Fig. 3. PLD is required for export site formation. (A) PLD is required for Sar1p-induced tubule formation. VSV-infected NRK cells were permeabilized and incubated (Aridor et al., 2001) in the absence of cytosol in the presence of 9 µg of Sar1p-GTP (a–i), 1-butanol (d–f, 1.5%) or tert-butanol (g–i, 1.5%). The distribution of Sar1p (b, e and h) and VSV-G (c, f and i) was determined using indirect immunofluorescence (merged images are shown in a, d and g). Arrows in (a) indicate VSV-G-containing Sar1p-dependent tubules. Butanol inhibits tubule formation but does not block Sar1 binding (d–f). Tert-butanol does not inhibit the Sar1-dependent tubule formation (g–i; arrows in g indicate tubules on the ER; the bar in f is 5 µm. All images are representative of at least three independent experiments. (B) Cells were untreated or treated with butanol or tert-butanol as indicated (described above) and the localization of VSV-G (c, f and i) or Bip (b, e and h) was analyzed by confocal microscopy (merged images are shown in a, d and g).