Figure 1.
In vitro characterization of SHIP164. (A) The fold prediction algorithm AlphaFold indicates that SHIP164 harbors an extended β-sheet folded to resemble a taco shell, as in VPS13 (Li et al., 2020). From left to right: (1) Ribbons representation for the taco shell “core,” predicted with high confidence. (2) The extended β-sheet is shown in two orientations as surface representation, with carbons shown white, oxygens red, nitrogens blue, and sulfurs yellow. The concave surface of the taco shell is predicted to be entirely hydrophobic, providing a binding site for lipid fatty acid moieties. (3) Additional secondary structure that may associate with the core is shown; segments discussed in the text are indicated. (B) SHIP164 co-purifies with phospholipids according to their abundance in cells as assessed by shotgun lipidomics. (C) SHIP164 co-migrates with NBD-PE in a native gel. Comparison of NBD fluorescence that co-migrates with SHIP164 versus with the well-characterized lipid transport module from E-Syt2 suggests that each SHIP164 molecule accommodates multiple (∼8) phospholipids. (D) Schematic drawing explaining the FRET-based lipid transfer and the dithionite-based control for the absence of liposome fusion. (E) Assay consistent with transfer of fluorescent lipids from donor to acceptor liposomes by SHIP164 but not by a tether construct lacking a lipid transfer module. An increase in fluorescence observed with donor liposomes only (red arrow) is due to lipid extraction by SHIP164. A larger increase is observed when lipids are subsequently transferred to the acceptor liposomes. (F) Dithionite addition excludes that SHIP164 facilitates membrane fusion, as the fluorescence decrease is the same in the SHIP164 sample and the no-protein control. In the case of protein-mediated fusion, NBD fluorescence would be increased with respect to the no-protein control. (G) 2D class averages from negatively stained SHIP164 and MBP-SHIP164, showing tail-to-tail dimerization. MBPs (arrows) are at the ends of the elongated dimer. (H) 2D class averages for the cryo-EM reconstruction show a channel running along the length of SHIP164. (I) Cryo-EM map of SHIP164 at a resolution of ∼8.3 Å, showing a long central cavity with or without the AlphaFold model (see A 1) superimposed onto it. The docking is approximate because the resolution of the reconstruction is low and because a high confidence model for SHIP164, beyond the β-sheet taco shell core, is not available. Experiments in E and F were performed in triplicate; SDs are indicated. mins, minutes; PC, phosphatidylcholine; PI, phosphatidylinisitol; PS, phosphatidylserine. Source data are available for this figure: SourceData F1.

In vitro characterization of SHIP164. (A) The fold prediction algorithm AlphaFold indicates that SHIP164 harbors an extended β-sheet folded to resemble a taco shell, as in VPS13 (Li et al., 2020). From left to right: (1) Ribbons representation for the taco shell “core,” predicted with high confidence. (2) The extended β-sheet is shown in two orientations as surface representation, with carbons shown white, oxygens red, nitrogens blue, and sulfurs yellow. The concave surface of the taco shell is predicted to be entirely hydrophobic, providing a binding site for lipid fatty acid moieties. (3) Additional secondary structure that may associate with the core is shown; segments discussed in the text are indicated. (B) SHIP164 co-purifies with phospholipids according to their abundance in cells as assessed by shotgun lipidomics. (C) SHIP164 co-migrates with NBD-PE in a native gel. Comparison of NBD fluorescence that co-migrates with SHIP164 versus with the well-characterized lipid transport module from E-Syt2 suggests that each SHIP164 molecule accommodates multiple (∼8) phospholipids. (D) Schematic drawing explaining the FRET-based lipid transfer and the dithionite-based control for the absence of liposome fusion. (E) Assay consistent with transfer of fluorescent lipids from donor to acceptor liposomes by SHIP164 but not by a tether construct lacking a lipid transfer module. An increase in fluorescence observed with donor liposomes only (red arrow) is due to lipid extraction by SHIP164. A larger increase is observed when lipids are subsequently transferred to the acceptor liposomes. (F) Dithionite addition excludes that SHIP164 facilitates membrane fusion, as the fluorescence decrease is the same in the SHIP164 sample and the no-protein control. In the case of protein-mediated fusion, NBD fluorescence would be increased with respect to the no-protein control. (G) 2D class averages from negatively stained SHIP164 and MBP-SHIP164, showing tail-to-tail dimerization. MBPs (arrows) are at the ends of the elongated dimer. (H) 2D class averages for the cryo-EM reconstruction show a channel running along the length of SHIP164. (I) Cryo-EM map of SHIP164 at a resolution of ∼8.3 Å, showing a long central cavity with or without the AlphaFold model (see A 1) superimposed onto it. The docking is approximate because the resolution of the reconstruction is low and because a high confidence model for SHIP164, beyond the β-sheet taco shell core, is not available. Experiments in E and F were performed in triplicate; SDs are indicated. mins, minutes; PC, phosphatidylcholine; PI, phosphatidylinisitol; PS, phosphatidylserine. Source data are available for this figure: SourceData F1.

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