the cm1 mutant showed a reduction in primary, secondary, and tertiary pigment granules in the adult eye. These findings provide evidence of a role for mu3 in the sorting processes required for pigment granule biogenesis in Drosophila.

The Cytoplasmic Coatomer Protein COPI: A POTENTIAL TRANSLATIONAL REGULATOR

Expression of the asialoglycoprotein receptor (ASGR) by the human hepatocellular carcinoma cell lines HepG2 and HuH-7 in response to intracellular cGMP concentrations was previously shown to be regulated at the translational level (1). Stable transfection of COS-7 cells with deletion constructs encoding the asialoglycoprotein receptor H2b subunit localized the cGMP-responsive cis-acting element to the mRNA 5'-untranslated region. Resolution by anion exchange chromatography of an S-100 isolated from human liver resulted in the partial purification of an RNA-binding protein specific to this cis-acting element. Northwestern analysis using the 5'-untranslated region as probe indicated that a 140-kDa protein was the potential RNA-binding protein. Sequence of tryptic peptides suggested that the 140-kDa protein was the alpha-COP subunit of coatomer protein COPI, usually associated with trans-Golgi network membrane traffic. Immunoblot analysis confirmed the presence of alpha-COP in the Mono-Q fraction as well as that of a second coatomer subunit, beta-COP. Antibody induced gel retardation supershift confirmed the identification of the RNA-binding proteins as alpha- and beta-COP. Although the RNA recognition motif appears to reside solely in alpha-COP, antibody-induced supershift strongly indicated that the entire coatomer complex was the trans-acting factor. Depletion of S-100 with the antibody to beta-COP confirmed that the coatomer was the sole protein binding to the ASGR mRNA 5'-untranslated region in liver cytosol and responsible for inhibition of in vitro translation of the asialoglycoprotein receptor.

COPI proteins: A model for their role in vesicle budding

COPI-coated vesicles are involved in intracellular trafficking between the endoplasmic reticulum and the Golgi complex. In the current model for COPI assembly the small GTP-binding protein ADP-ribosylation factor 1 is recruited from the cytoplasm to the Golgi membrane followed by binding of the hetero-oligomeric protein complex coatomer. However, the mechanism of subsequent vesicle budding is discussed controversially. This review summarizes the available experimental data on the COPI coat and discusses a model of how the major coat protein, coatomer, might act in vesicle budding.