Acute COG complex inactivation unveiled its immediate impact on Golgi and illuminated the nature of intra‐Golgi recycling vesicles

Abstract

Conserved Oligomeric Golgi (COG) complex controls Golgi trafficking and glycosylation, but the precise COG mechanism is unknown. The auxin-inducible acute degradation system was employed to investigate initial defects resulting from COG dysfunction. We found that acute COG inactivation caused a massive accumulation of COG-dependent (CCD) vesicles that carry the bulk of Golgi enzymes and resident proteins. v-SNAREs (GS15, GS28) and v-tethers (giantin, golgin84, and TMF1) were relocalized into CCD vesicles, while t-SNAREs (STX5, YKT6), t-tethers (GM130, p115), and most of Rab proteins remained Golgi-associated. Airyscan microscopy and velocity gradient analysis revealed that different Golgi residents are segregated into different populations of CCD vesicles. Acute COG depletion significantly affected three Golgi-based vesicular coats-COPI, AP1, and GGA, suggesting that COG uniquely orchestrates tethering of multiple types of intra-Golgi CCD vesicles produced by different coat machineries. This study provided the first detailed view of primary cellular defects associated with COG dysfunction in human cells.

Syntaxin‐5's flexibility in SNARE pairing supports Golgi functions

“…65 There are several explanations for the limited impact of GS28/ GS15 deletion on Golgi functions. Although Golgi glycosyltransferases are present in GS28/GS15 containing Golgi-derived COPI vesicles, 22,[66][67][68] enzymes could be recycled back to the Golgi independently of the STX5 SNARE complex. In support of this theory, a CDG-causing mutation in STX5, that abolishes expression of the short isoform of STX5, did not result in a dramatic mislocalization of Golgi enzymes despite producing lethal glycosylation abnormalities.…”

“…In COGdeficient cells the cellular abundance of SDF4 is significantly reduced. 22 GS28 KO did not show any effect on the abundance of SDF4, but affected the electrophoretic mobility and stability of TMEM165 (Figure 4C,D). These results indicate that GS28 is partially needed for efficient Golgi glycosylation, but its deletion does not affect glycosylation as severely as COG's deletion.…”

“…6 Acute KD or KO of any individual COG subunit results in mislocalization and depletion of every tested Golgi enzyme. 4,14,22,35,38 We used WB to measure the total cellular level of four endogenous enzymes involved in N-(MGAT1 and B4GALT) and O-(GALNT2 and GALNT3) glycosylation. Measurement of the total levels of glycosyltransferases in WT and SNARE KO cells, showed that the abundance of the tested enzyme was similar to WT in GS15 KO cells and somewhat reduced in GS28 KO, but this reduction was not as dramatic as in COG4 KO cells (Figure 4A,B).…”

“…9,20,21 COG-subunit deletions/mutations affect the steady-state levels of GS28 and GS15. Furthermore, depletion of COG3 by siRNA-mediated knockdown (KD) as well as acute depletion of COG4 results in the accumulation of small trafficking intermediates termed COG-complex dependent vesicles which contain GS28 and GS15, 14,22 indicating that both molecules act as v-SNAREs to form a SNARE complex with STX5 and YKT6 during vesicle fusion at the Golgi. Importantly, the abundance of STX5 and YKT6 is not regulated by the COG complex, 23 and these SNAREs are not accumulated on CCD vesicles, 22 indicating their role as t-SNAREs in the intra-Golgi trafficking.…”

“…15,25 While the exact temporal events in COG and SNARE-driven vesicle tethering and fusion at the Golgi is yet to be fully understood, the evidence so far indicates that the COG complex orchestrates intra-Golgi trafficking by capturing vesicles and modulating/proof-reading SNARE alignment that precedes fusion. 5,22,25 Therefore, we hypothesized that impairment in Golgi SNARE interactions due to COG malfunction is the major contributor to trafficking and glycosylation defects seen in COG mutants. Indeed, GS28 along with COG subunits are frequently identified as hits in CRISPR screens for Golgi trafficking and glycosylation players.…”