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The Golgi-associated retrograde protein (GARP) complex is proposed to tether endosome-derived transport vesicles, but the exact function and mechanism of GARP action are not completely understood. To uncover the GARP function in human cells, we employ CRISPR/Cas9 strategy and knock out (KO) the unique VPS54 subunit of the GARP complex. In this chapter, we describe the detailed method of generating CRISPR/Cas9-mediated VPS54-KO in hTERT-RPE1 cells, rescue of resulting KO cells with stable near-endogenous expression of myc-tagged VPS54, and validation of KO and rescued (KO-R) cells using Western blot and immunofluorescence approaches. This approach is helpful in uncovering new functions of the GARP and other vesicle tethering complexes., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Conserved oligomeric Golgi (COG) complex orchestrates intra-Golgi retrograde trafficking and glycosylation of macromolecules, but the detailed mechanism of COG action is unknown. Previous studies employed prolonged protein knockout and knockdown approaches which may potentially generate off-target and indirect mutant phenotypes. To achieve a fast depletion of COG subunits in human cells, the auxin-inducible degradation system was employed. This method of protein regulation allows a very fast and efficient depletion of COG subunits, which provides the ability to accumulate COG complex dependent (CCD) vesicles and investigate initial cellular defects associated with the acute depletion of COG complex subunits. This protocol is applicable to other vesicle tethering complexes and can be utilized to investigate anterograde and retrograde intracellular membrane trafficking pathways., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The conserved oligomeric Golgi (COG) complex is a key evolutionally conserved multisubunit protein machinery that regulates tethering and fusion of intra-Golgi transport vesicles. The Golgi apparatus specifically promotes sorting and complex glycosylation of glycoconjugates. Without proper glycosylation and processing, proteins and lipids will be mislocalized and/or have impaired function. The Golgi glycosylation machinery is kept in homeostasis by a careful balance of anterograde and retrograde trafficking to ensure proper localization of the glycosylation enzymes and their substrates. This balance, like other steps of membrane trafficking, is maintained by vesicle trafficking machinery that includes COPI vesicular coat proteins, SNAREs, Rabs, and both coiled-coil and multi-subunit vesicular tethers. The COG complex interacts with other membrane trafficking components and is essential for proper localization of Golgi glycosylation machinery. Here we describe using CRISPR-mediated gene editing coupled with a phenotype-based selection strategy directly linked to the COG complex's role in glycosylation homeostasis to obtain COG complex subunit knockouts (KOs). This has resulted in clonal KOs for each COG subunit in HEK293T cells and gives the ability to further probe the role of the COG complex in Golgi homeostasis.

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.

Staining of molecules such as proteins and glycoconjugates allows for an analysis of their localization within the cell and provides insight into their functional status. Glycosyltransferases, a class of enzymes which are responsible for glycosylating host proteins, are mostly localized to the Golgi apparatus, and their localization is maintained in part by a protein vesicular tethering complex, the conserved oligomeric Golgi (COG) complex. Here we detail a combination of fluorescent lectin and immuno-staining in cells depleted of COG complex subunits to examine the status of Golgi glycosyltransferases. The combination of these techniques allows for a detailed characterization of the changes in function and localization of Golgi glycosyltransferases with respect to transient COG subunit depletion.