title: Expression of functional Myc-tagged conserved oligomeric Golgi (COG) subcomplexes in mammalian cells.
abstract: Docking and fusion of transport carriers in eukaryotic cells are regulated by a family of multi-subunit tethering complexes (MTC) that sequentially and/or simultaneously interact with other components of vesicle fusion machinery, such as SNAREs, Rabs, coiled-coil tethers, and vesicle coat components. Probing for interactions of multi-protein complexes has relied heavily on the method of exogenously expressing individual proteins and then determining their interaction stringency. An obvious pitfall of this method is that the protein interactions are not occurring in their native multi-subunit state. Here, we describe an assay where we express all eight subunits of the conserved oligomeric Golgi (COG) complex that contain the same triple-Myc epitope tag and then an assay for the (sub) complex's interaction with known protein partners. The expression of all eight proteins allows for the assembled complex to interact with partner proteins, and by having the same tag on all eight COG subunits, we are able to very accurately quantify the interaction with each subunit. The use of this assay has highlighted a very important level of specificity of interactions between COG subcomplexes and their intracellular partners.
authors.affiliation: Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 W. Markham, Slot 505, Little Rock, AR, 72205, USA.
authors.forename: Rose A
authors.initials: RA
authors.lastname: Willett
authors.forename: Tetyana A
authors.initials: TA
authors.lastname: Kudlyk
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Lupashin
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D015870:Gene Expression; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D021381:Protein Transport; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins