Figure 2. Pil1 and Lsp1 directly bind... | Rockefeller University Press

Figure 2.

$Figure 2. Pil1 and Lsp1 directly bind PI(4,5)P2-containing membranes. (A) Pil1 and Lsp1 bind and tubulate PI(4,5)P2-containing liposomes. Negative staining and EM of recombinant Pil1 or Lsp1 incubated with liposomes containing PC/PS/PE (70%/15%/15%) or, in addition, 1.5% PI(4,5)P2. (B) Negative-stained samples of recombinant Pil1 or Lsp1 incubated with PC liposomes containing 1.5% PI or PI(4,5)P2. Insets show magnifications of Pil1 bound to liposomes. (A and B) Protein-covered membrane tubules are marked with yellow arrowheads. Bars, 100 nm. (C, top) Spin-down experiments of Lsp1 incubated with or without PC liposomes containing 1% PI or PI(4,5)P2 as indicated. Proteins bound to liposomes appear in the pellet (P). Lsp1 shows higher affinity to PI(4,5)P2 than to PI. S, supernatant. (bottom) Quantification of protein amounts in the pellet fractions from spin-down experiments represented in a box plot, consisting of the median (middle of the box), the upper and lower quartile (edges of the box), and whiskers at a 1.5–interquartile range distance from the upper and lower quartile. (D) Measurement of fluorescence from NBD-labeled pil1S45C (orange emission spectrum) alone as well as in the presence of PC/PS/PI liposomes (purple emission spectrum) or PC/PS/PI(4,5)P2 (green emission spectrum); the buffer control is shown in gray. n = 6.$

Pil1 and Lsp1 directly bind PI(4,5)P₂-containing membranes. (A) Pil1 and Lsp1 bind and tubulate PI(4,5)P₂-containing liposomes. Negative staining and EM of recombinant Pil1 or Lsp1 incubated with liposomes containing PC/PS/PE (70%/15%/15%) or, in addition, 1.5% PI(4,5)P₂. (B) Negative-stained samples of recombinant Pil1 or Lsp1 incubated with PC liposomes containing 1.5% PI or PI(4,5)P₂. Insets show magnifications of Pil1 bound to liposomes. (A and B) Protein-covered membrane tubules are marked with yellow arrowheads. Bars, 100 nm. (C, top) Spin-down experiments of Lsp1 incubated with or without PC liposomes containing 1% PI or PI(4,5)P₂ as indicated. Proteins bound to liposomes appear in the pellet (P). Lsp1 shows higher affinity to PI(4,5)P₂ than to PI. S, supernatant. (bottom) Quantification of protein amounts in the pellet fractions from spin-down experiments represented in a box plot, consisting of the median (middle of the box), the upper and lower quartile (edges of the box), and whiskers at a 1.5–interquartile range distance from the upper and lower quartile. (D) Measurement of fluorescence from NBD-labeled pil1S45C (orange emission spectrum) alone as well as in the presence of PC/PS/PI liposomes (purple emission spectrum) or PC/PS/PI(4,5)P₂ (green emission spectrum); the buffer control is shown in gray. n = 6.