Mol Biol Cell. 2025 May 21. mbcE24120556
Intracellular trafficking relies on small membrane intermediates that transport cargo between different compartments. However, the precise role of vesicles in preserving Golgi function remains uncertain. To clarify this, we induced acute inactivation of the Conserved Oligomeric Golgi (COG) complex and analyzed vesicles from the different Golgi compartments. Proteomic analysis of the resulting vesicles revealed distinct molecular profiles, indicating a robust recycling system for Golgi proteins. All glycosylation enzymes and sugar transporters were detected in immunoisolated vesicles. The abundance of glycosylation machinery in intra-Golgi vesicles significantly increased following acute COG malfunction. Vesicles isolated from wild-type cells retained various vesicular coats, which were detaching from COG complex-dependent (CCD) vesicles stalled in the untethered state. Additionally, COG depletion led to increased molecular overlap among different populations of vesicles, suggesting that defects in vesicle tethering disrupt intra-Golgi sorting. Notably, CCD vesicles were functional and could be specifically rerouted to mitochondria that ectopically express Golgi tethers. Our findings demonstrate that the entire Golgi glycosylation machinery recycles within vesicles in a COG-dependent manner, whereas secretory and ER-Golgi trafficking proteins were not enriched. These results support a model in which the COG complex orchestrates the multi-step recycling of glycosylation machinery, coordinated by specific coats, tethers, and SNAREs.