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. 2001 Aug 1;20(15):4035-40.
doi: 10.1093/emboj/20.15.4035.

Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion

M Kato  1 , W Wickner
Affiliations

Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion

M Kato et al. EMBO J. .

Abstract

In vitro homotypic fusion of yeast vacuoles occurs in three stages: priming, the Sec18 (NSF)-mediated changes that precede vacuole association; docking, the Ypt7 and SNARE-mediated pairing of vacuoles; and fusion, mediated by calmodulin/V0/t-SNARE interactions. Defects in catalysts of each stage result in fragmented (unfused) vacuoles. Strains with deletions in any of ERG genes 3-6, lacking normal ergosterol biosynthesis, have fragmented vacuoles. The ergosterol ligands filipin, nystatin and amphotericin B block the in vitro fusion of vacuoles from wild-type cells. Each of these inhibitors acts at the priming stage to inhibit Sec17p release from vacuoles. A reversible delay in Sec18p action prevents vacuoles from acquiring resistance to any of these three drugs, confirming that their action is on the normal fusion pathway. Ergosterol or cholesterol delivery to wild-type vacuoles stimulates their in vitro fusion, and the in vitro fusion of ergDelta vacuoles requires added sterol. The need for ergosterol for vacuole priming underscores the role of lipids in organizing the membrane elements of this complex reaction.

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Figures

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Fig. 1. Vacuole appearance in wild-type and ergΔ strains. Wild-type strain BY4742 (A) and isogenic erg3–6Δ strains (B–E) were grown in 0.2 ml of YPD with 200 µg/ml G418 in 15 ml culture tubes at 30°C. After 16 h, 0.4 ml of YPD with 3 µM FM4-64 (Vida and Emr, 1995) were added, incubation was continued for 4 h at 30°C, and cells were photographed as reported (L.Wang, W.Wickner and A.Merz, submitted).
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Fig. 2. (A) Fusion activity of vacuoles from ergΔ cells. Standard fusion reactions were performed with vacuoles from ERG5/pep4Δ, ERG5/Pho8Δ, erg5Δ/pep4Δ and erg5Δ/pho8Δ strains. (B) Vacuoles (3.75 µg) from each indicated strain were analyzed by immunoblotting.
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Fig. 3. Inhibition of the fusion of wild-type vacuoles by nystatin, filipin and amphotericin B. Standard reactions of vacuoles from BJ3505 and DKY6281 strains contained the indicated concentrations of nystatin, filipin or amphotericin B.
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Fig. 4. Cholesterol promotes the fusion of vacuoles from erg5Δ strains. Vacuoles from erg5Δ/pep4Δ and erg5Δ/pho8Δ strains [5 µg each, 0.3 mg/ml in buffer A (20 mM PIPES–KOH pH 6.8, 200 mM sorbitol, 125 mM KCl, 5 mM MgCl2)] were mixed, centrifuged (10 000 g, 4°C, 2 min), and resuspended in 30 µl of buffer A on ice in the presence or absence of methyl-β-cyclodextrin with 4 µg of cholesterol. After incubation on ice for 60 min, the samples were again centrifuged (10 000 g, 4°C, 2 min), resuspended in fusion reaction buffer with calmodulin (183.3 µg/ml), LMA1 (11.5 µg/ml) and Sec18p (26.6 µg/ml), incubated at 27°C for 90 min, and fusion activity was measured.
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Fig. 5. Ergosterol or cholesterol can promote the fusion of wild-type vacuoles. Mixed vacuoles from BJ3505 and DKY6281 strains (5 µg each, 0.3 mg/ml in fusion reaction buffer) were centrifuged (10 000 g, 4°C, 2 min), and resuspended in fusion reaction buffer (33 µl) with anti-Sec18 antibody (33.3 µg/ml) in the presence or absence of 0.16–16 µg of liposomes with 0.04–4 µg of ergosterol, respectively, or of methyl-β-cyclodextrin (MβCD; 0.4–4 mM) with 0.4–4 µg of cholesterol, respectively. After incubation at 27°C for 30 min, the samples were centrifuged (10 000 g, 4°C, 2 min), pellets were resuspended in fusion reaction buffer with calmodulin (183.3 µg/ml), LMA1 (11.5 µg/ml) and Sec18p (26.6 µg/ml), incubated at 27°C for 90 min in the presence or absence of anti-Sec17 antibody (66.7 µg/ml, lanes 8 and 15), anti-Vam3 antibody (66.7 µg/ml, lanes 9 and 16) or GTPγS (6.7 mM, lanes 10 and 17), and fusion activity was measured.
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Fig. 6. Nystatin, filipin and amphotericin B resistance is acquired with the same kinetics as resistance to anti-Sec18p antibody. Reactions (1350 µl) contained vacuoles from BJ3505 and DKY6281 strains in fusion reaction buffer. Aliquots (30 µl) were removed and placed on ice or added to inhibitors [anti-Sec17 antibody (66.7 µg/ml), anti-Vam3 antibody (66.7 µg/ml), nystatin (50 µg/ml), filipin (50 µg/ml) and amphotericin B (50 µg/ml)] at the indicated times. After 90 min, fusion activity was measured.
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Fig. 7. Resistance to nystatin, filipin or amphotericin B is not acquired until Sec18p has acted. Standard fusion reactions were started with (A) or without (B) 33.3 µg/ml anti-Sec18p antibody. After incubation for 30 min at 27°C, the samples received nystatin (250 µg/ml), filipin (50 µg/ml), amphotericin B (25 µg/ml) or buffer. After incubation for 5 min at 27°C, Sec18p (13.3 µg/ml) was added to reactivate the Sec18p pathway. A control sample received only buffer. After further incubation for 90 min at 27°C or on ice, fusion activity was measured. A standard reaction, kept on ice throughout the incubations, indicates the background.
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Fig. 8. Nystatin, filipin and amphotericin B inhibit Sec17p release from vacuoles. Samples equivalent to three standard fusion reactions were incubated at 27°C with Sec18p (13.3 µg/ml), apyrase (0.033 U/ml), anti-Sec18 antibody (66.7 µg/ml), nystatin (50 µg/ml), filipin (200 µg/ml) and amphotericin B (25 µg/ml), where indicated. After the incubation times, vacuoles were removed by centrifugation and samples of the supernatant were analyzed by immunoblotting with anti-Sec17p antibody.
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