The Vtc proteins in vacuole fusion: coupling NSF activity to V(0) trans-complex formation

Abstract

The fusion of cellular membranes comprises several steps; membrane attachment requires priming of SNAREs and tethering factors by Sec18p/NSF (N-ethylmaleimide sensitive factor) and LMA1. This leads to trans-SNARE pairing, i.e. formation of SNARE complexes between apposed membranes. The yeast vacuole system has revealed two subsequent molecular events: trans-complex formation of V-ATPase proteolipid sectors (V(0)) and release of LMA1 from the membrane. We have now identified a hetero-oligomeric membrane integral complex of vacuolar transporter chaperone (Vtc) proteins integrating these events. The Vtc complex associates with the R-SNARE Nyv1p and with V(0). Subunits Vtc1p and Vtc4p control the initial steps of fusion. They are required for Sec18p/NSF activity in SNARE priming, membrane binding of LMA1 and V(0) trans-complex formation. In contrast, subunit Vtc3p is required for the latest step, LMA1 release, but dispensible for all preceding steps, including V(0) trans-complex formation. This suggests that Vtc3p might act close to or at fusion pore opening. We propose that Vtc proteins may couple ATP-dependent NSF activity to a subset of V(0) sectors in order to activate them for V(0) trans-complex formation and/or control fusion pore opening.

Fig. 1. Co-immunoprecipitation with Vtc1p. (A) Vacuoles from wild type (OMY1) or a vtc1Δ strain (OMY2) were solubilized in CHAPS and incubated with immobilized affinity-purified antibodies to Vtc1p. Proteins eluted from the column were resolved by SDS–PAGE and stained with colloidal Coomassie Blue. The bands indicated were excised and identified by MALDI mass spectrometry. (B) An aliquot of the samples in (A) was used for western blotting with antibodies to Vtc1p and Nyv1p. (C) Vacuoles from strain SBY119 (chromosomally expressing Vph1p-His₆-HA₃) or from BJ3505 (non-tagged) were solubilized in Triton X-100 and incubated with monoclonal antibodies to HA and protein G–Sepharose. Absorbed proteins were analyzed by SDS–PAGE and western blotting with the antibodies indicated.

Fig. 2. The Vtc proteins are required for vacuole fusion. (A) Vacuoles from the wild type (DKY 6281) or vtc1Δ (OMY5) or vtc3Δ (OMY6) were labeled with the fluorescent dye FM4-64 at 30°C (Peters and Mayer, 1998) and visualized by fluorescence microscopy. Nomarski and fluorescence images were overlaid. (B) Standard fusion reactions without cytosol were performed with vacuoles from the wild type (OMY1/DKY6281 or SBY86/SBY85, respectively) or the indicated deletion mutants. Fusion of the wild-type vacuoles ranged from 3.5 to 6.2 U and was set to 100%. Three independent experiments were averaged. (C) Antibody inhibition. Standard fusion reactions without cytosol were incubated (10 min, 0°C) with the indicated amounts of affinity-purified antibodies to Vtc4p. All samples had been supplemented with control antibodies (protein G-purified from a non-immune serum) to the same final IgG concentration in every sample. After 70 min incubation at 27°C, fusion was assayed (n = 3). The fusion signal of the samples without specific antibodies (2.3–2.7 U) was set to 100%.

Fig. 3. LMA1 binding to vacuoles of different deletion strains. Vacuoles (30 µg) from the indicated strains were TCA precipitated and subjected to SDS–PAGE and western blotting with different antibodies. All strains were deleted for PEP4.

Fig. 4. Priming depends on Vtc1p and Vtc4p. Vacuoles were prepared from different vtc deletion mutants [strains OMY2 (B), OMY3 (C), OMY4 (D) and SBY83 (F)] and from their corresponding wild types [OMY1 (A) and SBY86 (E), respectively]. (A) Sec17p/α-SNAP release. Standard fusion reactions (3-fold volume) without cytosol were incubated (70 min) without ATP on ice (–fusion) or with ATP at 27°C (+fusion). Reactions were chilled on ice and centrifuged (10 000 g, 10 min, 4°C). The supernatants (S) were recovered and the pellets (P) resuspended in 90 µl of PS buffer. All samples were TCA-precipitated and processed for SDS–PAGE and western blotting with the antibodies indicated. Alkaline phosphatase (ALP) serves as a membrane integral vacuolar marker. (B) Effect of anti-Vtc4p on Sec17p release. Vacuoles (BJ3505) were used in standard fusion reactions without cytosol supplemented with affinity-purified antibodies to Vtc4p or with control antibodies (protein G-purified from a non-immune serum). After 10 min on ice, the ATP-regenerating system was added and the samples incubated for 15 min on ice (–fusion) or at 27°C (+fusion). The vacuoles were diluted with 0.5 ml of PS, 150 mM KCl, 1× PIC, 0.5 mM PMSF, re-isolated (10 000 g, 10 min, 4°C), resuspended in 0.2 ml of the same buffer, TCA-precipitated and analyzed as in (A). Antibody concentrations were 45 µM. (C) SNARE complex disruption. Standard fusion reactions (15-fold volume) without cytosol were incubated without the ATP-regenerating system on ice or with the ATP-regenerating system at 27°C for 10 min. SNARE complexes were recovered by immunoprecipitation and analyzed by SDS–PAGE and western blotting.

Fig. 5. Rescue of vtc1Δ vacuoles by exogenous Sec18p. (A) Fusion activity. Standard fusion reactions without cytosol were performed with vacuoles from vtc1Δ (OMY2, OMY5) or corresponding wild-type cells (OMY1, DKY 6281). The samples had been supplemented with 15 µg/ml Sec18p and the indicated amounts of LMA1. One hundred per cent fusion was 6.40 U. (B) As in (A), but the reactions were supplemented with the indicated amounts of Sec18p. (C and D) Priming. Vacuoles from strain OMY2 (vtc1Δ) were incubated in fusion reactions without cytosol, but supplemented with 15 µg/ml purified Sec18p and 0.5 µg/ml LMA1 or with control buffer only. Vacuoles were analyzed for SNARE complex disruption (C) after 10 min at 27°C as in Figure 4C. Sec17p and HOPS (Vps41p) release (D) were assayed at the end of the fusion reaction as in Figure 4A. (E) V₀ trans-complex formation was assayed as described (Peters et al., 2001). Fusion reactions (1 ml) without cytosol were incubated (27°C, 45 min) in the presence or absence of Sec18p (15 µg/ml) and LMA1 (0.5 µg/ml). They contained a mixture of vacuoles bearing either Vph1p-His₆-HA₃ or Vph1p-AU1 [strains OMY8/OMY9 (wt); OMY10/OMY11 (vtc1Δ)]. Vph1p-His₆- HA₃ was immunoprecipitated. Co-immunoprecipitated Vph1p-AU1 was analyzed by western blotting.

Fig. 6. Time course of inhibition by antibodies to Vtc4p. Standard fusion reactions without cytosol were started. After the indicated times at 27°C, inhibitors or control buffer only were added. The samples were left on ice for 10 min and then transferred to 27 or 0°C for the rest of the 70 min reaction period. Finally, fusion activity was assayed. Inhibitors used were 2 µM anti-Sec18p, 5 µM Gdi1p, 2 µM anti-Nyv1p and 80 µM anti-Vtc4p.

Fig. 7. Vtc3p affects a step between V₀ trans-complex formation and LMA1 release. (A) Effect of Sec18p and LMA1 on fusion of vtc1Δ and vtc3Δ vacuoles. Vacuoles prepared from vtc3Δ (strains OMY3/OMY6) or vtc1Δ (strains OMY2/OMY5) were used in standard fusion reactions with the indicated additions of Sec18p (15 µg/ml) and LMA1 (0.5 µg/ml). After 70 min at 27°C, fusion activity was assayed. Fusion of the mutant vacuoles without Sec18p and LMA1 was set as the 1× value for each strain (compare with Figure 2B; n = 3). (B) V₀ trans-complex formation was assayed as in Figure 5E, but without Sec18p and LMA1, using wild-type (OMY8/OMY9) or vtc3Δ (OMY12/OMY13) vacuoles.