Molecular Basis of Phosphatidylinositol 4-Phosphate and ARF1 GTPase Recognition by the FAPP1 Pleckstrin Homology (PH) Domain

Abstract

Four-phosphate-adaptor protein 1 (FAPP1) regulates secretory transport from the trans-Golgi network (TGN) to the plasma membrane. FAPP1 is recruited to the Golgi through binding of its pleckstrin homology (PH) domain to phosphatidylinositol 4-phosphate (PtdIns(4)P) and a small GTPase ADP-ribosylation factor 1 (ARF1). Despite the critical role of FAPP1 in membrane trafficking, the molecular basis of its dual function remains unclear. Here, we report a 1.9 Å resolution crystal structure of the FAPP1 PH domain and detail the molecular mechanisms of the PtdIns(4)P and ARF1 recognition. The FAPP1 PH domain folds into a seven-stranded ␤-barrel capped by an ␣-helix at one edge, whereas the opposite edge is flanked by three loops and the ␤4 and ␤7 strands that form a lipid-binding pocket within the ␤-barrel. The ARF1-binding site is located on the outer side of the ␤-barrel as determined by NMR resonance perturbation analysis, mutagenesis, and measurements of binding affinities. The two binding sites have little overlap, allowing FAPP1 PH to associate with both ligands simultaneously and independently. Binding to PtdIns(4)P is enhanced in an acidic environment and is required for membrane penetration and tubulation activity of FAPP1, whereas the GTP-bound conformation of the GTPase is necessary for the interaction with ARF1. Together, these findings provide structural and biochemical insight into the multivalent membrane anchoring by the PH domain that may augment affinity and selectivity of FAPP1 toward the TGN membranes enriched in both PtdIns(4)P and GTP-bound ARF1.

C2 domain membrane penetration by group IVA cytosolic phospholipase A2 induces membrane curvature changes

“…The FAPP1 and 2 PH domains require insertion of a turret loop adjacent to the PI(4)P binding pocket ( 42 ) to induce membrane remodeling where the inherent shape of FAPP1 or -2 may also play a critical role ( 30 ). However, unlike the ENTH and BAR domain, elegant models of membrane scaffolding and modes of membrane curvature induction for PH and C2 domains have not been investigated.…”

“…Thus, membrane penetration is necessary to induce membrane curvature changes, as observed in the EM, GUV, and membrane sheet assays. Likewise, phosphoinositides are necessary for the ENTH, PH, and ACCH domains to suffi ciently penetrate membranes and induce membrane deformation ( 31,39,42 ).…”

Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

“…PI4P lipids can be detected in intact cells using a plasmid encoding the PI4P sensor FAPP1- PH-GFP, a GFP-tagged pleckstrin-homology (PH) domain of FAPP1 (four-phosphate-adaptor protein 1). This PH domain contains a PI4P-binding pocket as well as an Arf1-binding site, which together induce localization of this PI4P sensor to the Golgi complex [33][34][35][36]. In mammalian cells, PI4P lipids present in the early secretory pathway can be produced by three of the four different isoforms of PI4K, namely PI4KIIα (localized to the Golgi), PI4KIIIα (ER), and PI4KIIIβ (Golgi).…”

Structural Basis for the Golgi Association by the Pleckstrin Homology Domain of the Ceramide Trafficking Protein (CERT)

“…In addition, most of the COFs family proteins are lipid transport proteins that are associated with the Golgi in a PtdIns(4)Pdependent manner (36,37,47). However, the proteins involved in signaling are usually specific to the di-or triphosphate forms of phosphoinositides (48 -51).…”