The Golgi-associated retrograde protein (GARP) complex plays an essential role in the maintenance of the Golgi glycosylation machinery

Abstract

The Golgi complex is a central hub for intracellular protein trafficking and glycosylation. Steady-state localization of glycosylation enzymes is achieved by a combination of mechanisms involving retention and recycling, but the machinery governing these mechanisms is poorly understood. Herein we show that the Golgi-associated retrograde protein (GARP) complex is a critical component of this machinery. Using multiple human cell lines, we show that depletion of GARP subunits impairs Golgi modification of N- and O-glycans, and reduces the stability of glycoproteins and Golgi enzymes. Moreover, GARP-KO cells exhibit reduced retention of glycosylation enzymes in the Golgi. A RUSH assay shows that, in GARP-KO cells, the enzyme beta-1,4-galactosyltransferase 1 is not retained at the Golgi complex but instead is missorted to the endolysosomal system. We propose that the endosomal system is part of the trafficking itinerary of Golgi enzymes or their recycling adaptors and that the GARP complex is essential for recycling and stabilization of the Golgi glycosylation machinery. [Media: see text]

Getting Sugar Coating Right! The Role of the Golgi Trafficking Machinery in Glycosylation

“…While coiled-coil tethers are mostly involved in maintaining Golgi structure, the Golgi MTCs, COG and GARP have been found to be primarily involved in delivering proper glycosylation enzymes by maintaining the trafficking in Golgi. The COG-CDGs have been well documented [ 131 ], and recently, our lab has demonstrated GARP’s requirement in the recycling of glycosylation enzymes [ 182 ]; however, we are yet to understand why COG and GARP mutations affect some organ systems more severely than others. A fundamental question that we are posed with is why do mutations affecting the Golgi trafficking machinery have various manifestations at the cellular, tissue, and organismal level?…”

“…These defects in the GARP complex alter the total level of the cis -Golgi protein GPP130, medial-trans -Golgi TMEM165, and the TGN-resident TGN46 Golgi proteins. In addition, lysosomal glycoprotein LAMP2, showed hypermobility on the SDS-PAGE of GARP deficient cells, indicating protein misglycosylation [ 143 , 182 ]. Misglycosylation of these Golgi glycoproteins can occur as a consequence of the reduction in Golgi enzymes that are responsible for N- and O- glycosylation [ 9 ].…”

“…The malfunction, mislocalization, and reduced stabilization of Golgi enzymes associated with a glycosylation defect were found to be rescued upon expression of the missing GARP subunit. Taken together, GARP plays a crucial role in normal Golgi glycosylation by mediating the maintenance of the Golgi glycosylation machinery [ 182 ]. While neurodevelopmental disorders are common in GARP mutated patients, it is crucial to investigate and characterize the role of the GARP complex in different cell types.…”

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

“…COG4-mAID-mCherry in pEntra1A were recombined into pLenti COG4 Neo DEST plasmid with COG4 promoter (Khakurel et al, 2021;Sumya, Pokrovskaya and Lupashin, 2021) hTERT RPE1 COG4 KO OsTIR1-9myc cells were plated in two wells of a 6-wells plate in complete medium to reach 90% confluency the next day. One of the wells was used as a control for antibiotic selection.…”

Development and Initial Characterization of Cellular Models for COG Complex-Related CDG-II Diseases

“…RPE1-Cas9 stable cells (Khakurel et al, 2021) were transfected by the mixture of TransEDIT-dual plasmids (Transomic) with the following target sequences.…”