Biomedical Engineering Reference
In-Depth Information
Chapter 13
Expression of Functional Myc-Tagged Conserved
Oligomeric Golgi (COG) Subcomplexes in Mammalian Cells
Rose A. Willett , Tetyana A. Kudlyk , and Vladimir V. Lupashin
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 indi-
vidual 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 specifi city
of interactions between COG subcomplexes and their intracellular partners.
Key words
COG, Conserved oligomeric Golgi complex, Golgi, SNARE, Vesicle tethering, Multi-
expression, Co-immunoprecipitation, Subcomplexes, Protein-protein interaction
1
Introduction
The conserved oligomeric Golgi (COG) complex is a peripheral
membrane protein complex in the subfamily of multi-subunit teth-
ering complexes (MTC) [
1
]. The COG complex functions to
tether retrograde intra-Golgi vesicles to the Golgi cisternae, a criti-
cal step in vesicle docking that occurs prior to SNARE-mediated
membrane fusion [
2
]. The COG complex is required for the proper
recycling of Golgi-localized glycosylation enzymes [
3
-
5
], with
defects in COG subunits resulting in a class of disorders known as
congenital disorders of glycosylation [
6
]. According to the current
“maturation” model of the Golgi [
7
], vesicle-mediated recycling
of Golgi enzymes is essential for proper glycosylation of glycocon-
jugates that traffi c through the Golgi apparatus. Vesicle tethering is
