pH-dependent binding of Ups1 to membranes reveals a shift in affinity across the physiological range of the IMS

(A) Ribbon diagram of His-tagged Ups1 in complex with Mdm35 as predicted by AlphaFold 3 (Abramson et al., 2024). (B) Schematic diagram of the flotation assay used to study protein membrane interactions (details see Methods). (C) Flotation analyses using untagged Ups1/Mdm35 and LUVs composed of 50% PC, 30% PA, 19.875% PE, 0.125% TF488-PE at indicated pH values. A representative image from three independent experiments is shown for each condition. (D) The percentage of membrane-bound Ups1 relative to total Ups1 quantified from Coomassie-stained gels of (C). Data represent the mean ± SD from three independent experiments. Significant differences relative to pH 5.5 are indicated. (E) Change in % membrane-bound Ups1 across the indicated pH intervals based on (D). Each data point represents the binding change between indicated pH values calculated using linear regression. Data are shown as mean slope ± SE from three independent experiments. (F) Quantification of membrane-bound His1-Ups1/Mdm35 from flotation assays using the same liposomes as in (C) at the indicated pH values. Data represent the mean ± SD from three independent experiments. Significant differences relative to pH 5.5. are indicated. Representative gels are shown in Supplementary Figure 1C. (G) Dissociation constant (KD) of Ups1/Mdm35 binding to membranes was obtained by a Langmuir fit of the optical thickness (red line).

Ups1 membrane binding is dependent on curvature and electrostatic interactions.

(A) Schematic illustration of the coarse-grained molecular dynamics simulation setup used to investigate the curvature-dependent binding of Ups1 to lipid membranes. The analytical membrane shape description is shown together with the Ups1/Mdm35 complex bound to the membrane. (B) Relative binding free energy (ΔΔF) of Ups1/Mdm35 as a function of membrane curvature. (C) Flotation analyses showing the binding of untagged Ups1/Mdm35 to liposomes extruded through membranes with indicated pore sizes and composed of 50% PC, 30% PA, 19.95% PE, 0.05% TF488-PE at pH 7.0. A representative image from three independent experiments is shown for each condition. (D) The percentage of membrane-bound Ups1 relative to total Ups1 quantified from Coomassie-stained gels of (C). Data represent the mean ± SD from three independent experiments. (E) Co-sedimentation analyses showing the binding of untagged Ups1/Mdm35 to liposomes extruded through membranes with the indicated pore sizes. Liposomes were composed of 50% PC, 30% PA, 19.95% PE and 0.05% TF488-PE at pH 7.0. A representative image from three independent experiments is shown for each condition. (F) The percentage of membrane-bound Ups1 relative to total Ups1 quantified from Coomassie-stained gels of (E). Data represent the mean ± SD from three independent experiments. (G) Flotation analyses showing the binding of untagged Ups1/Mdm35 to liposomes composed of 69.875% PC, 0.125% TF488-PE and 30% of either PE, PA, PS, PI, or PG, as indicated. CL liposomes were composed of 84.875% PC, 0.125% TF488-PE, and 15% CL. Liposomes were extruded through 50 nm membranes and the assay was performed at pH 7.0. A representative image from three independent experiments is shown for each condition. (H) The percentage of membrane-bound Ups1 relative to total Ups1 quantified from Coomassie-stained gels of (G). Data represent the mean ± SD from three independent experiments. (I) Schematic diagram of the co-sedimentation assay used to study protein membrane interactions (details see Methods).

Ups1 binding to membranes mimicking the MOM is curvature-dependent.

(A) Flotation analyses showing the binding of untagged Ups1/Mdm35 to liposomes extruded through membranes with indicated pore sizes, representing MOM-like membranes (46% PC, 32.95% PE, 10% PI, 1% PS, 6% CL, 4% PA, 0.05% TF488-PE) at pH 7.0. The percentage of membrane-bound Ups1 relative to total Ups1 was quantified from Coomassie-stained gels. Data represent the mean ± SD from three independent experiments. (B) Flotation analyses showing the binding of untagged Ups1/Mdm35 to liposomes representing MIM-like membranes (38% PC, 23.95% PE, 16% PI, 4% PS, 16% CL, 2% PA, 0.05% TF488-PE). The experiment and subsequent analysis were performed as in (A).

Curvature-dependent lipid partitioning and free energy landscapes reveal energetically favourable PA Extraction from positively curved membranes.

(A) Schematic illustration of the coarse-grained molecular dynamics simulation setup used to analyse curvature-dependent lipid partitioning. The analytical membrane shape description is shown along the arc length parameter s. (B) Lipid distributions along the arc length parameter s after the system was equilibrated to allow curvature-induced lipid partitioning. Errors from bootstrapping are smaller than the symbols. Dashed lines represent the mean of linear fits to every resampled lipid distribution. (C) Free energy landscapes of each lipid species along the arc length parameter s constructed from the mean of linear fits to the lipid distributions. (D) Relative free energy difference of the indicated lipid species in a positively curved compared to a flat membrane. (E) Relative energy differences required for lipid desorption form a positively curved compared to a flat membrane.

Lipid Transfer by Ups1/Mdm35 Depends on Donor Curvature.

(A) Schematic diagram of the transfer assay to study Ups/Mdm35 mediated PA transfer. (B-G) PA transfer between donor and acceptor liposomes extruded through membranes with the indicated pore sizes. Donor liposomes contained 8% NBD-PA, 2% Rhod-PE, 90% DOPC and acceptor liposomes 10% DOPA and 90% DOPC. (B) Size dependent spontaneous PA transfer between donor and acceptor liposomes in the absence of Ups1/Mdm35. Normalized NBD fluorescence is shown over time. Data represent the mean ± SD of three independent replicates (left). The transfer rate is determined from the slope of the linear NBD increase using linear regression (right). Data represent the mean ± SE of three independent replicates. (C) Size dependent PA transfer between donor and acceptor liposomes in the presence of Ups1/Mdm35. Same experimental setup and analyses as in (B), with Ups1/Mdm35 added at time point 0. (D+E) Effect of donor size on PA transfer by Ups1/Mdm35. Same experimental setup and analyses as in (C). (F+G) Effect of acceptor size on PA Transfer by Ups1/Mdm35. Same experimental setup and analyses as in (C). (H) Schematic diagram of the extraction assay to study Ups1/Mdm35 mediated PA extraction from donor liposomes. (I) PA extraction from donor liposomes with Ups1/Mdm35 added at time point 0. Normalized NBD fluorescence is shown over time. Data represent the mean ± SD of three independent replicates (left). The extraction rate is determined from the slope of the linear NBD increase using linear regression (right). Data represent the mean ± SE of three independent replicates.

Curvature-Dependent PA Binding and Its Role in Efficient PA Extraction by Ups1

(A) Ups1 has low binding affinity to flat membranes, where PA is in a low-energy state, making extraction energetically unfavourable. (B) In contrast, Ups1 preferentially binds to highly positively curved membranes, where PA is in a high-energy state, lowering the energy barrier for extraction and enabling more efficient lipid transfer.

Purification of Ups/Mdm35 and pH dependent binding of His-Ups1/Mdm35.

(A) Expression and purification of recombinant His-Ups1/Mdm35 complex analyzed by SDS-PAGE and colloidal Coomassie staining. (B) Expression and purification of recombinant His-TEV-Ups1/Mdm35, followed by TEV cleavage and subsequent purification of cleaved Ups1/Mdm35, analysed by SDS-PAGE and colloidal Coomassie staining. (C) Flotation analyses using His-Ups1/Mdm35 and LUVs composed of 50% PC, 30% PA, 19.875% PE, 0.125% TF488-PE at indicated pH values. A representative image from three independent experiments is shown for each condition.

Ups1 exhibits mild curvature-inducing activity.

(A) Electron micrographs of liposomes (LUVs) in the absence and presence of Ups1/Mdm35 and quantification of spherical and deformed shapes in control LUVs and in LUVs after incubation with Ups1/Mdm35. A minimum of 200 membranous structures were counted per condition. Scale bar corresponds to 100 nm. (B) Tubulation assay in the absence and presence of Ups1/Mdm35. Indicated lipid compositions were dried on a glass slide and rehydrated using assay buffer (50 mM Tris pH 7.0; 150 mM NaCl). Formed lipid membranes were visualized by DIC imaging at the indicated timepoints before or after the addition of 10 µM Ups1/Mdm35.

Uncropped SDS-PAGE gels.

Images of uncropped SDS-PAGE gels corresponding to the cropped gel images shown in Figures 1-3 and Supplementary Figure 1.