COG defects,birth and rise!

The COG complex is a cytosolic heteromeric Golgi complex constituted of 8 subunits (Cog1 to Cog8) and involved in retrograde vesicular Golgi trafficking. The involvement of this complex in glycosylation and more specifically in Golgi glycosyltransferases localization has been highlighted with the discovery of COG subunit deficiencies leading to CDG (Congenital Disorders of Glycosylation), a group of inherited disorders of glycosylation. To date, many COG deficient CDG patients have been discovered and this article reviews the birth and rise of this group of defects. The architecture of the COG complex and its cellular functions in Golgi trafficking and Golgi glycosylation are discussed.     

How Golgi glycosylation meets and needs trafficking: the case of the COG complex

Protein glycosylation is one of the major biosynthetic functions occurring in the endoplasmic reticulum and Golgi compartments. It requires an amazing number of enzymes, chaperones, lectins and transporters whose actions delicately secure the fidelity of glycan structures. Over the past 30 years, glycobiologists hammered that glycan structures are not mere decorative elements but serve crucial cellular functions. This becomes dramatically illustrated by a group of mostly severe, inherited human disorders named congenital disorders of glycosylation (CDG). To date, many types of CDG have been defined genetically and most of the time the defects impair the biosynthesis, transfer and remodeling of N-glycans. Recently, the identification of the several types of CDG caused by deficiencies in the conserved oligomeric Golgi (COG) complex, a complex involved in vesicular Golgi trafficking, expanded the field of CDG but also brought novel insights in glycosylation. The molecular mechanisms underlying the complex pathway of N-glycosylation in the Golgi are far from understood. The availability of COG-deficient CDG patients and patients' cells offered a new way to study how COG, and its different subunits, could influence the Golgi N-glycosylation machinery and localization. This review summarizes the recent findings on the implication of COG in Golgi glycosylation. It highlights the need for a dynamic, finely tuned balance between anterograde and retrograde trafficking for the correct localization of Golgi enzymes to assure the stepwise maturation of N-glycan chains.     

Retrograde transport on the COG railway

The conserved oligomeric Golgi (COG) complex is essential for establishing and/or maintaining the structure and function of the Golgi apparatus. The Golgi apparatus, in turn, has a central role in protein sorting and glycosylation within the eukaryotic secretory pathway. As a consequence, COG mutations can give rise to human genetic diseases known as congenital disorders of glycosylation. We review recent results from studies of yeast, worm, fly and mammalian COG that provide evidence that COG might function in retrograde vesicular trafficking within the Golgi apparatus. This hypothesis explains the impact of COG mutations by postulating that they impair the retrograde flow of resident Golgi proteins needed to maintain normal Golgi structure and function.     

COG-7-deficient Human Fibroblasts Exhibit Altered Recycling of Golgi Proteins

Recently, we reported that two siblings presenting with the clinical syndrome congenital disorders of glycosylation (CDG) have mutations in the gene encoding Cog7p, a member of the conserved oligomeric Golgi (COG) complex. In this study, we analyzed the localization and trafficking of multiple Golgi proteins in patient fibroblasts under a variety of conditions. Although the immunofluorescent staining pattern of several Golgi proteins was indistinguishable from normal, the staining of endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC)-53 and the vesicular-soluble N-ethylmaleimide-sensitive factor attachment protein receptors GS15 and GS28 was abnormal, and the steady-state level of GS15 was greatly decreased. Retrograde transport of multiple Golgi proteins to the ER in patient fibroblasts via brefeldin A-induced tubules was significantly slower than occurs in normal fibroblasts, whereas anterograde protein trafficking was much less affected. After prolonged treatment with brefeldin A, several Golgi proteins were detected in clusters that colocalize with the microtubule-organizing center in patient cells. All of these abnormalities were normalized in COG7-corrected patient fibroblasts. These results serve to better define the role of the COG complex in facilitating protein trafficking between the Golgi and ER and provide a diagnostic framework for the identification of CDG defects involving trafficking proteins.     

Role of the conserved oligomeric Golgi (COG) complex in protein glycosylation

The Golgi apparatus is a central hub for both protein and lipid trafficking/sorting and is also a major site for glycosylation in the cell. This organelle employs a cohort of peripheral membrane proteins and protein complexes to keep its structural and functional organization. The conserved oligomeric Golgi (COG) complex is an evolutionary conserved peripheral membrane protein complex that is proposed to act as a retrograde vesicle tethering factor in intra-Golgi trafficking. The COG protein complex consists of eight subunits, distributed in two lobes, Lobe A (Cog1-4) and Lobe B (Cog5-8). Malfunctions in the COG complex have a significant impact on processes such as protein sorting, glycosylation, and Golgi integrity. A deletion of Lobe A COG subunits in yeasts causes severe growth defects while mutations in COG1, COG7, and COG8 in humans cause novel types of congenital disorders of glycosylation. These pathologies involve a change in structural Golgi phenotype and function. Recent results indicate that down-regulation of COG function results in the resident Golgi glycosyltransferases/glycosidases to be mislocalized or degraded.     

More than just sugars: Conserved oligomeric Golgi complex deficiency causes glycosylation‐independent cellular defects

The conserved oligomeric Golgi (COG) complex controls membrane trafficking and ensures Golgi homeostasis by orchestrating retrograde vesicle trafficking within the Golgi. Human COG defects lead to severe multisystemic diseases known as COG‐congenital disorders of glycosylation (COG‐CDG). To gain better understanding of COG‐CDGs, we compared COG knockout cells with cells deficient to 2 key enzymes, Alpha‐1,3‐mannosyl‐glycoprotein 2‐beta‐N‐acetylglucosaminyltransferase and uridine diphosphate‐glucose 4‐epimerase (GALE), which contribute to proper N‐ and O‐glycosylation. While all knockout cells share similar defects in glycosylation, these defects only account for a small fraction of observed COG knockout phenotypes. Glycosylation deficiencies were not associated with the fragmented Golgi, abnormal endolysosomes, defective sorting and secretion or delayed retrograde trafficking, indicating that these phenotypes are probably not due to hypoglycosylation, but to other specific interactions or roles of the COG complex. Importantly, these COG deficiency specific phenotypes were also apparent in COG7‐CDG patient fibroblasts, proving the human disease relevance of our CRISPR knockout findings. The knowledge gained from this study has important implications, both for understanding the physiological role of COG complex in Golgi homeostasis in eukaryotic cells, and for better understanding human diseases associated with COG/Golgi impairment.      

COG complex-mediated recycling of Golgi glycosyltransferases is essential for normal protein glycosylation

Defects in conserved oligomeric Golgi (COG) complex result in multiple deficiencies in protein glycosylation. On the other hand, acute knock-down (KD) of Cog3p (COG3 KD) causes accumulation of intra-Golgi COG complex-dependent (CCD) vesicles. Here, we analyzed cellular phenotypes at different stages of COG3 KD to uncover the molecular link between COG function and glycosylation disorders. For the first time, we demonstrated that medial-Golgi enzymes are transiently relocated into CCD vesicles in COG3 KD cells. As a result, Golgi modifications of both plasma membrane (CD44) and lysosomal (Lamp2) glycoproteins are distorted. Localization of these proteins is not altered, indicating that the COG complex is not required for anterograde trafficking and accurate sorting. COG7 KD and double COG3/COG7 KD caused similar defects with respect to both Golgi traffic and glycosylation, suggesting that the entire COG complex orchestrates recycling of medial-Golgi-resident proteins. COG complex-dependent docking of isolated CCD vesicles was reconstituted in vitro, supporting their role as functional trafficking intermediates. Altogether, the data suggest that constantly cycling medial-Golgi enzymes are transported from distal compartments in CCD vesicles. Dysfunction of COG complex leads to separation of glycosyltransferases from anterograde cargo molecules passing along secretory pathway, thus affecting normal protein glycosylation.     

Comparative analyses of the Conserved Oligomeric Golgi (COG) complex in vertebrates

Background  

The Conserved Oligomeric Golgi (COG) complex is an eight-subunit assembly that localizes peripherally to Golgi membranes and is involved in retrograde vesicular trafficking. COG subunits are organized in two heterotrimeric groups, Cog2, -3, -4 and Cog5, -6, -7, linked by a dimeric group formed by Cog1 and Cog8. Dysfunction of COG complex in humans has been associated with new forms of Congenital Disorders of Glycosylation (CDG), therefore highlighting its essential role. In the present study, we intended to gain further insights into the evolution of COG subunits in vertebrates, using comparative analyses of all eight COG proteins.     

IntraGolgi distribution of the Conserved Oligomeric Golgi (COG) complex

The Conserved Oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in membrane trafficking and glycoconjugate synthesis. COG appears to participate in retrograde vesicular transport and is required to maintain normal Golgi structure and function. COG mutations interfere with normal transport, distribution, and/or stability of Golgi proteins associated with glycoconjugate synthesis and trafficking, and lead to failure of spermatogenesis in Drosophila melanogaster, misdirected migration of gonadal distal tip cells in Caenorhabditis elegans, and type II congenital disorders of glycosylation in humans. The mechanism by which COG influences Golgi structure and function is unclear. Immunogold electron microscopy was used to visualize the intraGolgi distribution of a functional, hemagglutinin epitope-labeled COG subunit, Cog1-HA, that complements the Cog1-deficiency in Cog1-null Chinese hamster ovary cells. COG was found to be localized primarily on or in close proximity to the tips and rims of the Golgi's cisternae and their associated vesicles and on vesicles and vesiculo-tubular structures seen on both the cis and trans-Golgi Network faces of the cisternal stacks, in some cases on COPI containing vesicles. These findings support the proposal that COG is directly involved in controlling vesicular retrograde transport of Golgi resident proteins throughout the Golgi apparatus.     

Differential effects of lobe A and lobe B of the Conserved Oligomeric Golgi complex on the stability of {beta}1,4-galactosyltransferase 1 and {alpha}2,6-sialyltransferase 1

Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged β1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structure.     

The Golgi puppet master: COG complex at center stage of membrane trafficking interactions

The central organelle within the secretory pathway is the Golgi apparatus, a collection of flattened membranes organized into stacks. The cisternal maturation model of intra-Golgi transport depicts Golgi cisternae that mature from cis to medial to trans by receiving resident proteins, such as glycosylation enzymes via retrograde vesicle-mediated recycling. The conserved oligomeric Golgi (COG) complex, a multi-subunit tethering complex of the complexes associated with tethering containing helical rods family, organizes vesicle targeting during intra-Golgi retrograde transport. The COG complex, both physically and functionally, interacts with all classes of molecules maintaining intra-Golgi trafficking, namely SNAREs, SNARE-interacting proteins, Rabs, coiled-coil tethers, vesicular coats, and molecular motors. In this report, we will review the current state of the COG interactome and analyze possible scenarios for the molecular mechanism of the COG orchestrated vesicle targeting, which plays a central role in maintaining glycosylation homeostasis in all eukaryotic cells.     

Deficiency of the Cog8 Subunit in Normal and CDG‐Derived Cells Impairs the Assembly of the COG and Golgi SNARE Complexes

Multiple mutations in different subunits of the tethering complex Conserved Oligomeric Golgi (COG) have been identified as a cause for Congenital Disorders of Glycosylation (CDG) in humans. Yet, the mechanisms by which COG mutations induce the pleiotropic CDG defects have not been fully defined. By detailed analysis of Cog8 deficiency in either HeLa cells or CDG‐derived fibroblasts, we show that Cog8 is required for the assembly of both the COG complex and the Golgi Stx5‐GS28‐Ykt6‐GS15 and Stx6‐Stx16‐Vti1a‐VAMP4 SNARE complexes. The assembly of these SNARE complexes is also impaired in cells derived from a Cog7‐deficient CDG patient. Likewise, the integrity of the COG complex is also impaired in Cog1‐, Cog4‐ and Cog6‐depleted cells. Significantly, deficiency of Cog1, Cog4, Cog6 or Cog8 distinctly influences the production of COG subcomplexes and their Golgi targeting. These results shed light on the structural organization of the COG complex and its subcellular localization, and suggest that its integrity is required for both tethering of transport vesicles to the Golgi apparatus and the assembly of Golgi SNARE complexes. We propose that these two key functions are generally and mechanistically impaired in COG‐associated CDG patients, thereby exerting severe pleiotropic defects.     

Genetic analysis of the subunit organization and function of the conserved oligomeric golgi (COG) complex: studies of COG5- and COG7-deficient mammalian cells

The conserved oligomeric Golgi (COG) complex is an eight-subunit (Cog1-8) peripheral Golgi protein involved in Golgi-associated membrane trafficking and glycoconjugate synthesis. We have analyzed the structure and function of COG using Cog1 or Cog2 null Chinese hamster ovary cell mutants, fibroblasts from a patient with Cog7-deficient congenital disorders of glycosylation, and stable Cog5-deficient HeLa cells generated by RNA interference. Although the dilation of some Golgi cisternae in Cog5-deficient cells resembled that observed in Cog1- or Cog2-deficient cells, their global glycosylation defects (less severe) and intracellular processing and function of low density lipoprotein receptors (essentially normal) differed from Cog1- and Cog2-deficient cells. Immunoblotting, gel filtration, and immunofluorescence microscopy analyses of the COG-deficient cells and cell extracts indicated that 1) Cog2-4 and Cog5-7 form stable subcomplexes, 2) Cog1 mediates Golgi association of a Cog2-4 plus Cog8 subcomplex, 3) Cog8 associates stably with both Cog5-7 and Cog1-4 subcomplexes, and thus 4) Cog8 helps assemble the Cog1-4 and Cog5-7 subcomplexes into the complete COG complex. This model of the subunit organization of COG is in excellent agreement with in vitro data presented in an accompanying paper (Ungar, D., Oka, T., Vasile, E., Krieger, M., and Hughson, F. M. (2005) J. Biol. Chem. 280, 32729-32735). Only one or two of the seven Cog1- or Cog2-dependent Golgi membrane proteins called GEARs are also sensitive to Cog5 or Cog7 deficiency, indicating that the COG subunits play distinctive roles in controlling Golgi structure and function.     

COG5-CDG: expanding the clinical spectrum

Background

The Conserved Oligomeric Golgi (COG) complex is involved in the retrograde trafficking of Golgi components, thereby affecting the localization of Golgi glycosyltransferases. Deficiency of a COG-subunit leads to defective protein glycosylation, and thus Congenital Disorders of Glycosylation (CDG). Mutations in subunits 1, 4, 5, 6, 7 and 8 have been associated with CDG-II. The first patient with COG5-CDG was recently described (Paesold-Burda et al. Hum Mol Genet 2009; 18:4350–6). Contrary to most other COG-CDG cases, the patient presented a mild/moderate phenotype, i.e. moderate psychomotor retardation with language delay, truncal ataxia and slight hypotonia.

Methods

CDG-IIx patients from our database were screened for mutations in COG5. Clinical data were compared. Brefeldin A treatment of fibroblasts and immunoblotting experiments were performed to support the diagnosis.

Results and conclusion

We identified five new patients with proven COG5 deficiency. We conclude that the clinical picture is not always as mild as previously described. It rather comprises a broad spectrum with phenotypes ranging from mild to very severe. Interestingly, on a clinical basis some of the patients present a significant overlap with COG7-CDG, a finding which can probably be explained by subunit interactions at the protein level.

     

Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery

Cell surface lectin staining, examination of Golgi glycosyltransferases stability and localization, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were employed to investigate conserved oligomeric Golgi (COG)-dependent glycosylation defects in HeLa cells. Both Griffonia simplicifolia lectin-II and Galanthus nivalus lectins were specifically bound to the plasma membrane glycoconjugates of COG-depleted cells, indicating defects in activity of medial- and trans-Golgi-localized enzymes. In response to siRNA-induced depletion of COG complex subunits, several key components of Golgi glycosylation machinery, including MAN2A1, MGAT1, B4GALT1 and ST6GAL1, were severely mislocalized. MALDI-TOF analysis of total N-linked glycoconjugates indicated a decrease in the relative amount of sialylated glycans in both COG3 KD and COG4 KD cells. In agreement to a proposed role of the COG complex in retrograde membrane trafficking, all types of COG-depleted HeLa cells were deficient in the Brefeldin A- and Sar1 DN-induced redistribution of Golgi resident glycosyltransferases to the endoplasmic reticulum. The retrograde trafficking of medial- and trans-Golgi-localized glycosylation enzymes was affected to a larger extent, strongly indicating that the COG complex regulates the intra-Golgi protein movement. COG complex-deficient cells were not defective in Golgi re-assembly after the Brefeldin A washout, confirming specificity in the retrograde trafficking block. The lobe B COG subcomplex subunits COG6 and COG8 were localized on trafficking intermediates that carry Golgi glycosyltransferases, indicating that the COG complex is directly involved in trafficking and maintenance of Golgi glycosylation machinery.     

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.     

Golgi inCOGnito: From vesicle tethering to human disease

The Conserved Oligomeric Golgi (COG) complex, a multi-subunit vesicle tethering complex of the CATCHR (Complexes Associated with Tethering Containing Helical Rods) family, controls several aspects of cellular homeostasis by orchestrating retrograde vesicle traffic within the Golgi. The COG complex interacts with all key players regulating intra-Golgi trafficking, namely SNAREs, SNARE-interacting proteins, Rabs, coiled-coil tethers, and vesicular coats. In cells, COG deficiencies result in the accumulation of non-tethered COG-complex dependent (CCD) vesicles, dramatic morphological and functional abnormalities of the Golgi and endosomes, severe defects in N- and O- glycosylation, Golgi retrograde trafficking, sorting and protein secretion. In humans, COG mutations lead to severe multi-systemic diseases known as COG-Congenital Disorders of Glycosylation (COG-CDG). In this report, we review the current knowledge of the COG complex and analyze COG-related trafficking and glycosylation defects in COG-CDG patients.     

C. elegans as a model system to study the function of the COG complex in animal development

The conserved oligomeric Golgi (COG) complex is an octameric protein complex associated with the Golgi apparatus and is required for proper sorting and glycosylation of Golgi resident enzymes and secreted proteins. Although COG complex function has been extensively studied at the cellular and subcellular levels, its role in animal development mostly remains unknown. Recently, mutations in the components of the COG complex were found to cause abnormal gonad morphogenesis in Caenorhabditis elegans. In C. elegans, the COG complex acts in the glycosylation of an ADAM (a disintegrin and metalloprotease) family protein, MIG-17, which directs migration of gonadal distal tip cells to lead gonad morphogenesis. This is the first link between the COG complex and the function of an ADAM protease that is directly involved in organ morphogenesis, demonstrating the potential of C. elegans as a model system to study COG function in animal development.     

Glycosylation disorders of membrane trafficking

During evolution from prokaryotic to eukaryotic cells, compartmentalization of cellular functions has been achieved with a high degree of complexity. Notably, all secreted and transmembrane proteins travel through endoplasmic reticulum (ER) and Golgi apparatus, where they are synthesized, folded and subjected to covalent modifications, most particularly glycosylation. N-glycosylation begins in the ER with synthesis and transfer of glycan onto nascent protein and proceeds in Golgi apparatus where maturation occurs. This process not only requires the precise localization of glycosyltransferases, glycosidases and substrates but also an efficient, finely regulated and bidirectional vesicular trafficking among membrane-enclosed organelles. Basically, it is no surprise that alterations in membrane transport or related pathways can lead to glycosylation abnormalities. During the last few years, this has particularly been highlighted in genetic diseases called CDG (Congenital Disorders of Glycosylation). Alterations in mechanisms of vesicle formation due to COPII coat component SEC23B deficiency, or in vesicles tethering, caused by defects of the COG complex, but also impaired Golgi pH homeostasis due to ATP6V0A2 defects have been discovered in CDG patients. This mini review will summarize these fascinating discoveries.     

Chlamydia trachomatis hijacks intra-Golgi COG complex-dependent vesicle trafficking pathway

Chlamydia spp. are obligate intracellular bacteria that replicate inside the host cell in a bacterial modified unique compartment called the inclusion. As other intracellular pathogens, chlamydiae exploit host membrane trafficking pathways to prevent lysosomal fusion and to acquire energy and nutrients essential for their survival and replication. The Conserved Oligomeric Golgi (COG) complex is a ubiquitously expressed membrane-associated protein complex that functions in a retrograde intra-Golgi trafficking through associations with coiled-coil tethers, SNAREs, Rabs and COPI proteins. Several COG complex-interacting proteins, including Rab1, Rab6, Rab14 and Syntaxin 6 are implicated in chlamydial development. In this study, we analysed the recruitment of the COG complex and GS15-positive COG complex-dependent vesicles to Chlamydia trachomatis inclusion and their participation in chlamydial growth. Immunofluorescent analysis revealed that both GFP-tagged and endogenous COG complex subunits associated with inclusions in a serovar-independent manner by 8 h post infection and were maintained throughout the entire developmental cycle. Golgi v-SNARE GS15 was associated with inclusions 24 h post infection, but was absent on the mid-cycle (8 h) inclusions, indicating that this Golgi SNARE is directed to inclusions after COG complex recruitment. Silencing of COG8 and GS15 by siRNA significantly decreased infectious yield of chlamydiae. Further, membranous structures likely derived from lysed bacteria were observed inside inclusions by electron microscopy in cells depleted of COG8 or GS15. Our results showed that C. trachomatis hijacks the COG complex to redirect the population of Golgi-derived retrograde vesicles to inclusions. These vesicles likely deliver nutrients that are required for bacterial development and replication.