Fig. 5. Gyp7p induces HOPS release by promoting GTP hydrolysis. (A) Release of HOPS and Ypt7p. Fusion reactions (180 µl) containing 36 µg of only BJ3505 vacuoles were incubated in the presence of reaction buffer with no inhibitor or with Gyp7-47(R458K)p, Gyp7-47p or Gdi1p at 27°C. At the indicated times, 30 µl were removed from each reaction and the vacuoles were collected by centrifugation (16 000 g for 5 min at 4°C). Supernatants (15 µl) were analyzed for the release of Vsp39p (top panel) and Ypt7p (bottom panel) by immunoblotting. (B) GTPγS reduces the Gdi1p-stimulated release of Ypt7p and HOPS. Fusion reactions containing only BJ3505 vacuoles were incubated with Gdi1p (lane 1), Gdi1p and GTPγS (lane 2), or GTPγS (lane 3) for 30 min at 27°C. Reactions containing Gdi1p or Gdi1p and GTPγS (lanes 1 and 2) were placed on ice while the reaction containing GTPγS only (lane 3) was given Gdi1p and incubated for an additional 30 min. The samples were centrifuged at 16 000g for 5 min and 15 µl of the supernatant were analyzed by immunoblotting for Vps39p release (top panel) and Ypt7p release (middle panel). An equal portion of the vacuolar pellet was analyzed for Ypt7p (bottom panel). (C) GTPγS reduces the Gyp7p-stimulated release of HOPS. The same experiment as described in (B) was performed, except that Gyp7p was substituted for Gdi1p. Ypt7p is not released by Gyp7p and therefore was not analyzed.