Excess vacuolar SNAREs drive lysis and Rab bypass fusion

Abstract

Although concentrated soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) drive liposome fusion and lysis, the fusion of intracellular membranes also requires Rab GTPases, Rab effectors, SM proteins, and specific regulatory lipids and is accompanied by little or no lysis. To rationalize these findings, we generated yeast strains that overexpress all four vacuolar SNAREs (4SNARE(++)). Although vacuoles with physiological levels of Rab, Rab effector/SM complex, and SNAREs support rapid fusion without Rab- and SNARE-dependent lysis, vacuoles from 4SNARE(++) strains show extensive lysis and a reduced need for the Rab Ypt7p or regulatory lipids for fusion. SNARE overexpression and the addition of pure homotypic fusion and vacuole protein sorting complex (HOPS), which bears the vacuolar SM protein, enables ypt7Delta vacuoles to fuse, allowing direct comparison of Rab-dependent and Rab-independent fusion. Because 3- to 40-fold more of each of the five components that form the SNARE/HOPS fusion complex are required for vacuoles from ypt7Delta strains to fuse at the same rate as vacuoles from wild-type strains, the apparent forward rate constant of 4SNARE/HOPS complex assembly is enhanced many thousand-fold by Ypt7p. Rabs function in normal membrane fusion by concentrating SNAREs, other proteins (e.g., SM), and key lipids at a fusion site and activating them for fusion without lysis.

Fig. 1.

The soluble vacuolar SNARE Vam7p promotes lysis during vacuole fusion. (A) Assay schematic; see Results for details. Vacuoles from BJ3505 GFP⁺ and DKY6281 were used to assay vacuole fusion and lysis (see Materials and Methods). Fusion (filled bars) and percent GFP release (open bars) were measured after 60 min. (B–F) Reactions bore: no inhibitors (B), reaction pathway inhibitors without additional fusion components (C), inhibitors with rSec18p (D), inhibitors with rVam7p and HOPS (E), and inhibitors with rSec18p and HOPS (F). See Materials and Methods for all reagent concentrations. Results are the mean of three independent experiments ± SD.

Fig. 2.

Vam7p promotes lysis through SNARE pairing. (A) The full-length Vam7p is needed to promote lysis. Full-length Vam7p, its Phox homology domain (PX), or its SNARE domain (SD) was added to standard fusion and lysis assays at the indicated concentrations. Fusion (filled bars) and lysis (open bars) were measured after 60 min. (B) A 3Q:1R SNARE 0-layer is as necessary for Vam7p promotion of lysis as for fusion. Fusion reactions without ATP (33), using vacuoles from BJ3505 GFP⁺ and DKY6281, were examined for fusion (filled symbols) and lysis (open symbols) upon the addition of either wild-type Vam7p (squares) or Vam7p^Q283R (triangles). Results in A and B are the mean of three independent experiments ± SD. (C) The kinetics of content mixing and GFP release in a synchronized vacuole fusion reaction. Fusion reactions containing 3 μg BJ3505 vacuoles bearing GFP and 3 μg DKY6281 vacuoles were incubated with affinity-purified anti-Sec17p (154 nM) for 20 min. After recombinant Vam7p (1 μM) was added, portions of the reactions (90 μl) received anti-Vam3p IgG (444 nM) or were placed on ice at indicated times (−0.5, 0.5, 1, 2, 4, 8, 16, 32, 48, 64, 80, or 96 min) and 30 μl was assayed for alkaline phosphatase activity as a measure of fusion (content mixing) after 96 min. For lysis, 30 μl was centrifuged (5,200 × g, 4°C, 6 min), and GFP fluorescence was measured in supernatants and resuspended pellets.

Fig. 3.

Immunoblot analysis of purified vacuoles. (A) Vacuoles were isolated from indicated yeast strains, and protein compositions were analyzed by immunoblot. Immunoblots for Nyv1p and Sec18p were from one gel, but lanes 1–4 were reordered for clarity. (B) Vacuoles were purified from BJ3505, BJ3505 ypt7Δ, BJ3505 4SNARE⁺⁺, and BJ3505 4SNARE⁺⁺ ypt7Δ, and 3 μg (lanes 1–4), 1 μg (lanes 5 and 6), or 0.33 μg (lanes 7 and 8) was analyzed by immunoblot.

Fig. 4.

Fusion and lysis of vacuoles with high levels of SNARE proteins. Standard fusion and lysis reactions using vacuoles from BJ3505 4SNARE⁺⁺ GFP and DKY6281 4SNARE⁺⁺. Fusion (filled bars) and percent GFP release (open bars) were measured after 60 min: no inhibitors (A), inhibitors with rSec18p (B), inhibitors with rVam7p and HOPS (C), and inhibitors with rSec18p and HOPS (D). The fusion data in B–D were normalized to uninhibited fusion levels as follows: 3.47 ± 1.3 units (B), 1.62 ± 0.71 units (C), or 3.52 ± 0.86 units (D). Anti-Vam3p-inhibited reactions contained 2.7 μM or 5.5 μM IgG (D). Reactions with anti-Sec17p contained 366 nM IgG. Results are the mean of three independent experiments ± SD.

Fig. 5.

Kinetics of fusion and lysis. Standard fusion and lysis reactions (×16 scale) bore vacuoles from BJ3505 GFP and DKY6281 or 4SNARE-overproducing strains BJ3505 4SNARE⁺⁺ GFP, DKY6281 4SNARE⁺⁺. Two 30-μl aliquots were withdrawn before incubation at 27°C for fusion and GFP release analysis (see Materials and Methods). Additional 30-μl aliquots were withdrawn at 10, 20, 40, 60, and 90 min. Aliquots withdrawn for fusion analysis remained at 0°C for the rest of the experiment, while the separation of aliquots into pellets and supernatants was performed immediately after each sample was withdrawn. Reactions contained either: no extra additions (■), rSec18p (▴), rSec18p and α-Vam3p IgG (▾), rSec18p and HOPS (♦), or rSec18p, HOPS, and α-Vam3p IgG (○). α-Vam3p-inhibited reactions only show 0- and 90-min assays for fusion and lysis. (A and B) Fusion (A) and lysis (B) for vacuoles with physiological levels of wild-type SNARE proteins. (C and D) Fusion (C) and lysis (D) for 4SNARE⁺⁺ vacuoles. α-Vam3p IgG was added to 2.7 μM for reactions containing 4SNARE⁺⁺ vacuoles. Results are the mean of three independent experiments ± SD.

Fig. 6.

Ypt7p Rab GTPase bypass fusion via 4SNARE overproduction requires trans-SNARE complexes. Fusion reactions (see Materials and Methods) with vacuoles isolated from BJ3505 NYV1⁺⁺ and DKY6281 NYV1⁺⁺ (dark gray bars), BJ3505 3Q⁺⁺ and DKY6281 3Q⁺⁺ (light gray bars), or BJ3505 3Q⁺⁺ and DKY6281 NYV1⁺⁺ (black bars) were incubated for 90 min on ice or at 27°C in the absence or presence of the indicated reagents. Results are the mean of three independent experiments ± SD.

Fig. 7.

Fusion and lysis of ypt7Δ, 4SNARE⁺⁺ vacuoles. Standard fusion and lysis reactions with vacuoles from BJ3505 4SNARE⁺⁺ GFP ypt7Δ and DKY6281 4SNARE⁺⁺ ypt7Δ. (A) Fusion (filled bars) and lysis (open bars) for uninhibited reactions. (B) Fusion and lysis reactions containing rSec18p and HOPS plus reaction inhibitors. (C) Reaction inhibitors in the presence of rSec18p, HOPS, and rVam7p. α-Vam3p-inhibited reactions contained 2.7 μM IgG. Results are the mean of three independent experiments ± SD.