GARP dysfunction results in COPI displacement, depletion of Golgi v-SNAREs and calcium homeostasis proteins

Abstract

Golgi-associated retrograde protein (GARP) is an evolutionary conserved heterotetrameric protein complex that tethers endosome-derived vesicles and is vital for Golgi glycosylation. Microscopy and proteomic approaches were employed to investigate defects in Golgi physiology in RPE1 cells depleted for the GARP complex. Both cis and trans-Golgi compartments were significantly enlarged in GARP-knock-out (KO) cells. Proteomic analysis of Golgi-enriched membranes revealed significant depletion of a subset of Golgi residents, including Ca2+ binding proteins, enzymes, and SNAREs. Validation of proteomics studies revealed that SDF4 and ATP2C1, related to Golgi calcium homeostasis, as well as intra-Golgi v-SNAREs GOSR1 and BET1L, were significantly depleted in GARP-KO cells. Finding that GARP-KO is more deleterious to Golgi physiology than deletion of GARP-sensitive v-SNAREs, prompted a detailed investigation of COPI trafficking machinery. We discovered that in GARP-KO cells COPI is significantly displaced from the Golgi and partially relocalized to the ER-Golgi intermediate compartment (ERGIC). Moreover, COPI accessory proteins GOLPH3, ARFGAP1, GBF1, and BIG1 are also relocated to off-Golgi compartments. We propose that the dysregulation of COPI machinery, along with the depletion of Golgi v-SNAREs and alteration of Golgi Ca2+ homeostasis, are the major driving factors for the depletion of Golgi resident proteins, structural alterations, and glycosylation defects in GARP deficient cells.

Role of GARP Vesicle Tethering Complex in Golgi Physiology

“…Immunofluorescence microscopy demonstrated the depletion of Golgi GOSR1 and BET1L signals in GARP-KOs. Consistently, the total protein abundance of v-SNAREs in the GARP-KOs was significantly decreased [ 61 ]. This indicates that the GARP is involved in regulating of at least two different SNARE complexes promoting the fusion of cargo vesicles not only in TGN but also in the early-Golgi compartments.…”

“…Although the majority of GARP-sensitive proteins are predicted to reside in trans -Golgi compartments [ 56 ], the proteomic analysis of Golgi-enriched membranes in hTERT-RPE1 cells lacking GARP subunits revealed significant depletion of cis / medial Golgi proteins such as GLG1/MG-160, GALNT1, and MAN1A2 [ 61 ]. Recent results from our lab revealed that in human GARP KO cells, COPI vesicular coat complex, which is involved in intra-Golgi and Golgi-ER retrograde trafficking [ 67 ], is partially mislocalized to the ERGIC compartment.…”

“…In VPS54 KO cells, there is an enlargement of the Golgi structure, indicating an increase in the distance between the cisternae. In VPS53 KO cells, the Golgi structure was round, swollen, and disrupted [ 61 ]. This suggests that GARP dysfunction impacts not just trans -Golgi compartments but also the entire Golgi complex ( Figure 4 ).…”

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

“…The deletion of GS28 resulted in delayed retrograde trafficking of SubAB toxin comparable to the effects of COG subunit deletion (Figure 3A,B), and GS28/GS15 DKO cells exhibited a phenotype similar to GS28 KO cells without any additional effects (Z.D., unpublished observation). Moreover, RPE1 cells lacking either GS28 or GS15 were viable 65 …”

“…Moreover, RPE1 cells lacking either GS28 or GS15 were viable. 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.…”