Purification of active HOPS complex reveals its affinities for phosphoinositides and the SNARE Vam7p

Abstract

Coupling of Rab GTPase activation and SNARE complex assembly during membrane fusion is poorly understood. The homotypic fusion and vacuole protein sorting (HOPS) complex links these two processes: it is an effector for the vacuolar Rab GTPase Ypt7p and is required for vacuolar SNARE complex assembly. We now report that pure, active HOPS complex binds phosphoinositides and the PX domain of the vacuolar SNARE protein Vam7p. These binding interactions support HOPS complex association with the vacuole and explain its enrichment at the same microdomains on docked vacuoles as phosphoinositides, Ypt7p, Vam7p, and the other SNARE proteins. Concentration of the HOPS complex at these microdomains may be a key factor for coupling Rab GTPase activation to SNARE complex assembly.

Figure 1

HOPS complex purification. (A) Lysates from BJ2168 (no tag) and CSY12 (Vps33-SBP) vacuoles were incubated with streptavidin Sepharose. Bound material was eluted in buffer containing Triton X-100 as described in Materials and methods and subjected to SDS–PAGE and silver staining. Lane 1, material purified from BJ2168 vacuoles; lane 2, material purified from CSY12 vacuoles. (B) Lysates from BJ3505 (lanes 1–3) and CSY12 (lanes 4–6) vacuoles were incubated with streptavidin Sepharose. Bound material was eluted in the presence of BSA and subjected to SDS–PAGE and immunoblotting. Lanes 1 and 4, vacuole lysate (representative of 10% of sample); lanes 2 and 5, material not bound to streptavidin Sepharose (representative of 10% of sample); lanes 3 and 6, material eluted from streptavidin Sepharose (representative of 100% of sample).

Figure 2

HOPS complex activity assay. Vps11-1^ts (CSY9 and CSY10) and VPS11 (BY4742 pep4Δ∷kanMX6 and BY4742 pho8Δ∷kanMX6) vacuoles were assayed for fusion in the presence of pure HOPS complex and rVam7p. HOPS buffer was added, so the total volume of pure HOPS and HOPS buffer was 9 μl. Filled arrows indicate the fusion signal observed with the concentrations of pure HOPS complex (2.2 nM) and rVam7p was (3.7 nM) used in the fusion assays in the remainder of this paper. Means and standard deviations for fusion signals observed in three independent experiments are shown. (A) Pure HOPS added to vps11-1^ts vacuoles, with the indicated concentrations of rVam7p. (B) Pure HOPS added to VPS11 vacuoles, with the indicated concentrations of rVam7p. (C) rVam7p added to vps11-1^ts vacuoles, with or without pure HOPS. (D) rVam7p added to VPS11 vacuoles, with or without pure HOPS.

Figure 3

Authentic fusion of vps11-1^ts vacuoles. Vps11-1^ts vacuoles supplemented with pure HOPS complex were assayed for fusion in the absence (black bars) or presence (gray bars) of rVam7p and with the indicated inhibitors. All reactions received HOPS except for the reactions labeled ‘no added HOPS', which received an equal volume of HOPS buffer.

Figure 4

Staging of HOPS complex function. Reactions with vps11-1^ts vacuoles and 3.7 nM rVam7p were divided into three sets. One set received inhibitors and pure HOPS at the beginning of the assay and HOPS buffer at 35 min. A second set received pure HOPS at the beginning of the assay, inhibitors at 30 min, and HOPS buffer at 35 min. The third set received HOPS buffer at the beginning of the assay, inhibitors at 30 min, and pure HOPS at 35 min. All reactions were assayed for fusion at 90 min. Means and standard deviations of the fusion in three independent experiments, expressed as a percentage of the fusion signal of each ‘complete' reaction (no inhibitor) before averaging, are shown.

Figure 5

The HOPS complex is required for vacuole tethering. Vps11-1^ts vacuoles (CSY10) were incubated in the presence or absence of pure HOPS and Gyp1-46 and GDI where indicated; no rVam7p was added. Samples were randomized and mounted on slides for fluorescence microscopy. Random fields were imaged. For each sample, the number of vacuoles in ∼100 vacuole clusters was counted. Data from three independent experiments were pooled for analysis. Bar, 5 μm. (A) Representative images of vacuoles from each sample in one experiment. The enlarged vacuoles in samples with HOPS but without Gyp1-46/GDI reflect fusion, and cause an underestimation of the number of vacuoles that had initially tethered in these clusters. (B) Cumulative distribution plot of the pooled data. (C) Median cluster sizes for the four treatments.

Figure 6

The HOPS complex binds phosphoinositides. (A) PIP MicroStrips (Echelon) were blocked with HBSTG (20 mM NaHEPES, pH 7.8, 3 mg/ml defatted BSA, 200 mM NaCl, 0.1% Tween-20, 5% glycerol) and incubated with pure HOPS (0.5 ml of 0.33 nM HOPS in HBSTG) at 4°C. Strips were washed four times with HBSTG and probed with antibodies in HBSTG, then washed four times with HBSTG; secondary detection was carried out with HRP-conjugated donkey anti-rabbit antibodies (GE). Strips were washed four times with HBSTG and bound antibody was detected by enhanced chemiluminescence (GE). (B) HOPS binding to liposomes was assayed by a modification of a described method (Matsuoka et al, 1998). Liposomes with PI were made by mixing CHCl₃ solutions of soy PC, soy PE, soy PI (Avanti), and rhodamine-DHPE (Invitrogen) at molar ratios of 50:25:25:0.025 and drying with N₂ gas and vacuum. Lipids were resuspended in 250 μl RB (20 mM NaHEPES, pH 7.8, 100 mM NaCl, 5% glycerol, 250 mM sorbitol; 8 mM total lipids), and subjected to freeze–thaw cycles (liquid N₂/bath sonication) until the suspension was clear. Liposomes with DOPA, brain PS (Avanti), or phosphoinositides (Echelon; in 1:2:0.8 CHCl₃:MeOH:H₂O) were prepared in the same way, with 20–25% of the PI replaced by the appropriate phospholipid. Liposomes (20 μl) or RB were mixed in 7 × 20 mm tubes (Beckman) with 10 μl pure HOPS (2.6 nM final) and 10 μl of HBSG (20 mM NaHEPES, pH 7.8, 200 mM NaCl, 5% glycerol, 250 mM sorbitol) with 1.8 M sucrose and 2 mg/ml defatted BSA, then incubated on ice for 30 min. This mixture was overlayed with 75 μl of HBSG with 0.5 M sucrose, 75 μl of HBSG with 0.3 M sucrose, and 50 μl of HBSG, then centrifuged (50 000 r.p.m., 1 h, 4°C, TLS-55 rotor). Liposomes (40 μl) were taken from the border between the top layers, and bound HOPS was quantified with a ChemiDoc system (UVP) after SDS–PAGE and immunoblotting for Vps33p, with a standard curve of pure HOPS. Liposome recovery was estimated using rhodamine-DHPE fluorescence (λ_ex/λ_em 510/580 nm); reported bound HOPS was corrected for liposome recovery. Mean percentages and standard deviations for at least three independent experiments are shown. ^*P<0.05 for differences relative to binding to PI liposomes, by one-way ANOVA. (C) HOPS release assay: fusion assays were supplemented as indicated and incubated on ice or at 27°C for 90 min, then sedimented (16 000 g, 15 min, 4°C). Supernatants and pellets were subjected to SDS–PAGE and immunoblotting for Vps33p. Blots were quantified by densitometry. Left, representative immunoblot. Right, mean percentages and standard deviations of Vps33p released in three independent experiments. ^**P<0.01, ^*P<0.05, for differences relative to release in the presence of GST, by one-way ANOVA.

Figure 7

The HOPS complex binds Vam7p and the Vam7p PX domain. (A) HOPS binds recombinant Vam7p. Vacuoles (1.2 mg) from BJ3505 yeast were sedimented (16 000 g, 10 min, 4°C), resuspended in 2 ml SB (20 mM HEPES–KOH, pH 7.4, 100 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, 20% glycerol, 0.46 μg/ml leupeptin, 3.5 μg/ml pepstatin, 2.4 μg/ml pefabloc-SC, 1 mM DTT) and incubated on ice for 20 min. Insoluble material was removed by centrifugation (TLA 120.2, 52 000 r.p.m., 11 min, 4°C). A sample of lysate was removed, and 200 μl of SB (lanes 4, 6, 8, 10, and 12) or vacuole lysate (lanes 5, 7, 9, 11, and 13) was incubated with 5 μg each of pure GST (lanes 4 and 5) or GST fused to the cytoplasmic residues of indicated SNAREs (lanes 6 and 7: Vti1p; lanes 8 and 9: Nyv1p; lanes 10 and 11: Vam3p; lanes 12 and 13: Vam7p). Lanes 1–3 represent 20, 5, and 1% of each lysate added. Fusion proteins were retrieved with glutathione Sepharose and bound material was analyzed by SDS–PAGE and immunoblotting (top) or staining with Coomassie blue (40% of eluted material, bottom). (B) The HOPS complex interacts with the Vam7p PX domain. GST (lane 4) or GST fusions to full-length Vam7p (lanes 5–7), the PX domain (lanes 8–10), or the SNARE domain (lanes 11–13) were added (5 μg for GST alone, and 5, 1, and 0.2 μg for Vam7p, PX, and SNARE domains) to vacuole lysates prepared in parallel to those in (A). Lanes 1–3 represent 20, 5, and 1% of each lysate added. Fusion proteins were retrieved with glutathione Sepharose (GE) and bound material was analyzed by SDS–PAGE. (C) The HOPS complex binds directly to the Vam7p PX domain. Pure HOPS (0.65 nM; lane 1) was incubated with glutathione Sepharose and buffer (lanes 2 and 6) or 5 μg GST (lanes 3 and 7), GST-Vam7 (lanes 4 and 8), or GST-Vam7 PX domain (lanes 5 and 9), in a total volume of 200 μl of SB; bound material was eluted by heating with SDS. Unbound (lanes 2–5) and bound (lanes 6–9) material was analyzed by SDS–PAGE and immunoblotting.