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.

Vesicular tethers and SNAREs are two key protein components of the intracellular membrane trafficking machinery. The COG (conserved oligomeric Golgi) complex has been implicated in the tethering of retrograde intra-Golgi vesicles. Here, using yeast two hybrid and co-immunoprecipitation approaches, we show that three COG subunits, namely COG4, 6, and 8, are capable of interacting with defined Golgi SNAREs, namely STX5, STX6, STX16, GS27, and SNAP29. Comparative analysis of COG8-STX16 and COG4-STX5 interactions by a COG-based mitochondrial re-localization assay reveals that the COG8 and COG4 proteins initiate the formation of two different tethering platforms that can facilitate the redirection of two populations of Golgi transport intermediates to the mitochondrial vicinity. Our results uncover a role for COG subcomplexes in defining the specificity of vesicular sorting within the Golgi.

Background: Delivery of the vesicle into the pre-fusion state during tethering is not understood. Results: Interactions between the COG complex, golgins and Rabs were mapped. Two ends of the golgin TMF both bind COG and different Rabs, the middle binds the target membrane. Conclusion: COG may reel the vesicle into docking along the golgin. Significance: Mechanistic link between tethering complex and coiled tether established.

Toxin trafficking studies provide valuable information about endogenous pathways of intracellular transport. Subtilase cytotoxin (SubAB) is transported in a retrograde manner through the endosome to the Golgi and then to the endoplasmic reticulum (ER), where it specifically cleaves the ER chaperone BiP/GRP78 (Binding immunoglobin protein/Glucose-Regulated Protein of 78 kDa). To identify the SubAB Golgi trafficking route, we have used siRNA-mediated silencing and immunofluorescence microscopy in HeLa and Vero cells. Knockdown (KD) of subunits of the conserved oligomeric Golgi (COG) complex significantly delays SubAB cytotoxicity and blocks SubAB trafficking to the cis Golgi. Depletion of Rab6 and β-COP proteins causes a similar delay in SubAB-mediated GRP78 cleavage and did not augment the trafficking block observed in COG KD cells, indicating that all three Golgi factors operate on the same 'fast' retrograde trafficking pathway. SubAB trafficking is completely blocked in cells deficient in the Golgi SNARE Syntaxin 5 and does not require the activity of endosomal sorting nexins SNX1 and SNX2. Surprisingly, depletion of Golgi tethers p115 and golgin-84 that regulates two previously described coat protein I (COPI) vesicle-mediated pathways did not interfere with SubAB trafficking, indicating that SubAB is exploiting a novel COG/Rab6/COPI-dependent retrograde trafficking pathway.

The Golgi complex is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits impairs Golgi modification of N- and O-glycans, and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-KO cells exhibit reduced retention of glycosylation enzymes in the Golgi. A RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi complex but instead is missorted to the endolysosomal system. We propose that the endosomal system is part of the trafficking itinerary of Golgi enzymes or their recycling adaptors and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery. [Media: see text]

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.

Vesicular tethers and SNAREs are two key protein components that govern docking and fusion of intracellular membrane carriers in eukaryotic cells. The COG (conserved oligomeric Golgi) complex has been specifically implicated in the tethering of retrograde intra-Golgi vesicles. Using yeast two hybrid and co-immunoprecipitation approaches, we show that the COG6 subunit of the COG complex is capable of interacting with a subset of Golgi SNAREs, namely STX5, STX6, GS27, and SNAP29. Interaction with SNAREs is accomplished via the universal SNARE-binding motif of COG6. Overexpression of COG6, or its depletion from cells, disrupts the integrity of the Golgi complex. Importantly, COG6 protein lacking the SNARE binding domain is deficient in Golgi binding, and is not capable of inducing Golgi complex fragmentation when overexpressed. These results indicate that COG6-SNARE interactions are important for both COG6 localization and Golgi integrity.

The conserved oligomeric Golgi (COG) complex is a peripheral membrane protein complex which orchestrates tethering of intra-Golgi vesicles. We found that COG1-4 (lobe A) and 5–8 (lobe B) protein assemblies are present as independent sub-complexes on cell membranes. Super-resolution microscopy demonstrates that COG sub-complexes are spatially separated on the Golgi with lobe A preferential localization on Golgi stacks and the presence of lobe B on vesicle-like structures, where it physically interacts with v-SNARE GS15. The localization and specific interaction of the COG sub-complexes with the components of vesicle tethering/fusion machinery suggests their different roles in the vesicle tethering cycle. We propose and test a novel model that employs association/disassociation of COG sub-complexes as a mechanism that directs vesicle tethering at Golgi membranes. We demonstrate that defective COG assembly or restriction of tethering complex disassembly by a covalent COG1-COG8 linkage is inhibitory to COG complex activity, supporting the model.