The COG complex is a multisubunit tethering complex that can be subdivided into two lobes: lobe A (COG1-4) and lobe B (COG5-8). COG tethers retrograde vesicles with Golgi membranes. This function is important in the recycling of resident Golgi enzymes, and also possibly with vesicles in endosome-to-Golgi retrieval. The COG complex tethers vesicles by interacting with the core trafficking machinery and their regulators: SNAREs, Rab-GTPases, tethers, SM proteins, coat proteins, and motor proteins. It is not known how and when the COG complex interacts with membranes during vesicle tethering and if COG function requires cycling between the membrane and the cytosol. To answer this question we took two directions: 1) remove COG-interacting proteins to dislodge the COG complex from Golgi membranes and disrupt COG function, and 2) permanently tether COG to the membrane and monitor COG function. For the later, we created cell lines lobe A or lobe B COG subunits permanently attached to Golgi membranes. Investigations of morphological, trafficking and glycosylation defects using using superresolution microscopy, cathepsin D sorting assay, and lecting binding assays revealed that membrane-attached COG subcomplexes are mostly functional indicating that COG detachment from membranes is not essential for its major functions. We also investigated COG complex interactions with two large classes of Golgi membrane proteins: transmembrane proteins (TMEM43, TMEM165) and Rab-GTPases (Rab30, Rab39, and Rab43). siRNA depletion or CRISPR/Cas9 mediated knockout of the these proteins revealed that, alone, they are dispensible for COG interaction with membranes. In all probability, the interactions with these proteins and the other trafficking components are complex in that COG interacts with more than one part of the machinery at a time in a yet unknown sequence. By permanently attaching a lobe A or a lobe B subunit to Golgi membranes we learn that 1) anchored-lobe A does not increase the affinity of lobe B with Golgi membranes compared with freely soluble lobe A, and 2) anchored-lobe B does increase the affinity of lobe A with the Golgi membranes. This suggests that lobe B dictates the interactions between the COG sub-complexes and suggests that some COG interactions enhance other interactions within COG and with the trafficking machinery.
|Advisor:||Lupashin, Vladimir V.|
|Commitee:||Abbott, Karen L., Franco, Aime T., Jennings, Michael L., Storrie, Brian|
|School:||University of Arkansas for Medical Sciences|
|Department:||Interdisciplinary Biomedical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 79/10(E), Dissertation Abstracts International|
|Keywords:||Cog complex, Golgi trafficking, Membrane attachment, Rab-gtpase, Tmem165|
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