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. 2010 Apr;11(4):279-84.
doi: 10.1038/embor.2010.28. Epub 2010 Mar 19.

Structural Basis of Wedging the Golgi Membrane by FAPP Pleckstrin Homology Domains

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Free PMC article

Structural Basis of Wedging the Golgi Membrane by FAPP Pleckstrin Homology Domains

Marc Lenoir et al. EMBO Rep. .
Free PMC article

Abstract

The mechanisms underlying Golgi targeting and vesiculation are unknown, although the responsible phosphatidylinositol 4-phosphate (PtdIns(4)P) ligand and four-phosphate-adaptor protein (FAPP) modules have been defined. The micelle-bound structure of the FAPP1 pleckstrin homology domain reveals how its prominent wedge independently tubulates Golgi membranes by leaflet penetration. Mutations compromising the exposed hydrophobicity of full-length FAPP2 abolish lipid monolayer binding and compression. The trafficking process begins with an electrostatic approach, phosphoinositide sampling and perpendicular penetration. Extensive protein contacts with PtdIns(4)P and neighbouring phospholipids reshape the bilayer and initiate tubulation through a conserved wedge with features shared by diverse protein modules.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Solution structures of FAPP1-PH determined by nuclear magnetic resonance and docked to a DPC micelle. (A) Backbone superposition of the 20 lowest energy structures and (B) ribbon of the representative structure with helices and β-strands labelled and coloured in aqua and red, respectively. (C) Structure of a representative FAPP1-PH micelle complex. The centre of the protein is depicted as a purple sphere, and the micelle surface and centre are coloured yellow and blue with the radius and distance from the protein indicated. The β1–β2 wedge inserts with an average distance of 8.3 Å between the micelle centre and deeply buried L12 Cδ2 group. The inset is rotated and expanded to show the interactions of the side chains with the DPC molecules. DPC, dodecylphosphocholine; FAPP, four-phosphate-adaptor protein; PH, pleckstrin homology.
Figure 2
Figure 2
Micelle insertion site mapped to the FAPP1-PH surface. The exposed FAPP1-PH β1–β2 loop residues exhibiting significant backbone or side-chain HN PREs in the presence of micelles spiked with 14-doxyl phosphocholine as well as CSPs induced by DPC are indicated in purple. Those exhibiting medium or large chemical shift changes but undetectable or insignificant PREs are shown on the surface in aqua or blue, thus defining the region experiencing conformational changes on insertion. CSPs, chemical shift perturbations; DPC, dodecylphosphocholine; FAPP, four-phosphate-adaptor protein; PC, phosphocholine; PH, pleckstrin homology; PREs, paramagnetic relaxation enhancements.
Figure 3
Figure 3
PtdIns(4)P docking to the FAPP1-PH structure. (A) The PI binding pocket of the protein is coloured according to the extent of CSPs induced by the addition of an eightfold excess of C6-PtdIns(4)P. (B) The docked headgroup and the hydrogen bonds are indicated in yellow and dashed lines, respectively. Ligand-binding residues are labelled and shown as sticks. CSPs, chemical shift perturbations; FAPP, four-phosphate-adaptor protein; PH, pleckstrin homology; PI, phosphoinositide.
Figure 4
Figure 4
FAPP-PH independently tubulates membrane sheets. (A) Membrane sheets composed of POPC and PtdIns(4)P (98:2 mol%) spontaneously formed dynamic ∼10 μM-diameter tubules on injection of wild-type FAPP1-PH (1 mg/ml), as monitored in real time by differential interference contrast microscopy (supplementary Movie S1 online). (B) Surface pressure changes (ΔΠ) induced in POPC and PtdIns(4)P (98:2 mol%) lipid monolayers after injection of full-length FAPP2 with Thr 11–Leu 12 replaced with GG and EE sequences as well as wild-type protein at the concentrations indicated, with the latter control as described previously (Cao et al, 2009). The ΔΠ of the monolayer was recorded after protein injection into the subphase every 5 min. The isotherm was normalized to the initial established Π (~30 mN/m). FAPP, four-phosphate-adaptor protein; PH, pleckstrin homology; POPC, palmitoyl-oleoyl-phosphatidylcholine; PtdIns(4)P, phosphatidylinositol 4-phosphate; wt, wild type.
Figure 5
Figure 5
Sequences alignment of FAPP-related PH domains with PtdIns(4)P binding activities. The FAPP1-PH secondary structures are shown; wedge residues are indicated under an orange bar, those with CSPs induced by PtdIns(4)P or exhibiting PREs are indicated by red dots and black squares, respectively. Most of the PtdIns(4)P and micelle contacts are mediated in residues in the consensus LXKΩTNΦΦXGΩQXRΩΦΦL motif in the β1–β2 loop, where X, Ω and Φ refer to any, aromatic and hydrophobic residues, respectively (Aasland et al, 2002). CERT, ceramide transfer protein; CSPs, chemical shift perturbations; DPC, dodecylphosphocholine; FAPP, four-phosphate-adaptor protein; OSBP, oxysterol-binding protein; PH, pleckstrin homology; PREs, paramagnetic relaxation enhancements.

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