Acute GARP depletion disrupts vesicle transport, leading to severe defects in sorting, secretion, and O-glycosylation

Abstract

The GARP complex is an evolutionarily conserved protein complex proposed to tether endosome-derived vesicles at the trans-Golgi network. Prolonged depletion of GARP leads to severe trafficking and glycosylation defects; however, the primary defects associated with GARP dysfunction remain unclear. In this study, the mAID degron strategy was used to degrade VPS54 in human cells, allowing for the acute disruption of GARP function. This acute dysfunction resulted in the partial mislocalization and degradation of a subset of Golgi-resident proteins, including TGN46, ATP7A, TMEM87A, CPD, C1GALT1, and GS15. Defects in enzyme recycling led to an early onset of O-glycosylation abnormalities. Additionally, the secretion of fibronectin and cathepsin D was altered, though mannose-6-phosphate receptors remained largely unaffected. COPI, AP1, and GGA coats were partially displaced, causing a significant accumulation of vesicle-like structures and large vacuoles. The detection of GARP-dependent vesicles by electron microscopy, along with the identification of specific cargo proteins associated with these trafficking carriers, provides direct experimental evidence for the GARP role as a vesicular tether. We conclude that the primary defects of GARP dysfunction include the mislocalization of vesicular coats, accumulation of GARP-dependent vesicles, degradation and mislocalization of specific Golgi proteins, and defects in O-glycosylation.