PI4P Promotes the Recruitment of the GGA Adaptor Proteins to the Trans-Golgi Network and Regulates Their Recognition of the Ubiquitin Sorting Signal

Abstract

Phosphatidylinositol 4 phosphate (PI4P) is highly enriched in the trans-Golgi network (TGN). Here we establish that PI4P is a key regulator of the recruitment of the GGA clathrin adaptor proteins to the TGN and that PI4P has a novel role in promoting their recognition of the ubiquitin (Ub) sorting signal. Knockdown of PI4KIIalpha by RNA interference (RNAi), which depletes the TGN's PI4P, impaired the recruitment of the GGAs to the TGN. GGAs bind PI4P primarily through their GAT domain, in a region called C-GAT, which also binds Ub but not Arf1. We identified two basic residues in the GAT domain that are essential for PI4P binding in vitro and for the recruitment of GGAs to the TGN in vivo. Unlike wild-type GGA, GGA with mutated GATs failed to rescue the abnormal TGN phenotype of the GGA RNAi-depleted cells. These residues partially overlap with those that bind Ub, and PI4P increased the affinity of the GAT domain for Ub. Because the recruitment of clathrin adaptors and their cargoes to the TGN is mediated through a web of low-affinity interactions, our results show that the dual roles of PI4P can promote specific GGA targeting and cargo recognition at the TGN.

GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion

“…By analogy to PI3P, which regulates early-stage autophagy by creating a platform to recruit specific effectors, PI4P may provide a platform for assembling the GABARAP interactome. There are many examples of deploying coincident detection to amplify low-affinity protein:lipid interactions (6). Therefore, our findings are compatible with the possibility that GABARAPs and PI4P have synergistic roles in tethering and fusion at physiological PI4P levels, even though this synergy may be less critical when PI4P is greatly increased by shuttling.…”

“…GABARAPs may direct trafficking of palmitoylated PI4KIIα from the Golgi to autophagosomes, analogous to their previously identified role as facilitators of GABA A trafficking to the plasma membrane (2). Second, PI4KIIα has already been implicated in constitutive exocytic, endo/lysosomal trafficking (6,15,16), and its recruitment to autophagosomes is decreased by GABARAP depletion. Third, there is emerging evidence that lipids enriched in raft microdomains may be involved in autophagosome maturation.…”

“…Under normal growth conditions (Ctrl), PI4KIIα is concentrated in the perinuclear region and colocalizes with TGN46, a trans Golgi network (TGN) protein (Fig. 1A) (6). Short-term nutrient starvation in Earle's balanced salt solution (EBSS) promoted autophagy and induced PI4KIIα exit from the TGN.…”

A phosphoinositide conversion mechanism for exit from endosomes

“…The Golgi complex, in particular the TGN, is characterized by PI(4)P, a PI that is of particular importance for the integrity of the Golgi, largely through its involvement in AP-1-and GGA-dependent vesicle formation at the TGN (Wang, Sun et al 2007). So far, the subcellular distribution of PI(5)P remains poorly understood, although a regulatory role in autophagosome biogenesis (Vicinanza, Korolchuk et al 2015), cell migration and enzymatic activity of PI(3,5)P 2 -metabolizing enzymes was reported (Haugsten, Oppelt et al 2013;Oppelt, Lobert et al 2013).…”

Conserved role for Gga proteins in phosphatidylinositol 4-kinase localization to the trans -Golgi network

“…At the TGN, phosphatidylinositol 4-phosphate (PtdIns4P) provides this function. The primary TGN adaptors-the heterotetrameric AP-1 (assembly protein-1) complex, GGA (Golgi-localized, gammaadaptin ear homology, Arf-binding) proteins, and epsin-related proteins (epsinR in mammals, Ent3 and Ent5 in yeast)-preferentially bind PtdIns4P and/or are dependent on PtdIns4P for optimal localization and dynamics (5)(6)(7)(8)(9)(10).…”