The Conserved Oligomeric Golgi (COG) complex is critical for efficient intra-Golgi trafficking and glycosylation. Prior research has demonstrated that COG dysfunction or RNAi-induced depletion leads to the accumulation of non-tethered COG complex-dependent (CCD) vesicles. However, the precise connection between COG deficiency, degradation of Golgi enzymes, and its impact on vesicular trafficking has not been fully elucidated. In this study, we conducted a comprehensive proteomic analysis of Golgi-derived vesicles from both wild-type and COG-depleted cells. We specifically analyzed three distinct populations of vesicles immunoisolated with antibodies targeting transmembrane proteins from the cis, medial, and trans-Golgi sub-compartments. Our findings reveal that, while the vesicle content encompasses the entire Golgi proteome, the molecular signatures of vesicles derived from wild- type cells were markedly distinct, underscoring a robust recycling mechanism for Golgi- dependent proteins. Notably, these vesicles retained various vesicular coats, and COG depletion significantly accelerated their uncoating. Furthermore, the increased overlap in molecular signatures upon COG depletion indicates that persistent defects in vesicle tethering severely compromise intra-Golgi sorting mechanisms. Crucially, our analysis highlights that the entire Golgi glycosylation machinery recycles within CCD vesicles in a COG-dependent manner, while secretory proteins and components involved in ER-Golgi and Golgi-endosome trafficking were not enriched. These results strongly support a model of multi-step intra-Golgi vesicular recycling of the glycosylation machinery, orchestrated by the COG complex in concert with a cisternae-specific array of vesicular coats, coiled-coil tethers, Rabs, and SNARE proteins.
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