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. 2019 Jun 18;17:101–118. doi: 10.1016/j.isci.2019.06.020

Figure 1.

Figure 1

Identification of ANKHD1 ARD as a Membrane Vesiculation Protein

(A) The 18 purified ARD fragments. The purity of these proteins was examined by SDS-PAGE with Coomassie brilliant blue staining (CBB). See also Figures S1–S3.

(B) Illustration of vesiculation by liposome sedimentation assay. When the liposomes were labeled with a small amount of fluorescent-labeled lipids such as rhodamine-PE, the small vesiculated liposomes were not effectively pelleted, which increased the fluorescence from labeled lipids in the supernatant.

(C–G) Percentage of rhodamine-PE in the supernatants in liposome sedimentation assay. Liposomes containing PC, PE, PS, and rhodamine-PE prepared at a weight ratio of 6:3:1:0.02 were incubated with the 18 proteins containing ARD fragments (1 μM) for 30 min and then subjected to ultracentrifugation at 109,000 × g. The supernatant (S) and the pellet (P) fractions were analyzed by SDS-PAGE, followed by visualization of the protein by CBB staining and the liposomes by rhodamine fluorescence. The percentages of rhodamine-PE in the supernatant are shown as the mean of three independent experiments. (C) ASAP1 443-737 aa and OSTF1 1-215 aa. (D) ACAP2 581-770 aa, CLPB 86-330 aa, EHMT1 684-1008 aa, EHMT2 628-968 aa, PPP1R12B 1-358 aa, and FEM1A 1-654 aa. (E) ANKHD1 195-1418 aa, ANKFY1 213-1169 aa, KANK1 1069-1360 aa, PPP1R12C 1-345 aa, RFXANK 1-259 aa, and CDKN2C 1-168 aa. (F) CDKN2D 1-166 aa, ANKRD11 140-297 aa, and ANKRD12 161-311 aa. (G) ANKRD17 224-1410 aa.

(H) Vesiculation by liposome sedimentation assay for ARD25 at 1 μM and 250 nM. The lipid compositions of the liposomes were PC:PE:PS:rhodamine-PE at a weight ratio of 6:3:1:0.02 and porcine brain lipids:rhodamine-PE at a weight ratio of 10:0.02.

(I) Quantification of liposomes in (H). Data represent the mean of three (250 nM protein) and four (1 μM protein) independent experiments.

(J and K) Electron micrographs of liposomes of PC:PE:PS at a weight ratio of 6:3:1 (J) and porcine brain lipids (K) after incubation with ARD25 (1 μM) for 20 min. Liposomes were visualized by negative staining. The boxed region presents an enlarged image. Scale bars, 100 nm.

All error bars represent SE. *p < 0.05, **p < 0.01. Statistical significance value was determined using the Student's t test.