Figure S3. DysF domains bind PA in... | Rockefeller University Press

Figure S3.

$DysF domains bind PA in vitro. (A) DysF and Opi1-PA-binding domains were expressed as fusion proteins to the epitope tags indicated and were detected with antibodies against the C-terminal tag. Bottom: size-exclusion chromatogram of the His-SUMO-Pex30-DysF-HA purification run. UV in arbitrary units represents the amount of protein based on the absorbance at 280 nm. (B) Both Opi1 and DysF domains bind to PA and more weakly to phosphoinositides. Purified proteins were incubated with the indicated lipids immobilized in a nitrocellulose membrane. Proteins were expressed as a fusion protein to the epitope tags indicated and were detected with antibodies against their C-terminal tag. IB, immunoblot. (C) Liposome flotation assay of His-Sumo as described in Fig. 3 B using liposomes containing different PA or PS concentrations. On the bottom of the top fraction (1), the lipids from the liposomes can be observed. The position of molecular weight markers (in kDa) is indicated. (D) Quantification of the percentage of DysF from Pex31 cofractionating with liposomes to the top fraction of experiments described as in Fig. 3 B. The bars represent the SD. (E) Quantification of the percentage of InnerDysF from human DysF cofractionating with liposomes to the top fraction of experiments described as in Fig. 3 B. The bars represent the SD. (F) Binding of the DysF domain of Pex30 to membrane systems with 40% of DOPA, DOPE, or DOPS in CG-MD simulations. (G) Insertion depth (Å) per residue of Pex30-DysF in PA-rich (top, orange) and PS-rich (green) membranes during the first and last 20 ns of AA-MD simulations. The dotted line denotes the surface of the membrane, while arrows indicate main variations in insertion depth over time. Blue, DysF; yellow, DOPC; orange, DOPA, and green, DOPS. (H) Steady-state levels of endogenously tagged Pex28-13xMyc, Pex29-V5, and Pex32-3HA in cells with the indicated genotype. Whole-cell extracts were prepared from exponentially growing cells, separated by SDS-PAGE, and analyzed by western blotting. Pex28-13xMyc, Pex29-V5, Pex30, and Pex32-3xHA were detected with anti-Myc, anti-V5, anti-Pex30, and anti-HA, respectively. Dpm1, used as a loading control, was detected with anti-Dpm1 antibody. Pex30DysF-5A corresponds to a variant of Pex30 with alanine mutations on W298, I301, K304, F392, and Y395. *, not specific band. IB, immunoblot. The position of molecular weight markers (in kDa) is indicated. (I) Localization of Spo2051–91-GFP-ER in pex30Δ cells. Cells were analyzed in the diauxic shift after overnight growth in SC medium, and LDs were stained with the neutral lipid dye MDH. Individual Z-planes corresponding to the center and the periphery of the cell are shown. Bar, 5 µm. (J) Distribution of lipid sensors for DAG (Ca1/b-PKD-GFP-ER), PI3P (mRFP-FYVE), PS (Lact-C2-GFP), and sterol (mCherry-D4H) in WT and pex30Δ cells during the exponential phase after overnight growth in SC medium. Bar, 5 µm. (K) Distribution of the PI(4)P lipid sensor GPD-mCherry-2xPHOsh2 in cells of the indicated genotype during the exponential phase after overnight growth in SC medium. As a control, sac1Δ cells, lacking the phosphoinositide phosphatase Sac1, were analyzed. Bar, 5 µm. Source data are available for this figure: SourceData FS3.$

DysF domains bind PA in vitro. (A) DysF and Opi1-PA-binding domains were expressed as fusion proteins to the epitope tags indicated and were detected with antibodies against the C-terminal tag. Bottom: size-exclusion chromatogram of the His-SUMO-Pex30-DysF-HA purification run. UV in arbitrary units represents the amount of protein based on the absorbance at 280 nm. (B) Both Opi1 and DysF domains bind to PA and more weakly to phosphoinositides. Purified proteins were incubated with the indicated lipids immobilized in a nitrocellulose membrane. Proteins were expressed as a fusion protein to the epitope tags indicated and were detected with antibodies against their C-terminal tag. IB, immunoblot. (C) Liposome flotation assay of His-Sumo as described in Fig. 3 B using liposomes containing different PA or PS concentrations. On the bottom of the top fraction (1), the lipids from the liposomes can be observed. The position of molecular weight markers (in kDa) is indicated. (D) Quantification of the percentage of DysF from Pex31 cofractionating with liposomes to the top fraction of experiments described as in Fig. 3 B. The bars represent the SD. (E) Quantification of the percentage of InnerDysF from human DysF cofractionating with liposomes to the top fraction of experiments described as in Fig. 3 B. The bars represent the SD. (F) Binding of the DysF domain of Pex30 to membrane systems with 40% of DOPA, DOPE, or DOPS in CG-MD simulations. (G) Insertion depth (Å) per residue of Pex30-DysF in PA-rich (top, orange) and PS-rich (green) membranes during the first and last 20 ns of AA-MD simulations. The dotted line denotes the surface of the membrane, while arrows indicate main variations in insertion depth over time. Blue, DysF; yellow, DOPC; orange, DOPA, and green, DOPS. (H) Steady-state levels of endogenously tagged Pex28-13xMyc, Pex29-V5, and Pex32-3HA in cells with the indicated genotype. Whole-cell extracts were prepared from exponentially growing cells, separated by SDS-PAGE, and analyzed by western blotting. Pex28-13xMyc, Pex29-V5, Pex30, and Pex32-3xHA were detected with anti-Myc, anti-V5, anti-Pex30, and anti-HA, respectively. Dpm1, used as a loading control, was detected with anti-Dpm1 antibody. Pex30^DysF-5A corresponds to a variant of Pex30 with alanine mutations on W298, I301, K304, F392, and Y395. *, not specific band. IB, immunoblot. The position of molecular weight markers (in kDa) is indicated. (I) Localization of Spo20^51–91-GFP-ER in pex30Δ cells. Cells were analyzed in the diauxic shift after overnight growth in SC medium, and LDs were stained with the neutral lipid dye MDH. Individual Z-planes corresponding to the center and the periphery of the cell are shown. Bar, 5 µm. (J) Distribution of lipid sensors for DAG (Ca1/b-PKD-GFP-ER), PI3P (mRFP-FYVE), PS (Lact-C2-GFP), and sterol (mCherry-D4H) in WT and pex30Δ cells during the exponential phase after overnight growth in SC medium. Bar, 5 µm. (K) Distribution of the PI(4)P lipid sensor GPD-mCherry-2xPH^Osh2 in cells of the indicated genotype during the exponential phase after overnight growth in SC medium. As a control, sac1Δ cells, lacking the phosphoinositide phosphatase Sac1, were analyzed. Bar, 5 µm. Source data are available for this figure: SourceData FS3.

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