Docking of yeast vacuoles is catalyzed by the Ras-like GTPase Ypt7p after symmetric priming by Sec18p (NSF)

Abstract

Vacuole inheritance in yeast involves the formation of tubular and vesicular "segregation structures" which migrate into the bud and fuse there to establish the daughter cell vacuole. Vacuole fusion has been reconstituted in vitro and may be used as a model for an NSF-dependent reaction of priming, docking, and fusion. We have developed biochemical and microscopic assays for the docking step of in vitro vacuole fusion and characterized its requirements. The vacuoles must be primed for docking by the action of Sec17p (alpha-SNAP) and Sec18p (NSF). Priming is necessary for both fusion partners. It produces a labile state which requires rapid docking in order to lead productively to fusion. In addition to Sec17p/Sec18p, docking requires the activity of the Ras-like GTPase Ypt7p. Unlike Sec17p/Sec18p, which must act before docking, Ypt7p is directly involved in the docking process itself.

Figure 1

Kinetics of dilution resistance and the requirements for Sec17p, Sec18p, and Ypt7p. Samples (7× the standard volume) were incubated at 27°C. At each time, one sample was divided into 30-μl standard reactions. Six aliquots were transferred to tubes containing 5 μl of the inhibitors, control buffer only, or 570 μl reaction mixture and incubated at 27°C for the rest of the 70-min reaction. Another aliquot, which received only 5 μl of control buffer, was set on ice. After 70 min, fusion was measured. The graphs present the average of four independent experiments with standard deviation. The curves for the samples which received buffer only, were set on ice or were diluted are displayed in both panels for comparison. To facilitate averaging, the data was normalized: The activity of the samples incubated at 27°C with addition of buffer only was set to 100%. The activity of the sample set on ice at 0 min is the 0% reference. In the four experiments, the maximal fusion activities in the samples with buffer only were: 1.8 ± 0.13 U, 2.1 ± 0.2 U, 1.7 ± 0.19 U and 2.2 ± 0.19 U. The background, given by samples set on ice at 0 min, varied from 0.16 to 0.29 U. Inhibitor concentrations were: Affinity-purified antibodies against Sec18p (70 μg/ml) Sec17p (70 μg/ml), or Ypt7p (120 μg/ml); Gdi1p (120 μg/ml).

Figure 2

Sec18p action is a prerequisite for dilution resistance. (A) Sec18p dependence. Two 3× fusion reactions were started at 27°C in the presence or absence of F_ab-fragments (150 μg/ml) prepared from the total immunoglobulin fraction from antiserum directed against Sec18p. After 20 min, the samples were chilled and made chemically equivalent by supplementation with antibodies to Sec18p. The samples were then split into three standard reactions. These were either left concentrated (Undil.) or diluted 20fold (Dil.) with reaction mixture. Each received 15 μg/ml purified Sec18p and 75 μM DTT and all were incubated at 27°C. An aliquot kept on ice serves as a measure of the background reaction which had occurred during the first incubation. After 70 min, all samples were chilled on ice and adjusted to the same volume with reaction mixture. The vacuoles were reisolated by centrifugation, resuspended in 30 μl ice-cold reaction mixture and assayed for fusion. The same results were obtained if Sec18p was added to the diluted sample to 0.75 μg/ml to satisfy only the stoichiometric requirement for saturation of the antibodies (not shown). (B) ATP dependence. Two 3× fusion reactions were incubated for 20 min at 27°C in the presence or absence of the ATP regenerating system. The samples were then chilled and the regenerating system was added to the sample lacking it. The samples were split into aliquots which were diluted with reaction mixture or left concentrated. Further incubation and analysis was as in A. (C) Temperature dependence. Fusion reactions equivalent to three standard reactions were incubated for 20 min at 0°C or 27°C. The samples were split into aliquots, diluted with reaction mixture where indicated, and further processed as in A.

Figure 3

Sec18p activity is required for visible clustering of vacuoles. (A) Biochemical inhibition of Sec18p. Vacuoles were prepared from the strain RSY249 and used in 30 μl fusion reactions for microscopic analysis as described in Materials and Methods. After adding apyrase (10 U/ ml; “no ATP”), F_ab fragments against Sec18p (125 μg/ml), control-F_ab against Carboxypeptidase Y (125 μg/ ml), or buffer only, the samples were preincubated for 5 min on ice. One sample, blocked with Sec18p-F_ab, then received purified Sec18p (35 μg/ml) to rescue the block imposed by the F_ab fragments. All samples were supplemented with 75 μM DTT and incubated for 15 min at 27°C or on ice. They were then chilled on ice and mixed with low melting agarose containing the vacuole stain FM4-64. The suspension was transferred onto a prechilled microscopy slide, left at 4°C for 5 min, and analyzed by fluorescence microscopy. Pictures of 10 fields were taken for each condition. (B) Clustering in sec17-1 and sec18-1 mutants. Wildtype (RSY249), sec17-1 (RSY270), and sec18-1 (RSY272) strains were grown at 25°C. Vacuoles and cytosols were prepared from these strains as described in Materials and Methods. Fusion reactions for microscopic analysis were performed as in A using cytosol and vacuoles from the same strains. In one sample, sec18-1 vacuoles were combined with wt cytosol to rescue the defect of Sec18p. Vacuoles from the sec17-1 strain could not be reactivated by wt cytosol (not shown). (C) Quantitation of the samples shown in A and B. The average number of contacts per vacuole in the focal plane was determined from the pictures.

Figure 3

Sec18p activity is required for visible clustering of vacuoles. (A) Biochemical inhibition of Sec18p. Vacuoles were prepared from the strain RSY249 and used in 30 μl fusion reactions for microscopic analysis as described in Materials and Methods. After adding apyrase (10 U/ ml; “no ATP”), F_ab fragments against Sec18p (125 μg/ml), control-F_ab against Carboxypeptidase Y (125 μg/ ml), or buffer only, the samples were preincubated for 5 min on ice. One sample, blocked with Sec18p-F_ab, then received purified Sec18p (35 μg/ml) to rescue the block imposed by the F_ab fragments. All samples were supplemented with 75 μM DTT and incubated for 15 min at 27°C or on ice. They were then chilled on ice and mixed with low melting agarose containing the vacuole stain FM4-64. The suspension was transferred onto a prechilled microscopy slide, left at 4°C for 5 min, and analyzed by fluorescence microscopy. Pictures of 10 fields were taken for each condition. (B) Clustering in sec17-1 and sec18-1 mutants. Wildtype (RSY249), sec17-1 (RSY270), and sec18-1 (RSY272) strains were grown at 25°C. Vacuoles and cytosols were prepared from these strains as described in Materials and Methods. Fusion reactions for microscopic analysis were performed as in A using cytosol and vacuoles from the same strains. In one sample, sec18-1 vacuoles were combined with wt cytosol to rescue the defect of Sec18p. Vacuoles from the sec17-1 strain could not be reactivated by wt cytosol (not shown). (C) Quantitation of the samples shown in A and B. The average number of contacts per vacuole in the focal plane was determined from the pictures.

Figure 4

Sec18p action is required on both fusion partners to activate them for docking and fusion. Separate samples of BJ3505 vacuoles (with pro-alkaline phosphatase) and DKY6281 vacuoles (carrying the maturation enzymes), each equivalent to three standard fusion reactions, were incubated at 27°C in the presence or absence of antibodies to Sec18p (70 μg/ml). After 15 min, the samples were chilled, supplemented with antibodies to Sec18p (to 70 μg/ml), and left on ice for 5 min. They were mixed pairwise to make combinations where only one of the fusion partners or neither of them had been preincubated with anti-Sec18p. The tubes were centrifuged briefly (2 min, 8,000 g, 4°C) and the vacuoles resuspended in fresh reaction mixture with 75 μM DTT. Each sample was split into aliquots which received either 1.5 μg/ml Sec18p to release the block of the Sec18p pathway or antibodies to Sec18p to maintain the block. The samples were incubated at 27°C or on ice (to indicate the background reaction which had occurred during the first incubation). After 70 min, fusion activity was determined. Control samples having both fusion partners already mixed during the first incubation were taken through the same procedure in parallel (right panel).

Figure 6

The requirement for Ypt7p cannot be fulfilled while the vacuoles are in separate tubes. For each time point, two separate samples, each equivalent to five standard fusion reactions, were incubated at 27°C. One contained BJ3505 vacuoles carrying the pro-alkaline phosphatase (B) whereas the other contained DKY6281 vacuoles harboring the maturation enzyme, proteinase A (D). At the indicated times, aliquots were withdrawn from the reactions, chilled, and transferred into tubes containing Gdi1p (final concentration 100 μg/ml), antibodies to either Ypt7p (120 μg/ ml), Sec18p (80 μg/ml), or Sec17p (80 μg/ml), or control buffer only. The tubes were centrifuged briefly (1 min, 8,000 g, 4°C) and the vacuoles were resuspended gently and incubated for 70 min at 27°C or on ice. Fusion was then determined.

Figure 5

Activation produces a labile state which requires docking for productive consumption. Fusion reactions were started in the presence or absence of ATP with 6 μg vacuoles in a total volume of 600 μl, i.e., with a 20-fold reduced vacuole concentration. After the indicated times at 27°C or on ice, the samples were centrifuged (5 min at 10,000 g, 2°C) and the vacuoles were resuspended in 30 μl complete reaction mixture, i.e., at standard vacuole concentration. After a total reaction time of 120 min at standard concentration and at 27°C, fusion activity was determined.

Figure 7

Visible clustering of vacuoles in the early reaction phase depends on Ypt7p. (A) Fusion reactions for microscopic analysis were performed as in Fig. 4. Samples were incubated for 15 min at 27°C in the presence of buffer, Gdi1p (100 μg/ ml), or F_ab fragments against Ypt7p (125 μg/ml) or carboxypeptidase Y (Control-F_ab; 125 μg/ml). (B) Quantitation of the data in A was performed as in Fig. 3.