Coatopathies: Genetic Disorders of Protein Coats.

Protein coats are supramolecular complexes that assemble on the cytosolic face of membranes to promote cargo sorting and transport carrier formation in the endomembrane system of eukaryotic cells. Several types of protein coats have been described, including COPI, COPII, AP-1, AP-2, AP-3, AP-4, AP-5, and retromer, which operate at different stages of the endomembrane system. Defects in these coats impair specific transport pathways, compromising the function and viability of the cells. In humans, mutations in subunits of these coats cause various congenital diseases that are collectively referred to as coatopathies. In this article, we review the fundamental properties of protein coats and the diseases that result from mutation of their constituent subunits.

The Evolution of Organellar Coat Complexes and Organization of the Eukaryotic Cell.

Eukaryotic cells possess a remarkably diverse range of organelles that provide compartmentalization for distinct cellular functions and are likely responsible for the remarkable success of these organisms. The origins and subsequent elaboration of these compartments represent a key aspect in the transition between prokaryotic and eukaryotic cellular forms. The protein machinery required to build, maintain, and define many membrane-bound compartments is encoded by several paralog families, including small GTPases, coiled-bundle proteins, and proteins with ß-propeller and α-solenoid secondary structures. Together these proteins provide the membrane coats and control systems to structure and coordinate the endomembrane system. Mechanistically and evolutionarily, they unite not only secretory and endocytic organelles but also the flagellum and nucleus. The ancient origins for these families have been revealed by recent findings, providing new perspectives on the deep evolutionary processes and relationships that underlie eukaryotic cell structure.

COPB2 loss of function causes a coatopathy with osteoporosis and developmental delay.

Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.

Genome-wide RNA interference screening reveals a COPI-MAP2K3 pathway required for YAP regulation.

The transcriptional regulator YAP, which plays important roles in the development, regeneration, and tumorigenesis, is activated when released from inhibition by the Hippo kinase cascade. The regulatory mechanism of YAP in Hippo-low contexts is poorly understood. Here, we performed a genome-wide RNA interference screen to identify genes whose loss of function in a Hippo-null background affects YAP activity. We discovered that the coatomer protein complex I (COPI) is required for YAP nuclear enrichment and that COPI dependency of YAP confers an intrinsic vulnerability to COPI disruption in YAP-driven cancer cells. We identified MAP2K3 as a YAP regulator involved in inhibitory YAP phosphorylation induced by COPI subunit depletion. The endoplasmic reticulum stress response pathway activated by COPI malfunction appears to connect COPI and MAP2K3. In addition, we provide evidence that YAP inhibition by COPI disruption may contribute to transcriptional up-regulation of PTGS2 and proinflammatory cytokines. Our study offers a resource for investigating Hippo-independent YAP regulation as a therapeutic target for cancers and suggests a link between YAP and COPI-associated inflammatory diseases.

COP1 SUPPRESSOR 6 represses the PIF4 and PIF5 action to promote light-inhibited hypocotyl growth.

Light signaling precisely controls photomorphogenic development in plants. PHYTOCHROME INTERACTING FACTOR 4 and 5 (PIF4 and PIF5) play critical roles in the regulation of this developmental process. In this study, we report CONSTITUTIVELY PHOTOMORPHOGENIC 1 SUPPRESSOR 6 (CSU6) functions as a key regulator of light signaling. Loss of CSU6 function largely rescues the cop1-6 constitutively photomorphogenic phenotype. CSU6 promotes hypocotyl growth in the dark, but inhibits hypocotyl elongation in the light. CSU6 not only associates with the promoter regions of PIF4 and PIF5 to inhibit their expression in the morning, but also directly interacts with both PIF4 and PIF5 to repress their transcriptional activation activity. CSU6 negatively controls a group of PIF4- and PIF5-regulated gene expressions. Mutations in PIF4 and/or PIF5 are epistatic to the loss of CSU6, suggesting that CSU6 acts upstream of PIF4 and PIF5. Taken together, CSU6 promotes light-inhibited hypocotyl elongation by negatively regulating PIF4 and PIF5 transcription and biochemical activity.

Dissection of GTPase-activating proteins reveals functional asymmetry in the COPI coat of budding yeast.

The Arf GTPase controls formation of the COPI vesicle coat. Recent structural models of COPI revealed the positioning of two Arf1 molecules in contrasting molecular environments. Each of these pockets for Arf1 is expected to also accommodate an Arf GTPase-activating protein (ArfGAP). Structural evidence and protein interactions observed between isolated domains indirectly suggest that each niche preferentially recruits one of the two ArfGAPs known to affect COPI, i.e. Gcs1/ArfGAP1 and Glo3/ArfGAP2/3, although only partial structures are available. The functional role of the unique non-catalytic domain of either ArfGAP has not been integrated into the current COPI structural model. Here, we delineate key differences in the consequences of triggering GTP hydrolysis through the activity of one versus the other ArfGAP. We demonstrate that Glo3/ArfGAP2/3 specifically triggers Arf1 GTP hydrolysis impinging on the stability of the COPI coat. We show that the Snf1 kinase complex, the yeast homologue of AMP-activated protein kinase (AMPK), phosphorylates the region of Glo3 that is crucial for this effect and, thereby, regulates its function in the COPI-vesicle cycle. Our results revise the model of ArfGAP function in the molecular context of COPI.This article has an associated First Person interview with the first author of the paper.

Hazara Nairovirus Requires COPI Components in both Arf1-Dependent and Arf1-Independent Stages of Its Replication Cycle.

Hazara nairovirus (HAZV) is an enveloped trisegmented negative-strand RNA virus classified within the Nairoviridae family of the Bunyavirales order and a member of the same subtype as Crimean-Congo hemorrhagic fever virus, responsible for fatal human disease. Nairoviral subversion of cellular trafficking pathways to permit viral entry, gene expression, assembly, and egress is poorly understood. Here, we generated a recombinant HAZV expressing enhanced green fluorescent protein and used live-cell fluorescent imaging to screen an siRNA library targeting genes involved in cellular trafficking networks, the first such screen for a nairovirus. The screen revealed prominent roles for subunits of the coat protein 1 (COPI)-vesicle coatomer, which regulates retrograde trafficking of cargo between the Golgi apparatus and the endoplasmic reticulum, as well as intra-Golgi transport. We show the requirement of COPI-coatomer subunits impacted at least two stages of the HAZV replication cycle: an early stage prior to and including gene expression and also a later stage during assembly and egress of infectious virus, with COPI-knockdown reducing titers by approximately 1,000-fold. Treatment of HAZV-infected cells with brefeldin A (BFA), an inhibitor of Arf1 activation required for COPI coatomer formation, revealed that this late COPI-dependent stage was Arf1 dependent, consistent with the established role of Arf1 in COPI vesicle formation. In contrast, the early COPI-dependent stage was Arf1 independent, with neither BFA treatment nor siRNA-mediated ARF1 knockdown affecting HAZV gene expression. HAZV exploitation of COPI components in a noncanonical Arf1-independent process suggests that COPI coatomer components may perform roles unrelated to vesicle formation, adding further complexity to our understanding of cargo-mediated transport.IMPORTANCE Nairoviruses are tick-borne enveloped RNA viruses that include several pathogens responsible for fatal disease in humans and animals. Here, we analyzed host genes involved in trafficking networks to examine their involvement in nairovirus replication. We revealed important roles for genes that express multiple components of the COPI complex, which regulates transport of Golgi apparatus-resident cargos. COPI components influenced at least two stages of the nairovirus replication cycle: an early stage prior to and including gene expression and also a later stage during assembly of infectious virus, with COPI knockdown reducing titers by approximately 1,000-fold. Importantly, while the late stage was Arf1 dependent, as expected for canonical COPI vesicle formation, the early stage was found to be Arf1 independent, suggestive of a previously unreported function of COPI unrelated to vesicle formation. Collectively, these data improve our understanding of nairovirus host-pathogen interactions and suggest a new Arf1-independent role for components of the COPI coatomer complex.

Functional comparison of the WD-repeat domains of SPA1 and COP1 in suppression of photomorphogenesis.

The Arabidopsis COP1/SPA complex acts as a cullin4-based E3 ubiquitin ligase to suppress photomorphogenesis in darkness. It is a tetrameric complex of two COP1 and two SPA proteins. Both COP1 and SPA are essential for the activity of this complex, and they both contain a C-terminal WD-repeat domain responsible for substrate recruitment and binding of DDB1. Here, we used a WD domain swap-approach to address the cooperativity of COP1 and SPA proteins. We found that expression of a chimeric COP1 carrying the WD-repeat domain of SPA1 mostly complemented the cop1-4-mutant phenotype in darkness, indicating that the WD repeat of SPA1 can replace the WD repeat of COP1. In the light, SPA1-WD partially substituted for COP1-WD. In contrast, expression of a chimeric SPA1 protein carrying the WD repeat of COP1 did not rescue the spa-mutant phenotype. Together, our findings demonstrate that a SPA1-type WD repeat is essential for COP1/SPA activity, while a COP1-type WD is in part dispensible. Moreover, a complex with four SPA1-WDs is more active than a complex with only two SPA1-WDs. A homology model of SPA1-WD based on the crystal structure of COP1-WD uncovered two insertions and several amino acid substitutions at the predicted substrate-binding pocket of SPA1-WD.

Recycling of Golgi glycosyltransferases requires direct binding to coatomer.

The glycosyltransferases of the mammalian Golgi complex must recycle between the stacked cisternae of that organelle to maintain their proper steady-state localization. This trafficking is mediated by COPI-coated vesicles, but how the glycosyltransferases are incorporated into these transport vesicles is poorly understood. Here we show that the N-terminal cytoplasmic tails (N-tails) of a number of cis Golgi glycosyltransferases which share a ϕ-(K/R)-X-L-X-(K/R) sequence bind directly to the δ- and ζ-subunits of COPI. Mutations of this N-tail motif impair binding to the COPI subunits, leading to mislocalization of the transferases to lysosomes. The physiological importance of these interactions is illustrated by mucolipidosis III patients with missense mutations in the N-tail of GlcNAc-1-phosphotransferase that cause the transferase to be rapidly degraded in lysosomes. These studies establish that direct binding of the N-tails of mammalian cis Golgi glycosyltransferases with COPI subunits is essential for recycling within the Golgi.

cTAGE5/MEA6 plays a critical role in neuronal cellular components trafficking and brain development.

Normal neural development is essential for the formation of neuronal networks and brain function. Cutaneous T cell lymphoma-associated antigen 5 (cTAGE5)/meningioma expressed antigen 6 (MEA6) plays a critical role in the secretion of proteins. However, its roles in the transport of nonsecretory cellular components and in brain development remain unknown. Here, we show that cTAGE5/MEA6 is important for brain development and function. Conditional knockout of cTAGE5/MEA6 in the brain leads to severe defects in neural development, including deficits in dendrite outgrowth and branching, spine formation and maintenance, astrocyte activation, and abnormal behaviors. We reveal that loss of cTAGE5/MEA6 affects the interaction between the coat protein complex II (COPII) components, SAR1 and SEC23, leading to persistent activation of SAR1 and defects in COPII vesicle formation and transport from the endoplasmic reticulum to the Golgi, as well as disturbed trafficking of membrane components in neurons. These defects affect not only the transport of materials required for the development of dendrites and spines but also the signaling pathways required for neuronal development. Because mutations in cTAGE5/MEA6 have been found in patients with Fahr's disease, our study potentially also provides insight into the pathogenesis of this disorder.

The p24 Complex Contributes to Specify Arf1 for COPI Coat Selection.

Golgi trafficking depends on the small GTPase Arf1 which, upon activation, drives the assembly of different coats onto budding vesicles. Two related types of guanine nucleotide exchange factors (GEFs) activate Arf1 at different Golgi sites. In yeast, Gea1 in the cis-Golgi and Gea2 in the medial-Golgi activate Arf1 to form COPI-coated vesicles for retrograde cargo sorting, whereas Sec7 generates clathrin/adaptor-coated vesicles at the trans-Golgi network (TGN) for forward cargo transport. A central question is how the same activated Arf1 protein manages to assemble different coats depending on the donor Golgi compartment. A previous study has postulated that the interaction between Gea1 and COPI would channel Arf1 activation for COPI vesicle budding. Here, we found that the p24 complex, a major COPI vesicle cargo, promotes the binding of Gea1 with COPI by increasing the COPI association to the membrane independently of Arf1 activation. Furthermore, the p24 complex also facilitates the interaction of Arf1 with its COPI effector. Therefore, our study supports a mechanism by which the p24 complex contributes to program Arf1 activation by Gea1 for selective COPI coat assembly at the cis-Golgi compartment.

Formation of COPI-coated vesicles at a glance.

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.

A genome-wide analysis of coatomer protein (COP) subunits of apicomplexan parasites and their evolutionary relationships.

BACKGROUND: Protein secretion is an essential process in all eukaryotes including organisms belonging to the phylum Apicomplexa, which includes many intracellular parasites. The apicomplexan parasites possess a specialized collection of secretory organelles that release a number of proteins to facilitate the invasion of host cells and some of these proteins also participate in immune evasion. Like in other eukaryotes, these parasites possess a series of membrane-bound compartments, namely the endoplasmic reticulum (ER), the intermediate compartments (IC) or vesicular tubular clusters (VTS) and Golgi complex through which proteins pass in a sequential and vectorial fashion. Two sets of proteins; COPI and COPII are important for directing the sequential transfer of material between the ER and Golgi complex. RESULTS: Here, using in silico approaches, we identify the components of COPI and COPII complexes in the genome of apicomplexan organisms. The results showed that the COPI and COPII protein complexes are conserved in most apicomplexan genomes with few exceptions. Diversity among the components of COPI and COPII complexes in apicomplexan is either due to the absence of a subunit or due to the difference in the number of protein domains. For example, the COPI epsilon subunit and COPII sec13 subunit is absent in Babesia bovis, Theileria parva, and Theileria annulata genomes. Phylogenetic and domain analyses for all the proteins of COPI and COPII complexes was performed to predict their evolutionary relationship and functional significance. CONCLUSIONS: The study thus provides insights into the apicomplexan COPI and COPII coating machinery, which is crucial for parasites secretory network needed for the invasion of host cells.

Transient N-glycosylation abnormalities likely due to a de novo loss-of-function mutation in the delta subunit of coat protein I.

Accurate glycosylation of proteins is essential for their function and their intracellular transport. Numerous diseases have been described, where either glycosylation or intracellular transport of proteins is impaired. Coat protein I (COPI) is involved in anterograde and retrograde transport of proteins between endoplasmic reticulum and Golgi, where glycosylation takes place, but no association of defective COPI proteins and glycosylation defects has been described so far. We identified a patient whose phenotype at a first glance was reminiscent of PGM1 deficiency, a disease that also affects N-glycosylation of proteins. More detailed analyses revealed a different disease with a glycosylation deficiency that was only detectable during episodes of acute illness of the patient. Trio-exome analysis revealed a de novo loss-of-function mutation in ARCN1, coding for the delta-COP subunit of COPI. We hypothesize that the capacity of flow through Golgi is reduced by this defect and at high protein synthesis rates, this bottleneck also manifests as transient glycosylation deficiency.

Discovery Proteomics Identifies a Molecular Link between the Coatomer Protein Complex I and Androgen Receptor-dependent Transcription.

Aberrant androgen receptor (AR)-dependent transcription is a hallmark of human prostate cancers. At the molecular level, ligand-mediated AR activation is coordinated through spatial and temporal protein-protein interactions involving AR-interacting proteins, which we designate the "AR-interactome." Despite many years of research, the ligand-sensitive protein complexes involved in ligand-mediated AR activation in prostate tumor cells have not been clearly defined. Here, we describe the development, characterization, and utilization of a novel human LNCaP prostate tumor cell line, N-AR, which stably expresses wild-type AR tagged at its N terminus with the streptavidin-binding peptide epitope (streptavidin-binding peptide-tagged wild-type androgen receptor; SBP-AR). A bioanalytical workflow involving streptavidin chromatography and label-free quantitative mass spectrometry was used to identify SBP-AR and associated ligand-sensitive cytosolic proteins/protein complexes linked to AR activation in prostate tumor cells. Functional studies verified that ligand-sensitive proteins identified in the proteomic screen encoded modulators of AR-mediated transcription, suggesting that these novel proteins were putative SBP-AR-interacting proteins in N-AR cells. This was supported by biochemical associations between recombinant SBP-AR and the ligand-sensitive coatomer protein complex I (COPI) retrograde trafficking complex in vitro Extensive biochemical and molecular experiments showed that the COPI retrograde complex regulates ligand-mediated AR transcriptional activation, which correlated with the mobilization of the Golgi-localized ARA160 coactivator to the nuclear compartment of prostate tumor cells. Collectively, this study provides a bioanalytical strategy to validate the AR-interactome and define novel AR-interacting proteins involved in ligand-mediated AR activation in prostate tumor cells. Moreover, we describe a cellular system to study how compartment-specific AR-interacting proteins influence AR activation and contribute to aberrant AR-dependent transcription that underlies the majority of human prostate cancers.

Key components of COPI and COPII machineries are required for chikungunya virus replication.

The infection of CHIKV is associated with cellular membranes; however whether early secretory pathways are involved in CHIKV replication remains unclear. In the present study, we have provided initial evidences that CHIKV requires both COPI and COPII for its replication. Small interfering RNAs against COPI components, including coatomer, ARFs or GBF1, suppress CHIKV replication. Moreover, CHIKV infection is abolished by the presence of ARF1 inhibitor brefeldin A or GBF1 inhibitor golgicide A. In addition, perturbation of COPII by silencing key components of COPII pathways leads to a reduction in CHIKV replication. Collectively, these observations demonstrate the importance of functional secretory pathways in the infectivity of CHIKV.

Reconstitution of the targeting of Rab6A to the Golgi apparatus in semi-intact HeLa cells: A role of BICD2 in stabilizing Rab6A on Golgi membranes and a concerted role of Rab6A/BICD2 interactions in Golgi-to-ER retrograde transport.

Rab is a small GTP-binding protein family that regulates various pathways of vesicular transport. Although more than 60 Rab proteins are targeted to specific organelles in mammalian cells, the mechanisms underlying the specificity of Rab proteins for the respective organelles remain unknown. In this study, we reconstituted the Golgi targeting of Rab6A in streptolysin O (SLO)-permeabilized HeLa cells in a cytosol-dependent manner and investigated the biochemical requirements of targeting. Golgi-targeting assays identified Bicaudal-D (BICD)2, which is reportedly involved in the dynein-mediated transport of mRNAs during oogenesis and embryogenesis in Drosophila, as a cytosolic factor for the Golgi targeting of Rab6A in SLO-permeabilized HeLa cells. Subsequent immunofluorescence analyses indicated decreased amounts of the GTP-bound active form of Rab6 in BICD2-knockdown cells. In addition, fluorescence recovery after photobleaching (FRAP) analyses revealed that overexpression of the C-terminal region of BICD2 decreased the exchange rate of GFP-Rab6A between the Golgi membrane and the cytosol. Collectively, these results indicated that BICD2 facilitates the binding of Rab6A to the Golgi by stabilizing its GTP-bound form. Moreover, several analyses of vesicular transport demonstrated that Rab6A and BICD2 play crucial roles in Golgi tubule fusion with the endoplasmic reticulum (ER) in brefeldin A (BFA)-treated cells, indicating that BICD2 is involved in coat protein I (COPI)-independent Golgi-to-ER retrograde vesicular transport.

Evolution of modular intraflagellar transport from a coatomer-like progenitor.

The intraflagellar transport (IFT) complex is an integral component of the cilium, a quintessential organelle of the eukaryotic cell. The IFT system consists of three subcomplexes [i.e., intraflagellar transport (IFT)-A, IFT-B, and the BBSome], which together transport proteins and other molecules along the cilium. IFT dysfunction results in diseases collectively called ciliopathies. It has been proposed that the IFT complexes originated from vesicle coats similar to coat protein complex (COP) I, COPII, and clathrin. Here we provide phylogenetic evidence for common ancestry of IFT subunits and α, ß', and ε subunits of COPI, and trace the origins of the IFT-A, IFT-B, and the BBSome subcomplexes. We find that IFT-A and the BBSome likely arose from an IFT-B-like complex by intracomplex subunit duplication. The distribution of IFT proteins across eukaryotes identifies the BBSome as a frequently lost, modular component of the IFT. Significantly, loss of the BBSome from a taxon is a frequent precursor to complete cilium loss in related taxa. Given the inferred late origin of the BBSome in cilium evolution and its frequent loss, the IFT complex behaves as a "last-in, first-out" system. The protocoatomer origin of the IFT complex corroborates involvement of IFT components in vesicle transport. Expansion of IFT subunits by duplication and their subsequent independent loss supports the idea of modularity and structural independence of the IFT subcomplexes.