Figure 3.

In vitro homotypic ER fusion is Rab independent. (A) Homotypic ER fusion is insensitive to Rab inhibition by Gdi1p/Gyp1p. Gluc1 and Gluc2 microsomes were incubated on ice or at 27°C with ATP/GTP in the absence or presence of his₆-Gdi1p and his₆-Gyp1p for 90 min. For 1×Gdi1p/Gyp1p, the concentrations of his₆-Gdi1p and his₆-Gyp1p were 1.5 and 2.5 µM, respectively; the corresponding concentrations for 4×Gdi1p/Gyp1p were 6 and 10 µM, respectively. Data represent the means ± SEM (error bars; n = 3). ***, P < 0.001, Tukey’s test. (B) Ypt1p is efficiently removed from microsomes by the addition of Gdi1p/Gyp1p. ER fusion reactions were performed as described in A in the absence or presence of his₆-Gdi1p (1.5 µM) and his₆-Gyp1p (2.5 µM), and then centrifuged. Ypt1p partitioning between the soluble (supernatant) and insoluble (pellet) fractions was assessed by immunoblotting using anti-Ypt1p and anti-Sey1p antibodies. (C) In vitro yeast vacuole fusion reactions were efficiently prevented by Gdi1p/Gyp1p, but were completely resistant to anti-Sey1p and anti-Sec22p antibodies. Vacuoles isolated from BJ3505 and DKY6281 were mixed and incubated in fusion reaction buffer (see Materials and methods for details) at 27°C. After 90 min, alkaline phosphatase activity was measured, and fusion values (%) were normalized to those obtained in reactions performed without an inhibitor at 27°C. Data represent the means ± SEM (error bars; n = 3). Lowercase letters indicate statistically different groups (P < 0.001, Tukey’s test).