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Understanding the cellular processes that lead to Alzheimer's disease (AD) is critical, and one key lies in the genetics of families with histories of AD. Mutations a complex known as COPI were found in families with AD. The COPI complex is involved in protein processing and trafficking. Intriguingly, several recent publications have found components of the COPI complex can affect the metabolism of pathogenic AD proteins. We reduced levels of the COPI subunit α-COP, altering maturation and cleavage of amyloid precursor protein (APP), resulting in decreased release of Aβ-42 and decreased accumulation of the AICD. Depletion of α-COP reduced uptake of proteopathic Tau seeds and reduces intracellular Tau self-association. Expression of AD-associated mutant α-COP altered APP processing, resulting in increased release of Aβ-42 and increased intracellular Tau aggregation and release of Tau oligomers. These results show that COPI coatomer function modulates processing of both APP and Tau, and expression of AD-associated α-COP confers a toxic gain of function, resulting in potentially pathogenic changes in both APP and Tau., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. Several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. A collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. Here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. Network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. We also identified the core cellular process subnetworks that are targeted by all the viruses. Integration with functional RNA interference (RNAi) datasets showed that a large proportion of the targets are required for viral replication. Furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis C virus and human metapneumovirus. Altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape., (Copyright © 2019 Bösl, Ianevski, Than, Andersen, Kuivanen, Teppor, Zusinaite, Dumpis, Vitkauskiene, Cox, Kallio-Kokko, Bergqvist, Tenson, Merits, Oksenych, Bjørås, Anthonsen, Shum, Kaarbø, Vapalahti, Windisch, Superti-Furga, Snijder, Kainov and Kandasamy.)

COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed α-, β-, β'-, γ-, δ-, ε-, and ζ-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The β'-, γ-, and δ-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of β'-, γ-, and δ-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of β'-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.

Eukaryotic cells require selective sorting and transport of cargo between intracellular compartments. This is accomplished at least in part by vesicles that bud from a donor compartment, sequestering a subset of resident protein "cargos" destined for transport to an acceptor compartment. A key step in vesicle formation and targeting is the recruitment of specific proteins that form a coat on the outside of the vesicle in a process requiring the activation of regulatory GTPases of the ARF family. Like all such GTPases, ARFs cycle between inactive, GDP-bound, and membrane-associated active, GTP-bound, conformations. And like most regulatory GTPases the activating step is slow and thought to be rate limiting in cells, requiring the use of ARF guanine nucleotide exchange factor (GEFs). ARF GEFs are characterized by the presence of a conserved, catalytic Sec7 domain, though they also contain motifs or additional domains that confer specificity to localization and regulation of activity. These domains have been used to define and classify five different sub-families of ARF GEFs. One of these, the BIG/GBF1 family, includes three proteins that are each key regulators of the secretory pathway. GEF activity initiates the coating of nascent vesicles via the localized generation of activated ARFs and thus these GEFs are the upstream regulators that define the site and timing of vesicle production. Paradoxically, while we have detailed molecular knowledge of how GEFs activate ARFs, we know very little about how GEFs are recruited and/or activated at the right time and place to initiate transport. This review summarizes the current knowledge of GEF regulation and explores the still uncertain mechanisms that position GEFs at "budding ready" membrane sites to generate highly localized activated ARFs.

The nuclear envelope (NE) is a specific extension of the endoplasmic reticulum (ER) that wraps around the nucleus and enables the spatial separation of gene transcription and protein translation, one of the signature features of eukaryotes. Rather than being completely closed, the double lipid bilayer of the NE is perforated at sites where the inner and outer nuclear membranes fuse, resulting in circular openings lined with sharply bent membranes. These openings are filled with nuclear pore complexes (NPCs), enormous protein assemblies that facilitate nuclear transport. The scaffold components of the NPC surprisingly share interesting similarities with elements of coat protein complexes, which have general implications for function and evolution of these membrane-coating complexes. Here I discuss, from a structural perspective, what these findings might teach us.

The coatomer protein complex, COPI, mediates retrograde vesicle transport from the Golgi apparatus to the ER. Here, we investigated the meiotic phenotype of Drosophila melanogaster spermatocytes expressing dsRNA of 52 genes encoding membrane-trafficking-related factors. We identified COPI as an essential factor for male meiosis. In Drosophila male meiotic divisions, COPI is localized in the ER-Golgi intermediate compartment of tER-Golgi units scattered throughout the spermatocyte cytoplasm. Prior to chromosome segregation, the vesicles assemble at the spindle pole periphery through a poleward movement, mediated by minus-end motor dynein along astral microtubules. At the end of each meiotic division, COPI-containing vesicles are equally partitioned between two daughter cells. Our present data strongly suggest that spermatocytes possess a regulatory mechanism for equal inheritance of several types of membrane vesicles. Using testis-specific knockdown of COPI subunits or the small GTPase Arf or mutations of the γCOP gene, we examined the role of COPI in male meiosis. COPI depletion resulted in the failure of cytokinesis, through disrupted accumulation of essential proteins and lipid components at the cleavage furrow region. Furthermore, it caused a reduction in the number of overlapping central spindle microtubules, which are essential for cytokinesis. Drosophila spermatocytes construct ER-based intracellular structures associated with astral and spindle microtubules. COPI depletion resulted in severe disruption of these ER-based structures. Thus, we propose that COPI plays an important role in Drosophila male meiosis, not only through vesicle transport to the cleavage furrow region, but also through the formation of ER-based structures.

Coat proteins orchestrate membrane budding and molecular sorting during the formation of transport intermediates. Coat protein complex I (COPI) vesicles shuttle between the Golgi apparatus and the endoplasmic reticulum and between Golgi stacks. The formation of a COPI vesicle proceeds in four steps: coat self-assembly, membrane deformation into a bud, fission of the coated vesicle and final disassembly of the coat to ensure recycling of coat components. Although some issues are still actively debated, the molecular mechanisms of COPI vesicle formation are now fairly well understood. In this review, we argue that physical parameters are critical regulators of COPI vesicle formation. We focus on recent real-time in vitro assays highlighting the role of membrane tension, membrane composition, membrane curvature and lipid packing in membrane remodelling and fission by the COPI coat.

Many proteins mature within the secretory pathway by the acquisition of glycans. Failure to maintain the proper distribution of the glycosylation machinery might lead to disease. High expression levels of the ubiquitous Golgi protein estrogen receptor-binding fragment-associated gene 9 (EBAG9) in human tumors correlate with poor clinical prognosis, and EBAG9 overexpression in epithelial cell lines induces truncated glycans, typical of many carcinomas. Here, we addressed the pathogenetic link between EBAG9 expression and the alteration of the cellular glycome. We applied confocal microscopy, live imaging, pulse-chase labeling in conjunction with immunoprecipitation, and enzymatic activity assays in a variety of EBAG9-overexpressing or depleted epithelial tumor cell lines. EBAG9 shuttles between the ER-Golgi intermediate compartment and the cis-Golgi, and we demonstrate association of EBAG9 with coat protein complex I (COPI)-coated transport vesicles. EBAG9 overexpression imposes delay of endoplasmic reticulum-to-Golgi transport and mislocalizes components of the ER quality control and glycosylation machinery. Conversely, EBAG9 down-regulation accelerates glycoprotein transport through the Golgi and enhances mannosidase activity. Thus, EBAG9 acts as a negative regulator of a COPI-dependent ER-to-Golgi transport pathway in epithelial cells and represents a novel pathogenetic principle in which interference with intracellular membrane trafficking results in the emergence of a tumor-associated glycome.

Because the functional borders of the intermediate compartment (IC) are not well defined, the spatial map of the transport machineries operating between the endoplasmic reticulum (ER) and the Golgi apparatus remains incomplete. Our previous studies showed that the IC consists of interconnected vacuolar and tubular parts with specific roles in pre-Golgi trafficking. Here, using live cell imaging, we demonstrate that the tubules containing the GTPase Rab1A create a long-lived membrane compartment around the centrosome. Separation of this pericentrosomal domain of the IC from the Golgi ribbon, due to centrosome motility, revealed that it contains a distinct pool of COPI coats and acts as a temperature-sensitive way station in post-ER trafficking. However, unlike the Golgi, the pericentrosomal IC resists the disassembly of COPI coats by brefeldin A, maintaining its juxtaposition with the endocytic recycling compartment, and operation as the focal point of a dynamic tubular network that extends to the cell periphery. These results provide novel insight into the compartmental organization of the secretory pathway and Golgi biogenesis. Moreover, they reveal a direct functional connection between the IC and the endosomal system, which evidently contributes to unconventional transport of the cystic fibrosis transmembrane conductance regulator to the cell surface.

The coat protein I (COPI) complex is considered to be one of the best-characterized coat complexes. Studies on how it functions in vesicle formation have provided seminal contributions to the general paradigm in vesicular transport that the ADP-ribosylation factor (ARF) small GTPases are key regulators of coat complexes. Here, we discuss emerging evidence that suggests the need to revise some long-held views on how COPI vesicle formation is achieved.

The sorting and concentration of cargo proteins within ER exit sites (ERESs) is a fundamental function of the secretory machinery. The mechanism by which peripheral coat complexes and their small GTPase effectors mediate this function with export membrane domains is only partially understood. The secretory-machinery-mediated sorting to ERESs is a process that counters the entropy-driven even distribution of membrane proteins within organellar membranes. Here, for the first time, we quantified the dynamic properties of GFP-VSVG sorting to ERESs in living cells by uncoupling it from later translocation steps using microtubule depolymerization. The dynamics of the ER to ERES redistribution of cargo proteins was quantified in single cells by measuring changes in fluorescence-intensity variance after shift to the permissive temperature. Cargo concentration within ERESs continued in cells overexpressing the GTP-locked ARF1Q71L or in the presence of brefeldin A. In the absence of COPI and microtubules, ERESs transformed from tubulovesicular to spherical membranes that actively accumulated secretory cargo and excluded ER-membrane markers. We found sorting to ERESs to be a slow and diffusion-unlimited process. Our findings exclude COPI, and identify the COPII protein complex to be directly involved in the secretory cargo sorting and redistribution functions of ERESs.

Cystic fibrosis (CF) is caused by defects in the CF transmembrane conductance regulator (CFTR) that functions as a chloride channel in epithelial cells. The most common cause of CF is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the plasma membrane (PM) is important. The coat protein complex I (COPI) has been implicated in the anterograde and retrograde transport of proteins and lipids between the endoplasmic reticulum and the Golgi. Here, we investigated the role of COPI in CFTR trafficking. Blocking COPI recruitment to membranes by expressing an inactive form of the GBF1 guanine nucleotide exchange factor for ADP-ribosylation factor inhibits CFTR trafficking to the PM. Similarly, inhibiting COPI dissociation from membranes by expressing a constitutively active ADP-ribosylation factor 1 mutant arrests CFTR within disrupted Golgi elements. To definitively explore the relationship between COPI and CFTR in epithelial cells, we depleted beta-COP from the human colonic epithelial cell HT-29Cl.19A using small interfering RNA. Beta-COP depletion did not affect CFTR synthesis but impaired its trafficking to the PM. The arrest occurred pre-Golgi as shown by reduced level of glycosylation. Importantly, decreased trafficking of CFTR had a functional consequence as cells depleted of beta-COP showed decreased cAMP-activated chloride currents. To explore the mechanism of COPI action in CFTR traffic we tested whether CFTR was COPI cargo. We discovered that the alpha-, beta-, and gamma-subunits of COPI co-immunoprecipitated with CFTR. Our results indicate that the COPI complex plays a critical role in CFTR trafficking to the PM.

Ubiquitylation appears to be involved in the membrane trafficking system including endocytosis, exocytosis, and ER-to-Golgi transport. We found that PIRH2, which was identified as an interacting protein for androgen receptor or p53, interacts with and ubiquitylates the epsilon-subunit of coatmer complex, epsilon-COP. PIRH2 promotes the ubiquitylation of epsilon-COP in vitro and in vivo and consequently promotes the degradation of epsilon-COP. The interaction between PIRH2 and epsilon-COP is affected by the presence of androgen, and PIRH2 in the presence of androgen promotes ubiquitylation of epsilon-COP in vivo. Furthermore, overexpression of the wild type of PIRH2 in prostate cancer cells causes downregulation of the secretion of prostate-specific antigen (PSA), a secretory protein in prostate epithelial cells and one of diagnostic markers for prostate cancer. Our results indicate that PIRH2 functions as a regulator for COP I complex.

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.

Coated vesicles represent vital transport intermediates in all eukaryotic cells. While the basic mechanisms of membrane exchange are conserved through the kingdoms, the unique topology of the plant endomembrane system is mirrored by several differences in the genesis, function and regulation of coated vesicles. Efforts to unravel the complex network of proteins underlying the behaviour of these vesicles have recently benefited from the application in planta of several molecular tools used in mammalian systems, as well as from advances in imaging technology and the ongoing analysis of the Arabidopsis genome. In this review, we provide an overview of the roles of coated vesicles in plant cells and highlight salient new developments in the field.

Emerging experimental evidence favours the existence of cargo sorting occurring upon the endoplasmic reticulum (ER) exit. Recent studies revealed that, in contrast to the conventional secretory marker ts-O45-G, procollagen (PC I) exits the ER at sites not coated with coat protein II and is transported to the Golgi complex in carriers devoid of coat protein I. Here, we investigated whether PC I trafficking requires a different molecular machinery in comparison with the ts-O45-G. By combining colocalization of the cargoes with endogenous markers, downregulation of transport machinery by RNA interference and knock-ins by complementary DNA over-expression, we provide strong evidence that PC I and ts-O45-G have common but also different molecular requirements during pre- and post-Golgi trafficking events.

Eukaryotic cells use an elaborate machinery involving the COPI coat complex to control protein trafficking in the secretory pathway. Although individual components of this complex are well known and their roles in deforming lipid membranes into coated carriers are well described, the precise sequence of molecular events by which these components assemble into and release from the COPI coat lattice remains unclear. Here, we present images and movies characterizing the dynamics of protein components of the COPI coat in living cells. We discuss the self-assembly of these coat components into a molecular machine for sorting and trafficking membranes.

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.

When higher eukaryotic cells transition into mitosis, the nuclear envelope, nuclear pore complexes, and nuclear lamina are coordinately disassembled. The COPI coatomer complex, which plays a major role in membrane remodeling at the Golgi, has been implicated in the process of nuclear envelope breakdown and requires interactions at the nuclear pore complex for recruitment to this new site of action at mitosis. Nup153, a resident of the nuclear pore basket, was found to be involved in COPI recruitment, but the molecular nature of the interface between COPI and the nuclear pore has not been fully elucidated. To better understand what occurs at the nuclear pore at this juncture, we have probed the role of the nucleoporin Nup358/RanBP2. Nup358 contains a repetitive zinc finger domain with overall organization similar to a region within Nup153 that is critical to COPI association, yet inspection of these two zinc finger domains reveals features that also clearly distinguish them. Here, we found that the Nup358 zinc finger domain, but not a zinc finger domain from an unrelated protein, binds to COPI and dominantly inhibits progression of nuclear envelope breakdown in an assay that robustly recapitulates this process in vitro. Moreover, the Nup358 zinc finger domain interferes with COPI recruitment to the nuclear rim. Consistent with a role for this pore protein in coordinating nuclear envelope breakdown, Nup358-specific antibodies impair nuclear disassembly. Significantly, targeting either Nup153 or Nup358 for inhibition perturbs nuclear envelope breakdown, supporting a model in which these nucleoporins play nonredundant roles, perhaps contributing to COPI recruitment platforms on both the nuclear and cytoplasmic faces of the pore. We found that an individual zinc finger is the minimal interface for COPI association, although tandem zinc fingers are optimal. These results provide new information about the critical components of nuclear membrane remodeling and lay the foundation for a better understanding of how this process is regulated.

COPI-coated vesicles are protein and liquid carriers that mediate transport within the early secretory pathway. In this Topical Review, we present their main protein components and discuss current models for cargo sorting. Finally, we describe the striking similarities that exist between the COPI system and the two other characterized types of vesicular carriers: COPII- and clathrin-coated vesicles.

Cdc42, a member of the Rho subfamily of small GTPases, is highly conserved in both sequence and function across eukaryotic species. In budding yeast, Cdc42 triggers polarized growth necessary for bud emergence via rearrangement of the actin cytoskeleton. It has been shown that the role of Cdc42 in bud emergence requires both Cdc28-Cln (G1) kinase and the passage through START. In this report, we show that Cdc42 also serves an essential function in the establishment of bud site prior to START by catalyzing the translocation of bud-site components such as Spa2 to the cell cortex. Our analysis of various conditional alleles of CDC42 suggests that these two functions (bud site establishment and bud emergence) are genetically separable. Surprisingly, the role of Cdc42 in the cortical localization of Spa2 appears to be independent of its well known GTP/GDP exchange factor Cdc24. We also provide evidence that this role of Cdc42 requires the function of the COPI coatomer complex.

The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)-dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.

Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.

The conserved oligomeric Golgi (COG) complex is a soluble hetero-octamer associated with the cytoplasmic surface of the Golgi. Mammalian somatic cell mutants lacking the Cog1 (ldlB) or Cog2 (ldlC) subunits exhibit pleiotropic defects in Golgi-associated glycoprotein and glycolipid processing that suggest COG is involved in the localization, transport, and/or function of multiple Golgi processing proteins. We have identified a set of COG-sensitive, integral membrane Golgi proteins called GEARs (mannosidase II, GOS-28, GS15, GPP130, CASP, giantin, and golgin-84) whose abundances were reduced in the mutant cells and, in some cases, increased in COG-overexpressing cells. In the mutants, some GEARs were abnormally localized in the endoplasmic reticulum and were degraded by proteasomes. The distributions of the GEARs were altered by small interfering RNA depletion of epsilon-COP in wild-type cells under conditions in which COG-insensitive proteins were unaffected. Furthermore, synthetic phenotypes arose in mutants deficient in both epsilon-COP and either Cog1 or Cog2. COG and COPI may work in concert to ensure the proper retention or retrieval of a subset of proteins in the Golgi, and COG helps prevent the endoplasmic reticulum accumulation and degradation of some GEARs.

The endoplasmic reticulum (ER)-Golgi system has been studied using biochemical, genetic, electron and light microscopic techniques. We now understand many aspects of trafficking from the ER to the Golgi apparatus, including some of the signals and mechanisms for selective retention and retrieval of ER resident proteins and export of cargo proteins. Proteins that leave the ER emerge in 'export complexes' or ER 'exit sites' and accumulate in pleiomorphic transport carriers referred to sometimes as VTCs or intermediate compartments. These structures then transit from the ER to the Golgi apparatus along microtubules using the dynein/dynactin motor and fuse with the cis cisterna of the Golgi apparatus. Many proteins (including vSNAREs, ERGIC53/p58 and the KDEL receptor) must cycle back to the ER from pre-Golgi intermediates or the Golgi. We will discuss both the currently favored model that this cycling occurs via 50-nm COPI-coated vesicles and in vivo evidence that suggests retrograde trafficking may occur via tubular structures.

Since its discovery, the Golgi complex has attracted the attention of cell biologists because of its 'fashionable' morphology and central position within the secretory system of the cell. Here, we discuss how the three-dimensional architecture of the Golgi complex relates to its multiple functions in protein sorting and processing, and how an analysis of the morphology of the Golgi complex can help to provide an understanding of the mechanisms involved in transport through this unique organelle.

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.

COP I and COP II coat proteins direct protein and membrane trafficking in between early compartments of the secretory pathway in eukaryotic cells. These coat proteins perform the dual, essential tasks of selecting appropriate cargo proteins and deforming the lipid bilayer of appropriate donor membranes into buds and vesicles. COP II proteins are required for selective export of newly synthesized proteins from the endoplasmic reticulum (ER). COP I proteins mediate a retrograde transport pathway that selectively recycles proteins from the cis-Golgi complex to the ER. Additionally, COP I coat proteins have complex functions in intra-Golgi trafficking and in maintaining the normal structure of the mammalian interphase Golgi 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.

It is unknown how Legionella pneumophila cells escape the degradative lysosomal pathway after phagocytosis by macrophages and replicate in an organelle derived from the endoplasmic reticulum. Here we show that, after internalization, L. pneumophila-containing phagosomes recruit early secretory vesicles. Once L. pneumophila phagosomes have intercepted early secretory vesicles they begin to acquire proteins residing in transitional and rough endoplasmic reticulum. The functions of Sar1 and ADP-ribosylation factor-1 are important for biogenesis of the L. pneumophila replicative organelle. These data indicate that L. pneumophila intercepts vesicular traffic from endoplasmic-reticulum exit sites to create an organelle that permits intracellular replication and prevents destruction by the host cell.

We have examined the ER-to-Golgi transport of procollagen, which, when assembled in the lumen of the ER, is thought to be physically too large to fit in classically described 60-80 nm COPI- and COPII-coated transport vesicles. We found that procollagen exits the ER via COPII- coated ER exit sites and is transported to the Golgi along microtubules in defined transport complexes. These procollagen-containing transport complexes are, however, distinct from those containing other cargo proteins like ERGIC-53 and ts-045-G. Furthermore, they do not label for the COPI coat complex in contrast to those containing ts-045-G. Inhibition of COPII or COPI function before addition of ascorbate, which is required for the folding of procollagen, inhibits export of procollagen from the ER. Inactivation of COPI coat function after addition of ascorbate results in the localisation of procollagen to transport complexes that now also contain ERGIC-53 and are inhibited in their transport to the Golgi complex. These data reveal the existence of an early COPI-dependent, pre-Golgi cargo sorting step in mammalian cells.

Inhibition of the putative coatomer protein I (COPI) vesicle tethering complex, giantin-p115-GM130, may contribute to mitotic Golgi breakdown. However, neither this, nor the role of the giantin-p115-GM130 complex in the maintenance of Golgi structure has been demonstrated in vivo. Therefore, we generated antibodies directed against the mapped binding sites in each protein of the complex and injected these into mammalian tissue culture cells. Surprisingly, the injected anti-p115 and antigiantin antibodies caused proteasome-mediated degradation of the corresponding antigens. Reduction of p115 levels below detection led to COPI-dependent Golgi fragmentation and apparent accumulation of Golgi-derived vesicles. In contrast, neither reduction of giantin below detectable levels, nor inhibition of p115 binding to GM130, had any detectable effect on Golgi structure or Golgi reassembly after cell division or brefeldin A washout. These observations indicate that inhibition of p115 can induce a mitotic-like Golgi disassembly, but its essential role in Golgi structure is independent of its Golgi-localized binding partners giantin and GM130.

Recent evidence suggests that extension of pseudopods during phagocytosis requires localized insertion of endomembrane vesicles. The nature of these vesicles and the processes mediating their release and insertion are unknown. COPI plays an essential role in the budding and traffic of membrane vesicles in intracellular compartments. We therefore assessed whether COPI is also involved in phagosome formation. We used ldlF cells, a mutant line derived from Chinese hamster ovary cells that express a temperature-sensitive form of epsilonCOP. To confer phagocytic ability to ldlF cells, they were stably transfected with Fc receptors type IIA (FcgammaRIIA). In the presence of functional COPI, FcgammaRIIA-transfected ldlF cells effectively internalized opsonized particles. In contrast, phagocytosis was virtually eliminated after incubation at the restrictive temperature. Similar results were obtained impairing COPI function in macrophages using brefeldin A. Notably, loss of COPI function preceded complete inhibition of phagocytosis, suggesting that COPI is indirectly required for phagocytosis. Despite their inability to internalize particles, COPI-deficient cells nevertheless expressed normal levels of FcgammaRIIA, and signal transduction appeared unimpeded. The opsonized particles adhered normally to COPI-deficient cells and were often found on actin-rich pedestals, but they were not internalized due to the inability of the cells to extend pseudopods. The failure to extend pseudopods was attributed to the inability of COPI-deficient cells to mobilize endomembrane vesicles, including a VAMP3-containing compartment, in response to the phagocytic stimulus.

Now that whole genome sequences are available for many eukaryotic organisms from yeast to man, we can form broad hypotheses on the basis of the relative expansion of protein families. To investigate the molecular mechanisms responsible for the organization of membrane compartments, we identified members of the SNARE, coat complex, Rab and Sec1 protein families in four eukaryotic genomes. Of these families only the Rab family expanded from the unicellular yeast to the multicellular fly and worm. All families were expanded in humans, where we find 35 SNAREs, 60 Rabs and 53 coat complex subunits. In addition, we were able to resolve the SNARE class of proteins into four distinct subfamilies.

Cargo molecules have to be included in carrier vesicles of different forms and sizes to be transported between organelles. During this process, a limited set of proteins, including the coat proteins COPI, COPII and clathrin, carries out a programmed set of sequential interactions that lead to the budding of vesicles. A general model to explain the formation of coated vesicles is starting to emerge but the picture is more complex than we had imagined.

The COPI coatomer is thought to be required in yeast directly for retrograde transport from the Golgi to the endoplasmic reticulum (ER), and directly or indirectly for ER-to-Golgi transport. Unexpectedly, the secretory glycoproteins Hsp150 and invertase have been found not to require COPI for ER exit. The features according to which cargo proteins are selected for the COPI-independent pathway are not known. The ER form of Hsp150 has three distinct domains: an N-terminal fragment of 54 amino acids (subunit I) is followed by 11 repeats of a 19 amino acid peptide plus a unique C-terminal fragment of 114 amino acids (subunit II). By fusing heterologous proteins to different Hsp150 domains and expressing them in sec21-1 and sec21-3 mutants with temperature-sensitive mutations in the gamma-COPI subunit, we show here that the repeats of subunit II function as sorting determinants for COPI-independent ER exit. The C-terminal fragment of Hsp150 could be replaced by E. coli beta-lactamase or rat nerve growth factor receptor ectodomain (NGFRe), and subunit I could be deleted, without inhibiting COPI-independent transport. However, when the repetitive region was omitted and beta-lactamase was fused directly to the C terminus of subunit I, COPI was required for efficient ER exit. Mass spectroscopic analysis demonstrated that both subunit I and II of Hsp150 were extensively O-glycosylated, suggesting that the O-glycosylation pattern was not decisive for cargo selection.