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.
Highlights
Efficient docking and fusion of intracellular transport carriers in eukaryotic cells is tightly regulated by a family of multi-subunit tethering complexes (MTC) that sequentially and/or simultaneously interact with other components of vesicle docking and fusion machinery (Lupashin and Sztul, 2005; Yu and Hughson, 2010)
Our group has previously shown that Conserved Oligomeric Golgi (COG) complex subunit knock downs (KD) in HeLa and HEK293T cells cause altered binding of several lectins due to impaired glycosylation of plasma membrane glycoconjugates while in the Golgi
As a result of this work a complete set of HEK293T cell lines void of individual COG subunits was created. These cells are an improvement on past KD and mutant studies as they are all in the same cell type
Summary
Efficient docking and fusion of intracellular transport carriers in eukaryotic cells is tightly regulated by a family of multi-subunit tethering complexes (MTC) that sequentially and/or simultaneously interact with other components of vesicle docking and fusion machinery (Lupashin and Sztul, 2005; Yu and Hughson, 2010). The COG complex is Characterization of COG KO Cell Lines a hetero-octamer and a member of the CATCHR (complexes associated with tethering containing helical rods) family of proteins (Yu and Hughson, 2010) It is comprised of eight different protein subunits (named Cog1–8) (Whyte and Munro, 2001; Ungar et al, 2002) that are organized into two functionally distinct sub complexes or lobes: Cog in lobe A and Cog in lobe B (Fotso et al, 2005; Ungar et al, 2005). Though these approaches have proved useful, they fail to allow for study of the long term effects of COG loss
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