Fig. 2: Two conserved hydrophobic motifs in VPS37A1–148 interact with the membrane. | Communications Biology

Fig. 2: Two conserved hydrophobic motifs in VPS37A1–148 interact with the membrane.

From: Identification of membrane curvature sensing motifs essential for VPS37A phagophore recruitment and autophagosome closure

Fig. 2

a Overlay of 15N-labeled VPS37A1–148 TROSY spectra in the absence (black) and presence (red) of bicelles (DMPC:DMPG:DHPC = 4:1:10, molar ratio, q = 0.5). Several perturbed resonances are labeled with their assignments. Unassigned resonances are labeled with n1 to n4. b Diagrams of VPS37A1–148 constructs for in vitro study. c Gel images of liposome flotation assays for VPS37A1–148 wildtype (WT), Mut1, Mut2, and Mut3 mixed with sonicated liposomes (POPC:DOPG:DOPE = 3:2:5, protein:lipid = 1:400, molar ratio). T, M, and B represent the top, middle, and bottom layers after centrifugation. The protein marker is indicated by m. The arrow indicates the lipid band. d Plots of VPS37A1–148 WT, Mut1, Mut2, and Mut3 in the top layer relative to the bottom layer in flotation experiments (c) using sonicated liposomes (POPC:DOPG:DOPE = 3:2:5). The T/B ratios of mutants are normalized to the ratio of VPS37A1–148 WT using the same batch of sonicated liposomes. The amount of protein in the top layer relative to the bottom layer (T/B) is quantitated by ImageJ. Data are presented as mean ± SD from three independent experiments (n = 3 for each construct). e Gel images of liposome flotation assays for VPS37A1–148 WT, Mut4, and Mut5 mixed with sonicated liposomes (POPC:DOPG:DOPE = 3:2:5, protein:lipid = 1:400, molar ratio). T, M, and B represent the top, middle, and bottom layers after centrifugation. The arrow indicates the lipid band. f Gel images of liposome flotation assays for Mut4, Mut5, Mut6, Mut7, and Mut8 mixed with sonicated liposomes (POPC:DOPG:DOPE = 3:2:5, protein:lipid = 1:400, molar ratio). T, M, and B represent the top, middle, and bottom layers after centrifugation. Protein marker is indicated by m. g Plots of VPS37A1–148 WT, Mut4, Mut5, Mut6, Mut7, and Mut8 in the top relative to the bottom layer (T/B) in flotation experiments (e, f) using sonicated liposomes (POPC:DOPG:DOPE = 3:2:5). The T/B ratios of mutants are normalized to the ratio of VPS37A1–148 WT using the same batch of sonicated liposomes. The amount of protein in the top layer relative to the bottom layer (T/B) is quantitated by ImageJ. Data are presented as mean ± SD from three independent experiments (n = 3 for each construct).

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