COG Complex Complexities: Detailed Characterization of a Complete Set of HEK293T Cells Lacking Individual COG Subunits

Abstract

The Conserved Oligomeric Golgi complex is an evolutionarily conserved multisubunit tethering complex (MTC) that is crucial for intracellular membrane trafficking and Golgi homeostasis. The COG complex interacts with core vesicle docking and fusion machinery at the Golgi; however, its exact mechanism of action is still an enigma. Previous studies of COG complex were limited to the use of CDGII (Congenital disorders of glycosylation type II)-COG patient fibroblasts, siRNA mediated knockdowns, or protein relocalization approaches. In this study we have used the CRISPR approach to generate HEK293T knock-out (KO) cell lines missing individual COG subunits. These cell lines were characterized for glycosylation and trafficking defects, cell proliferation rates, stability of COG subunits, localization of Golgi markers, changes in Golgi structure, and N-glycan profiling. We found that all KO cell lines were uniformly deficient in cis/medial-Golgi glycosylation and each had nearly abolished binding of Cholera toxin. In addition, all cell lines showed defects in Golgi morphology, retrograde trafficking and sorting, sialylation and fucosylation, but severities varied according to the affected subunit. Lobe A and Cog6 subunit KOs displayed a more severely distorted Golgi structure, while Cog2, 3, 4, 5, and 7 knock outs had the most hypo glycosylated form of Lamp2. These results led us to conclude that every subunit is essential for COG complex function in Golgi trafficking, though to varying extents. We believe that this study and further analyses of these cells will help further elucidate the roles of individual COG subunits and bring a greater understanding to the class of MTCs as a whole.

Keywords: COG complex; CRISPR; Golgi apparatus; glycan processing; glycosylation; toxin trafficking; vesicle tethering.

Figure 1

COG KO validation. Left column: Sequence alignment of mutant and control DNA. Chromosomal DNA was amplified at the CRISPR target region by high fidelity PCR. The expected cut site based on the guide RNA is highlighted in yellow. The central column shows plasma membrane staining of WT HEK293T and COG KO cells with Galanthus nivalis lectin (GNL-pink). Nuclei stained with DAPI (blue). Right column: cells were analyzed using flow cytometry for GNL staining (wild-type cells are in black, COG KO cells are in white).

Figure 2

Growth and rescue of COG KO cells. (A) Growth of WT and KO cells. Cells were plated in 24 well plates in triplicate at 100,000 cells per well (Day 0). Cells were counted at the indicated time points over a week and cell counts were plotted. (B) The average growth in a 24 h period was calculated by (# of cells on day n/ # of cells on day n-1)^*100 to get percent growth per day. Growth percentages over the week for each cell line were averaged. (C) Western blot analysis for each COG subunit KO cell line. β-actin is used as a loading control. Asterisks indicate non-specific bands. (D) Rescue of COG dependent glycosylation defect. Missing COG subunits (green) were transfected into KO cells. Seventy two hours later cells were fixed and stained with GNL-Alexa 647 (pink). Note that GNL binding was significantly reduced in cells expressing COG subunits.

Figure 3

COG protein stability upon loss of other COG proteins. (A) All COG KO cells were probed by western blot for Cog3, 4, 5, 6, 7, and 8 protein levels. β-actin used as a loading control. (B) Quantification of 2 separate western blot analyses. Each Cog protein amount was first standardized to β-actin then compared to WT levels. (C) A model of COG subunit interactions. lobe A is shown in shades of red and lobe B is shown in shades of green. Cog1 and Cog8 are lighter in color to show that they also gain stability from one another in addition to their lobe partners.

Figure 4

Golgi structure is severely distorted in COG KO cells. Cells were grown on carbon and collagen coated sapphire disk then subjected to high pressure freezing and freeze substitution. Samples were stained with tannic acid and osmium before embedding then uranyl acetate and lead citrate staining post sectioning. Arrows indicate Golgi stacks. Arrowheads indicate autophagosomal like structures.

Figure 5

Golgi and other secretory markers are largely undisturbed in Cog KO cells. Immunofluorescence of Lamp2, Giantin, ERGIC53, TGN46, Vamp3, and M6PR. Asterisks in Cog7 KO indicate Lamp2-positive, large, endocytic-like structures, Cog4 structures not pictured.

Figure 6

COG KO cells are altered for the binding of RCA-I and CTxB to plasma membrane. (A) Pulse chase with labeled toxins was done at 37°C for 30 min before fixing and staining with GM130 antibodies. Cells were mounted in DAPI containing media. Coverslips were analyzed using a confocal microscope. (B) Cells were incubated with the toxins on ice for 30 min then processed using a flow cytometer and analyzed in FlowJo.

Figure 7

Retrograde trafficking of SubAB is impaired in COG subunit KO cells. (A) Time course of SubAB-dependent cleavage of GRP78 in control and COG KO cells. Western blot. (B) Quantification of GRP78 cleavage. (C) Time required for SubAB-dependent 50% cleavage of GRP78 in control and COG KO cells.

Figure 8

Pro-cathepsin D is secreted from COG subunit KO cells but not WT cells. Cells at 80–100% confluency were placed in chemically defined serum free media. Media was collected at 24 h and then analyzed by western blot for Cathepsin D.

Figure 9

Lamp2 is hypoglycosylated in COG subunit KO cells, but still localizes to lysosome like structures. (A) Cell lysates were analyzed by western blot for Lamp2 mobility. (B) WT and COG 2 KO cells were transfected with Lamp2-mCherry and GFP-Rab7a. Arrows represent colocalization. Asterisks represent large, late endocytic inclusions. Images taken of live cells.

Figure 10

COG KO cells have global changes in N-linked glycans structures. N-glycans prepared from cell lysates were profiled by mass spectrometry. The normalized intensities for individual glycans grouped into different classes as indicated were summed. Error bars show SEM for n = 4 (WT, Cog2KO), n = 5 (Cog4KO), n = 8 (Cog7 KO). (A) Oligomannose composition changes in KO cells vs. WT cells. (B) Sialyation changes in KO cells vs. WT cells. ^**denotes p < 0.01. (C) Fucosylation changes in KO cell vs. WT cells ^*denotes p < 0.05. For (B,C) one-way ANOVA with a Sidak-Holm post hoc statistical significance test was utilized.