Bypass fusion. (A) Recombinant Vam7 protein stimulates vacuole fusion in the absence of ATP. Reactions were under standard conditions (Materials and methods). Where indicated, ATP and creatine kinase/creatine phosphate were omitted and reactions had 5 mM MgCl₂ instead of 6 mM MgCl₂. Recombinant Vam7p was added to a final concentration of 3.1 μM where indicated. (B) The indicated amounts of recombinant Vam7p (by Bradford assay, with albumin standard) and mixed BJ3505 and DKY6281 vacuoles were analyzed by SDS–PAGE and immunoblotting with anti-Vam7p antibodies (1:2000 dilution). The immunoblot was quantified by densitometry. (C) BSA promotes rVam7p-stimulated fusion. BSA and rVam7p were added at the indicated final concentrations to ‘bypass' fusion reactions with 10 μM coenzyme A (CoA). (D) SNARE specificity of bypass fusion. Assays contained the indicated concentrations of either rVam7p or recombinant soluble domains of Vam3p, Vti1p, or Nyv1p.

Sensitivities to fusion inhibitors. See Materials and methods for inhibitor concentrations. Reactions labeled ‘+ATP+Vam7p' had 170 nM rVam7p.

Added Vam7p enters SNARE complexes. (A) GST-Vam7p stimulates vacuole fusion and associates with Vam3p under ‘Bypass' reaction conditions. Fusion reactions (180 μl) with indicated amounts of GST-Vam7p were incubated for 70 min at 27°C. Samples (30 μl) were withdrawn and assayed for vacuole fusion by alkaline phosphatase activity. (B) Vacuoles were reisolated from 5 × scale bypass fusion reactions with GST-Vam7p, solubilized, and Vam3p was immunoprecipitated from detergent extracts as described in Materials and methods. Samples representing 10% of the input (Total) and the α-Vam3p immunoprecipitated material (IP) were separated by SDS–PAGE and transferred to nitrocellulose membranes for immunoblot. (C) GST-Vam7p-driven bypass fusion and Vam7:Vam3 associations are sensitive to α-Vps33p. ‘Bypass' fusion reactions (180 μl) containing 165 nM GST-Vam7p and the indicated inhibitors were incubated (27°C, 70 min) and mature alkaline phosphatase activity was assayed. Vacuoles from the remainder (150 μl) were reisolated, solubilized, and GST-Vam7p was retrieved by glutathione–Sepharose. Samples of the extracts (Total) and bead-bound material (GSH) were analyzed by SDS–PAGE and immunoblotting.

PI(3)P and bypass fusion. (A) Quantitative analysis of vacuole surface PI(3)P. Vacuoles were incubated under standard or bypass conditions at 27°C for the indicated times, transferred to ice, and mixed with 3 μM Cy3-labeled FVYE₂. Samples were centrifuged (5 min, 4°C, 13 000 g) and vacuoles were resuspended in 30 μl PS buffer and solubilized with 100 μl 4 mM polydoconol detergent. Equal amounts of polydoconol were added to the supernatant fractions. Samples were analyzed using an ISS K2 fluorimeter (ISS Inc., Champaign, IL), exciting at 546 nm and recording emission at 570 nm. (B) Standard reactions were incubated for 30 min in the presence or absence of inhibitors and labeled as described above. To inhibit PI(3)P production, reactions were incubated at 4°C or 27°C in the presence of the phosphatidylinositol 3-kinase inhibitor LY294002 (200 μM). A separate sample was incubated at 27°C for 28 min in the absence of inhibitors and then mixed with Vam7p, hexokinase, and glucose. This sample was further incubated at 27°C for 2 min to allow Vam7p binding to PI(3)P prior to Cy3-FYVE labeling as described above. (C) FYVE₂ and PX were added to standard fusion reactions, standard reactions+170 nM rVam7p, or bypass fusion reactions, and fusion was assayed after 90 min at 27°C. (D) Bypass fusion reactions of 90 μl (3 × ) bypass reactions were prepared with 39, 19, or 9.7 nM GST-Vam7p (substituted for the rVam7p) in the presence of the indicated inhibitors. Reactions also contained 550 nM IB2. Reactions were incubated for 90 min at 27°C, and then 60 μl of each reaction was transferred to a fresh tube and centrifuged at 20 000 g, 4°C, for 10 min. The supernatants were removed and pellets were resuspended in SDS sample loading buffer. Samples were electrophoresed on a 10% gel and immunoblotted using a biotinylated Vam7p antibody as primary and neutravidin–HRP as secondary.

Recombinant Vam7p reduces the levels of Ypt7p needed for fusion. (A) Bypass fusion reactions without rVam7p were preincubated for 10 min on ice with either no inhibitor, Gdi1p, Gyp1-46p, or Gdi1p and Gyp1-46p (see Materials and methods for concentrations), and then mixed with the indicated concentrations of rVam7p. (B, C) Four large reactions (13 × ) containing vacuoles, reaction salts, BSA, and either glucose/hexokinase for the bypass reactions or coenzyme A for the standard reaction (see Materials and methods for concentrations of reagents) were preincubated with Gdi1p or PS buffer for 10 min at 27°C. Reactions were returned to ice and 30 μl aliquots were added to tubes containing mixtures of anti-Vam3 IgG with either 1.5 μM rVam7p or ATP and then incubated at 27°C for 90 min; these samples represent the 0-time addition of anti-Vam3p. Vam7p (1.5 μM final) or ATP was added to the remaining large reactions on ice and 30 μl of each reaction was removed for a 0-time immunoblot analysis of the pellet (13 000 rpm, 5 min, 4°C in a microfuge). The remaining four large reactions were then moved to 27°C. Aliquots were removed at 5, 10, 20, 40, and 90 min for immunoblot analysis of pellets (C) and other 30 μl aliquots were transferred to tubes containing anti-Vam3 IgG before returning to 27° for the remaining 90 min (B).

Recombinant Vam7p supports Ca²⁺ efflux and fusion in the absence of priming. (A) The indicated proteins were added to standard fusion reactions prior to incubation at 27°C. (B) Standard fusion reactions without inhibitor, with antibody to Sec17p, or bypass fusion reactions were performed with the indicated concentrations of added rVam7p. All reactions had 10 mg/ml BSA and 2% (v/v) dimethylsulfoxide. (C) Standard Ca²⁺ release reactions (Merz and Wickner, 2004) with ATP were initiated in the presence of the indicated reagents: 10 nM Sec18p, 1 μM rVam7p, 380 nM anti-Sec17p IgG, and 200 nM affinity-purified anti-Vam3p antibody. When used in combination, reagents were added in the sequence specified in the labels. (D) Standard or bypass fusion reactions were performed in the presence of the indicated Ca²⁺ chelator.

The Vo domain of the vacuolar ATPase is needed for vacuole fusion in the absence of ATP. Vacuoles were isolated from BJ3505 and DKY6281 strains and their vph1-Δ derivatives and assayed for fusion. Fusion reactions were performed under the standard or bypass fusion conditions (+ATP or −ATP, respectively) with or without 11 μM Vac8p and the following pure components (PCs): 12 nM His₆-Sec18p, 240 nM LMA1, and 6 μM calmodulin. Vacuoles were premixed in equal concentrations in the pairs BJ/DKY, BJ/DKY vph1-Δ, BJvph1-Δ/DKY, or BJvph1-Δ/DKYvph1-Δ and added to complete mixtures of the other reaction components on ice. Alkaline phosphatase activity was assayed after 90 min at 27°C.

Recombinant Vam7p permits Ykt6p to substitute for Nyv1p in vacuole fusion. Vacuoles were isolated from BJ3505 and DKY6281 wild-type (NYV1) and nyv1-Δ strains and assayed for fusion. Assays contained 125 mM KCl, 6 mM MgCl₂, 2% EtOH, 10 mg/ml BSA, 15 μM coenzyme A, 12 nM His₆-Sec18p, 550 nM IB2, 1 mM ATP, 29 mM creatine phosphate, and 1 mg/ml creatine kinase. Recombinant Vam7p was added at the indicated concentrations. See Materials and methods for concentrations of inhibitory proteins. Affinity-purified antibody to His₆-Ykt6p (preparation A, lanes 9 and 25) was added at 330 nM. For affinity isolation of this antibody, MBP-Ykt6p was purified on amylose resin (NE Biolabs) and immobilized on Affigel-15 (Bio-Rad). An independent preparation of antibody to Ykt6p (preparation B, lane 10), purified on protein A–Sepharose and added at 4 μM, was the generous gift of Dr C Ungermann.