GOLPH3 and GOLPH3L are broad-spectrum COPI adaptors for sorting into intra-Golgi transport vesicles

Abstract

Glycosylation is a diverse and abundant modification of proteins, lipids and RNA. The fidelity of glycosylation is, in part, assured by the correct compartmentalisation of Golgi-resident glycosylation enzymes within the Golgi stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention in the Golgi. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterisation of the clientele of GOLPH3 and its paralogue GOLPH3L has been lacking. The role of GOLPH3 is of particular interest as it is frequently amplified in several solid tumour types. Here, we combine two orthogonal proteomic analyses to identify a diverse range of GOLPH3+3L clients and find that they act in a wide spectrum of glycosylation pathways, or have other roles in the Golgi. Using binding studies, bioinformatics and an in vivo Golgi retention assay, we show that GOLPH3+3L interact with the cytoplasmic tails of their clients through membrane-proximal positively-charged residues. Furthermore, deletion of GOLPH3+3L causes diverse defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi.

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

“…Microscopy analysis of COG4‐mAID cells revealed that both β′COP/COPB2 and γCOP/COPG1 were Golgi located in control cells and become severely displaced from Golgi perinuclear region as early as the 1 h of COG4 depletion (Figure 9A,B). COPI selects recycling proteins into vesicles either directly, 76 or indirectly, using additional adaptors like GOLPH3 and GOLPH3L 77–79 . In agreement with this model, GOLPH3 was rapidly displaced from the Golgi region at the early onset of COG4‐mAID depletion (Figure S9A,B).…”

“…COPI selects recycling proteins into vesicles either directly, 76 or indirectly, using additional adaptors like GOLPH3 and GOLPH3L. [77][78][79] In agreement with this model, GOLPH3 was rapidly displaced from the Golgi region at the early onset of COG4-mAID depletion (Figure S9A (Figure 9E). In summary, we have uncovered that COG complex acute depletion caused a buildup of multiple types of nontethered recycling vesicles that likely to be formed from different Golgi cisternae with the help of COPI, AP1, and GGA vesicle budding/cargo sorting machineries.…”

GARP complex controls Golgi physiology by stabilizing COPI machinery and Golgi v-SNAREs

“…GOLPH3 plays a crucial role in retrograde intra-Golgi trafficking of glycosyltransferases [46]. It is shown to bind the cytoplasmic tails of Golgi enzymes and packages them into recycling COPI vesicles [47]. ARFGAP1 promotes the formation of COPI vesicles [48, 49].…”

“…Two COPI associated proteins, GOLPH3 and ARFGAP1 are also displaced from the Golgi in GARP-KO cells, not to the ERGIC, but likely dissociate to cytoplasm. GOLPH3 plays a crucial role in sorting of a subset of Golgi resident glycosyltransferases back to Golgi by binding to the cytoplasmic tails of Golgi glycosyltransferases to package them into recycling COPI vesicles [46,47,[70][71][72][73], and its displacement from the Golgi in GARP deficient cells could be responsible for some glycosylation defects in mutant cells. Displacement of GOLPH3, however, could not explain the instability of B4GALT1 and MGAT1 in GARP-KO cells [42], since GOLPH3 is not responsible for their Golgi retention.…”

Sorting of secretory proteins at the trans-Golgi network by human TGN46

“…Adding to these earlier reports, our data suggest that the cytosolic tail of TGN46 also determines its fine intra-Golgi localization. Factors such as Vps74/GOLPH3 or coat proteins such as COPI [74][75][76] , influence enzyme-cargo interaction/segregation by recognizing cytoplasmic signals and thereby inducing protein retention by fast recycling. In addition, SM metabolism can also contribute to laterally organize the Golgi membranes at the nanoscale for efficient cargoenzyme interactions 52,53 .…”