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. 1998 Jan 12;140(1):61-9.
doi: 10.1083/jcb.140.1.61.

A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion

C Ungermann  1 , B J Nichols, H R Pelham, W Wickner
Affiliations

A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion

C Ungermann et al. J Cell Biol. .

Abstract

Homotypic vacuole fusion in yeast requires Sec18p (N-ethylmaleimide-sensitive fusion protein [NSF]), Sec17p (soluble NSF attachment protein [alpha-SNAP]), and typical vesicle (v) and target membrane (t) SNAP receptors (SNAREs). We now report that vacuolar v- and t-SNAREs are mainly found with Sec17p as v-t-SNARE complexes in vivo and on purified vacuoles rather than only transiently forming such complexes during docking, and disrupting them upon fusion. In the priming reaction, Sec18p and ATP dissociate this v-t-SNARE complex, accompanied by the release of Sec17p. SNARE complex structure governs each functional aspect of priming, as the v-SNARE regulates the rate of Sec17p release and, in turn, Sec17p-dependent SNARE complex disassembly is required for independent function of the two SNAREs. Sec17p physically and functionally interacts largely with the t-SNARE. (a) Antibodies to the t-SNARE, but not the v-SNARE, block Sec17p release. (b) Sec17p is associated with the t-SNARE in the absence of v-SNARE, but is not bound to the v-SNARE without t-SNARE. (c) Vacuoles with t-SNARE but no v-SNARE still require Sec17p/Sec18p priming, whereas their fusion partners with v-SNARE but no t-SNARE do not. Sec18p thus acts, upon ATP hydrolysis, to disassemble the v-t-SNARE complex, prime the t-SNARE, and release the Sec17p to allow SNARE participation in docking and fusion. These studies suggest that the analogous ATP-dependent disassembly of the 20-S complex of NSF, alpha-SNAP, and v- and t-SNAREs, which has been studied in detergent extracts, corresponds to the priming of SNAREs for docking rather than to the fusion of docked membranes.

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Figures

Figure 1
Figure 1
The vacuolar v- and t-SNAREs are in a complex that is dissociated by ATP hydrolysis. (A) Vacuolar v–t-SNARE complexes are present on the vacuole. Isolated vacuoles (100 μg) from BJ3505 (v,t) and BJ3505, with deletion in the v-SNARE (t) or the t-SNARE (v) were solubilized in 1 ml of 1.5% Triton X-100, 100 mM NaPi, pH 7.4, 150 mM NaCl, 1 mM PMSF (TX buffer). A portion (5%) of the total detergent extract was TCA precipitated before the immunoprecipitation analysis. Coprecipitation with immobilized antibodies to Vam3p was performed overnight at 4°C. In lane 7, detergent extracts of both deletion vacuoles were mixed before addition of the protein A–Sepharose immobilized α-Vam3p. Proteins were removed from beads by suspension in 200 μl of 100 mM glycine/Cl, pH 2.2, and precipitated with TCA before SDS-PAGE analysis. Proteins were transferred to nitrocellulose and decorated with antibodies to Vam3p and Nyv1p. (B) Vacuolar v–t-SNARE complexes are dissociated by the action of Sec18p and ATP. Vacuoles were isolated from sec18-1 cells and incubated for 10 min in PS buffer on ice or at 37°C, and then mixed with reaction buffer without (lane 2) or with 1 mM Mg-ATP (lane 3), and then incubated for 10 min at 25°C. After reisolation, vacuoles were solubilized in detergent, coprecipitated with α-Vam3p and processed as in A. (C) v–t-SNARE complexes are abundant in vivo. Cells carrying the sec18-1 mutation were converted to spheroplasts at 25°C (Lewis et al., 1997), and incubated at either 25° or 37°C for 30 min before lysis. After the incubation, spheroplasts were lysed in TX buffer, which either contained 1 mM Mg-ATP (lanes 3 and 4) or did not contain ATP (lanes 5 and 6). Coprecipitation analysis with antibodies to Vam3p was as in A. (D) Nyv1p is only found in a complex with Sec17p in the presence of the t-SNARE. Vacuoles from wild-type cells or from cells with only a vacuolar t-SNARE or v-SNARE were lysed and coprecipitated with an immobilized antibody to Sec17p as described in the Materials and Methods section. 10% of the detergent extract was TCA-precipitated before the coprecipitation analysis. Proteins were dissolved in sample buffer, resolved by SDS-PAGE and transferred to nitrocellulose. The membrane was decorated with antibodies to Sec17p, Vam3p, and Nyv1p. The efficiency of coprecipitation was not altered when Triton X-100 was used instead of digitonin (data not shown).
Figure 2
Figure 2
Functional separation of the v- and t-SNAREs during priming. Two double-scale reactions containing either BJ3505 or DKY6281 were incubated on ice without ATP or at 27°C with ATP. All tubes were then placed on ice and α-Vam3p IgGs were added to one tube containing each vacuole type. Reactions were kept on ice for 10 min, allowing the antibody to bind. Ice-cold PS buffer (350 μl) was then added, vacuoles were reisolated (3 min; 8,000 g) and resuspended in the original volume of reaction mixture containing ATP and cytosol. A 15-μl portion of vacuole sample was combined with the corresponding partner such that neither partner, one partner, or both saw the antibody in the second incubation. Vacuoles were incubated for an additional 90 min at 27°C with cytosol and analyzed for fusion as described in the Materials and Methods section. Cytosol, but not LMA1 and Sec18p (not shown), was essential in the last step to insure a normal fusion signal, suggesting that an additional cytosolic factor might be necessary after these preincubations. The lower overall signal in the primed samples is because of the lability of the vacuolar membrane induced by this treatment (Mayer et al., 1997; Xu et al., 1997).
Figure 3
Figure 3
ATP-dependent dissociation of the Sec17p/ Vam3p complex is enhanced in the absence of the v-SNARE. For each time point, 80 μg of vacuoles (BJ3505 [v,t] or BJ3505 Δnyv1 [t]) in 400-μl fusion reaction mixtures were incubated for 10 min at 27°C without nucleotide or with 1 mM Mg-ATP. PS buffer (400 μl) was then added, vacuoles were reisolated (10 min; 8,000 g) and suspended in 200 μl of PS buffer, and then reisolated again (5 min; 8,000 g). Vacuoles were solubilized in digitonin buffer and processed for coprecipitation with protein A–Sepharose immobilized Sec17p-IgGs as described in Materials and Methods. Proteins were dissociated from the beads by addition of sample buffer, resolved on SDS-PAGE, transferred to nitrocellulose, and then decorated with antibodies to Sec17p and Vam3p. Vam3p bands of the enhanced chemiluminescence–developed film were quantitated by laser densitometry. The stability of the Sec17p/Vam3p complex was unaltered when Triton X-100 was used instead of digitonin (not shown).
Figure 4
Figure 4
Sensitivity of the stages of the vacuole fusion reaction to antibody to SNAREs. (A) Resistance to anti-Nyv1p and anti-Vam3p is coincident during the fusion reaction. A 30× scale fusion reaction was started in the presence of ATP at 27°C. Aliquots (30 μl) were removed at indicated times, added to antibodies to Sec17p, Vam3p, or Nyv1p, and then incubated at 27°C or placed on ice. Fusion reactions were incubated for a total of 90 min at 27°C before being assayed for alkaline phosphatase activity. (B) Sec18p action must precede that of the t-SNARE in the fusion assay. Two sixfold reactions containing vacuoles from BJ3505 and DKY6801 were started in the presence of cytosol, ATP, and 1.5 mM GTPγS. Half received α-Sec18p. A 30-μl portion of each was removed and placed on ice. The rest was incubated for 30 min at 27°C, and then vacuoles were reisolated, resuspended in the previous volume of reaction buffer containing ATP and cytosol, and divided into 30-μl reactions. Where indicated, IgGs to Vam3p or Ypt7p were added. One reaction was left on ice, the others were returned to 27°C. After 5 min, 400 ng of pure Sec18p was added to each and incubations were continued for 90 min at 27°C and analyzed for fusion as described above. The background activity (0.25 U) was subtracted in all lanes.
Figure 5
Figure 5
Sec17p release depends on Sec18p and ATP, and is blocked by t-SNARE antibodies. For each condition, a fourfold scale reaction was incubated for 10 min at 27°C. Where indicated, vacuoles were preincubated with IgGs for 3 min on ice at the following concentrations: α-Sec18p (100 μg/ml), α-Vam3p, α-Nyv1p, or α-Ypt7p (each 300 μg/ml). After the incubation, vacuoles were reisolated at 8,000 g and assayed for bound Sec17p as described by Mayer et al. (1996).
Figure 6
Figure 6
Vacuoles with the t-SNARE, but not those with the v-SNARE, require Sec17p/Sec18p. A double-scale reaction containing cytosol was started with vacuoles from BJ3505 Δvam3 (v) and DKY6281 Δnyv1 (t) in separate tubes. Reactions were either kept on ice in the presence of antibodies to Sec18p or were incubated for 5 min at 27°C in the presence of ATP. Then 15 μl of each reaction was combined with 15 μl of the other partner in the presence of α-Sec18p such that either both, one, or none had been primed. Samples were then incubated for an additional 90 min at 27°C, and analyzed for fusion as before. The background activity (0.2 U) was subtracted in all lanes. Similar results were obtained with DKY6281 Δvam3 and BJ3505 Δnyv1 (data not shown). (B) Binding of Sec17p and Sec18p to the vacuole is independent of the Nyv1p and Vam3p. Vacuoles from BJ3505 (v,t), and the parental strain with deletion in the v-SNARE (t), the t-SNARE (v), or both SNAREs (−) were isolated as described in Materials and Methods. Isolated vacuoles (20 μg) were suspended in SDS–sample buffer, separated by SDS-PAGE, transferred to nitrocellulose, and then decorated with the indicated antibodies.
Figure 7
Figure 7
Working model for homotypic vacuolar fusion. The box on Sec18p indicates a hypothetical Sec18p receptor. Abbreviations are as follows: Sec17p (17), Sec18p (18), Nvy1p (v), Vam3p (t), pro-alkaline phosphatase (Pro-ALP), and alkaline phosphatase (ALP). A star on the t-SNARE indicates the activated state of the protein after priming. See text for details.
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