Pull-down analysis of APPL1-Rab interaction. GST fusion proteins of Rab5, Rab21, and Rab22 were used to pull down His-tagged APPL1 (5−385) and (5−419) fragments in the presence of GDP or GTP analog (GppNHp). The results were analyzed with SDS–PAGE and anti-His Western blot.

Crystal structure of the APPL1 BAR-PH dimer. (A) The overall structure of the dimer. The left view is along the direction of the dyad symmetry and on the concave surface, and the right view differs by 90°. One protomer is shown in ribbon diagram, and the other is shown in molecular surface model. Helix α1 is colored yellow, α2 magenta, α3 green, α4 blue, and PH domain red. (B, C) A close view of the BAR–PH interface. These images were generated using the program PyMol.

Electrostatic potential distribution of APPL1. Electrostatic potentials of APPL1 BAR-PH protomer (left) and dimer (middle and right) are mapped on their molecular surfaces. Negatively charged regions (⩽−0.5 V) are colored red, positively charged regions (⩾+0.5 V) blue, and neutral regions gray. The right view is looking down along the dyad axis of the dimer at the concave surface, and the side view differs by 90°. Clusters of acidic residues which are potentially important in Rab5 binding are circled. This figure was generated with the program CCP4mg.

Putative complex model of the APPL1 BAR-PH dimer and Rab5. (A) APPL1–Rab5 interaction. The two BAR-PH protomers are shown in molecular surface models and colored gray and cyan, respectively. Positions of APPL1 mutations are colored similarly to Figure 5B. Overlaying APPL1, Rab5 is shown in a green backbone trace and positioned according to the mutagenesis data; the N-and C-termini of its GTPase domain are labeled. Rab5 mutants that affect binding are marked with red spheres, and those having no effect are marked with blue spheres. Positions of the reversal mutation pair are labeled. (B) Membrane recruitment of APPL1 mediated by Rab5. The APPL1 BAR-PH protomers are colored gray and yellow, and APPL1 C-terminal peptides are represented by ovals. Rab5 molecules are shown in green molecular surface models, and their membrane anchored C-terminal tails are represented by red curves.

APPL1 domain packing. (A, B), Stereoviews of two major interacting regions between the PH and BAR domains. The PH domain from one APPL1 protomer is colored gray, and the BAR domain from the dimer-mate is colored yellow. Helix backbones are shown in ribbon representation, otherwise in ropes. Side-chain and/or main-chain atoms of selected residues are shown in stick models. Nitrogen atoms are colored blue, oxygen red, and sulfur green. Hydrogen bonds (<3.0 Å) are shown as dash-lines. Note that most residues displayed are highly conserved among the known BAR-PH containing proteins (Supplementary Figure 1). (C) Stereoview of interactions of the APPL1 N-terminal region. The two protomers are colored gray and yellow, respectively. This figure was generated with the programs Molscript and Raster3D.

Mutational analyses of Rab5. (A) Pull-down assay on Rab5 variants. Point mutations in switch regions were introduced in the full-length Rab5-Q79L background of the GST fusion construct. Each mutant was expressed in E. coli, purified, and equal amounts of each Rab5 sample was used to pull down recombinant proteins of His-tagged WT APPL1 (5−419) (top panel) and His-tagged rabaptin5 (551−862) (bottom panel). The results were visualized by Coomassie blue stain. (B) Molecular surface model of Rab5 GTPase domain. Its N-terminus, switch I (SW1) and switch II (SW2) regions are labeled. Rab5 mutations that have effects and have no effect on binding are labeled red and blue, respectively. The position showing reversed binding property with the APPL1 N308D mutant is colored yellow.

Mutational analyses of APPL1. (A) Quantifying the Rab binding ability of APPL1 variants. The Rab5- and Rab21 binding abilities of APPL1 variants relative to that of WT APPL1 (5−419) were estimated based on chemiluminescence-labeled Western blot (see the Materials and methods) and shown as the white and gray bars, respectively. Standard deviations calculated from multiple experiments were represented by the thin lines. (B) Mutational effects on binding to Rab5 and Rab21. Distribution of point mutations is shown on the molecular surface of the BAR-PH dimer where the two protomers are colored gray and light green, respectively. APPL1 mutations that affect binding strongly (i.e., <30% binding comparing to WT) are colored red; otherwise, the mutants are colored blue. The position showing reversed binding property is colored yellow. Mutation positions are selectively labeled; note that the PH domain contains residues 276−379. (C) Mutational effects on APPL1 targeting to Rab5-positive early endosomes in the cell. RFP−Rab5-Q79L was coexpressed with GFP−APPL1 (full length, FL; BAR-PH domain; or BAR-PH mutant) in PC12 cells as indicated, followed by confocal fluorescence microscopy. Shown are typical confocal microscopic images indicating the RFP−Rab5-Q79L labeled early endosomes (red) and the colocalization of GFP−APPL1 or mutants (green) in the same cells. Scale bar, 16 μm.