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. 2010 Feb 23;107(8):3511-6.
doi: 10.1073/pnas.0914658107. Epub 2010 Feb 2.

Two closely related endocytic proteins that share a common OCRL-binding motif with APPL1

Laura E Swan  1 , Livia Tomasini, Michelle Pirruccello, Joël Lunardi, Pietro De Camilli
Affiliations

Two closely related endocytic proteins that share a common OCRL-binding motif with APPL1

Laura E Swan et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations of the inositol 5' phosphatase oculocerebrorenal syndrome of Lowe (OCRL) give rise to the congenital X-linked disorders oculocerebrorenal syndrome of Lowe and Dent disease, two conditions giving rise to abnormal kidney proximal tubule reabsorption, and additional nervous system and ocular defects in the case of Lowe syndrome. Here, we identify two closely related endocytic proteins, Ses1 and Ses2, which interact with the ASH-RhoGAP-like (ASPM-SPD-2-Hydin homology and Rho-GTPase Activating Domain-like) domain of OCRL. The interaction is mediated by a short amino acid motif similar to that used by the rab-5 effector APPL1 (Adaptor Protein containing pleckstrin homology [PH] domain, PTB domain and Leucine zipper motif 1) APPL1 for OCRL binding. Ses binding is mutually exclusive with APPL1 binding, and is disrupted by the same missense mutations in the ASH-RhoGAP-like domain that also disrupt APPL1 binding. Like APPL1, Ses1 and -2 are localized on endosomes but reside on different endosomal subpopulations. These findings define a consensus motif (which we have called a phenylalanine and histidine [F&H] motif) for OCRL binding and are consistent with a scenario in which Lowe syndrome and Dent disease result from perturbations at multiple sites within the endocytic pathway.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
(A) Domain cartoon of Ses proteins. Vertebrates have two Ses genes, Ses1/FAM109a and Ses2/Fam109b, whereas Drosophila (DmSes, CG12393) and all invertebrates examined to date have a single Ses gene. Ses proteins harbor a PH domain immediately followed by a putative coiled-coil region and then by a predicted unfolded C-terminal portion containing several PxxP motifs and a short highly conserved sequence (OCRL-binding site) close to the C terminus. (B ) The C-terminal portion of Ses1 is necessary and sufficient for OCRL binding. HA-tagged fragments of Ses1 (Right) were overexpressed in Cos7 cells and immunoprecipitated (IP) from cell extracts (Input) with antibodies directed against the HA tag. The Western blots shown (Left) demonstrate that endogenous OCRL coprecipitated with both full-length (FL) Ses1 and the C-terminal (CT) region of Ses1. Clathrin heavy chain (CHC) is used as a negative control. (C) Western blots of GST pulldowns from Cos7 cells expressing either HA-tagged human Ses1 or HA-tagged Ses2. Both HA-Ses1 and HA-Ses2, like endogenous APPL1 (20), strongly interact with a GST fusion of the ASH-RhoGAP–like domain of OCRL but not with the GST-ASH domain alone, the GST-RhoGAP–like domain alone, or GST. Both the ASH-RhoGAP–like domain and the RhoGAP–like domain alone bind clathrin, as expected (20).
Fig. 2.
Fig. 2.
Ses1 colocalizes with OCRL on endocytic structures. (A) Spinning disk confocal image showing a section of a Cos7 cell expressing TagRFP-T-Ses1 and GFP-OCRL. (Scale bar, 5 μm.) (B) TagRFP-T-Ses1 and GFP-OCRL also colocalize on macropinosomes. (Scale bar, 4 μm.) (C) TagRFP-T-Ses1–positive macropinosomes are positive for GFP-EEA1 and GFP-WDFY2, two markers of PI3P-positive, Rab5-positive endosomes (35, 41). (Scale bar, 2 μm.)
Fig. 3.
Fig. 3.
Minimal OCRL-binding sequence of Ses1 and similarity with the minimal OCRL-binding peptide of APPL1. (A) The C-terminal 26 amino acids of human Ses1 contain a sequence (pink box) well conserved among Ses proteins. (B) Deletion of this sequence abolishes the ability of HA-Ses1 to immunoprecipitate endogenous OCRL from Cos7 cells. Clathrin was used as a negative control. (C) Weighted matrix of a conserved 13 amino acid peptide (highlighted in orange in A) recovered from the C-termini of the 55 Ses proteins found in sequenced genomes. Inspection of the peptide reveals heavily conserved phenylalanine, histidine, and hydrophobic residues (red) at positions that also are conserved in the minimal OCRL-binding peptide of APPL1. The last two positions, which include a conserved hydrophobic residue in Ses proteins (blue), are not conserved and in the APPL1 peptide are not needed for OCRL binding. (D) A GST fusion of the conserved 13 amino acid peptide sequence of human Ses1 (C), but not the same peptide lacking the last two amino acids, is sufficient to bind OCRL from a rat brain extract. (E and F) Mutations to alanine of the conserved phenylalanine at position 224 and histidine at position 228 of full-length HA-tagged human Ses1 abolishes OCRL binding. The figure shows Western blots for the HA epitope, endogenous OCRL, and endogenous clathrin (as a control) of anti-HA immunoprecipitates (and starting extracts) from transfected Cos7 cells.
Fig. 4.
Fig. 4.
APPL1 and Ses1 binding to the ASH-RhoGAP–like domain is similarly abolished in a subset of mutations that cause Lowe syndrome. Western blots of GST pulldowns from mouse brain extracts (APPL1 and clathrin; Upper Panels) and transfected Cos7 cells (HA-Ses1 and HA-Ses2; Lower Panels) on GST fusions of WT and mutant human ASH-RhoGAP–like domain. In common with all other Lowe syndrome mutations of the ASH-RhoGAP–like domain tested (20, 29), the recently discovered patient mutations N591K, L634P, P799L, and P801L abolish APPL1 binding while preserving clathrin binding. P799L mutation causes Dent disease. All mutants deficient for APPL1 binding also are deficient for HA-Ses1 and HA-Ses2 binding; detectable but strongly decreased Ses2 binding occurred for the N591K mutant, including all mutants previously tested negative for APPL1 binding (Fig. S2) (20, 29). Two mutations (F668V and A861T) preserve both APPL1 and Ses1/2 binding. Collectively, these results indicate that APPL1 and Ses proteins use a binding modality similar to that of the OCRL ASH-RhoGAP–like domain.
Fig. 5.
Fig. 5.
Direct binding of the Ses1 minimal 13 amino acid peptide to the OCRL ASH-RhoGAP–like domain. ITC was used to measure the affinity of the minimal OCRL-binding peptides of APPL1 (11 amino acids;12 ± 2 μM affinity) and Ses1 (13 amino acids; 0.7 ± 0.08 μM). As in the case of the APPL1 peptide (20), Ses1 binding critically depended on a conserved phenylalanine residue. The similarity between Ses- and APPL1-binding properties suggested a competitive mechanism for OCRL binding. Accordingly, a Ses peptide:OCRL ASH-RhoGAP complex saturated for Ses binding did not bind the APPL1 peptide.
Fig. 6.
Fig. 6.
APPL1 and Ses2 are localized at different sites along the endocytic pathway. (A) Widefield microscopy of a transfected Cos7 cell showing the different localization of GFP-APPL1–positive and TagRFP-T-Ses2–positive spontaneously occurring macropinosomes. Rectangles define the same regions, primarily occupied by APPL1 vesicles and Ses2 vesicles, respectively, in the two channels. The corresponding movie (Movie S1) shows that GFP-APPL1–positive micropinosomes disappear or mature to become TagRFP-T-Ses2 positive. (Scale bar, 8 μm.) (B) APPL1 precedes Ses2 on the same macropinosome. Selected frames at 1-min intervals of a spinning disk-confocal movie. The micropinosome acquired APPL1, then shed it and acquired Ses2. APPL1 and Ses2 did not overlap. (Scale bar, 2 μm.) (C) The association of Ses2 with endosomes requires the presence of PI3P. Inhibition of PI 3′ kinases with wortmannin induces the dissociation of Ses2 from endosomes which then acquire APPL1. (Scale bar, 10 μm.)
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