Dismantling the Golgi's Tethering and Fusion Machinery: An Investigation of the Functional Interplay Between Key Players of the Vesicle Tethering and Fusion Machinery at the Golgi-Endosome Interface

Abstract

The Golgi apparatus is a dynamic organelle crucial for protein and lipid glycosylation, a process essential for proper protein function. Dysregulation of glycosylation is linked to pathogenic conditions such as cancer and Congenital Disorders of Glycosylation. Proper localization of glycosylation machinery is crucial for efficient modification of proteins and lipids. This localization is partially achieved through retrograde vesicular trafficking in a trans-medial-cis fashion, as the secretory cargo moves forward by the cisternal maturation of Golgi subcompartments. However, the precise mechanisms underlying the maintenance of Golgi’s glycosylation machinery in the context of the cisternal maturation model remain unclear. To investigate the contribution of key players driving Golgi trafficking and glycosylation, this study used a CRISPR-Cas9 knockout (KO) strategy to generate cell lines deficient in one or more evolutionary conserved proteins in the vesicle tethering and fusion machinery. Deletion of the Conserved Oligomeric Golgi (COG) complex subunits resulted in severe glycosylation defects and altered Golgi/endolysosomal trafficking, leading to the degradation of Golgi vesicular SNAREs and accumulation of enlarged endolysosomal structures (EELSs). However, depletion of two evolutionary conserved vesicular SNAREs, GS28 and GS15, did not phenocopy the severe Golgi and glycosylation defects of COG knockout. STX5-GS28-GS15-YKT6 is the major SNARE complex in the Golgi. Single or double KOs of vesicular SNAREs did not significantly affect glycosylation, while downregulation of STX5 and YKT6 was detrimental for Golgi physiology, suggesting a substitution mechanism for Golgi SNAREs. Using native co-immunoprecipitation coupled with label-free mass-spectrometry, this study found two novel Golgi SNARE complexes, STX5-SNAP29-VAMP7 and STX5-VTI1B-STX8-YKT6 that compensate the loss of GS28 and GS15. Analysis of the EELS compartment, a hallmark of COG knockout, revealed VAMP7 and STX8 on the EELSs, indicating a tight link between Golgi and endosomal membrane trafficking. Indeed, the superresolution microscopy analysis of EELS revealed their hybrid TGN-endosomal origin and specific function in degradation of mistargeted Golgi resident proteins. In summary, this study provides novel insights into the mechanisms underlying Golgi trafficking and glycosylation. The results provide evidence of the remarkable plasticity in Golgi SNAREs, indicating a complex interplay between endosomal and Golgi trafficking.