HOPS initiates vacuole docking by tethering membranes before trans-SNARE complex assembly

Abstract

Vacuole homotypic fusion has been reconstituted with all purified components: vacuolar lipids, four soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Sec17p, Sec18p, the Rab Ypt7p, and the hexameric homotypic fusion and vacuole protein sorting complex (HOPS). HOPS is a Rab-effector with direct affinity for SNAREs (presumably via its Sec1-Munc18 homologous subunit Vps33p) and for certain vacuolar lipids. Each of these pure vacuolar proteins was required for optimal proteoliposome clustering, raising the question of which was most directly involved. We now present model subreactions of clustering and fusion that reveal that HOPS is the direct agent of tethering. The Rab and vacuole lipids contribute to tethering by supporting the membrane association of HOPS. HOPS indirectly facilitates trans-SNARE complex formation by tethering membranes, because the synthetic liposome tethering factor polyethylene glycol can also stimulate trans-SNARE complex formation and fusion. SNAREs further stabilize the associations of HOPS-tethered membranes. HOPS then protects newly formed trans-SNARE complexes from disassembly by Sec17p/Sec18p.

Figure 1.

HOPS stimulates trans-SNARE complex assembly between proteoliposomes but does not affect the assembly of SNAREs in detergent. (A) Schematic representation of the SNAREs used in this study. (B) Schematic representation for the topology of the SNAREs used to drive trans-SNARE complex formation in the absence of cis-SNARE complex disassembly. (C) Proteoliposomes bearing Qab-SNAREs (acceptor) and R-SNARE (donor) were incubated with HOPS (90 nM) or HOPS buffer but without Vam7p for 10 min at 27°C. All reactions contained 0.5 mM MgCl₂. Triton X-100 (in RB150) or RB150 (no Triton X-100) was added where indicated, immediately followed by Vam7-3Δp (200 nM) addition. After 15 min, RIPA buffer was added, Vam3p was immunoprecipitated, and Vam3p and Nyv1p were detected by immunoblot (see Materials and Methods). % total refers to the indicated percentage of the starting proteoliposome mixture, to allow evaluation of the efficiency of SNARE coisolation. Data shown are representative of three independent experiments. See Supplemental Figure S2 for mean ± SD of three experiments.

Figure 2.

HOPS directly tethers liposomes of vacuolar lipid composition. (A) Protein-free liposomes (450 μM lipids) of vacuolar lipid composition were incubated as in lipid mixing assays except that all liposomes bore fluorescent NBD-PE and RH-PE (“donors”), the final volume was 10 μl, the reactions were in microcentrifuge tubes, and the incubations were performed in a water bath. All reactions contained 0.5 mM MgCl₂. After 25 min at 27°C, each reaction was diluted sixfold in RB150 + 0.5 mM MgCl₂ and assayed by fluorescence microscopy. Images were thresholded at 50 by using Photoshop (Adobe Systems, Mountain View, CA). (B) Particle sizes for the reactions in A were measured with ImageJ (see Materials and Methods) using 10 fields per reaction. HOPS (90 nM; squares) or HOPS buffer (circles) were added as indicated. (C) POPC/POPS liposomes were incubated as described in A, and particle sizes were measured as described in B. B and C are each representative of three independent experiments. See Supplemental Table S1 for the reproducibility and statistical significance of the clustering reactions.

Figure 3.

Ypt7p enhances HOPS-mediated tethering. (A) Liposomes (450 μM lipids) of vacuolar lipid composition plus Ypt7p (450 nM) were incubated as in lipid mixing assays except that all liposomes bore fluorescent NBD-PE and RH-PE (“donors”), the final volume was 10 μl, the reactions were in microcentrifuge tubes, and the incubations were performed in a water bath. All reactions contained 0.5 mM MgCl₂. HOPS (90 nM) or HOPS buffer were added as indicated. After 25 min at 27°C, each reaction was diluted sixfold in RB150 + 0.5 mM MgCl₂ and assayed by fluorescence microscopy. Images were thresholded at 50 by using Photoshop. (B) Particle sizes for the reactions in A were measured with ImageJ (see Materials and Methods) using 10 fields per reaction. (C) Liposomes of POPC/POPS with Ypt7p (225 nM) were incubated as in A and particle sizes were measured as in B. (D) Liposomes of POPC/POPS with Ypt7p at 6 times the concentration in C were incubated as described in A, and particle sizes were measured as described in B. B–D are each representative of three independent experiments. See Supplemental Table S1 for the reproducibility and statistical significance of the clustering reactions. (E) The association of HOPS (90 nM) with liposomes of the indicated compositions was assessed by liposome floatation as described previously (Hickey et al., 2009). Results are displayed as the mean ± SD of triplicates.

Figure 4.

cis-4-SNARE complexes do not increase HOPS-mediated tethering. (A) Liposomes of vacuolar lipid composition which had no proteins or bearing the four vacuolar SNAREs were incubated as in lipid mixing assays except all liposomes bore fluorescent NBD-PE and RH-PE (“donors”), the final volume was 10 μl, the reactions were in microcentrifuge tubes, and the incubations were performed in a water bath. HOPS (90 nM) or HOPS buffer (−HOPS), Sec17p (600 nM), and Sec18p (600 nM) were added as indicated. All reactions contained 1.5 mM MgCl₂ and 1 mM ATP. After 25 min at 27°C, each reaction was diluted sixfold in RB150 + 0.5 mM MgCl₂ and assayed by fluorescence microscopy. Particle sizes were measured with ImageJ (see Materials and Methods) using 10 fields per reaction. (B) Liposomes of vacuolar lipid composition bearing the four vacuolar SNAREs but with Vam7-3Δp were incubated as described in A. Images were taken and particles sizes were measured using 10 fields per reaction. A and B are each representative of three independent experiments. See Supplemental Table S1.

Figure 5.

Synergy between HOPS-dependent tethering and SNARE-complex completion of docking. (A) Clustering of Qab-SNAREs (acceptor) and R-SNARE (donor) proteoliposomes by HOPS in absence or presence of Vam7p or Vam7-3Δp. After incubation as in lipid mixing assays (see B), 4 μl of each reaction was used to capture images. Particle sizes were measured with ImageJ (see Materials and Methods) using 10 fields per reactions. Data are representative of three independent experiments (see Supplemental Table S1). (B) Lipid mixing between Qab-SNAREs (acceptor) and R-SNARE (donor) proteoliposomes requires HOPS and Vam7p. HOPS (40 nM) was present as indicated. Vam7p (200 nM) or Vam7-3Δp (200 nM) was added as indicated at time 0, which followed a preincubation of the plate at 27°C for 10 min. All reactions contained 0.5 mM MgCl₂. Results are displayed as the mean ± SD of three independent experiments.

Figure 6.

PEG-mediated tethering supports SNARE complex assembly and fusion. (A) PEG alone clusters liposomes. PC/PS liposomes were incubated with or without 5% PEG in 10 μl for 25 min at 27°C. Both reactions contained 0.5 mM MgCl₂. The reaction without PEG was diluted sixfold in RB150 + 0.5 mM MgCl₂, and the reaction with PEG was diluted sixfold in RB150 + 0.5 mM MgCl₂ + 5% PEG. Both reactions were assayed by fluorescence microscopy. Particle sizes were measured with ImageJ (see Materials and Methods) using 10 fields per reaction. Results are representative of three independent experiments. (B) PEG-mediated clustering. After lipid mixing (see D), 4 μl of each reaction was used to capture images. Particle sizes were measured with ImageJ (see Materials and Methods) using seven fields per reactions. (C) SNARE complex assembly. Reactions and immunoprecipitations were performed as described in Figure 1C, except that 200 nM Vam7p (*; lane 4) or 200 nM Vam7-3Δp (all others) was added at time 0, and PEG (6% final) was added just before Vam7p addition. Input samples and lanes 1 and 2 are from Figure 1C. Lanes 3 and 4 were used to generate lanes 7 and 8 in Supplemental Figure S2. See Supplemental Figure S2 for mean ± SD of three experiments. (D) Lipid mixing between Qab-SNAREs (acceptor) and R-SNARE (donor) proteoliposomes supported by PEG. All reactions contained 0.5 mM MgCl₂. PEG was present as indicated. Vam7p (300 nM) was added to all wells at time 0, which followed a pre-incubation of the plate at 27°C for 10 min. (E) Lipid mixing between liposomes bearing Qab-SNAREs (acceptor) and R-SNARE (donor). PEG (6%) was added to both reactions just before Vam7p. Vam7p (200 nM) or Vam7-3Δp (200 nM) was added at time 0, which followed a preincubation of the plate at 27°C for 10 min. These lipid mixing data are from the same reactions used for the trans-SNARE assay in C. Results are displayed as the mean ± SD of three independent experiments. (F) Lipid mixing between liposomes bearing Qab-SNAREs (acceptor) and R-SNARE (donor). All reactions contained 0.5 mM MgCl₂. PEG (3% final) was added where indicated 10 min before time 0, and then the plate was incubated at 27°C. HOPS (90 nM) or HOPS buffer were added where indicated 5 min before time 0, and the plate was returned to 27°C. Vam7p (300 nM) was added to all wells at time 0. Results are representative of three independent experiments.

Figure 7.

The synergy between HOPS and Sec17/Sec18 is not solely due to HOPS tethering activity. Lipid mixing between liposomes bearing Qab-SNAREs (acceptor) and R-SNARE (donor). All reactions contained 1.5 mM MgCl₂ and 1 mM ATP. HOPS (40 nM) followed by PEG (3% final) were added as indicated followed by incubation of the plate at 27°C for 5 min. Sec17p (1.2 μM) and Sec18p (1.4 μM) were added as indicated followed by further incubation of the plate at 27°C for 5 min. Vam7p (300 nM) was added to all reactions at time 0. Results are the mean ± SD of three assays.