Acute COG complex inactivation unveiled its immediate impact on Golgi and illuminated the nature of intra-Golgi recycling vesicles.

Conserved Oligomeric Golgi (COG) complex controls Golgi trafficking and glycosylation, but the precise COG mechanism is unknown. The auxin-inducible acute degradation system was employed to investigate initial defects resulting from COG dysfunction. We found that acute COG inactivation caused a massive accumulation of COG-dependent (CCD) vesicles that carry the bulk of Golgi enzymes and resident proteins. v-SNAREs (GS15, GS28) and v-tethers (giantin, golgin84, and TMF1) were relocalized into CCD vesicles, while t-SNAREs (STX5, YKT6), t-tethers (GM130, p115), and most of Rab proteins remained Golgi-associated. Airyscan microscopy and velocity gradient analysis revealed that different Golgi residents are segregated into different populations of CCD vesicles. Acute COG depletion significantly affected three Golgi-based vesicular coats-COPI, AP1, and GGA, suggesting that COG uniquely orchestrates tethering of multiple types of intra-Golgi CCD vesicles produced by different coat machineries. This study provided the first detailed view of primary cellular defects associated with COG dysfunction in human cells.

Biallelic missense variants in COG3 cause a congenital disorder of glycosylation with impairment of retrograde vesicular trafficking.

Biallelic variants in genes for seven out of eight subunits of the conserved oligomeric Golgi complex (COG) are known to cause recessive congenital disorders of glycosylation (CDG) with variable clinical manifestations. COG3 encodes a constituent subunit of the COG complex that has not been associated with disease traits in humans. Herein, we report two COG3 homozygous missense variants in four individuals from two unrelated consanguineous families that co-segregated with COG3-CDG presentations. Clinical phenotypes of affected individuals include global developmental delay, severe intellectual disability, microcephaly, epilepsy, facial dysmorphism, and variable neurological findings. Biochemical analysis of serum transferrin from one family showed the loss of a single sialic acid. Western blotting on patient-derived fibroblasts revealed reduced COG3 and COG4. Further experiments showed delayed retrograde vesicular recycling in patient cells. This report adds to the knowledge of the COG-CDG network by providing collective evidence for a COG3-CDG rare disease trait and implicating a likely pathology of the disorder as the perturbation of Golgi trafficking.

Disorder of sex development associated with a novel homozygous nonsense mutation in COG6 expands the phenotypic spectrum of COG6-CDG.

Congenital disorders of glycosylation (CDG) are an expanding group of metabolic disorders that result from abnormal protein glycosylation. A special subgroup of CDG type II comprises defects in the Conserved Oligomeric Golgi Complex (COG). In order to further delineate the genotypic and phenotypic spectrum of COG complex defect, we describe a novel variant of COG6 gene found in homozygosity in a Moroccan patient with severe presentation of COG6-CDG (OMIM #614576). We compared the phenotype of our patient with other previously reported COG6-CDG cases. Common features in COG6-CDG are facial dysmorphism, growth retardation, microcephaly, developmental disability, liver or gastrointestinal disease, recurrent infections, hypohidrosis/hyperthermia. In addition to these phenotypic features, our patient exhibited a disorder of sexual differentiation, which has rarely been reported in COG6-CDG. We hypothesize that the severe COG6 gene mutation interferes with glycosylation of a disintegrin and metalloprotease family members, inhibiting the correct gonadal distal tip cells migration, fundamental for the genitalia morphogenesis. This report broadens the genetic and phenotypic spectrum of COG6-CDG and provides further supportive evidence that COG6-CDG can present as a disorder of sexual differentiation.

Hypothesis: determining phenotypic specificity facilitates understanding of pathophysiology in rare genetic disorders.

In the rapidly growing group of rare genetic disorders, data scarcity demands an intelligible use of available data, in order to improve understanding of underlying pathophysiology. We hypothesize, based on the principle that clinical similarities may be indicative of shared pathophysiology, that determining phenotypic specificity could provide unsuspected insights in pathophysiology of rare genetic disorders. We explored our hypothesis by studying subunit deficiencies of the conserved oligomeric Golgi (COG) complex, a subgroup of congenital disorders of glycosylation (CDG). In this systematic data assessment, all 45 reported patients with COG-CDG were included. The vocabulary of the Human Phenotype Ontology was used to annotate all phenotypic features and to assess occurrence in other genetic disorders. Gene occurrence ratios were calculated by dividing the frequency in the patient cohort over the number of associated genes, according to the Human Phenotype Ontology. Prioritisation based on phenotypic specificity was highly informative and captured phenotypic features commonly associated with glycosylation disorders. Moreover, it captured features not seen in any other glycosylation disorder, among which episodic fever, likely reflecting underappreciated other cellular functions of the COG complex. Interestingly, the COG complex was recently implicated in the autophagy pathway, as are more than half of the genes underlying disorders that present with episodic fever. This suggests that whereas many phenotypic features in these patients are caused by disrupted glycosylation, episodic fever might be caused by disrupted autophagy. Thus, we here demonstrate support for our hypothesis that determining phenotypic specificity could facilitate understanding of pathophysiology in rare genetic disorders.

Defective mucin-type glycosylation on α-dystroglycan in COG-deficient cells increases its susceptibility to bacterial proteases.

Deficiency in subunits of the conserved oligomeric Golgi (COG) complex results in pleiotropic defects in glycosylation and causes congenital disorders in humans. Insight regarding the functional consequences of this defective glycosylation and the identity of specific glycoproteins affected is lacking. A chemical glycobiology strategy was adopted to identify the surface glycoproteins most sensitive to altered glycosylation in COG-deficient Chinese hamster ovary (CHO) cells. Following metabolic labeling, an unexpected increase in GalNAz incorporation into several glycoproteins, including α-dystroglycan (α-DG), was noted in cog1-deficient ldlB cells. Western blotting analysis showed a significantly lower molecular weight for α-DG in ldlB cells compared with WT CHO cells. The underglycosylated α-DG molecules on ldlB cells are highly vulnerable to bacterial proteases that co-purify with V. cholerae neuraminidase, leading to rapid removal of the protein from the cell surface. The purified bacterial mucinase StcE can cleave both WT and ldlB α-DG but did not cause rapid degradation of the fragments, implicating other V. cholerae proteases in the final proteolysis of the fragments. Extending terminal glycosylation on the existing mucin-type glycans of ldlB α-DG stabilized the resulting fragments, indicating that fragment stability, but not the initial fragmentation of the protein, is influenced by the glycosylation status of the cell. This discovery highlights a functional importance for mucin-type O-glycans found on α-DG and reinforces a growing role for these glycans as regulators of extracellular proteolysis and protein stability.

Hypothesis: lobe A (COG1-4)-CDG causes a more severe phenotype than lobe B (COG5-8)-CDG.

The conserved oligomeric Golgi (COG) complex consists of eight subunits organized in two lobes: lobe A (COG1-4) and lobe B (COG5-8). The different functional roles of COG lobe A and lobe B might result in distinct clinical phenotypes in patients with COG-CDG (congenital disorders of glycosylation). This hypothesis is supported by three observations. First, knock-down of COG lobe A components affects Golgi morphology more severely than knock-down of COG lobe B components. Second, nearly all of the 27 patients with lobe B COG-CDG had bi-allelic truncating mutations, as compared with only one of the six patients with lobe A COG-CDG. This represents a frequency gap which suggests that bi-allelic truncating mutations in COG lobe A genes might be non-viable. Third, in support, large-scale exome data of healthy adults (Exome Aggregation Consortium (ExAC)) underline that COG lobe A genes are less tolerant to genetic variation than COG lobe B genes. Thus, comparable molecular defects are more detrimental in lobe A COG-CDG than in lobe B COG-CDG. In a larger perspective, clinical phenotypic severity corresponded nicely with tolerance to genetic variation. Therefore, genomic epidemiology can potentially be used as a photographic negative for mutational severity.

Dissecting functions of the conserved oligomeric Golgi tethering complex using a cell-free assay.

Vesicle transport sorts proteins between compartments and is thereby responsible for generating the non-uniform protein distribution along the eukaryotic secretory and endocytic pathways. The mechanistic details of specific vesicle targeting are not yet well characterized at the molecular level. We have developed a cell-free assay that reconstitutes vesicle targeting utilizing the recycling of resident enzymes within the Golgi apparatus. The assay has physiological properties, and could be used to show that the two lobes of the conserved oligomeric Golgi tethering complex play antagonistic roles in trans-Golgi vesicle targeting. Moreover, we can show that the assay is sensitive to several different congenital defects that disrupt Golgi function and therefore cause glycosylation disorders. Consequently, this assay will allow mechanistic insight into the targeting step of vesicle transport at the Golgi, and could also be useful for characterizing some novel cases of congenital glycosylation disorders.

Novel coiled-coil proteins regulate exocyst association with cortical microtubules in xylem cells via the conserved oligomeric golgi-complex 2 protein.

Xylem vessel cells develop secondary cell walls in distinct patterns. Cortical microtubules are rearranged into distinct patterns and regulate secondary cell wall deposition; however, it is unclear how exocytotic membrane trafficking is linked to cortical microtubules. Here, we show that the novel coiled-coil proteins vesicle tethering 1 (VETH1) and VETH2 recruit EXO70A1, an exocyst subunit essential for correct patterning of secondary cell wall deposition, to cortical microtubules via the conserved oligomeric Golgi complex (COG) 2 protein. VETH1 and VETH2 encode an uncharacterized domain of an unknown function designated DUF869, and were preferentially up-regulated in xylem cells. VETH1-green fluorescent protein (GFP) and VETH2-GFP co-localized at novel vesicle-like small compartments, which exhibited microtubule plus-end-directed and end-tracking dynamics. VETH1 and VETH2 interacted with COG2, and this interaction promoted the association between cortical microtubules and EXO70A1 These results suggest that the VETH-COG2 complex ensures the correct secondary cell wall deposition pattern by recruiting exocyst components to cortical microtubules.

Key features and clinical variability of COG6-CDG.

The conserved oligomeric Golgi (COG) complex consists of eight subunits and plays a crucial role in Golgi trafficking and positioning of glycosylation enzymes. Mutations in all COG subunits, except subunit 3, have been detected in patients with congenital disorders of glycosylation (CDG) of variable severity. So far, 3 families with a total of 10 individuals with biallelic COG6 mutations have been described, showing a broad clinical spectrum. Here we present 7 additional patients with 4 novel COG6 mutations. In spite of clinical variability, we delineate the core features of COG6-CDG i.e. liver involvement (9/10), microcephaly (8/10), developmental disability (8/10), recurrent infections (7/10), early lethality (6/10), and hypohidrosis predisposing for hyperthermia (6/10) and hyperkeratosis (4/10) as ectodermal signs. Regarding all COG6-related disorders a genotype-phenotype correlation can be discerned ranging from deep intronic mutations found in Shaheen syndrome as the mildest form to loss-of-function mutations leading to early lethal CDG phenotypes. A comparison with other COG deficiencies suggests ectodermal changes to be a hallmark of COG6-related disorders. Our findings aid clinical differentiation of this complex group of disorders and imply subtle functional differences between the COG complex subunits.

Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay.

Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was tagged with either CFP or YFP. Given that GalT is recycled to the cisterna where it is located at steady state, GalT-containing vesicles target GalT-containing cisternal membranes. Golgi membranes were therefore isolated from GalT-CFP expressing cells, while vesicles were prepared from GalT-YFP expressing ones. Incubating CFP-labelled Golgi with YFP-labelled vesicles in the presence of cytosol and an energy regeneration mixture at 37 °C produced a significant increase in CFP-YFP co-localization upon fluorescent imaging of the mixture compared to incubation on ice. The assay was validated to require energy, proteins and physiologically important trafficking components such as Rab GTPases and the conserved oligomeric Golgi tethering complex. This assay is useful for the investigation of both physiological and pathological changes that affect the Golgi trafficking machinery, in particular, vesicle tethering.

Creating Knockouts of Conserved Oligomeric Golgi Complex Subunits Using CRISPR-Mediated Gene Editing Paired with a Selection Strategy Based on Glycosylation Defects Associated with Impaired COG Complex Function.

The conserved oligomeric Golgi (COG) complex is a key evolutionally conserved multisubunit protein machinery that regulates tethering and fusion of intra-Golgi transport vesicles. The Golgi apparatus specifically promotes sorting and complex glycosylation of glycoconjugates. Without proper glycosylation and processing, proteins and lipids will be mislocalized and/or have impaired function. The Golgi glycosylation machinery is kept in homeostasis by a careful balance of anterograde and retrograde trafficking to ensure proper localization of the glycosylation enzymes and their substrates. This balance, like other steps of membrane trafficking, is maintained by vesicle trafficking machinery that includes COPI vesicular coat proteins, SNAREs, Rabs, and both coiled-coil and multi-subunit vesicular tethers. The COG complex interacts with other membrane trafficking components and is essential for proper localization of Golgi glycosylation machinery. Here we describe using CRISPR-mediated gene editing coupled with a phenotype-based selection strategy directly linked to the COG complex's role in glycosylation homeostasis to obtain COG complex subunit knockouts (KOs). This has resulted in clonal KOs for each COG subunit in HEK293T cells and gives the ability to further probe the role of the COG complex in Golgi homeostasis.

Defects in the COG complex and COG-related trafficking regulators affect neuronal Golgi function.

The Conserved Oligomeric Golgi (COG) complex is an evolutionarily conserved hetero-octameric protein complex that has been proposed to organize vesicle tethering at the Golgi apparatus. Defects in seven of the eight COG subunits are linked to Congenital Disorders of Glycosylation (CDG)-type II, a family of rare diseases involving misregulation of protein glycosylation, alterations in Golgi structure, variations in retrograde trafficking through the Golgi and system-wide clinical pathologies. A troublesome aspect of these diseases are the neurological pathologies such as low IQ, microcephaly, and cerebellar atrophy. The essential function of the COG complex is dependent upon interactions with other components of trafficking machinery, such as Rab-GTPases and SNAREs. COG-interacting Rabs and SNAREs have been implicated in neurodegenerative diseases like Alzheimer's disease and Parkinson's disease. Defects in Golgi maintenance disrupts trafficking and processing of essential proteins, frequently associated with and contributing to compromised neuron function and human disease. Despite the recent advances in molecular neuroscience, the subcellular bases for most neurodegenerative diseases are poorly understood. This article gives an overview of the potential contributions of the COG complex and its Rab and SNARE partners in the pathogenesis of different neurodegenerative disorders.

Target silencing of components of the conserved oligomeric Golgi complex impairs HIV-1 replication.

All viruses require host cell factors to replicate. A large number of host factors have been identified that participate at numerous points of the human immunodeficiency virus 1 (HIV-1) life cycle. Recent evidence supports a role for components of the trans-Golgi network (TGN) in mediating early steps in the HIV-1 life cycle. The conserved oligomeric Golgi (COG) complex is a heteroctamer complex that functions in coat protein complex I (COPI)-mediated intra-Golgi retrograde trafficking and plays an important role in the maintenance of Golgi structure and integrity as well as glycosylation enzyme homeostasis. The targeted silencing of components of lobe B of the COG complex, namely COG5, COG6, COG7 and COG8, inhibited HIV-1 replication. This inhibition of HIV-1 replication preceded late reverse transcription (RT) but did not affect viral fusion. Silencing of the COG interacting protein the t-SNARE syntaxin 5, showed a similar defect in late RT product formation, strengthening the role of the TGN in HIV replication.