RhoGAP6 interacts with COPI to regulate protein transport.

RhoGAP6 is the most highly expressed GTPase-activating protein (GAP) in platelets specific for RhoA. Structurally RhoGAP6 contains a central catalytic GAP domain surrounded by large, disordered N- and C-termini of unknown function. Sequence analysis revealed three conserved consecutive overlapping di-tryptophan motifs close to the RhoGAP6 C-terminus which were predicted to bind to the mu homology domain (MHD) of δ-COP, a component of the COPI vesicle complex. We confirmed an endogenous interaction between RhoGAP6 and δ-COP in human platelets using GST-CD2AP which binds an N-terminal RhoGAP6 SH3 binding motif. Next, we confirmed that the MHD of δ-COP and the di-tryptophan motifs of RhoGAP6 mediate the interaction between both proteins. Each of the three di-tryptophan motifs appeared necessary for stable δ-COP binding. Proteomic analysis of other potential RhoGAP6 di-tryptophan motif binding partners indicated that the RhoGAP6/δ-COP interaction connects RhoGAP6 to the whole COPI complex. 14-3-3 was also established as a RhoGAP6 binding partner and its binding site was mapped to serine 37. We provide evidence of potential cross-regulation between 14-3-3 and δ-COP binding, however, neither δ-COP nor 14-3-3 binding to RhoGAP6 impacted RhoA activity. Instead, analysis of protein transport through the secretory pathway demonstrated that RhoGAP6/δ-COP binding increased protein transport to the plasma membrane, as did a catalytically inactive mutant of RhoGAP6. Overall, we have identified a novel interaction between RhoGAP6 and δ-COP which is mediated by conserved C-terminal di-tryptophan motifs, and which might control protein transport in platelets.

An extended motif in the SARS-CoV-2 spike modulates binding and release of host coatomer in retrograde trafficking.

ß-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.

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.

Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly.

BACKGROUND: Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as "coatopathies". METHODS: Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (ß-COP). To investigate Family 1's splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2's missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2's mutation. RESULTS: We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between ß-COP and ß'-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant ß-COP, with the mutant protein being retarded in the Golgi. CONCLUSIONS: This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.

Crystal structure of truncated human coatomer protein complex subunit ζ1 (Copζ1).

The majority of modern anticancer approaches target DNA/protein targets involved in tumour-cell proliferation. Such approaches have a major drawback, as nonproliferating cancer cells remain unaffected and may cause relapse or remission. Human coatomer protein complex I (COPI) subunit ζ (Copζ), a component of the coat protein involved in cell apoptosis and intracellular trafficking, has recently been proposed as a potential anticancer drug target. Previous studies have shown that two different isoforms of the Copζ subunit exist in mammalian cells. While normal cells express both Copζ1 and Copζ2 isoforms, various types of tumour cells display a loss of Copζ2 expression and rely solely on Copζ1 for growth and survival. Subsequent knockdown of Copζ1 results in specific inhibition of both proliferating and dormant tumour-cell populations, with no adverse growth effects on normal cells. Therefore, a Copζ1-targeting therapy was proposed to bypass the problem of dormant cancer cells that are resistant to conventional antiproliferative drugs, which is the major cause of tumour relapse. In order to aid in structure-based inhibitor design, a crystal structure is required. In this article, the recombinant expression, purification, crystallization and crystal structure of Copζ1, as well as the expression and purification of Copζ2, are reported.

RNA Editing TUTase 1: structural foundation of substrate recognition, complex interactions and drug targeting.

Terminal uridyltransferases (TUTases) execute 3' RNA uridylation across protists, fungi, metazoan and plant species. Uridylation plays a particularly prominent role in RNA processing pathways of kinetoplastid protists typified by the causative agent of African sleeping sickness, Trypanosoma brucei In mitochondria of this pathogen, most mRNAs are internally modified by U-insertion/deletion editing while guide RNAs and rRNAs are U-tailed. The founding member of TUTase family, RNA editing TUTase 1 (RET1), functions as a subunit of the 3' processome in uridylation of gRNA precursors and mature guide RNAs. Along with KPAP1 poly(A) polymerase, RET1 also participates in mRNA translational activation. RET1 is divergent from human TUTases and is essential for parasite viability in the mammalian host and the insect vector. Given its robust in vitro activity, RET1 represents an attractive target for trypanocide development. Here, we report high-resolution crystal structures of the RET1 catalytic core alone and in complex with UTP analogs. These structures reveal a tight docking of the conserved nucleotidyl transferase bi-domain module with a RET1-specific C2H2 zinc finger and RNA recognition (RRM) domains. Furthermore, we define RET1 region required for incorporation into the 3' processome, determinants for RNA binding, subunit oligomerization and processive UTP incorporation, and predict druggable pockets.

Structural characterization of coatomer in its cytosolic state.

Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.

Sequence evidence for common ancestry of eukaryotic endomembrane coatomers.

Eukaryotic cells are defined by compartments through which the trafficking of macromolecules is mediated by large complexes, such as the nuclear pore, transport vesicles and intraflagellar transport. The assembly and maintenance of these complexes is facilitated by endomembrane coatomers, long suspected to be divergently related on the basis of structural and more recently phylogenomic analysis. By performing supervised walks in sequence space across coatomer superfamilies, we uncover subtle sequence patterns that have remained elusive to date, ultimately unifying eukaryotic coatomers by divergent evolution. The conserved residues shared by 3,502 endomembrane coatomer components are mapped onto the solenoid superhelix of nucleoporin and COPII protein structures, thus determining the invariant elements of coatomer architecture. This ancient structural motif can be considered as a universal signature connecting eukaryotic coatomers involved in multiple cellular processes across cell physiology and human disease.

A novel RING finger in the C-terminal domain of the coatomer protein α-COP.

The C-terminal domain of α-COP, an essential subunit of the COPI coatomer complex, is composed of an all α-helical region and a small ß-sheet domain. We show that this ß-sheet domain is a Really Interesting New Gene (RING)-like treble clef zinc finger. The zinc-binding residues are substituted by other aminoacids in many homologs including the structurally-characterized proteins from Saccharomyces cerevisiae and Bos taurus. This RING-like domain is possibly related to those of other vesicle membrane-associated complexes, such as CORVET, HOPS and SEA, and likely mediates interactions with Dsl1p and assist in coat oligomerization.

Structural basis for the binding of tryptophan-based motifs by δ-COP.

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding ßγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αß'ε-COP B-subcomplex. We present the structure of the C-terminal µ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP µ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1-6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.

Inter-Golgi transport mediated by COPI-containing vesicles carrying small cargoes.

A core prediction of the vesicular transport model is that COPI vesicles are responsible for trafficking anterograde cargoes forward. In this study, we test this prediction by examining the properties and requirements of inter-Golgi transport within fused cells, which requires mobile carriers in order for exchange of constituents to occur. We report that both small soluble and membrane-bound secretory cargo and exogenous Golgi resident glycosyl-transferases are exchanged between separated Golgi. Large soluble aggregates, which traverse individual stacks, do not transfer between Golgi, implying that small cargoes (which can fit in a typical transport vesicle) are transported by a different mechanism. Super-resolution microscopy reveals that the carriers of both anterograde and retrograde cargoes are the size of COPI vesicles, contain coatomer, and functionally require ARF1 and coatomer for transport. The data suggest that COPI vesicles traffic both small secretory cargo and steady-state Golgi resident enzymes among stacked cisternae that are stationary. DOI:http://dx.doi.org/10.7554/eLife.01296.001.

Rules for the recognition of dilysine retrieval motifs by coatomer.

Cytoplasmic dilysine motifs on transmembrane proteins are captured by coatomer α-COP and ß'-COP subunits and packaged into COPI-coated vesicles for Golgi-to-ER retrieval. Numerous ER/Golgi proteins contain K(x)Kxx motifs, but the rules for their recognition are unclear. We present crystal structures of α-COP and ß'-COP bound to a series of naturally occurring retrieval motifs-encompassing KKxx, KxKxx and non-canonical RKxx and viral KxHxx sequences. Binding experiments show that α-COP and ß'-COP have generally the same specificity for KKxx and KxKxx, but only ß'-COP recognizes the RKxx signal. Dilysine motif recognition involves lysine side-chain interactions with two acidic patches. Surprisingly, however, KKxx and KxKxx motifs bind differently, with their lysine residues transposed at the binding patches. We derive rules for retrieval motif recognition from key structural features: the reversed binding modes, the recognition of the C-terminal carboxylate group which enforces lysine positional context, and the tolerance of the acidic patches for non-lysine residues.

Molecular basis for recognition of dilysine trafficking motifs by COPI.

COPI mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER) and within the Golgi stack, sorting transmembrane proteins bearing C-terminal KKxx or KxKxx motifs. The structure of KxKxx motifs bound to the N-terminal WD-repeat domain of ß'-COP identifies electrostatic contacts between the motif and complementary patches at the center of the ß'-COP propeller. An absolute requirement of a two-residue spacing between the terminal carboxylate group and first lysine residue results from interactions of carbonyl groups in the motif backbone with basic side chains of ß'-COP. Similar interactions are proposed to mediate binding of KKxx motifs by the homologous α-COP domain. Mutation of key interacting residues in either domain or in their cognate motifs abolishes in vitro binding and results in mistrafficking of dilysine-containing cargo in yeast without compromising cell viability. Flexibility between ß'-COP WD-repeat domains and the location of cargo binding have implications for COPI coat assembly.

Size doesn't matter! The importance of size variability in coated vesicles.

Coated vesicles are key components of the machinery of vesicular transport used by eukaryotic cells. In a recent story published in Science, Faini et al. (2012) report on the detailed 3D structure of a synthetically generated COPI vesicle coat obtained using cryoelectron tomography.

Crystallization and preliminary X-ray analysis of the C-terminal domain of δ-COP, a medium-sized subunit of the COPI complex involved in membrane trafficking.

Coat protein I (COPI) is a protein complex composed of seven subunits that mediates retrograde transport of proteins and lipids from the cis-Golgi network to the endoplasmic reticulum and intra-Golgi membranes. The medium-sized δ subunit of COPI (δ-COP) is a 57 kDa protein with a C-terminal domain (CTD) and an N-terminal longin domain. Here, the δ-COP CTD was successfully cloned, purified and crystallized. Diffraction data were collected from native and selenomethionyl crystals of δ-COP CTD to resolutions of 2.60 and 2.30 Å, respectively. Both crystals belonged to space group P2(1)2(1)2, with similar unit-cell parameters. The native crystals had unit-cell parameters a = 100.23, b = 136.77, c = 44.39 Å.

The kiss-and-run model of intra-Golgi transport.

The Golgi apparatus (GA) is the main station along the secretory pathway. Mechanisms of intra-Golgi transport remain unresolved. Three models compete with each other for the right to be defined as the paradigm. The vesicular model cannot explain the following: (1) lipid droplets and aggregates of procollagen that are larger than coatomer I (COPI)-dependent vesicles are transported across the GA; and (2) most anterograde cargoes are depleted in COPI vesicles. The compartment progression/maturation model has the following problems: (1) most Golgi-resident proteins are depleted in COPI vesicles; (2) there are no COPI vesicles for the recycling of the resident proteins in the trans-most-Golgi cisterna; and (3) different proteins have different rates of intra-Golgi transport. The diffusion model based on permanent inter-cisternal connections cannot explain the existence of lipid, ionic and protein gradients across the Golgi stacks. In contrast, the kiss-and-run model has the potential to explain most of the experimental observations. The kiss-and-run model can be symmetric when fusion and then fission occurs in the same place, and asymmetric when fusion takes place in one location, whereas fission takes place in another. The asymmetric kiss-and-run model resembles the carrier maturation mechanism, and it can be used to explain the transport of large cargo aggregates.

The structures of COPI-coated vesicles reveal alternate coatomer conformations and interactions.

Transport between compartments of eukaryotic cells is mediated by coated vesicles. The archetypal protein coats COPI, COPII, and clathrin are conserved from yeast to human. Structural studies of COPII and clathrin coats assembled in vitro without membranes suggest that coat components assemble regular cages with the same set of interactions between components. Detailed three-dimensional structures of coated membrane vesicles have not been obtained. Here, we solved the structures of individual COPI-coated membrane vesicles by cryoelectron tomography and subtomogram averaging of in vitro reconstituted budding reactions. The coat protein complex, coatomer, was observed to adopt alternative conformations to change the number of other coatomers with which it interacts and to form vesicles with variable sizes and shapes. This represents a fundamentally different basis for vesicle coat assembly.

Effects of mutations in the WD40 domain of α-COP on its interaction with the COPI coatomer in Saccharomyces cerevisiae.

Replacement of glycine 227 in the fifth WD40 motif of α-COP/Ret1p/Soo1p by charged or aromatic amino acids is responsible for the temperature-dependent osmo-sensitivity of Saccharomyces cerevisiae, while truncations of WD40 motifs exerted a reduction in cell growth rate and impairment in assembly of cell-wall associated proteins such as enolase and Gas1p. Yeast two-hybrid analysis revealed that the ret1-1/soo1-1 mutation of α-COP abolished the interaction with ß- and ɛ-COP, respectively, and that the interaction between α-COP and ß-COP relied on the WD40 domain of α-COP. Furthermore, although the WD40 domain is dispensable for interaction of α-COP with ɛ-COP, structural alterations in the WD40 domain could impair the interaction.

Full-length structural model of RET3 and SEC21 in COPI: identification of binding sites on the appendage for accessory protein recruitment motifs.

COPI, a 600 kD heptameric complex (consisting of subunits α, ß, γ, δ, ε, ζ, and ß') "coatomer," assembles non-clathrin-coated vesicles and is responsible for intra-Golgi and Golgi-to-ER protein trafficking. Here, we report the three-dimensional structures of the entire sequences of yeast Sec21 (γ-COPI mammalian ortholog), yeast Ret3 (ζ-COPI mammalian ortholog), and the results of successive molecular dynamics investigations of the subunits and assembly based on a protein-protein docking experiment. The three-dimensional structures of the subunits in their complexes indicate the residues of the two subunits that impact on assembly, the conformations of Ret3 and Sec21, and their binding orientations in the complexed state. The structure of the appendage domain of Sec21, with its two subdomains--the platform and the ß-sandwich, was investigated to explore its capacity to bind to accessory protein recruitment motifs. Our study shows that a binding site on the platform is capable of binding the Eps15 DPF and epsin DPW2 peptides, whereas the second site on the platform and the site on the ß-sandwich subdomain were found to selectively bind to the amphiphysin FXDXF and epsin DPW1 peptides, respectively. Identifying the regions of both the platform and sandwich subdomains involved in binding each peptide motif clarifies the mechanism through which the appendage domain of Sec21 engages with the accessory proteins during the trafficking process of non-clathrin-coated vesicles.