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The evolution of eukaryotic cellular complexity is interwoven with the extensive diversification of many protein families. One key family is the ARF GTPases that act in eukaryote-specific processes, including membrane traffic, tubulin assembly, actin dynamics, and cilia-related functions. Unfortunately, our understanding of the evolution of this family is limited. Sampling an extensive set of available genome and transcriptome sequences, we have assembled a dataset of over 2,000 manually curated ARF family genes from 114 eukaryotic species, including many deeply diverged protist lineages, and carried out comprehensive molecular phylogenetic analyses. These reconstructed as many as 16 ARF family members present in the last eukaryotic common ancestor (LECA), nearly doubling the previously inferred ancient system complexity. Evidence for the wide occurrence and ancestral origin of Arf6, Arl13 and Arl16 is presented for the first time. Moreover, Arl17, Arl18 and SarB, newly described here, are absent from well-studied model organisms and as a result their function(s) remain unknown. Analyses of our dataset revealed a previously unsuspected diversity of membrane association modes and domain architectures within the ARF family. We detail the step-wise expansion of the ARF family in the metazoan lineage, including discovery of several new animal-specific family members. Delving back to its earliest evolution in eukaryotes, the resolved relationship observed between the ARF family paralogs sets boundaries for scenarios of vesicle coat origins during eukaryogenesis. Altogether, our work fundamentally broadens the understanding of the diversity and evolution of a protein family underpinning the structural and functional complexity of the eukaryote cells.SignificanceARF Family GTPases are crucial regulations of a diversity of cellular compartments and processes and as such the extent of this system in eukaryotes reflects both cellular complexity in modern eukaryotes and its evolution. Strikingly, a comprehensive comparative genomic analysis of the protein family is lacking, leaving its recent and ancient evolution poorly resolved. We performed a comprehensive molecular evolutionary analysis, reconstructing a highly complex ARF family complement in the Last Eukaryotic Common Ancestor, including a number of paralogs never before identified as such, and we find resolved relationships between the paralogs. This work has implications for cellular evolution from eukaryogenesis to cellular complexity in metazoans.

Dysregulation of collagen production and secretion contributes to aging and tissue fibrosis of major organs. How procollagen proteins in the endoplasmic reticulum (ER) route as specialized cargos for secretion remains to be fully elucidated. Here, we report that TMEM39, an ER-localized transmembrane protein, regulates production and secretory cargo trafficking of procollagen. We identify the C. elegans ortholog TMEM-39 from an unbiased RNAi screen and show that deficiency of tmem-39 leads to striking defects in cuticle collagen production and constitutively high ER stress response. RNAi knockdown of the tmem-39 ortholog in Drosophila causes similar defects in collagen secretion from fat body cells. The cytosolic domain of human TMEM39A binds to Sec23A, a vesicle coat protein that drives collagen secretion and vesicular trafficking. TMEM-39 regulation of collagen secretion is independent of ER stress response and autophagy. We propose that the roles of TMEM-39 in collagen secretion and ER homeostasis are likely evolutionarily conserved., Author summary As the most abundant protein in animals, collagen plays diverse roles and its dysregulation impacts aging and many fibrotic disorders. It is important to understand how premature collagen proteins in the ER are processed and secreted, as many other aspects of collagen regulation have been elucidated in mechanistic detail. In this paper, we have characterized a novel conserved family of TMEM39 proteins, including human TMEM39A and C. elegans tmem-39 that regulates ER stress response and collagen secretion. Human TMEM39A directly interacts with SEC23A, a core component of the COPII vesicle coating complex responsible for vesicular cargo secretion to the Golgi apparatus. The function of TMEM-39 proteins in collagen secretion appears highly conserved and independent to the ER stress response and the autophagy pathway. Our results provide insights into functions and mechanisms of TMEM39 proteins in collagen secretion and suggest it as a plausible target for tissue fibrotic diseases.

The delta opioid receptor (DOR), a physiologically relevant prototype for G protein–coupled receptors, is retained in intracellular compartments in neuronal cells. This retention is mediated by a nerve growth factor (NGF)-regulated checkpoint that delays the export of DOR from the trans-Golgi network. How DOR is selectively retained in the Golgi, in the midst of dynamic membrane transport and cargo export, is a fundamental unanswered question. Here we address this by investigating sequence elements on DOR that regulate DOR surface delivery, focusing on the C-terminal tail of DOR that is sufficient for NGF-mediated regulation. By systematic mutational analysis, we define conserved dual bi-arginine (RXR) motifs that are required for NGF- and phosphoinositide-regulated DOR export from intracellular compartments in neuroendocrine cells. These motifs were required to bind the coatomer protein I (COPI) complex, a vesicle coat complex that mediates primarily retrograde cargo traffic in the Golgi. Our results suggest that interactions of DOR with COPI, via atypical COPI motifs on the C-terminal tail, retain DOR in the Golgi. These interactions could provide a point of regulation of DOR export and delivery by extracellular signaling pathways.

Plant cells show many signs of a unique evolutionary history. This is seen in the system of intracellular organelles and vesicle transport pathways plants use to traffic molecular cargo. Bioinformatic and cell biological work in this area is beginning to tackle the question of how plant cells have evolved, and what this tells us about the evolution of other eukaryotes. Key protein families with membrane trafficking function, including Rabs, SNAREs, vesicle coat proteins, and ArfGAPs, show patterns of evolution that indicate both specialization and conservation in plants. These changes are accompanied by changes at the level of organelles and trafficking pathways between them. Major specializations include losses of several ancient Rabs, novel functions of many proteins, and apparent modification of trafficking in endocytosis and cytokinesis. Nevertheless, plants show extensive conservation of ancestral membrane trafficking genes, and conservation of their ancestral function in most duplicates. Moreover, plants have retained several ancient membrane trafficking genes lost in the evolution of animals and fungi. Considering this, plants such as Arabidopsis are highly valuable for investigating not only plant-specific aspects of membrane trafficking, but also general eukaryotic mechanisms.

Transport of late endosomes and lysosome-related organelles (LE/LROs) in axons is essential for supplying synaptic cargoes and for removing damaged macromolecules. Defects in this system are implicated in a range of human neurodegenerative and neurodevelopmental disorders. The findings reported here identify a novel mechanism regulating LE/LRO transport based on the coordinated coupling of microtubule motors and vesicle coat proteins to the neuron-enriched, transmembrane protein calcyon (Caly). We found that the cytoplasmic C-terminus of Caly pulled down proteins involved in microtubule-dependent transport (DIC, KIF5A, p150Glued, Lis1) and organelle biogenesis (AP-1 and AP-3) from the brain. In addition, RNA interference–mediated knockdown of Caly increased the percentage of static LE/LROs labeled by LysoTracker in cultured dorsal root ganglion axons. In contrast, overexpression of Caly stimulated movement of organelles positive for LysoTracker or the AP-3 cargo GFP-PI4KIIα. However, a Caly mutant (ATEA) that does not bind AP-3 was unable to pull down motor proteins from brain, and expression of the ATEA mutant failed to increase either LE/LRO flux or levels of associated dynein. Taken together, these data support the hypothesis that Caly is a multifunctional scaffolding protein that regulates axonal transport of LE/LROs by coordinately interacting with motor and vesicle coat proteins.

BACKGROUND RNA interference (RNAi) triggered by maize plants expressing RNA hairpins against specific western corn rootworm (WCR) transcripts have proven to be effective at controlling this pest. To provide robust crop protection, mRNA transcripts targeted by double-stranded RNA must be sensitive to knockdown and encode essential proteins. RESULTS Using WCR adult feeding assays, we identified Sec23 as a highly lethal RNAi target. Sec23 encodes a coatomer protein, a component of the coat protein (COPII) complex that mediates ER-Golgi transport. The lethality detected in WCR adults was also observed in early instar larvae, the life stage causing most of the crop damage, suggesting that WCR adults can serve as an alternative to larvae for dsRNA screening. Surprisingly, over 85% transcript inhibition resulted in less than 40% protein knockdown, suggesting that complete protein knockdown is not necessary for Sec23 RNAi-mediated mortality. The efficacy of Sec23 dsRNA for rootworm control was confirmed in planta; T0 maize events carrying rootworm Sec23 hairpin transgenes showed high levels of root protection in greenhouse assays. A reduction in larval survival and weight were observed in the offspring of WCR females exposed to Sec23 dsRNA LC25 in diet bioassays. CONCLUSION We describe Sec23 as RNAi target for in planta rootworm control. High mortality in exposed adult and larvae and moderate sublethal effects in the offspring of females exposed to Sec23 dsRNA LC25 , suggest the potential for field application of this RNAi trait and the need to factor in responses to sublethal exposure into insect resistance management programs. © 2019 Society of Chemical Industry.

The coat protein complex I (COPI) allows the precise sorting of lipids and proteins between Golgi cisternae and retrieval from the Golgi to the ER. This essential role maintains the identity of the early secretory pathway and impinges on key cellular processes, such as protein quality control. In this Cell Science at a Glance and accompanying poster, we illustrate the different stages of COPI-coated vesicle formation and revisit decades of research in the context of recent advances in the elucidation of COPI coat structure. By calling attention to an array of questions that have remained unresolved, this review attempts to refocus the perspectives of the field.

Vesicular transport was key to the evolution of eukaryotes, and is essential for eukaryotic life today. All modern eukaryotes have a set of vesicle coat proteins, which couple cargo selection to vesicle budding in the secretory and endocytic pathways. Although these coats share common features (e.g. recruitment via small GTPases, β-propeller-α-solenoid proteins acting as scaffolds), the relationships between them are not always clear. Structural studies on the coats themselves, comparative genomics and cell biology in diverse eukaryotes, and the recent discovery of the Asgard archaea and their 'eukaryotic signature proteins' are helping us to piece together how coats may have evolved during the prokaryote-to-eukaryote transition.

Protein kinase C epsilon (PKCε) contributes to multiple signaling pathways affecting human disease. The function of PKCε requires it to undergo changes in subcellular distribution in response to signaling events. While the mechanisms underlying this translocation are incompletely understood, it involves the receptor for activated C kinase protein (RACK2/β′-COP). This receptor also functions as a vesicle coat protein in the secretory pathway where it is regulated by the small GTP-binding protein ADP-ribosylation factor, ARF1. We inhibited ARF1 activation to test the requirement for RACK2/β′-COP in PKCε localization in NIH3T3 fibroblasts. We found that steady-state localization of PKCε at the Golgi complex requires ARF1-regulated RACK2/β′-COP function. By contrast, we did not observe any defects in phorbol ester-induced translocation when ARF1 was inhibited. We also found that PKCε bound to isolated membranes through two distinct mechanisms. One mechanism was dependent upon RACK2/β′-COP while a second was RACK2/β′-COP-independent and stimulated by phorbol esters. Finally, we show that RACK2/β′-COP affects the subcellular distribution of a constitutively active form of PKCε, in a manner similar to what we observed for wild-type PKCε. Together, our data support a role for RACK2/β′-COP in the steady-state localization of PKCε at the Golgi apparatus, which may be independent of its role during PKCε translocation to the cell surface.

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1−/− cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions. Refereed/Peer-reviewed

Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways. In this study the subunits of adaptor protein complex-1 (AP-1) of silkworm Bombyx mori were molecularly characterized. All coding genes for the four subunits were cloned and sequenced. Phylogenic tree for each adaptin was constructed and all subunits were found to be conserved in respective group among organisms. The mRNA expression pattern for each adaptin was similar among tissues. Alternative splicing event was observed in genes encoding both the heavy chain gamma and beta adaptin and the light chain subunit, which could generate other possible adaptin forms. GFP-tagged fusion proteins indicated that AP-1 located in the peripheral plasma area. Furthermore, the BmNPV infection in B. mori cells had differentiated effect on the expression level of AP-1 subunits.

Packaging of proteins from the endoplasmic reticulum into COPII vesicles is essential for secretion. In cells, most COPII vesicles are approximately 60-80 nm in diameter, yet some must increase their size to accommodate 300-400 nm procollagen fibres or chylomicrons. Impaired COPII function results in collagen deposition defects, cranio-lenticulo-sutural dysplasia, or chylomicron retention disease, but mechanisms to enlarge COPII coats have remained elusive. Here, we identified the ubiquitin ligase CUL3-KLHL12 as a regulator of COPII coat formation. CUL3-KLHL12 catalyses the monoubiquitylation of the COPII-component SEC31 and drives the assembly of large COPII coats. As a result, ubiquitylation by CUL3-KLHL12 is essential for collagen export, yet less important for the transport of small cargo. We conclude that monoubiquitylation controls the size and function of a vesicle coat.

An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, 70-kDa heat-shock cognate protein (Hsc70), and the J-domain-containing protein, auxilin, in an ATP-dependent process. We present a detailed mechanistic analysis of clathrin disassembly catalyzed by Hsc70 and auxilin, using loss of perpendicular light scattering to monitor the process. We report that a single auxilin per clathrin triskelion is required for maximal rate of disassembly, that ATP is hydrolyzed at the same rate that disassembly occurs, and that three ATP molecules are hydrolyzed per clathrin triskelion released. Stopped-flow measurements revealed a lag phase in which the scattering intensity increased owing to association of Hsc70 with clathrin cages followed by serial rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped-flow data to several physically plausible mechanisms showed the best fit to a model in which sequential hydrolysis of three separate ATP molecules is required for the eventual release of a triskelion from the clathrin–auxilin cage.

Spinal muscular atrophy (SMA), an inherited disease of motor neuron dysfunction, results from insufficient levels of the survival motor neuron (SMN) protein. Movement of the SMN protein as granules within cultured axons suggests that the pathogenesis of SMA may involve defects in neuronal transport, yet the nature of axon transport vesicles remains enigmatic. Here we show that SMN directly binds to the α-subunit of the coat protein I (COPI) vesicle coat protein. The α-COP protein co-immunoprecipitates with SMN, small nuclear ribonucleoprotein-associated assembly factors and β-actin mRNA. Although typically Golgi associated, in neuronal cells α-COP localizes to lamellipodia and growth cones and moves within the axon, with a subset of these granules traveling together with SMN. Depletion of α-COP resulted in mislocalization of SMN and actin at the leading edge at the lamellipodia. We propose that neurons utilize the Golgi-associated COPI vesicle to deliver cargoes necessary for motor neuron integrity and function.

Small G proteins play a central role in the organization of secretory and endocytotic pathways. The recruitment of some effectors, including vesicle coat proteins, is mediated by the ADP-ribosylation factor (Arf) family. Arf proteins have distinct subcellular localizations. ArfGAPs (Arf GTPase-activating proteins) regulate Arf GTPase activity. Thus, each ArfGAP is distinctly localized to allow it to maintain a specific interaction with its target Arf(s). However, the domains that regulate the subcellular localization of ArfGAPs and the way in which these subcellular localizations affect the target specificities of ArfGAPs remain unclear. Recently, we identified two novel ArfGAPs, SMAP1 (Small ArfGAP protein 1) and SMAP2. In the current study, we identified sequences in the carboxy-terminal region of SMAP2 that are critical for its specific subcellular localization and its specificity for Arf proteins.

The secretory pathway is of vital importance for eukaryotic cells and has a pivotal role in the synthesis, sorting, processing and secretion of a large variety of bioactive molecules involved in intercellular communication. One of the key processes in the secretory pathway concerns the transport of cargo proteins from the ER (endoplasmic reticulum) to the Golgi. Type-I transmembrane proteins of approximately 24 kDa are abundantly present in the membranes of the early secretory pathway, and bind the COPI and COPII coat complexes that cover vesicles travelling between the membranes. These p24 proteins are thought to play an important role in the selective transport processes at the ER-Golgi interface, although their exact functioning is still obscure. One model proposes that p24 proteins couple cargo selection in the lumen with vesicle coat recruitment in the cytosol. Alternatively, p24 proteins may furnish subcompartments of the secretory pathway with the correct subsets of machinery proteins. Here we review the current knowledge of the p24 proteins and the various roles proposed for the p24 family members.

Item does not contain fulltext The p24 family is thought to be somehow involved in endoplasmic reticulum-to-Golgi protein transport, and its members are major constituents of transport vesicles and bind to the vesicle coat protein complexes COPI and COPII. A subset of the p24 proteins (p24alpha(3), -beta(1), -gamma(3) and -delta(2)) is upregulated when Xenopus laevis intermediate pituitary melanotrope cells are physiologically activated to produce vast amounts of their major secretory cargo, the prohormone proopiomelanocortin (POMC). To investigate the role of the COP-binding motifs of p24 proteins in POMC biosynthesis, we here generated and analysed Xenopus with stable, melanotrope cell-specific transgene expression of p24delta(2)-GFP mutated in its COPI- or COPII-binding motif. In contrast to what has been found previously for wild-type (wt) p24delta(2)-GFP, the p24delta(2) mutations prevented the Golgi localisation of the transgene products and caused a reduced rate of POMC cleavage, but did not lead to a reduction of the endogenous p24 proteins nor to aberrations in POMC glycosylation and sulphation. We conclude that p24delta(2) requires the presence of the COPI- and COPII-binding sites to allow proper POMC processing. Thus, the p24 proteins fulfil their role in secretory protein biosynthesis via COPI- or COPII-coated transport vesicles.

The eukaryotic endomembrane system is responsible for the biosynthesis and transport of proteins and lipids, and for the definition of the major subcellular compartments. Recent work indicates that the endomembrane system is ancient, with near modern complexity predating the radiation of the major eukaryotic lineages. The challenge is to look beyond the last eukaryotic common ancestor and to attempt to deduce the evolutionary steps in the rise of membrane-trafficking complexity. Relationships between the endomembrane coatomer complexes and their evolutionary connection to the nuclear pore complex are emerging. These studies, plus the realization of a role for the ESCRT complex as an alternate, but equally ancient, system for membrane deformation are providing insight into the earliest stages of endomembrane evolution.

Two hundred years after Darwin's birth, our understanding of genetic mechanisms and cell biology has advanced to a level unimaginable in the 19th century. We now know that eukaryotic cells contain a huge variety of internal compartments, each with their own function, identity and history. For the compartments that together form the membrane-trafficking system, one of the central questions is how that identity is encoded and how it evolved. Here we review the key components involved in membrane-trafficking events, including SNAREs, Rabs, vesicle coats, and tethers and what is known about their evolutionary history. Our current understanding suggests a possible common mechanism by which the membrane-trafficking organelles might have evolved. This model of increased organellar complexity by gene duplication and co-evolution of multiple, interacting, specificity-encoding proteins could well be applicable to other non-endosymbiotic organelles as well. The application of basic evolutionary principles well beyond their original scope has been exceedingly powerful not only in reconstructing the history of cellular compartments, but for medical and applied research as well, and underlines the contributions of Darwin's ideas in modern biology.

The movement of most transmembrane proteins between the various membranes of the cell is directed by short linear amino acid sequences in their cytoplasmic portions. These sequence motifs are bound by protein components of transport vesicle coats thereby incorporating the transmembrane proteins as vesicle cargo. In the case of clathrin-coated vesicles (CCVs) the motifs are recognised by the AP complexes or GGA clathrin adaptors. The clathrin adaptor AP2 recognizes two major classes of endocytic motifs including an acidic dileucine motif. In this study, Kelly et al. present the crystal structure of AP2 in a complex with the dileucine motif of a cargo protein thereby revealing the mechanism of cargo-adaptor recognition. During clathrin-mediated endocytosis, cargo proteins are recognized by clathrin adaptors. The clathrin adaptor AP2 recognizes two major classes of endocytic motifs, including an acidic dileucine motif. This study presents the crystal structure of AP2 in complex with the diceucine motif of a cargo protein, thereby revealing the mechanism of cargo–adaptor recognition. Most transmembrane proteins are selected as transport-vesicle cargo through the recognition of short, linear amino-acid motifs in their cytoplasmic portions by vesicle coat proteins. For clathrin-coated vesicles, the motifs are recognized by clathrin adaptors. The AP2 adaptor complex (subunits α, β2, μ2 and σ2) recognizes both major endocytic motifs: YxxΦ motifs1 (where Φ can be F, I, L, M or V) and [ED]xxxL[LI] acidic dileucine motifs. Here we describe the binding of AP2 to the endocytic dileucine motif from CD4 (ref. 2). The major recognition events are the two leucine residues binding in hydrophobic pockets on σ2. The hydrophilic residue four residues upstream from the first leucine sits on a positively charged patch made from residues on the σ2 and α subunits. Mutations in key residues inhibit the binding of AP2 to ‘acidic dileucine’ motifs displayed in liposomes containing phosphatidylinositol-4,5-bisphosphate, but do not affect binding to YxxΦ motifs through μ2. In the ‘inactive’ AP2 core structure3 both motif-binding sites are blocked by different parts of the β2 subunit. To allow a dileucine motif to bind, the β2 amino terminus is displaced and becomes disordered; however, in this structure the YxxΦ-binding site on μ2 remains blocked.

Small G proteins play a central role in the organization of the secretory and endocytic pathways. The majority of such small G proteins are members of the Rab family, which are anchored to the bilayer by C-terminal prenyl groups. However, the recruitment of some effectors, including vesicle coat proteins, is mediated by a second class of small G proteins that is unique in having an N-terminal amphipathic helix that becomes available for membrane insertion upon GTP binding. Sar1, Arf1, and Arf6 are the best-characterized members of this ADP-ribosylation factor (Arf) family. In addition, all eukaryotes contain additional distantly related G proteins, often called Arf like, or Arls. The complete Arf family in humans has 29 members. The roles of these related G proteins are poorly understood, but recent work has shown that some are involved in membrane traffic or organizing the cytoskeleton. Here we review what is known about all the members of the Arf family, along with the known regulatory molecules that convert them between GDP- and GTP-bound states.

All identified basolateral sorting signals of integral membrane proteins are cytoplasmically disposed, suggesting that basolateral targeting is mediated exclusively by direct interaction with vesicle coat components. Here, we report that GPP130, a cis-Golgi protein that undergoes endosome-to-Golgi retrieval using the late endosome-bypass pathway in nonpolarized cells, cycles via the basolateral membrane in polarized MDCK cells. Significantly, the membrane-proximal lumenal domain of GPP130, which mediates GPP130 localization and trafficking in nonpolarized cells, was both necessary and sufficient for basolateral cycling in MDCK cells. The use of lumenal determinants for both basolateral cycling and endosome-to-Golgi retrieval suggests that a novel receptor-mediated mechanism operates at both the trans-Golgi network and distal sites to sort GPP130 along the late-endosome-bypass retrieval pathway in polarized cells.

The retrograde pathway is defined by the transport of proteins and lipids from the plasma membrane through endosomes to the Golgi complex, and is essential for a variety of cellular activities. Recycling endosomes are important sorting stations for some retrograde cargo. SMAP2, a GTPase-activating protein (GAP) for Arf1 with a putative clathrin-binding domain, has previously been shown to participate in the retrograde transport of the cholera toxin B-subunit (CTxB) from recycling endosomes. Here, we found that clathrin, a vesicle coat protein, and clathrin adaptor protein complex 1 (AP-1) were present at recycling endosomes and were needed for the retrograde transport of CTxB from recycling endosomes to the Golgi, but not from the plasma membrane to recycling endosomes. SMAP2 immunoprecipitated clathrin and AP-1 through a putative clathrin-binding domain and a CALM-binding domain, and SMAP2 mutants that did not interact with clathrin or AP-1 could not localize to recycling endosomes. Moreover, knockdown of Arf1 suppressed the retrograde transport of CTxB from recycling endosomes to the Golgi. These findings suggest that retrograde transport is mediated by clathrin-coated vesicles from recycling endosomes and that the role of the coat proteins is in the recruitment of Arf GAP to transport vesicles.

Docking and fusion of transport carriers in eukaryotic cells are regulated by a family of multi-subunit tethering complexes (MTC) that sequentially and/or simultaneously interact with other components of vesicle fusion machinery, such as SNAREs, Rabs, coiled-coil tethers, and vesicle coat components. Probing for interactions of multi-protein complexes has relied heavily on the method of exogenously expressing individual proteins and then determining their interaction stringency. An obvious pitfall of this method is that the protein interactions are not occurring in their native multi-subunit state. Here, we describe an assay where we express all eight subunits of the conserved oligomeric Golgi (COG) complex that contain the same triple-Myc epitope tag and then an assay for the (sub) complex's interaction with known protein partners. The expression of all eight proteins allows for the assembled complex to interact with partner proteins, and by having the same tag on all eight COG subunits, we are able to very accurately quantify the interaction with each subunit. The use of this assay has highlighted a very important level of specificity of interactions between COG subcomplexes and their intracellular partners.

Calcium has been implicated in regulating vesicle fusion reactions, but its potential role in regulating other aspects of protein transport, such as vesicle assembly, is largely unexplored. We find that treating cells with the membrane-permeable calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), leads to a dramatic redistribution of the vesicle coat protein, coatomer, in the cell. We have used the cell-free reconstitution of coat-protomer I (COPI) vesicle assembly to characterize the mechanisms of this redistribution. We find that the recovery of COPI-coated Golgi vesicles is inhibited by the addition of BAPTA to the cell-free vesicle budding assay. When coatomer-coated membranes are incubated in the presence of calcium chelators, the membranes “uncoat,” indicating that calcium is necessary for maintaining the integrity of the coat. This uncoating is reversed by the addition of calcium. Interestingly, BAPTA, a calcium chelator with fast binding kinetics, is more potent at uncoating the coatomer-coated membrane than EGTA, suggesting that a calcium transient or a calcium gradient is important for stabilizing COPI vesicle coat. The primary target for the effects of calcium on coatomer recruitment is a step that occurs after ADP-ribosylation factor binding to the membrane. We suggest that a calcium gradient may serve to regulate the timing of vesicle uncoating.

The trans-Golgi network (TGN) is a major secretory pathway sorting station that directs newly synthesized proteins to different subcellular destinations. The TGN also receives extracellular materials and recycled molecules from endocytic compartments. In this review, we summarize recent progress on understanding TGN structure and the dynamics of trafficking to and from this compartment. Protein sorting into different transport vesicles requires specific interactions between sorting motifs on the cargo molecules and vesicle coat components that recognize these motifs. Current understanding of the various targeting signals and vesicle coat components that are involved in TGN sorting are discussed, as well as the molecules that participate in retrieval to this compartment in both yeast and mammalian cells. Besides proteins, lipids and lipid-modifying enzymes also participate actively in the formation of secretory vesicles. The possible mechanisms of action of these lipid hydrolases and lipid kinases are discussed. Finally, we summarize the fundamentally different apical and basolateral cell surface delivery mechanisms and the current facts and hypotheses on protein sorting from the TGN into the regulated secretory pathway in neuroendocrine cells.

Clathrin is a vesicle coat protein involved in the assembly of membrane and cargo into transport vesicles at the plasma membrane and on certain intracellular organelles. Recently, crystal structures of two separate parts of the clathrin heavy chain, a fragment of the proximal leg and the N-terminal domain, have been analysed, providing the first high-resolution data for a vesicle coat protein. Viewing these structures in the context of a hexagonal barrel coat, recently determined to 21 A by cryo-electron microscopy, provides new insights into the assembly of clathrin coats.

Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat. To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (epsilon, beta4, mu4, and sigma4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 is localized to approximately 10-20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF. Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The mu4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.

Forward transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex depends on COPII, a membrane coat that forms ER-derived vesicles. Based on experimental observations, a series of integrated events must be accomplished during the formation of COPII coated vesicles. First, the subunits of the COPII coat must be recruited to the correct site on the surface of the ER. Second, soluble and integral membrane cargo proteins destined for the Golgi complex are concentrated into nascent buds. Third, a set of molecules that must cycle between the ER and Golgi compartments (such as SNARE proteins) are incorporated into vesicles. And fourth, the COPII coat is disassembled after release of ER-derived vesicles thus allowing vesicle fusion and recycling of COPII components. Incorporation of soluble cargo infers the existence of membrane spanning receptor molecules that link lumenal cargo to the vesicle coat. Some candidate proteins have been identified (including the p24 family) that appear to participate in the selection of soluble cargo; however, the mechanistic details of this selection procedure remain obscure. This review will focus on the molecular constituents of the COPII coat and emerging interactions of the coat subunits with proteins involved in selective export from the ER.

Nuclear pore complexes intuitively seem functionally analogous to transmembrane channels. Recent bioinformatic analysis, however, suggests a quite different evolutionary relationship: one between nuclear pore complexes and vesicle coat components. This has led to the provocative hypothesis that these distinct structures share a common role in bending membranes.

ER to Golgi transport requires the function of two distinct vesicle coat complexes, termed COPI (coatomer) and COPII, whose assembly is regulated by the small GTPases ADP-ribosylation factor 1 (ARF1) and Sar1, respectively. To address their individual roles in transport, we have developed a new assay using mammalian microsomes that reconstitute the formation of ER-derived vesicular carriers. Vesicles released from the ER were found to contain the cargo molecule vesicular stomatitis virus glycoprotein (VSV-G) and p58, an endogenous protein that continuously recycles between the ER and pre-Golgi intermediates. Cargo was efficiently sorted from resident ER proteins during vesicle formation in vitro. Export of VSV-G and p58 were found to be exclusively mediated by COPII. Subsequent movement of ER-derived carriers to the Golgi stack was blocked by a trans-dominant ARF1 mutant restricted to the GDP-bound state, which is known to prevent COPI recruitment. To establish the initial site of coatomer assembly after export from the ER, we immunoisolated the vesicular intermediates and tested their ability to recruit COPI. Vesicles bound coatomer in a physiological fashion requiring an ARF1-guanine nucleotide exchange activity. These results suggest that coat exchange is an early event preceding the targeting of ER-derived vesicles to pre-Golgi intermediates.

We have recently described a system that recreates in vitro the generation of post-Golgi vesicles from purified Golgi fractions obtained from virus-infected MDCK cells in which the vesicular stomatitis virus-G envelope glycoprotein had been allowed to accumulate in vivo in the TGN. Vesicle formation, monitored by the release of the viral glycoprotein, was shown to require the activation of a GTP-binding ADP ribosylation factor (ARF) protein that promotes the assembly of a vesicle coat in the TGN, and to be regulated by a Golgi-associated protein kinase C (PKC)-like activity. We have now been able to dissect the process of post-Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and another of vesicle scission, neither of which requires an ATP supply. The first stage can occur at 20 degrees C, and includes the GTP-dependent activation of the ARF protein, which can be effected by the nonhydrolyzable nucleotide analogue GTP gamma S, whereas the second stage is nucleotide independent and can only occur at a higher temperature of incubation. Cytosolic proteins are required for the vesicle scission step and they cannot be replaced by palmitoyl CoA, which is known to promote, by itself, scission of the coatomer-coated vesicles that mediate intra-Golgi transport. We have found that PKC inhibitors prevented vesicle generation, even when this was sustained by GTP gamma S and ATP levels reduced far below the K(m) of PKC. The inhibitors suppressed vesicle scission without preventing coat assembly, yet to exert their effect, they had to be added before coat assembly took place. This indicates that a target of the putative PKC is activated during the bud assembly stage of vesicle formation, but only acts during the phase of vesicle release. The behavior of the PKC target during vesicle formation resembles that of phospholipase D (PLD), a Golgi-associated enzyme that has been shown to be activated by PKC, even in the absence of the latter's phosphorylating activity. We therefore propose that during coat assembly, PKC activates a PLD that, during the incubation at 37 degrees C, promotes vesicle scission by remodeling the phospholipid bilayer and severing connections between the vesicles and the donor membrane.

COPI and COPII are vesicle coat complexes whose assembly is regulated by the ARF1 and Sar1 GTPases, respectively. We show that COPI and COPII coat complexes are recruited separately and independently to ER (COPII), pre-Golgi (COPI, COPII), and Golgi (COPI) membranes of mammalian cells. To address their individual roles in ER to Golgi transport, we used stage specific in vitro transport assays to synchronize movement of cargo to and from pre-Golgi intermediates, and GDP- and GTP-restricted forms of Sar1 and ARF1 proteins to control coat recruitment. We find that COPII is solely responsible for export from the ER, is lost rapidly following vesicle budding and mediates a vesicular step required for the build-up of pre-Golgi intermediates composed of clusters of vesicles and small tubular elements. COPI is recruited onto pre-Golgi intermediates where it initiates segregation of the anterograde transported protein vesicular stomatitis virus glycoprotein (VSV-G) from the retrograde transported protein p58, a protein which actively recycles between the ER and pre-Golgi intermediates. We propose that sequential coupling between COPII and COPI coats is essential to coordinate and direct bi-directional vesicular traffic between the ER and pre-Golgi intermediates involved in transport of protein to the Golgi complex.

Clathrin polymerization into a polyhedral vesicle coat drives receptor sorting at cellular membranes during endocytosis and organelle biogenesis. To study clathrin self-assembly, we expressed the C-terminal third of the clathrin heavy chain in bacteria. The recombinant fragment trimerized, bound clathrin light chains, and morphologically resembled the hub domain of the triskelion-shaped clathrin molecule. Self-assembly of recombinant hubs demonstrated a regulatory role for clathrin light chains and for the distal portions of triskelion legs in clathrin coat formation. Deletion mutagenesis of the hub localized a domain mediating light chain binding and clathrin self-assembly and mapped a transferable trimerization domain. These studies define molecular interactions controlling clathrin self-assembly and establish a recombinant system for future analysis.

Clathrin is considered the prototype vesicle coat protein whose self-assembly mediates sorting of membrane cargo and recruitment of lipid modifiers. Detailed knowledge of clathrin biochemistry, structure, and interacting proteins has accumulated since the first observation, almost 50 years ago, of its role in receptor-mediated endocytosis of yolk protein. This review summarizes that knowledge, and focuses on properties of the clathrin heavy and light chain subunits and interaction of the latter with Hip proteins, to address the diversity of clathrin function beyond conventional receptor-mediated endocytosis. The distinct functions of the two human clathrin isoforms (CHC17 and CHC22) are discussed, highlighting CHC22's specialized involvement in traffic of the GLUT4 glucose transporter and consequent role in human glucose metabolism. Analysis of clathrin light chain function and interaction with the actin-binding Hip proteins during bacterial infection defines a novel actin-organizing function for CHC17 clathrin. By considering these diverse clathrin functions, along with intracellular sorting roles and influences on mitosis, further relevance of clathrin function to human health and disease is established.

High voltage electron microscopy and computer axial tomography have been used to study the 3-D structure of trans-Golgi cisternae and trans-Golgi networks (TGNs) in NRK cells. Both structures were specifically labeled by photoconversion of a fluorescent analogue of ceramide using a modification of the techique of Pagano et al. (J. Cell Biol. 1991. 113: 1267-1279). Regions of the Golgi ribbon in fixed, stained cells were cut in 250-nm sections and analyzed by tilt series microscopy and subsequent tomographic reconstruction. Resolution of the reconstructions ranged from 6 to 10 nm. The size and structure of the TGN varied considerably throughout the Golgi ribbon; all reconstructions were made from regions with pronounced TGN. Most regions analyzed contained multiple (2-4) Golgi cisternae that stain with ceramide. These "peel off" from the closely stacked cisternae and are continuous at their ends with tubules that contribute to the TGN. Most vesicular profiles visualized in the TGN are connected to TGN tubules. The budding of vesicles appears to occur synchronously along the length of a TGN tubule. Two distinct coats were visualized on budding vesicles: clathrin cages and a novel, lace-like structure. Individual TGN tubules produce vesicles of only one coat type. These observations lead to the following predictions: (a) sorting of molecules must occur prior to the formation of TGN tubules; (b) vesicle formation takes place almost synchronously along a given TGN tubule; and (c) lace-like coats form an exocytic vesicles.

Vesicle trafficking is a highly regulated process that transports proteins and other cargoes through eukaryotic cells while maintaining cellular organization and compartmental identity. In order for cargo to reach the correct destination, each step of trafficking must impart specificity. During vesicle formation, this is achieved by coat proteins, which selectively incorporate cargo into the nascent vesicle. Classically, vesicle coats are thought to dissociate shortly after budding. However, recent studies suggest that coat proteins can remain on the vesicle en route to their destination, imparting targeting specificity by physically and functionally interacting with Rab-regulated tethering systems. This review focuses on how interactions among Rab GTPases, tethering factors, SNARE proteins, and vesicle coats contribute to vesicle targeting, fusion, and coat dynamics.

The evolutionarily conserved target of rapamycin complex 1 (TORC1) controls cell growth in response to nutrient availability and growth factors. TORC1 signaling is hyperactive in cancer, and regulators of TORC1 signaling may represent therapeutic targets for human diseases. To identify novel regulators of TORC1 signaling, we performed a genome-scale RNA interference screen on microarrays of Drosophila melanogaster cells expressing human RPS6, a TORC1 effector whose phosphorylated form we detected by immunofluorescence. Our screen revealed that the TORC1–S6K–RPS6 signaling axis is regulated by many subcellular components, including the Class I vesicle coat (COPI), the spliceosome, the proteasome, the nuclear pore, and the translation initiation machinery. Using additional RNAi reagents, we confirmed 70 novel genes as significant on-target regulators of RPS6 phosphorylation, and we characterized them with extensive secondary assays probing various arms of the TORC1 pathways, identifying functional relationships among those genes. We conclude that cell-based microarrays are a useful platform for genome-scale and secondary screening in Drosophila, revealing regulators that may represent drug targets for cancers and other diseases of deregulated TORC1 signaling., National Institutes of Health (U.S.) (R01 GM072555), National Institutes of Health (U.S.) (CA103866), National Institutes of Health (U.S.) (R01 GM089652), United States. Dept. of Defense (DOD TSC Research Program (W81XWH-05-1-0318-DS)), LAM Foundation, Society for Biomolecular Sciences, Whitehead Institute for Biomedical Research, Broad Institute

The RZZ complex recruits dynein to kinetochores. We investigated structure, topology, and interactions of the RZZ subunits (ROD, ZWILCH, and ZW10) in vitro, in vivo, and in silico. We identify neuroblastoma-amplified gene (NAG), a ZW10 binder, as a ROD homolog. ROD and NAG contain an N-terminal β propeller followed by an α solenoid, which is the architecture of certain nucleoporins and vesicle coat subunits, suggesting a distant evolutionary relationship. ZW10 binding to ROD and NAG is mutually exclusive. The resulting ZW10 complexes (RZZ and NRZ) respectively contain ZWILCH and RINT1 as additional subunits. The X-ray structure of ZWILCH, the first for an RZZ subunit, reveals a novel fold distinct from RINT1's. The evolutionarily conserved NRZ likely acts as a tethering complex for retrograde trafficking of COPI vesicles from the Golgi to the endoplasmic reticulum. The RZZ, limited to metazoans, probably evolved from the NRZ, exploiting the dynein-binding capacity of ZW10 to direct dynein to kinetochores. © 2010 Elsevier Ltd.

Clathrin-coated vesicles mediate cellular endocytosis of nutrients and molecules that are involved in a variety of biological processes. Basic components of the vesicle coat are clathrin heavy chain (Chc) and clathrin light chain molecules. In Drosophila melanogaster the chc gene function has been analyzed in a number of previous studies mainly using genetic approaches. However, the chc mRNA and protein expression patterns have not been studied systematically. We have generated an antibody that specifically recognizes Chc and we have analyzed chc RNA and protein expression patterns throughout embryonic and larval stages. We found that chc mRNA and protein are highly expressed from early stages of embryogenesis onwards, consistent with genetic studies predicting a maternal contribution of the gene function. During subsequent stages mRNA and protein are co-expressed in all embryonic cells; however we found an up-regulation in specific tissues including the gut, the salivary glands, tracheal system and the epidermis. In addition the central nervous system and the nephrocyte-like garland cells show strong Chc expression at late embryogenesis. In larvae Chc is highly expressed in garland cells, imaginal discs, fat body, salivary glands and the ring gland. Subcellularly, we found Chc protein in a vesicle-like pattern within the cytoplasm and at the plasma membrane. Co-labeling studies show that Chc is partially in contact with the trans-Golgi network and co-localizes with markers for early endocytosis. Together, the antibody may serve as a new tool to study the function of Chc in clathrin-dependent cellular processes, such as endocytosis.

ADP-ribosylation factor (Arf) and related small GTPases play crucial roles in membrane traffic within the exo- and endocytic pathways. Arf proteins in their GTP-bound state are associated with curved membrane buds and tubules, frequently together with effector coat proteins to which they bind. Here we report that Arf1 is found on membrane tubules originating from the Golgi complex where it colocalizes with COPI and GGA1 vesicle coat proteins. Arf1 also induces tubulation of liposomes in vitro. Mutations within the amino-terminal amphipathic helix (NTH) of Arf1 affect the number of Arf1-positive tubules in vivo and its property to tubulate liposomes. Moreover, hydrophilic substitutions within the hydrophobic part of its NTH impair Arf1-catalyzed budding of COPI vesicles in vitro. Our data indicate that GTP-controlled local induction of high curvature membranes is an important property of Arf1 that might be shared by a subgroup of Arf/Arl family GTPases.

Background Trypanosomatids utilise polycistronic transcription for production of the vast majority of protein-coding mRNAs, which operates in the absence of gene-specific promoters. Resolution of nascent transcripts by polyadenylation and trans-splicing, together with specific rates of mRNA turnover, serve to generate steady state transcript levels that can differ in abundance across several orders of magnitude and can be developmentally regulated. We used a targeted oligonucleotide microarray, representing the strongly developmentally-regulated T. brucei membrane trafficking system and ~10% of the Trypanosoma brucei genome, to investigate both between-stage, or differentiation-dependent, transcriptome changes and within-stage flexibility in response to various challenges. Results 6% of the gene cohort are developmentally regulated, including several small GTPases, SNAREs, vesicle coat factors and protein kinases both consistent with and extending previous data. Therefore substantial differentiation-dependent remodeling of the trypanosome transcriptome is associated with membrane transport. Both the microarray and qRT-PCR were then used to analyse transcriptome changes resulting from specific gene over-expression, knockdown, altered culture conditions and chemical stress. Firstly, manipulation of Rab5 expression results in co-ordinate changes to clathrin protein expression levels and endocytotic activity, but no detectable changes to steady-state mRNA levels, which indicates that the effect is mediated post-transcriptionally. Secondly, knockdown of clathrin or the variant surface glycoprotein failed to perturb transcription. Thirdly, exposure to dithiothreitol or tunicamycin revealed no evidence for a classical unfolded protein response, mediated in higher eukaryotes by transcriptional changes. Finally, altered serum levels invoked little transcriptome alteration beyond changes to expression of ESAG6/7, the transferrin receptor. Conclusion While trypanosomes regulate mRNA abundance to effect the major changes accompanying differentiation, a given differentiated state appears transcriptionally inflexible. The implications of the absence of a transcriptome response in trypanosomes for both virulence and models of life cycle progression are discussed.

Coatomer coated (COPI) vesicles play a pivotal role for multiple membrane trafficking steps throughout the eukaryotic cell. Our focus is on betaCOP, one of the most well known components of the COPI multi-protein complex. Amino acid differences in betaCOP may dictate functional divergence across species during the course of evolution, especially with regards to the evolutionary pressures on obligate intracellular parasites. A bioinformatic analysis of betaCOP amino acid sequences was conducted for 49 eukaryotic species. Cloning and sequence analysis of the Toxoplasma gondii betaCOP homologue revealed several amino acid insertions unique to T. gondii and one C-terminal insertion that is unique to apicomplexan parasites. These findings led us to investigate the possibility that betaCOP experienced functional divergence during the course of its evolution. Bayesian phylogenetic analysis revealed a tree consistent with pan eukaryote distribution and long-branch lengths were observed among the apicomplexans. Further analysis revealed that kinetoplast betaCOP underwent the most amount of change, leading to perhaps an overall change of function. In comparison, T. gondii exhibited subtle yet specific amino acid changes. The amino acid substitutions did not occur in the same places as other lineages, suggesting that TgbetaCOP has a role specific to the apicomplexans. Our work identifies 48 residues that are likely to be functionally important when comparing apicomplexan, kinetoplastid, and fungal betaCOP.

Arginine-based endoplasmic reticulum (ER)-localization signals are sorting motifs that are involved in the biosynthetic transport of multimeric membrane proteins. After their discovery in the invariant chain of the major histocompatibility complex class II, several hallmarks of these signals have emerged. They occur in polytopic membrane proteins that are subunits of membrane protein complexes; the presence of the signal maintains improperly assembled subunits in the ER by retention or retrieval until it is masked as a result of heteromultimeric assembly. A distinct consensus sequence and their position independence with respect to the distal termini of the protein distinguish them from other ER-sorting motifs. Recognition by the coatomer (COPI) vesicle coat explains ER retrieval. Often, di-leucine endocytic signals occur close to arginine-based signals. Recruitment of 14-3-3 family or PDZ-domain proteins can counteract ER-localization activity, as can phosphorylation. This, and the occurrence of arginine-based signals in alternatively spliced regions, implicates them in the regulated surface expression of multimeric membrane proteins in addition to their function in quality control.

Arf1 regulates membrane trafficking at several membrane sites by interacting with at least seven different vesicle coat proteins. Here, we test the hypothesis that Arf1-dependent coats are independently regulated by specific interaction with Arf GAPs. We find that the Arf GAP AGAP1 directly associates with and colocalizes with AP-3, a coat protein complex involved in trafficking in the endosomal-lysosomal system. Binding is mediated by the PH domain of AGAP1 and the delta and sigma3 subunits of AP-3. Overexpression of AGAP1 changes the cellular distribution of AP-3, and reduced expression of AGAP1 renders AP-3 resistant to brefeldin A. AGAP1 overexpression does not affect the distribution of other coat proteins, and AP-3 distribution is not affected by overexpression of other Arf GAPs. Cells overexpressing AGAP1 also exhibit increased LAMP1 trafficking via the plasma membrane. Taken together, these results support the hypothesis that AGAP1 directly and specifically regulates AP-3-dependent trafficking.

Arf GTP-binding proteins regulate membrane traffic and actin remodeling. Similar to other GTP-binding proteins, a complex of Arf-GTP with an effector protein mediates Arf function. Arf interacts with at least three qualitatively different types of effectors. First, it interacts with structural proteins, the vesicle coat proteins. The second type of effector is lipid-metabolizing enzymes, and the third comprises those proteins that bind to Arf-GTP but whose biochemical or biological functions are not yet clearly defined. Arf interacts with two other families of proteins, the exchange factors and the GTPase-activating proteins. Recent work examining the functional relationships among the diverse Arf interactors has led to reconsideration of the prevailing paradigms for Arf action.

Assembly of the coat protein I (COPI) vesicle coat is controlled by the small GTPase ADP ribosylation factor 1 (ARF1) and its GTPase-activating protein, ARFGAP1. Here, we investigate the diffusional behaviours of coatomer, the main component of the coat, and also those of ARF1 and ARFGAP1. Using fluorescence-correlation spectroscopy, we found that most ARF1 and ARFGAP1 molecules are highly mobile in the cytosol (diffusion constant D approximately equal to 15 microm(2) s(-1)), whereas coatomer diffuses 5-10 times more slowly than expected (D approximately equal to 1 microm(2) s(-1)). This slow diffusion causes diffusion-limited binding kinetics to Golgi membranes, which, in FRAP (fluorescence recovery after photobleaching) experiments, translates into a twofold slower binding rate. The addition of aluminium fluoride locks coatomer onto Golgi membranes and also decreases the binding kinetics of both ARF1 and ARFGAP1, suggesting that these proteins function in concert to mediate sorting and vesicle formation.

Although the actin cytoskeleton is widely believed to play an important role in intracellular protein transport, this role is poorly understood. Recently, progress has been made toward identifying specific actin-binding proteins and signaling molecules involved in regulating actin structures that function in the secretory pathway. Studies on coat protomer I (COPI)-mediated transport at the Golgi apparatus and on clathrin-mediated endocytosis have been particularly informative in identifying such mechanisms. Important similarities between actin regulation at the Golgi and at the plasma membrane have been uncovered. The studies reveal that ADP-ribosylation factor and vesicle coat proteins are able to act through the Rho-family GTP-binding proteins, Cdc42 and Rac, and several specific actin-binding proteins to direct actin assembly through the Arp2/3 complex. Efficient function of the secretory pathway is likely to require precise temporal regulation among transport-vesicle assembly, vesicle scission, and the targeting machinery. It is proposed that numerous actin regulatory mechanisms and the connections between actin signaling and vesicle-coat formation are employed to provide such temporal regulation.

This review includes 16 structures of vesicle coat components and accessory proteins and a description of their roles in vesicle budding or coat disassembly.

International audience; Clathrin-adaptor complexes (APs) are vesicle coat components that participate in cargo selectivity and transport vesicle formation. Here we cloned and characterized apm1, apm3 and apm4 cDNAs encoding AP medium chains (mu) in D. discoideum. Amino acid comparison suggested that predicted proteins were homologous to known mu1, mu3 and mu4 subunits of mammalian APs as they shared 69, 51, and 26% identity with mouse mu1A, human mu3A and human mu4, respectively. In all chains, amino acid residues predicted to interact with tyrosine based sorting signals were conserved. Southern blot analysis indicated only one copy of each gene in D. discoideum genome. Expression of apm1 and apm3 mRNAs stayed relatively constant during vegetative growth and throughout development. In contrast, apm4 was poorly expressed in amoebae but became well detectable by RT-PCR upon cell differentiation. This regulated expression of coat proteins enlightens the importance of intracellular membrane transport vesicles during development in D. discoideum and strengthens this attractive model organism for studying the function of coat complexes in vivo.Clathrin-adaptor complexes (APs) are vesicle coat components that participate in cargo selectivity and transport vesicle formation. Here we cloned and characterized apm1, apm3 and apm4 cDNAs encoding AP medium chains (mu) in D. discoideum. Amino acid comparison suggested that predicted proteins were homologous to known mu1, mu3 and mu4 subunits of mammalian APs as they shared 69, 51, and 26% identity with mouse mu1A, human mu3A and human mu4, respectively. In all chains, amino acid residues predicted to interact with tyrosine based sorting signals were conserved. Southern blot analysis indicated only one copy of each gene in D. discoideum genome. Expression of apm1 and apm3 mRNAs stayed relatively constant during vegetative growth and throughout development. In contrast, apm4 was poorly expressed in amoebae but became well detectable by RT-PCR upon cell differentiation. This regulated expression of coat proteins enlightens the importance of intracellular membrane transport vesicles during development in D. discoideum and strengthens this attractive model organism for studying the function of coat complexes in vivo.