Volume 43, Issue 4, Pages 522-529.e4 (November 2017) A Coincidence Detection Mechanism Controls PX-BAR Domain-Mediated Endocytic Membrane Remodeling via an Allosteric Structural Switch  Wen-Ting Lo, Andreja Vujičić Žagar, Fabian Gerth, Martin Lehmann, Dymtro Puchkov, Oxana Krylova, Christian Freund, Leonardo Scapozza, Oscar Vadas, Volker Haucke  Developmental Cell  Volume 43, Issue 4, Pages 522-529.e4 (November 2017) DOI: 10.1016/j.devcel.2017.10.019 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Autoinhibition of SNX9-Mediated Membrane Remodeling by Association of the Conserved Linker Region with the PX-BAR Domain (A) Multiple sequence alignments of vertebrate SNX9 linker. Conservation is colored from white to dark blue for most conserved. Red bar indicates conserved acidic stretch. (B) SNX9 domain organization and schemes of constructs used. Linker (L), amino acids 71–204; red, conserved acidic stretch (AS). (C) Potent plasma membrane tubule-forming activity of SNX9 (Δ103-171) expressed in HeLa cells. Mean ± SEM, N = 4, ∗∗∗∗p < 0.0001, unpaired t test. (D) Fluorescence-activated cell sorting analysis of transferrin-CME in HeLa cells overexpressing SNX9 wild-type (WT) or (Δ103-171). Cells with equal expression of EGFP or EGFP-SNX9 were analyzed (Figures S4C and S4D). Mean ± SEM, N = 3, ∗∗∗∗p < 0.0001, one-way ANOVA. (E and F) Association of GST-SNX9-SH3 or -SH3-linker (SH3-L) variants (amino acid numbers in brackets) with purified recombinant PX-BAR (SNX9 204–595). Samples were analyzed by SDS-PAGE and staining with Coomassie blue. (G) HDX-MS mapping of the dynamic exchange of protons of SNX9 (WT) versus the isolated PX-BAR (SNX9 204–595) (left) or mutant full-length SNX9 in which the AS has been mutationally inactivated (AS, compare also Figure 2A) (right). Differences in deuteration levels are color-coded. See Figures S1 and S2. Developmental Cell 2017 43, 522-529.e4DOI: (10.1016/j.devcel.2017.10.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Structural Basis for AP-2 Binding to the SNX9-Linker (A) Schematic view of the SNX9 linker (L) sequence and mutants or peptides used. (B and C) Association of WT or mutant GST-SNX9 SH3-linker (SH3-L) variants mutated in the sites indicated in (A) with purified recombinant AP2α-ear (B) or PX-BAR (C). WT or mutant GST-SH3-linker fusion proteins were immobilized on beads and used as bait. Samples were analyzed by SDS-PAGE and staining with Coomassie blue; ∗ in (C) degradation product. Relative binding of mutants relative to WT SH3-L (set to 1) were quantified and are depicted as mean ± SEM N = 3 for both experiments, ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p ≤ 0.05, one-way ANOVA. (D) PX-BAR and AP2α-ear compete for binding to SNX9-SH3-L. GST-L (71–204) was immobilized on beads either mock-treated, incubated with PX-BAR, or with PX-BAR in the presence of a 4-fold molar excess of AP2α-ear. Samples were analyzed by SDS-PAGE and staining with Coomassie blue. (E) HDX-MS comparison of D2O incorporation into SNX9 or SNX9 complex with AP2α-ear. Association with AP2α-ear significantly reduced HDX rates of SNX9 in the linker regions. Red dashed lines indicate AP-2 binding sites within the SNX9-linker. (F and G) NMR analysis of chemical shift changes within the AP2α-ear (PDB: 1QPT, gray) upon complex formation with SNX9 linker-derived DPW-containing peptides (F) or AS-containing peptides (G). The DPW peptide (F) via hydrophobic interactions binds to the top site on the platform subdomain of the AP2α-ear. The AS peptide (G) associates with both the top site and, more weakly, with the side site of the AP2α-ear. Binding residues for DPW (blue) or AS peptides (red) are color-coded. See Figures S3 and S4. Developmental Cell 2017 43, 522-529.e4DOI: (10.1016/j.devcel.2017.10.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Endocytic Membrane Remodeling Requires Release from Autoinhibition by AP-2-Dependent Sequestration of the SNX9-Linker (A) Model for the conversion of SNX9 (PDB: 2RAJ) between its closed inactive and its active open conformation triggered by association of its linker with AP2 (PDB: 1QPT). (B) Membrane-tubulating activity of EGFP-SNX9 WT or the indicated mutants (APA, AS, W2A2, or APA/W2A2) in live HeLa cells monitored by total internal reflection (TIRF) microscopy. Dashed lines indicate cell boundaries. (C) Quantification of data in (B) (mean ± SEM, N = 5 experiments with >90 cells/experiment, ∗∗∗∗p < 0.0001, one-way ANOVA). (D) Electron micrograph illustrating extensive plasma membrane tubule formation (indicated by the arrows) in HeLa cells overexpressing SNX9 (APA/W2A2). Note the presence of multiple elongated U-shaped CCPs that apparently have failed to undergo membrane fission. (E) Role of AP2 in modulating SNX9-mediated plasma membrane tubulation, representative dual-color TIRF images of HeLa cells transfected with control (siCtrl), or AP2αA/X small interfering RNA (siRNA), and expressing either SNX9 (WT) or APA/W2A2-mutant SNX9 (green) and immunostained for AP2α (red). Depletion of AP2αA/X abrogates membrane tubule formation by SNX9 (WT), but does not affect extensive tubulation induced by constitutively active open APA/W2A2-mutant SNX9. Dashed lines indicate cell boundaries. (F) Quantification of results shown in (D). Mean ± SEM, N = 4, ∗∗∗p < 0.001, one-way ANOVA. Scale bars, 10 μm, and 2 μm (inset). Developmental Cell 2017 43, 522-529.e4DOI: (10.1016/j.devcel.2017.10.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 PX-BAR-Mediated Endocytic Membrane Remodeling Depends on Coincident Detection of PI(3,4)P2 at Endocytic Sites (A) Partitioning of EGFP-SNX9 (WT) and AS or APA/W2A2 mutant versions between soluble and membrane fractions in overexpressing HEK293 cells assessed by immunoblotting for GFP. Mean ± SEM, N = 3, ∗∗p < 0.01, ∗p ≤ 0.05, single column t test and one-way ANOVA. (B) HDX-MS analysis of SNX9 (PX-BAR) with or without di-C8-PI(3,4)P2. Region 416–445 shows reduced dynamics (blue) in presence of PI(3,4)P2. (C) Representative TIRF images of HeLa cells transfected with control or PI3KC2α siRNA and expressing the AS or APA/W2A2 mutants of SNX9. Depletion of PI3KC2α caused a near complete loss of EGFP-SNX9 membrane tubule formation. Dashed lines indicate cell boundaries. Scale bars, 10 μm, and 2 μm inset. (D) Quantification of data shown in (C) (mean ± SEM, N = 4, ∗∗∗∗p < 0.0001). (E) Immunoblot analysis of control and PI3KC2α depleted HeLa cells confirming efficient down regulation of PI3KC2α expression by siRNA (Posor et al., 2013). (F) Electron micrographs showing enhanced membrane tubule formation from PI(3,4)P2-containing liposomes by purified constitutively active open SNX9 (APA/W2A2) compared with SNX9 (WT). (G) Quantification of data shown in (F). Developmental Cell 2017 43, 522-529.e4DOI: (10.1016/j.devcel.2017.10.019) Copyright © 2017 Elsevier Inc. Terms and Conditions