Reconstitution of lipid transfer and contact formation by PITPβ. Quantitative data are shown as mean ± SD, with the number of independent experiments indicated. Statistics was performed using the two-tailed Student’s t test: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. (A) Schematic of the in vitro reconstitution system; La, donor liposome with ER-like composition of lipids. Lb, acceptor liposome with Golgi-like composition of lipids. 6x his-tagged VAP-A was attached to La liposomes through nickel-bound lipids. Contact formation was initiated by adding PITPβ and lipid transfer was tracked by measuring the fluorescence signal of NBD-PC over time. (B) EM examination confirming the formation of membrane contact among liposomes in the lipid transfer assay, n = 4. Representative images of liposomes having contact or not are shown on left, bar = 200 nm. Quantitation is shown in right. (C) PC transfer requires the catalytic activity of PITPβ, n = 5. (D) PITPβ preferentially transfers PC with shorter acyl chains, n = 5. (E) PC transfer by PITPβ is enhanced by incorporating PI into acceptor (Lb) liposomes, n = 5. (F) Tracking PI transfer by PITPβ by measuring the fluorescence of Topfluor-PI. Schematics of experimental design is shown on the left. PI transfer from the Golgi liposomes to the ER liposomes is shown on the right, n = 6.