Cytoplasmic Coat Research Articles

Impact of live cell imaging on coated vesicle research

The role of membrane traffic is to transfer cargo between distinct subcellular compartments. Each individual trafficking event involves the creation, transport and fusion of vesicular and tubular carriers that are formed and regulated via cytoplasmic coat protein complexes. The dynamic nature of this process is therefore highly suitable for studying using live cell imaging techniques. Although these approaches have raised further questions for the field, they have also been instrumental in providing essential new information, in particular relating to the morphology of transport carriers and the exchange kinetics of coat proteins and their regulators on membranes. Here, we present an overview of live cell-imaging experiments that have been used in the study of coated-vesicle transport, and provide specific examples of their impact on our understanding of coat function.

Molecular mechanisms of COPII vesicle formation

The first step in protein secretion from eukaryotic cells is mediated by COPII vesicles, known for the cytoplasmic coat proteins that are the minimal machinery required to generate these small transport carriers. The five COPII coat components coordinate to create a vesicle by locally generating membrane curvature and populating the incipient bud with the appropriate cargo. This review describes the molecular details of how the COPII coat sculpts vesicles from the endoplasmic reticulum and highlights some unresolved questions regarding the regulation of this process in the complex environment of the eukaryotic cell.

HIV-1 Nef stabilizes AP-1 on membranes without inducing ARF1-independent de novo attachment

HIV-1 Nef affects the trafficking of numerous cellular proteins to optimize viral replication and evade host defenses. The adaptor protein (AP) complexes, which form part of the cytoplasmic coat of endosomal vesicles, are key cellular co-factors for Nef. Nef binds these complexes and alters their physiologic cycle of attachment and release from membranes. Specifically, while AP-1 normally becomes cytosolic when attachment events are blocked by inhibition of the GTPase cycle of ADP-ribosylation factor-1 (ARF1), the complex remains membrane-associated in Nef-expressing cells. To investigate the mechanism of this effect, we used a permeabilized cell system to detect the de novo attachment of exogenous AP-1 to endosomal membranes. Nef did not mediate de novo attachment independently of ARF1, despite its ability to maintain the association of AP-1 with endosomal membranes when the activity of ARF1 was blocked. We conclude that Nef stabilizes AP complexes on endosomal membranes after ARF1-dependent attachment. This stabilization may facilitate coat formation and stimulate the trafficking of multiple cellular proteins.

An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade bodies of endothelial cells

Clathrin provides an external scaffold to form small 50–100-nm transport vesicles. In contrast, formation of much larger dense-cored secretory granules is driven by selective aggregation of internal cargo at the trans-Golgi network; the only known role of clathrin in dense-cored secretory granules formation is to remove missorted proteins by small, coated vesicles during maturation of these spherical organelles. The formation of Weibel-Palade bodies (WPBs) is also cargo driven, but these are cigar-shaped organelles up to 5 μm long. We hypothesized that a cytoplasmic coat might be required to make these very different structures, and we found that new and forming WPBs are extensively, sometimes completely, coated. Overexpression of an AP-180 truncation mutant that prevents clathrin coat formation or reduced AP-1 expression by small interfering RNA both block WPB formation. We propose that, in contrast to other secretory granules, cargo aggregation alone is not sufficient to form immature WPBs and that an external scaffold that contains AP-1 and clathrin is essential.

Coated pits bring in the yolk

In the early 1960s, while analyzing electron micrographs of mosquito oocytes in search of hints about chromosomal structure, Thomas Roth's roving eye became more interested in the cell surface, which was covered with pit structures lined by a cytoplasmic coat of “bristles.” When he showed the

GTP/GDP exchange by Sec12p enables COPII vesicle bud formation on synthetic liposomes.

The generation of COPII vesicles from synthetic liposome membranes requires the minimum coat components Sar1p, Sec23/24p, Sec13/31p, and a nonhydrolyzable GTP analog such as GMP-PNP. However, in the presence of GTP and the full complement of coat subunits, nucleotide hydrolysis by Sar1p renders the coat insufficiently stable to sustain vesicle budding. In order to recapitulate a more authentic, GTP-dependent budding event, we introduced the Sar1p nucleotide exchange catalyst, Sec12p, and evaluated the dynamics of coat assembly and disassembly by light scattering and tryptophan fluorescence measurements. The catalytic, cytoplasmic domain of Sec12p (Sec12DeltaCp) activated Sar1p with a turnover 10-fold higher than the GAP activity of Sec23p stimulated by the full coat. COPII assembly was stabilized on liposomes incubated with Sec12DeltaCp and GTP. Numerous COPII budding profiles were visualized on membranes, whereas a parallel reaction conducted in the absence of Sec12DeltaCp produced no such profiles. We suggest that Sec12p participates actively in the growth of COPII vesicles by charging new Sar1p-GTP molecules that insert at the boundary between a bud and the surrounding endoplasmic reticulum membrane.

A novel cell-cell junction system: the cortex adhaerens mosaic of lens fiber cells.

The anucleate prismoid fiber cells of the eye lens are densely packed to form a tissue in which the plasma membranes and their associated cytoplasmic coat form a single giant cell-cell adhesive complex, the cortex adhaerens. Using biochemical and immunoprecipitation methods in various species (cow, pig, rat), in combination with immunolocalization microscopy, we have identified two different major kinds of cortical complex. In one, the transmembrane glycoproteins N-cadherin and cadherin-11 [which also occur in heterotypic ('mixed') complexes] are associated with alpha- and beta-catenin, plakoglobin (proportions variable among species), p120ctn and vinculin. The other complex contains ezrin, periplakin, periaxin and desmoyokin (and so is called the EPPD complex), usually together with moesin, spectrin(s) and plectin. In sections through lens fiber tissue, the short sides of the lens fiber hexagons appear to be enriched in the cadherin-based complexes, whereas the EPPD complexes also occur on the long sides. Moreover, high resolution double-label fluorescence microscopy has revealed, on the short sides, a finer, almost regular mosaicism of blocks comprising the cadherin-based, catenin-containing complexes, alternating with patches formed by the EPPD complexes. The latter, a new type of junctional plaque ensemble of proteins hitherto known only from certain other cell types, must be added to the list of major lens cortex proteins. We here discuss its possible functional importance for the maintenance of lens structure and functions, notably clear and sharp vision.

The AP-1 clathrin-adaptor is required for lysosomal enzymes sorting and biogenesis of the contractile vacuole complex in Dictyostelium cells.

Adaptor protein complexes (AP) are major components of the cytoplasmic coat found on clathrin-coated vesicles. Here, we report the molecular and functional characterization of Dictyostelium clathrin-associated AP-1 complex, which in mammalian cells, participates mainly in budding of clathrin-coated vesicles from the trans-Golgi network (TGN). The gamma-adaptin AP-1 subunit was cloned and shown to belong to a Golgi-localized 300-kDa protein complex. Time-lapse analysis of cells expressing gamma-adaptin tagged with the green-fluorescent protein demonstrates the dynamics of AP-1-coated structures leaving the Golgi apparatus and rarely moving toward the TGN. Targeted disruption of the AP-1 medium chain results in viable cells displaying a severe growth defect and a delayed developmental cycle compared with parental cells. Lysosomal enzymes are constitutively secreted as precursors, suggesting that protein transport between the TGN and lysosomes is defective. Although endocytic protein markers are correctly localized to endosomal compartments, morphological and ultrastructural studies reveal the absence of large endosomal vacuoles and an increased number of small vacuoles. In addition, the function of the contractile vacuole complex (CV), an osmoregulatory organelle is impaired and some CV components are not correctly targeted.

Cargo selection into COPII vesicles is driven by the Sec24p subunit.

Transport of secretory proteins out of the endoplasmic reticulum (ER) is mediated by vesicles generated by the COPII coat complex. In order to understand how cargo molecules are selected by this cytoplasmic coat, we investigated the functional role of the Sec24p homolog, Lst1p. We show that Lst1p can function as a COPII subunit independently of Sec24p on native ER membranes and on synthetic liposomes. However, vesicles generated with Lst1p in the absence of Sec24p are deficient in a distinct subset of cargo molecules, including the SNAREs, Bet1p, Bos1p and Sec22p. Consistent with the absence of any SNAREs, these vesicles are unable to fuse with Golgi membranes. Furthermore, unlike Sec24p, Lst1p fails to bind to Bet1p in vitro, indicating a direct correlation between cargo binding and recruitment into vesicles. Our data suggest that the principle role of Sec24p is to discriminate cargo molecules for incorporation into COPII vesicles.

Enter a novel COP I interactor: DSL1

Vesicular traffic in the secretory and endocytic pathways is mediated by the regulated assembly of cytoplasmic coat proteins on donor membranes. Once formed, transport vesicles dock at appropriate acceptor organelle membranes, the coat is shed and SNARE-mediated membrane fusion proceeds. To date, several coat proteins have been well characterized, including the hetero-tetrameric adaptor complexes (AP-1, AP-2, AP-3) and clathrin, and the COP I and COP II coats. COP I vesicles are involved in Golgi-to-endoplasmic reticulum and intra-Golgi retrograde traffic, COP II is in charge of ER-to-Golgi transport, and adaptors and clathrin mediate trafficking from the trans-Golgi complex and from the plasma membrane. All these coats are organized as multisubunit complexes, and their individual subunits are strictly conserved in evolution. In recent years, a large and growing number of auxiliary proteins have also been characterized that associate with subunits of the adaptors AP-1 and AP-2, or with clathrin, to effect cargo inclusion into coated vesicles or to regulate the vesicle budding event. Additionally, for some transport steps, ‘tethering factors’ have been identified that are required for vesicle delivery and thus contribute to the fidelity of fusion between membranes.

The GGAs pick up their coat

Intracellular transport in the secretory and endocytic pathways involves the regulated assembly of cytoplasmic coat proteins on appropriate donor membranes. ARF proteins, constituting a family of small GTPases, are crucial for the membrane recruitment of coat proteins such as COP I and the adaptor complexes AP-1, AP-3 and AP-4. ARFs cycle between a GDP-bound, cytosolic form and a GTP-bound, active form that is associated with membranes. The ARF GTP–GDP cycle is regulated by GDP–GTP exchange factors (GEFs) that load ARFs with GTP and by GTPase-activating proteins (GAPs) that induce hydrolysis of GTP bound to ARFs, but the mechanistic details of this regulation remain sketchy. GGAs (Golgi-associated, γ-adaptin homologous, ARF-interacting proteins) constitute a recently discovered family of conserved proteins that associate with membranes of the trans-Golgi network (TGN) and are implicated in regulating ARF-dependent trafficking events 1xThe GGAs promote ARF-dependent recruitment of clathrin to the TGN. Puertollano, R. et al. Cell. 2001; 105: 93–102Abstract | Full Text | Full Text PDF | PubMed | Scopus (185)See all References, 2xA family of proteins with γ-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome. Hirst, J. et al. J. Cell Biol. 2000; 149: 67–79Crossref | PubMed | Scopus (242)See all References, 3xA selective transport route from Golgi to late endosomes that requires the yeast GGA proteins. Black, M. and Pelham, H. J. Cell Biol. 2000; 151: 587–600Crossref | PubMed | Scopus (110)See all References. In yeast, deletion of both the genes encoding GGAs results in missorting of the vacuolar enzyme carboxypeptidase Y and of the yeast syntaxin Pep12p, and a defective vacuolar morphology phenotype 2xA family of proteins with γ-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome. Hirst, J. et al. J. Cell Biol. 2000; 149: 67–79Crossref | PubMed | Scopus (242)See all References, 3xA selective transport route from Golgi to late endosomes that requires the yeast GGA proteins. Black, M. and Pelham, H. J. Cell Biol. 2000; 151: 587–600Crossref | PubMed | Scopus (110)See all References.Mammalian and yeast GGAs have a modular structure, comprising a VHS, GAT, hinge and γ-adaptin ear domain. The GAT domain of the GGAs binds directly to the GTP-bound form of several members of the ARF family and is sufficient to target a GFP reporter protein to the Golgi complex. The VHS domain is implicated in interactions with vesicle cargo. Puertollano et al. have now performed a structure–function analysis of the GAT and hinge domains of GGAs, focusing on human GGA3 1xThe GGAs promote ARF-dependent recruitment of clathrin to the TGN. Puertollano, R. et al. Cell. 2001; 105: 93–102Abstract | Full Text | Full Text PDF | PubMed | Scopus (185)See all

Exiting the endoplasmic reticulum

The movement of nascent proteins from sites of synthesis to final cellular or extracellular destinations involves their transport through a distinct series of vesicular compartments. Vesicle biogenesis is regulated by specific proteins and co-factors that control distinct steps including budding, transport, docking, and fusion with target membranes. Budding requires assembly of a coat protein complex on the membrane, membrane deformation and the subsequent cleavage of the nascent vesicle from donor membrane. Coat proteins may also mediate vesicle interactions with the cytoskeleton or insulate the vesicles from fusion with unwanted compartments. Three classes of cytoplasmic coats have been identified. (1) Clathrin, interacting with different adaptor proteins, participates in endocytosis, lysosome biogenesis and as yet unidentified vesicular transport processes that arise in the trans-Golgi region of cells [reviewed in (Kreis, T.E., Lowe, M., Pepperkok, R., 1995. COPs regulating membrane traffic. Ann. Rev. Cell. Dev. Biol. 11, 677–706.)]. (2) The COPI coatomer is involved in retrograde traffic within the Golgi and from the cis-Golgi region to the endoplasmic reticulum (ER). It may also participate in anterograde transport from the ER [reviewed in (Aridor, M., Balch, W.E., 1999. Integration of endoplasmic reticulum signaling in health and disease. Nature 5, 745–751.)]. (3) COPII coats mediate anterograde transport of cargo out of the ER [Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.F., Ravazazola, M., Amherdt, M., Schekman, R., 1994. COPII: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77, 895–907; Scales, S.J., Gomez, M., Kreis, T.E., 2000. Coat proteins regulating membrane traffic. Int. Rev. Cytol. 195, 67–144.]. The COPII coat is required for budding from the ER and ER to Golgi trafficking. Further, COPII proteins also participate in cargo selection and concentrate some nascent proteins in the budding vesicle. Recent studies have shown that human disease may result from mutations that affect proteins in COPII vesicles.

AP-3: what color's your coat?

Signal-mediated delivery of proteins in several intracellular trafficking pathways requires the adaptor complexes AP-1, AP-2 and AP-3. Adaptors are heterotetrameric protein complexes that serve in cargo sorting through binding to di-leucine or tyrosine-based signals present in the cytoplasmic tails of cargo proteins. Adaptors team up with cytoplasmic coat proteins to effect inclusion of cargo into transport vesicles. AP-1 associates with vesicles budding from the trans-side of the Golgi complex, and AP-2 with endocytic vesicles budding from the plasma membrane. Both AP-1 and AP-2 cooperate with clathrin, a complex of three heavy chains and three light chains, which polymerizes to form the scaffold of the coat. The AP-3 adaptor mediates selective transport to lysosomes and lysosome-related organelles and has important biological roles in organisms as diverse as humans, flies, mice and yeast. In yeast, AP-3 function is required for the ALP (alkaline phosphatase) pathway to the vacuole. Mutations in AP-3 subunits in mice result in coat color defects and bleeding disorders and, in Drosophila, result in defects in eye pigmentation. In humans, mutations in the AP-3 β3A subunit cause an inherited disorder, Hermansky–Pudlak syndrome (HPS), in which patients show deficiencies in skin and eye pigmentation and a complete lack of dense granules in platelets, resulting in impaired blood clotting.

The AP-3 complex required for endosomal synaptic vesicle biogenesis is associated with a casein kinase Ialpha-like isoform.

The formation of small vesicles is mediated by cytoplasmic coats the assembly of which is regulated by the activity of GTPases, kinases, and phosphatases. A heterotetrameric AP-3 adaptor complex has been implicated in the formation of synaptic vesicles from PC12 endosomes (). When the small GTPase ARF1 is prevented from hydrolyzing GTP, we can reconstitute AP-3 recruitment to synaptic vesicle membranes in an assembly reaction that requires temperatures above 15 degrees C and the presence of ATP suggesting that an enzymatic step is involved in the coat assembly. We have now found an enzymatic reaction, the phosphorylation of the AP-3 adaptor complex, that is linked with synaptic vesicle coating. Phosphorylation occurs in the beta3 subunit of the complex by a kinase similar to casein kinase 1alpha. The kinase copurifies with neuronal-specific AP-3. In vitro, purified casein kinase I selectively phosphorylates the beta3A and beta3B subunit at its hinge domain. Inhibiting the kinase hinders the recruitment of AP-3 to synaptic vesicles. The same inhibitors that prevent coat assembly in vitro also inhibit the formation of synaptic vesicles in PC12 cells. The data suggest, therefore, that the mechanism of AP-3-mediated vesiculation from neuroendocrine endosomes requires the phosphorylation of the adaptor complex at a step during or after AP-3 recruitment to membranes.

Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function.

Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.

P230 is associated with vesicles budding from the trans-Golgi network.

Transport vesicle formation requires the association of cytosolic proteins with the membrane. We have previously described a brefeldin-A sensitive, hydrophilic protein (p230), containing a very high frequency of heptad repeats, found in the cytosol and associated with Golgi membranes. We show here that p230 is localised on the trans-Golgi network, by immunogold labeling of HeLa cell cryosections using alpha 2,6 sialyltransferase as a compartment-specific marker. The role of G protein activators on the binding of p230 to Golgi membranes and in vesicle biogenesis has been investigated. Treatment of streptolysin-O permeabilised HeLa cells with either GTP gamma S or AlF4- resulted in accumulation of p230 on Golgi membranes. Furthermore, immunolabeling of isolated Golgi membranes treated with AlF4-, to induce the accumulation of vesicles, showed that p230 is predominantly localised to the cytoplasmic surface of trans-Golgi network-derived budding structures and small coated vesicles. p230-labeled vesicles have a thin (approximately 10 nm) electron dense cytoplasmic coat and could be readily distinguished from clathrin-coated vesicles. Dual immunogold labeling of perforated cells, or of cryosections of treated Golgi membranes, revealed that p230 and the trans-Golgi network-associated p200, which we show here to be distinct molecules, appear to be localised on separate populations of vesicles budding from the trans-Golgi network. These results strongly suggest the presence of distinct populations of non-clathrin coated vesicles derived from the trans-Golgi network. As p230 recycles between the cytosol and buds/vesicles of TGN membranes, a process regulated by G proteins, we propose that p230 is involved in the biogenesis of a specific population of non-clathrin coated vesicles.

Molecular machinery mediating vesicle budding, docking and fusion.

A general machinery buds and fuses transport vesicles which connect intracellular compartments with each other and allow communication with the extracellular environment. Cytoplasmic coat proteins deform membranes to bud vesicles and interact directly or indirectly with cargo molecules. Compartment-specific SNAREs (SNAP receptors) on vesicles and target membranes dock vesicles and provide a scaffolding for the general fusion machinery to initiate lipid bilayer fusion.

Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein.

Movement of material between intracellular compartments takes place through the production of transport vesicles derived from donor membranes. Vesicle budding that results from the interaction of cytoplasmic coat proteins (coatomer and clathrin) with intracellular organelles requires a type of GTP-binding protein termed ADP-ribosylation factor (ARF). The GTPase cycle of ARF proteins that allows the uncoating and fusion of a transport vesicle with a target membrane is mediated by ARF-dependent GTPase-activating proteins (GAPs). A previously identified yeast protein, Gcs1, exhibits structural similarity to a mammalian protein with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as substrates. Furthermore, Gcs1 function is needed for the efficient secretion of invertase, as expected for a component of vesicle transport. The in vivo role of Gcs1 as an ARF GAP is substantiated by genetic interactions between mutations in the ARF1/ARF2 redundant pair of yeast ARF genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 proteins are markedly impaired for growth compared with cells missing either protein. Moreover, cells with decreased levels of Arf1 or Arf2 protein, and thus with decreased levels of GTP-Arf, are markedly inhibited for growth by increased GCS1 gene dosage, presumably because increased levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus by both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.

Molecular machinery mediating vesicle budding, docking and fusion.

A general machinery buds and fuses transport vesicles which connect intracellular compartments with each other and allow communication with the extracellular environment. Cytoplasmic coat proteins deform membranes to bud vesicles and interact directly or indirectly with cargo molecules. Compartment-specific SNAREs on vesicles and target membranes dock vesicles and provide a scaffolding for the general fusion machinery to initiate lipid bilayer fusion.

Cytoplasmic coat proteins involved in endosome function

Endosomes are intermediates for a complex series of sorting and transport events that occur during receptor-mediated endocytosis. These involve the recognition of targeting determinants on the cytoplasmic domains of many membrane proteins as well as the formation of specific transport vesicles. Accordingly, endosome function is likely to be governed by the regulated assembly of cytoplasmic coat complexes. We have found that, in vitro, endosomes recruit a characteristic set of cytoplasmic proteins in a GTPγS-stimulated and brefeldin A-sensitive fashion. Among these are members of the COP-1 and ARF families of coat proteins. In addition, endosomes were also found to assemble distinct, clathrin-like coats. Since micro-injection of antibodies to β-COP inhibits the entry of enveloped viruses via the endocytic pathway, it is apparent that the recruitment of COP-I or COP-I-related proteins plays an important role in the function of endosomes in intact cells.