Fig. 1. Rho1p and Cdc42p are essential for vacuole fusion. (A) Anti-Rho1p antibodies inhibit vacuole fusion. Anti-Vam3p, anti-Rho1p or anti-SecF IgG fraction or affinity-purified anti-Ypt7p antibodies were added to standard fusion reactions. Aliquots were heated to 95°C for 10 min for heat inactivation. (B and C) Vacuole-containing rho1-ts or cdc42-ts proteins are thermolabile for fusion. Vacuoles isolated from strain (B) GEY6048 (RHO1) (circles) and GEY1658 (rho1-104) (squares) or (C) GEY6038 (CDC42) (circles) and GEY2298 (cdc42-1) (squares) were incubated at 37°C for the indicated times before adding to a fusion reaction with vacuoles from strain BJ3505. (D) Deletion of the RHO2, 3, 4 and 5 genes does not affect vacuole fusion. Vacuoles isolated from strains GEY6028 (WT), GEY0908 (ΔRHO2), GEY2288 (ΔRHO3) and GEY1808 (ΔRHO5), which lack alkaline phosphatase, were tested for fusion with vacuoles from strain BJ3505. Vacuoles isolated from strain GEY0554 (ΔRHO4), which lacks proteinase A, were tested for fusion with vacuoles from strain DKY6281. (E and F) In vivo vacuole morphology in RHO mutant and parental strains. The vacuoles from strains TD4 (CDC42), DJTD2-16A (cdc42-1), ONHY1 (RHO1) and NHY21 (rho1-104) were stained with FM4-64. Cells were grown at 27°C to an OD₆₀₀ ∼ 1. An aliquot of each was removed and incubated at 37°C for 90 min prior to observation.

Fig. 3. Rdi1p extracts Rho1p and Cdc42p from vacuoles. (A) GTPase extractions. Standard fusion reactions (90 µl) with no inhibitor (None), 3 mM GTPγS or 3 mM BAPTA were incubated for 30 min at 27°C. Aliquots (30 µl) then received PS buffer (–), Gdi1p or Rdi1p and were further incubated for 60 min at 27°C. Reactions were centrifuged (5 min, 10 000 g) and equal fractions of supernatants and vacuole pellets were analyzed by immunoblotting for Rho1p, Cdc42p and Ypt7p. (B) GTPγS blocks Gdi1p but not Rdi1p inhibition. Fusion reactions were incubated with no inhibitor, Gdi1p, Rdi1p or anti-Vam3p antibodies in the absence (striped bars) or presence (gray bars) of GTPγS. After 30 min at 27°C, vacuoles were re-isolated, supplied with fresh reaction buffer without inhibitors and incubated for 60 min. Control samples without GTPγS (black bars) were not re-isolated.

Fig. 5. Rho GTPases can only act after Sec18p and Ypt7p but vacuoles blocked for fusion with the calcium chelator BAPTA are past Rho function. Fusion reactions were incubated at 27°C in the presence of (A) anti-Sec18p antibodies, (B) anti-Ypt7 peptide antibodies or (C) BAPTA. After 30 min the inhibition was reversed by the addition of (A) His₆-Sec18p, (B) Ypt7p peptide or (C) calcium, in the presence of a second inhibitor or PS buffer (none) as indicated. Lane 2, no reversal of inhibition. Reactions were incubated for an additional 60 min before assaying for alkaline phosphatase.

Fig. 2. Rdi1p (RhoGDI) blocks fusion. (A) Addition of GST-tagged Rdi1p (triangles), heat-denatured (95°C incubation for 10 min) GST–Rdi1p (circles) or GST (squares) to standard fusion reactions. (B) Inhibition by Rdi1p of pro-alkaline phosphatase processing is not due to vacuole lysis. Standard fusion reactions were incubated at 27°C for 90 min in the absence or presence of Gdi1p or Rdi1p. Reactions were centrifuged (5 min, 10 000 g), supernatants were removed from pelleted vacuoles, and equal fractions were analyzed by immunoblotting for proteinase A and alkaline phosphatase.

Fig. 4. Kinetic analysis of Rdi1p inhibition. Fusion reactions (270 µl) were incubated at 27°C. At the indicated times, aliquots (30 µl) were removed and added to tubes on ice or containing PS buffer, anti-Sec18p antibodies, Gdi1p or Rdi1p. Reactions were incubated for 90 min (total) and then assayed for alkaline phosphatase.

Fig. 6. Docked vacuoles, stripped of Ypt7p, Rho1p and Cdc42p, are still sensitive to GTPγS. (A) Docked vacuoles no longer require Ypt7p, Rho1p or Cdc42p yet are still sensitive to GTPγS. Fusion reactions (180 µl) were incubated at 27°C in the presence of BATPA for 30 min (docked vacuoles). Lane 1, 30 µl was removed and incubated for 60 min at 27°C. Twenty-five microliters of Gdi1p, 5 µl of Gyp7p and 30 µl of Rdi1p were added to the rest of the reaction and incubated for 15 min at 27°C. The reaction was centrifuged (5 min, 10 000 g) and the vacuole pellet was resuspended in 150 µl of PS buffer supplemented with 125 mM KCl, 5 mM MgCl₂, 30 µM Ca²⁺, 10 µM CoA. Aliquots (30 µl) were either placed on ice or added to tubes containing PS buffer, anti-Vam3p IgG, GTPγS or BAPTA and incubated at 27°C for 60 min prior to assaying for alkaline phosphatase. (B) Ypt7p, Rho1p and Cdc42p are extracted from docked vacuoles by Gdi1p, Gyp7p and Rdi1p. Fusion reactions (120 µl) were incubated at 27°C in the presence of BAPTA for 30 min (docked vacuoles). Samples (30 µl) were added to tubes containing 6 µl of PS buffer, 5 µl of Gdi1p and 1 µl of Gyp7p, 6 µl of Rdi1p or 5 µl of Gdi1p, 1 µl of Gyp7p and 6 µl of Rdi1p, and incubated at 27°C for 15 min. Samples were centrifuged for 5 min at 10 000 g and equal portions of the supernatant and pellet were analyzed by immunoblot analysis.