The yeast vacuolar ABC transporter Ybt1p regulates membrane fusion through Ca²⁺ transport modulation

Abstract

Ybt1p is a class C ABC transporter (ATP-binding cassette transporter) that is localized to the vacuole of Saccharomyces cerevisiae. Although Ybt1p was originally identified as a bile acid transporter, it has also been found to function in other capacities, including the translocation of phosphatidylcholine to the vacuole lumen, and the regulation of Ca²⁺ homoeostasis. In the present study we found that deletion of YBT1 enhanced in vitro homotypic vacuole fusion by up to 50 % relative to wild-type vacuoles. The increased vacuole fusion was not due to aberrant protein sorting of SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) or recruitment of factors from the cytosol such as Ypt7p and the HOPS (homotypic fusion and vacuole protein sorting) tethering complex. In addition, ybt1Δ vacuoles displayed no observable differences in the formation of SNARE complexes, interactions between SNAREs and HOPS, or formation of vertex microdomains. However, the absence of Ybt1p caused significant changes in Ca²⁺ transport during fusion. One difference was the prolonged Ca²⁺ influx exhibited by ybt1Δ vacuoles at the start of the fusion reaction. We also observed a striking delay in SNARE-dependent Ca²⁺ efflux. As vacuole fusion can be inhibited by high Ca²⁺ concentrations, we suggest that the delayed efflux in ybt1Δ vacuoles leads to the enhanced SNARE function.

INTRODUCTION

Maintaining eukaryotic cellular homoeostasis requires the trafficking of membrane-bound cargo through the endocytic and secretory pathways using mechanisms that are conserved in all eukaryotes [1]. The delivery of cargo to its destination is finalized by the fusion of two membranes that is driven through a series of regulated stages. In the present study we used vacuoles (lysosomes) from Saccharomyces cerevisiae to examine the regulation of membrane fusion. Yeast vacuole fusion requires numerous proteins, including the Q-SNAREs {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein] receptors} Vam3p, Vti1p, Vam7p and the R-SNARE Nyv1p. In addition, vacuole fusion requires the Rab GTPase Ypt7p and its effector complex HOPS (homotypic fusion and vacuole protein sorting), actin, as well as the lipids ergosterol, diacylglycerol, phosphatidic acid and phosphoinositides [2].

The final catalysts of fusion are SNARE proteins, however, at the start of the cascade, SNAREs are held in inactive cis complexes on a single membrane. The vacuole fusion pathway is initiated when cis-SNARE complexes are dissociated by the NSF homologue Sec18p and its co-chaperone α-SNAP/Sec17p in an ATP-dependent mechanism [3]. Membranes with activated SNAREs then tether and dock with partner membranes in a mechanism driven by Ypt7p and its effector complex HOPS [4,5]. During the docking stage, SNARE complexes form in trans between partner membranes where one membrane donates Nyv1p and the partner membrane donates Vam3p, Vti1p and Vam7p as a preformed 3Q-SNARE complex. In conjunction with these events, vacuoles are drawn together and become tightly apposed, causing the deformation of vesicles and the formation of a specialized membrane-raft microdomain called the vertex ring where the proteins and lipids that catalyse fusion laterally accumulate to create the site of fusion [6–8].

Although the core fusion machinery has been identified, there is a growing collection of regulatory factors that modulate fusion by various means. These regulators include the protein kinase Yck3p [9] and the lipid modifiers Pah1p [10] and Vps34p [11], as well as the Na⁺/H⁺ exchanger Nhx1p [12]. Additional regulation may occur through controlling Ca²⁺ transport or the translocation of lipids across the membrane bilayer. The latter function is executed by polytopic membrane proteins that are referred to as lipid ‘flippases’ and ‘floppases’ that establish lipid asymmetry between the inner and outer leaflets of a membrane [13]. Some of the putative flip/floppases belong to the ABC transporter (ATP-binding cassette transporter) superfamily [14].

The ABC transporter superfamily is present in all organisms and is responsible for actively transporting a wide range of substrates across lipid bilayers. Loss-of-function mutations in ABC transporters result in a number of inherited human diseases, including the lung and digestive disorder cystic fibrosis, the cholesterol transport disorder Tangier’s disease and the elastic tissue disorder pseudoxanthoma elasticum [15]. ABC transporters are formed from two homologous halves that contain an NBD (nucleotide-binding domain) and an MSD (membrane-spanning domain) consisting of six transmembrane spans. Human ABC transporters are divided into seven sub-families (ABCA–ABCG). Many of the best characterized ABC transporters belong to the Class C subfamily (ABCC). With the exception of CFTR (cystic fibrosis transconductance regulator), ABCC family members, also known as MRPs (multidrug resistance-associated proteins), contain an N-terminal extension composed of five transmembrane motifs and a short cytosolic loop. The yeast vacuole harbours five ABCC transporters: the yeast cadmium factor, Ycf1p [16,17]; two bile acid and pigment transporters, Bpt1p and Ybt1p [18,19]; and two less well-characterized transporters, Vmr1p and Nft1p [15]. Ybt1p was first described as an ATP-dependent bile acid transporter [19] and is similar in structure to the human MRP transporter. Ybt1p is highly expressed on the yeast vacuole [20] and also plays a part in ade2 pigment transport [21]. Recently a novel function was described for Ybt1p showing that it translocates phosphatidylcholine from the outer leaflet of the vacuole to the inner leaflet for degradation and choline recycling [14].

MATERIALS AND METHODS

Reagents

Reagents were dissolved in PS buffer (20 mM Pipes/KOH, pH 6.8, and 200 mM sorbitol). Antibodies against Vam3p [22], Sec17p [23], Nyv1p [24], Sec18p [25] and Ypt7p [4] were described previously. The recombinant proteins His₆–Gyp1-56 [8], GDI (guanine-nucleotide-dissociation inhibitor) [26], GST (glutathione transferase)–FYVE [27], MED [MARCKS (myristoylated alanine-rich C-kinase substrate) effector domain] [6], His₆–MTM1 (myotubularin 1) [28] and GST–Vam7p [29,30] were prepared as described previously and stored in PS buffer with 125 mM KCl.

Strains

BJ3505 [MATα pep4::HIS3 prb1-Δ1.6R his3-200 lys2-801 trp1Δ101 (gal3) ura3-52 gal2 can1] and DKY6281 (MATα leu2-3 leu 2-112 ura3-52 his3-Δ200 trp1-Δ901 lys2-801) were used for fusion assays [31]. BJ3505 CBP (calmodulin-binding peptide)–Vam3 nyv1Δ was used for trans-SNARE complex isolation [32] (Table 1). YBT1 was deleted from BJ3505, DKY6281 and BJ3505 CBP–Vam3 nyv1Δ by homologous recombination. YBT1 was deleted from BJ3505 and DKY6281 with the kanMX6 cassette using PCR products amplified from pFA6a-kanMx6 [33] with homology flanking the YBT1 coding sequence with the forward primer 5′-GTGTGCGCATCTGCAAAGAACGTACGTTGTGACTAATGAACGGATCCCCGGGTTAATTAA-3′ and the reverse primer 5′-TCAGTAAAAGTTCATTGGATCAGATTTCCTTCAAAGACGCGAATTCGAGCTCGTTTAAAC-3′. The PCR product was transformed into BJ3505 and DKY6281 by standard lithium acetate methods and plated on YPD medium [1% (w/v) yeast extract, 2 % (w/v) peptone and 2 % (w/v) glucose] containing G418 (250 μg/μl) to generate BJ3505 ybt1Δ::kanMX6 (RFY28) and DKY6281 ybt1Δ::kanMX6 (RFY29). YBT1 was deleted from BJ3505 CBP–Vam3 nyv1Δ with the hghMX4 cassette using PCR product amplified from pAG32 [34] with homology flanking the YBT1 coding sequence with the forward primer 5′-GTGTGCGCATCTGCAAAGAACGTACGTTGTGACTAATGAAATAGGCCACTAGTGGATCTG-3′ and reverse primer 5′-TCAGTAAAAGTTCATTGGATCAGATTTCCTTCAAAGACGCTCAGCTGAAGCTTCGTACGC-3′. The PCR product was transformed into BJ3505 CBP–Vam3 nyv1Δ by standard lithium acetate methods and plated on to YPD medium containing hygromycin (250 μg/ml) to generate BJ3505 CBP–Vam3 nyv1Δ ybt1::hghMX4 (RFY30). For vacuole localization studies YBT1 was fused in-frame to GFP (green fluorescent protein) by homologous recombination. DKY6281 was transformed with a PCR product amplified from pFA6a-GFP-kanMX6 [33] with the forward primer 5′-TAAAAAAGCCTTTGTGGAAAAATTGAACTCTAAAAAGGACCGGATCCCCGGGTTAATTAA-3′ and the reverse primer 5′-ATATATATATATATATATATATACTTTAGCATCGAAACAGGAATTCGAGCTCGTTTAAAC-3′ with homology flanking the stop codon of the YBT1 gene to make RFY31 (DKY6281 YBT1::GFP).

Table 1.

Yeast strains used in the present study

Strain	Genotype	Source
BJ3505	MAT α pep4::HIS3 prb1-Δ1.6R his3-200 lys2-801 trp1Δ101 (gal3) ura3-52 gal2 can1	[48]
DKY6281	MAT α pho8::TRP1 leu2-3 leu 2-112 ura3-52 his3-Δ200 trp1-Δ901 lys2-801	[49]
BJ3505 CBP–Vam3 nyv1Δ	BJ3505, CBP-VAM3::Kanr nyv1Δ::natr	[32]
RFY28	BJ3505, ybt1Δ::kanMX6	The present study
RFY29	DKY6281, ybt1Δ::kanMX6	The present study
RFY30	BJ3505 CBP-VAM3 nyv1Δ ybt1Δ::hghMX4	The present study
RFY31	DKY6281, YBT1::GFP	The present study

Vacuole isolation and in vitro vacuole fusion

Vacuoles were isolated by floatation as described previously [31]. Standard in vitro fusion reactions (30 μl) contained 3 μg each of vacuoles from BJ3505 and DKY6281 backgrounds, fusion reaction buffer (20 mM Pipes/KOH, pH 6.8, 200 mM sorbitol, 125 mM KCl and 5 mM MgCl₂), ATP-regenerating system (1 mM ATP, 0.1 mg/ml creatine kinase and 29 mM creatine phosphate), 10 μM CoA and 283 nM IB₂ (inhibitor of protease B). Reactions were incubated at 27 °C and Pho8p activity was assayed in 250 mM Tris/HCl, pH 8.5, 0.4 % Triton X-100, 10 mM MgCl₂ and 1 mM p-nitrophenyl phosphate. Fusion units were measured by determining the p-nitrophenolate produced in min⁻¹ · μg⁻¹ pep4Δ vacuole and absorbance was detected at 400 nm.

GST–Vam7p SNARE complex isolation and bypass fusion

SNARE complex isolation was performed as described previously using GST–Vam7p [29,30]. Briefly, large-scale 6× fusion reactions (180 μl) were incubated with 85 μg/ml anti-Sec17p IgG to block priming. After 15 min, 43 μg/ml anti-Vam3p IgG was added to selected reactions and incubated for an additional 5 min before adding 400 nM GST–Vam7p. After a total of 90 min, reactions were placed on ice for 5 min and 30 μl aliquots were removed to measure Pho8p activity. The remaining 150 μl reactions were sedimented (11 000 g, 10 min, 4 °C), and the supernatants were removed before extracting vacuoles with SB (solubilization buffer: 20 mM Hepes/KOH, pH 7.4, 100 mM NaCl, 2 mM EDTA, 20 %glycerol, 0.5 %Triton X-100 and 1 mM dithiothreitol) with protease inhibitors (1 mM PMSF, 10 μM Pefabloc-SC, 5 μM pepstatin A and 1 μM leupeptin). Vacuole pellets were overlaid with 100 μl of SB and resuspended gently. An additional 100 μl of SB was added, gently mixed, and incubated on ice for 20 min. Insoluble debris was sedimented (16 000 g, 10 min, 4 °C) and 176 μl of supernatants were removed and placed in chilled tubes. Next, 16 μl was removed from each reaction as 10 % total samples, mixed with 8 μl of 3× SDS-loading buffer and heated (95 °C, 5 min). Equilibrated glutathione–Sepharose 4B beads (30 μl) were incubated with the remaining extracts (15 h, 4 °C, nutation). Beads were sedimented and washed five times with 1 ml of SB (4000 g, 2 min, 4 °C), and bound material was eluted with 40 μl of 1× SDS loading buffer. Protein complexes were examined by immunoblotting. Secondary antibodies conjugated to alkaline phosphatase were used with ECF reagent (GE Healthcare).

trans-SNARE complex assay

Analysis of trans-SNARE complex formation was conducted as described previously with some modifications [32,35]. Complex formation was compared between reactions containing vacuoles from RFY29 and RFY30 relative to those with BJ3505 CBP–Vam3p nyv1Δ and DKY6281 vacuoles. The trans-SNARE assays were performed using 16× large-scale reactions (480 μl) containing 48 μg of vacuoles each from BJ3505 CBP–Vam3 nyv1Δ and DKY6281 backgrounds and incubated at 27 °C for 60 min. After incubation, reactions were placed on ice for 5 min and 30 μl was withdrawn from each sample to assay Pho8p activity. The remaining 450 μl samples were centrifuged (13 000 g, 15 min, 4 °C) and the supernatants were decanted. Vacuole pellets were overlaid with 200 μl of ice-cold vacuole solubilization buffer (20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 0.5 % Nonidet P40 alternative and 10 % glycerol) with protease inhibitors (0.46 μg/ml leupeptin, 3.5 μg/ml pepstatin, 2.4 μg/ml Pefabloc^®-SC and 1 mM PMSF) and gently resuspended. Vacuole solubilization buffer was added to a final volume of 600 μl and extracts were mixed (20 min, 4 °C, nutation). Detergent-insoluble debris was removed by centrifugation (16 000 g, 20 min, 4 °C). Supernatants were transferred to fresh tubes and 10 % of the extract was removed for input samples. The remaining extracts were brought to 2 mM CaCl₂. CBP–Vam3p complexes were incubated with 50 μl of equilibrated calmodulin–Sepharose 4B (GE Healthcare) (4 °C, 12 h, nutation). Beads were collected by centrifugation (4000 g, 2 min, 4 °C) and suspended four times with the solubilization buffer followed by bead sedimentation. Bound proteins were eluted with SDS sample buffer containing 5 mM EGTA and heated at 95 °C for 5 min. The samples were used for SDS/PAGE and immunoblotting.

Lipid mixing

Lipid mixing assays were conducted using Lissamine rhodamine B (Rh-PE; Invitrogen) as described previously with minor modifications [10]. Briefly, BJ3505 background vacuoles (300 μg) were incubated in 400 μl of PS buffer containing 150 μM Rh-PE (10 min, 4 °C, nutating). Samples were mixed with 15 % (w/v) Ficoll in PS buffer and transferred to a polyallomer tube (11 mm×60 mm). Samples were overlaid with 1.0 ml each of 8 %, 4 % and 0 % Ficoll. Labelled vacuoles were re-isolated by centrifugation using a SW60 Ti rotor (105 200 g, 25 min, 4 °C) and recovered from the 0–4 % Ficoll interface. Lipid mixing assays (90 μl) contained 2 μg of Rh-PE-labelled vacuoles and 16 μg of unlabelled vacuoles in fusion buffer. Reaction mixtures were transferred to a black half-volume 96-well flat-bottom microtitre plate with non-binding surface (Corning). Rhodamine fluorescence (λ_ex = 544 nm; λ_em = 590 nm) was measured using a POLARstar Omega fluorescence plate reader (BMG Labtech) at 27 °C. Measurements were taken every min for 75 min, yielding fluorescence values at the onset (F₀) and during the reaction (F_t). The final ten measurements of a sample after adding 0.33 % Triton X-100 were averaged and used as a value for the fluorescence after infinite dilution (F_TX100). The relative total fluorescence change ΔF_t/F_TX100 = (F_t − F₀)/F_TX100 was calculated.

Microscopy

Vacuole morphology was monitored by incubating yeast cells with YPD medium containing the vital dye FM4-64 (Invitrogen). Cultures were grown overnight to saturation, diluted to ~ 0.2 D₆₀₀ in YPD containing 5 μM FM4-64, grown for 1 h in a 30 °C shaker, washed with PBS, resuspended in YPD, incubated at 30 °C for 3 h, washed in PBS, concentrated by centrifugation, mixed with 0.6 % agarose, and mounted on to glass slides for observation. Images were acquired using a Zeiss Axio Observer Z1 inverted microscope equipped with an X-Cite 120XL light source, Plan Apochromat 63× oil objective (numerical aperture 1.4), and an AxioCam CCD (charge-coupled-device) camera. For vertex assembly detection, docking assays were performed. Docking reactions (30 μl) contained 6 μg of Ybt1p–GFP vacuoles, docking buffer (20 mM Pipes/KOH, pH 6.8, 200 mM sorbitol, 100 mM KCl and 0.5 mM MgCl₂), ATP-regenerating system, 20 μM CoA and 283 nM IB₂. PtdIns3P was labelled with 0.2 μM Cy5 (indodicarbocyanine)–FYVE and used as a marker for vertex microdomains [10]. Reactions were incubated at 27 °C for 30 min and placed on ice and stained with 3 μM FM4-64. Reactions were next mixed with 50 μl of 0.6 % low-melt agarose (in PS buffer), vortex-mixed to disrupt spurious clustering, and mounted on to slides for observation by fluorescence microscopy.

Ca²⁺ efflux assay

SNARE-dependent Ca²⁺ efflux was measured as described previously [36] with some modifications. Fusion reactions (60 μl) contained 20 μg of vacuoles isolated from BJ3505 backgrounds, fusion reaction buffer with 10 μM CoA and 283 nM IB₂. In lieu of the luminescent aequorin Ca²⁺ detection system, we used the low-affinity fluorescent probe Fluo-4-dextran at 200 μM (Invitrogen). Reaction mixtures were transferred to a black half-volume 96-well flat-bottom microtitre plate with non-binding surface (Corning). ATP-regenerating system was added and reactions were incubated at 27 °C while monitoring Fluo-4 fluorescence (λ_ex = 488 nm; λ_em = 520 nm).

Statistical analysis

Significant differences were calculated using Student’s t test and P values less than 0.05 were considered significant.

RESULTS

Ybt1p is localized to the vacuole

Ybt1p has been reported to transport bile acids into the vacuole lumen of Saccharomyces cerevisiae in an ATP-dependent manner [19]. To verify the localization of Ybt1p in yeast, we constructed a strain expressing Ybt1p–GFP. We incubated the strain with the vital dye FM4-64, which accumulates in the vacuole [37], and found that Ybt1p–GFP primarily localized to the vacuole (Figures 1a–1d), which is consistent with previous findings [14]. We next examined the vacuole morphology in ybt1Δ cells and found that the morphology and size of the vacuoles were similar to those in WT (wild-type) cells (Figures 1e–1g). As fusion regulators often accumulate at the vertices of docked vacuoles [6–8], we next examined the distribution of Ybt1p–GFP on isolated vacuoles during docking. Figure 2 shows that Ybt1p–GFP was evenly dispersed throughout the limiting membrane of the vacuole along with FM4-64. To probe for vertex microdomains, docking reactions were incubated with a Cy5-conjugated FYVE domain. The FYVE domain specifically binds to the regulatory lipid PtdIns3P [27], a critical lipid that accumulates at the site of fusion in vertex microdomains [6]. In Figure 2(c), the vertices of docked vacuoles were marked with Cy5–FYVE.

Figure 1. Ybt1–GFP is localized to the vacuole.

WT yeast cells harbouring Ybt1p–GFP were incubated with 5 μM FM4-64 to label vacuoles. Cells were washed with PBS and grown for 1 h in label-free YPD to chase the dye into the vacuole. Cells were washed with PBS and mounted for microscopy. (a and b) GFP images were acquired using a 38 HE GFP shift-free filter set and FM4-64 images were acquired using a 43 HE Cy3 (indocarbocyanine) shift-free filter set. (c) Cells were photographed using differential interference contrast (DIC). (d) Images were merged using Adobe Photoshop. (e and f) ybt1Δ yeast were incubated with FM4-64 as described above and imaged using DIC and Cy3 filters. (g) Quantification and box plot of vacuole size in WT and ybt1Δ cells. Scale bar, 4 μm.

Figure 2. Ybt1–GFP is not enriched at the vertex ring microdomain of docked vacuoles.

Purified vacuoles isolated from cells harbouring Ybt1p–GFP (a) were incubated at 27 °C for 30 min. The limiting membranes were stained with 5 μM FM4-64 (b) and vertex microdomains were labelled with Cy5-conjugated FYVE domain to label PtdIns3P (c). (d) Images were merged using Adobe Photoshop. Scale bar, 4 μm.

Ybt1p is a negative regulator of vacuole fusion

To determine the possible role of this ABCC transporter in the fusion process, we next examined the effect of deleting YBT1 on homotypic vacuole fusion. Fusion was measured by a content mixing assay in which proPho8p (pro-alkaline phosphatase) is cleaved by the protease Pep4p to yield a mature alkaline phosphatase. The level of fusion directly correlates with Pho8p activity. Equal quantities of the strains DKY6281 (PEP4 pho8Δ) and BJ3505 (pep4Δ PHO8) were mixed to assay content mixing. We deleted YBT1 from these strains to generate RFY28 and RFY29 and found that fusion was augmented and showed an approximate 50 % increase compared with WT vacuoles (Figure 3a). This suggests that Ybt1p acts as a negative regulator for vacuole fusion.

Figure 3. Vacuoles from ybt1Δ yeast show enhanced fusion.

Vacuoles were harvested from WT and ybt1Δ yeast and tested for fusion activity. (a) Standard fusion reactions used equal amounts of reporter (PHO8 pep4Δ) and effector (pho8Δ PEP4) vacuoles for WT (●) and ybt1Δ (○). Error bars represent S.E.M. (n = 3). (b) Lipid mixing assays measuring fusion were employed by labelling isolated vacuoles with Rh-PE. Labelled vacuoles were incubated with an 8-fold excess of unlabelled vacuoles and dequenching was measured using a fluorescence plate reader. (c) Analysis of core fusion components. Vacuoles were isolated from WT and ybt1Δ BJ3505 and DK6281 strains. Vacuoles (5 μg by protein) were mixed with 2× SDS loading buffer, separated by SDS/PAGE and transferred on to nitrocellulose. Immunoblotting was performed using the indicated antibodies and bands were detected using ECF. (d) Quantification of Pep4p and Pho8p in WT and ybt1Δ vacuoles. (e) Measurements of vacuole diameters after incubation (27 °C, 90 min). Error bars are S.E.M. (n = 3). *P < 0.0001

As changes in fusion could be due to dysregulated Pho8p reporting, we also used a real-time lipid-dequenching assay that is independent of Pho8p activity [12,38,39]. In the present study, the outer leaflets of vacuole-limiting membranes were labelled with Rh-PE, which is self-quenched at high concentrations. When labelled donor vacuoles were incubated with an 8-fold excess of unlabelled acceptor vacuoles, Rh-PE was dequenched. WT vacuoles showed a characteristic rapid dequenching that was inhibited with anti-Vam3 IgG (Figure 3b, circles). When ybt1Δ vacuoles were examined, we found that the initial dequenching was identical with WT, after which the ybt1Δ vacuoles surpassed WT fusion starting at 15 min (Figure 3b). This illustrates that the initial rates of fusion were the same for WT and ybt1Δ vacuoles, yet the V _max of ybt1Δ fusion was significantly higher. To determine whether the increase in fusion of ybt1Δ vacuoles was due to elevated levels of the fusion machinery (e.g. SNAREs), Western blot analysis was performed. No significant differences were observed in the fusion machinery of mutant vacuoles relative to their WT counterparts (Figure 3c). It should also be noted that the increased fusion was not due to defects in trafficking of Pho8p or Pep4p (Figure 3d). To further verify that the deletion of YBT1 caused an increase in fusion, visual measurements of vacuole diameters were measured. Fusion reactions containing either WT or ybt1Δ vacuoles were carried out as in Figure 3(a). After 90 min of incubation, vacuoles were placed on ice and stained with FM4-64. Aliquots of each reaction were transferred to microscope slides and images were captured for analysis. Figure 3(e) shows a box plot of vacuole diameter ranges for reactions treated with either buffer or anti-Sec17 IgG to inhibit priming, and incubated on ice or at 27 °C. We found that ybt1Δ vacuole diameters (7.13 ± 0.19 μm) were markedly larger than those of WT vacuoles (4.88 ± 0.23 μm).

To determine whether the augmented fusion seen with ybt1Δ vacuoles was regulated by the core fusion machinery, we examined the sensitivity of fusion to a panel of well-characterized fusion inhibitors that targeted SNAREs, Ypt7p and phosphoinositides. SNARE function was inhibited with antibodies against Nyv1p, Vam3p, Sec18p and Sec17p. Ypt7p function was inhibited with anti-Ypt7p IgG, the GTPase-activating protein Gyp1-56p, or the Rab GDI. PtdIns3P function was inhibited by ligation with the FYVE domain or modification with the phosphoinositide 3-phosphatase, MTM1. MED was used to bind multi-phosphorylated phosphoinositides, including PtdIns(4,5)P₂. We found that both WT and ybt1Δ fusion were similarly sensitive to the panel of inhibitors, indicating that the enhanced fusion seen with ybt1Δ vacuoles was not due to an undefined mechanism (Figure 4).

Figure 4. The augmented fusion of ybt1Δ vacuoles is dependent on the standard fusion machinery.

Fusion reactions were performed using WT or ybt1Δ vacuoles. Individual reactions were treated with PS buffer, 14 μg/ml anti-Nyv1p IgG, 27 μg/ml anti-Vam3p IgG, 12 μg/ml anti-Sec18p IgG, 67 μg/ml anti-Sec17p IgG, 8 μg/ml anti-Ypt7p, 0.5 μM Gyp1-56, 0.5 μM GDI, 2 μM GST–FYVE, 10 μM MED or 2 μM His₆–MTM1. Reactions were incubated for 90 min at 27 °C and tested for fusion by content mixing and Pho8p activity. Error bars are S.E.M. (n = 3).

YBT1 deletion does not alter SNARE complex formation

Dysregulation of fusion is often coupled with changes in SNARE complex formation. Thus we tested whether the absence of Ybt1p altered the SNARE/HOPS interactions by two methods. First, we performed a Vam7p bypass assay when SNARE priming was inhibited with anti-Sec17p IgG [29,30]. Fusion was then rescued with the addition of 400 nM GST–Vam7p and incubated for an additional 70 min. Selected reactions were treated with anti-Vam3p antibody prior to adding Vam7p. Fusion was restored when WT and mutant vacuoles were treated with exogenous GST–Vamp7p (Figure 5a). GST–Vam7p complexes were isolated and examined by immunoblotting. GST–Vam7p complexes included the SNAREs Nyv1p and Vam3p, the HOPS subunit Vps33p, the SNARE chaperone Sec17p and actin (Figure 5b). As a control we also probed for the presence of Ypt7p, which does not co-purify with the SNARE/HOPS complexes. SNARE complex formation was blocked by the anti-Vam3p antibody. We found that GST–Vam7p complexes from ybt1Δ vacuoles contained a similar SNARE makeup relative to WT vacuoles.

Figure 5. Vam7p forms complexes with SNAREs and HOPS on ybt1Δ vacuoles.

(a) Large-scale fusion reactions (6×) were incubated with anti-Sec17p IgG to block priming. After 15 min, anti-Vam3 IgG was added to the indicated reactions and incubated for 5 min prior to adding 400 nM GST–Vam7p. Reactions were incubated for an additional 70 min. After incubation, reactions were placed on ice and 30 μl was removed to measure Pho8p activity. (b) The remaining fractions were extracted with SB with protease inhibitors for 20 min while on ice. Insoluble debris was sedimented by centrifugation and supernatants were removed and incubated with equilibrated glutathione–Sepharose. Beads were washed with SB and protein complexes were eluted with 2× SDS/PAGE buffer. Proteins were resolved by SDS/PAGE, transferred on to nitrocellulose and probed with antibodies against Nyv1p, Vam3p, Vam7p, Sec17p, Vps33p, actin and Ypt7p. Error bars are S.E.M. (n = 3).

We next determined whether endogenous SNAREs could form trans complexes when priming is not blocked [32,35]. YBT1 was deleted from the trans-SNARE reporter strain BJ3505 CBP–Vam3p nyv1Δ to create RFY30. Vacuoles isolated from RFY29 (pho8Δ ybt1Δ VAM3 NYV1) and RFY30 were incubated and trans-SNARE complexes were isolated through the CBP integrated into Vam3p. CBP–Vam3p complexes were isolated using calmodulin–Sepharose beads and examined by immunoblotting. As shown previously, ybt1Δ vacuoles show increased fusion relative to WT vacuoles (Figure 6a). Both WT and ybt1Δ fusion reactions were sensitive to anti-Vam3p IgG and to the inactivation of Ypt7p with the combination of Gyp1-56p and GDI. We found that ybt1Δ vacuoles formed trans-SNARE complexes in a similar manner as their WT counterparts indicating that the enhanced fusion was not due to an augmented number of SNARE complexes (Figures 6b and 6c). Similarly, the association of SNAREs with the HOPS subunit Vps33p was not altered on ybt1Δ vacuoles. The lack of a notable difference between WT and mutant vacuoles suggest that fusion was not altered due to the efficiency of complex formation.

Figure 6. trans-SNARE complex formation.

Vacuoles harbouring CBP–Vam3p in a nyv1Δ background were incubated with vacuoles harbouring a full complement of SNAREs. Large-scale reactions (16×) were incubated at 27 °C for 60 min. After incubating, reactions were placed on ice for 5 min and 30 μl was withdrawn from each sample to assay Pho8p activity (a). (b) Membranes were pelleted and then resuspended in SB containing protease inhibitors. CBP–Vam3p complexes were isolated with calmodulin–Sepharose beads and protein complexes were resolved by SDS/PAGE. Complexes were probed for Nyv1p, Vam3p, Vps11p and Vps33p by immunoblot. (c) Quantification of efficiency of Nyv1p binding to CBP–Vam3p. Error bars are S.E.M. (n = 3).

Ybt1p regulates the release of lumenal Ca²⁺ stores

From Figure 3(b) we found that mutant fusion was increased after 15 min, which coincides with the formation of trans-SNARE pairing [32]. The formation of trans-SNARE complexes is linked to the release of lumenal Ca²⁺ stores from the vacuole [40,41]. As Ybt1p interacts with the Ca²⁺ ATPase Pmc1p [42], we next examined the effect of deleting YBT1 on Ca²⁺ transport. Fusion reactions (2×) were prepared containing 20 μg of either BJ3505 (WT) or RFY28 (ybt1Δ) in the presence of standard fusion reaction components and the fluorescent calcium indicator Fluo4-dextran. Reactions were started by the addition of buffer or ATP-regenerating system. Priming was blocked by the addition of anti-Sec17p IgG where indicated and fluorescence was monitored. Upon the addition of ATP, Ca²⁺ was transported into the vacuole, causing a drop in fluorescence. Ca²⁺ is transported into the vacuole via the Ca²⁺ -ATPase Pmc1p and the Vcx1p H⁺/Ca²⁺ exchanger [43]. After approximately 15 min, when trans-SNAREs have formed, we observed an increase in fluorescence indicating that Ca²⁺ was being effluxed from the vacuole lumen (Figure 7a, ●), which is in keeping with previous studies using the aequorin reporter system [40]. When ybt1Δ vacuoles were observed, we found that there were four differences relative to WT reactions. First, there was a prolonged influx of Ca²⁺ early in the reaction, suggesting that Pmc1p and/or Vcx1p activity may be augmented. Secondly, we found that the trans-SNARE complex-dependent efflux was markedly delayed in ybt1Δ vacuoles (Figure 7b). These observations suggest that Ybt1p regulates Ca²⁺ transport in vacuoles and that the enhanced fusion may be linked to alterations in Ca²⁺ homoeostasis. In addition, the relative amount of Ca²⁺ in ybt1Δ vacuoles was nearly twice as much as seen with WT vacuoles. We also found that WT vacuoles reabsorb Ca²⁺ after docking, resulting in a return to basal ‘pre-docking’ levels of extraluminal Ca²⁺, whereas ybt1Δ fails to reabsorb the effluxed Ca²⁺.

Figure 7. Calcium efflux is delayed in ybt1Δ vacuoles.

Vacuoles were harvested from WT BJ3503 and RFY28 (ybt1Δ). Fusion reactions (2×) were prepared containing 20 μg of either WT or ybt1Δ vacuoles and 200 μM Fluo-4-dextran. Immediately after addition of ATP or PS buffer, reactions were incubated at 27 °C and fluorescence was measured for 90 min. (a) A representative time course of Ca²⁺ efflux. (b) The average times at which the start and peaks of efflux occurred in WT and ybt1Δ vacuoles. Error bars are S.E.M. (n = 3).

DISCUSSION

In the present study we found that the ABCC transporter Ybt1p acts as a negative regulator of vacuole fusion. This was evident by the enhanced fusion observed with vacuoles from YBT1-deleted strains. Using a battery of experiments that tested different stages of the fusion pathway, we found that the defect in fusion occurred after the docking stage when trans-SNARE complexes are formed. The enhancement in fusion correlated with a delay in Ca²⁺ efflux, an event that is triggered by the formation of trans-SNARE complexes [40]. This suggested that Ybt1p might regulate the transport of ions across the membrane. Although Ybt1p itself does not transport Ca²⁺, the observed changes might be attributed in part to the interaction of Ybt1p with Pmc1p, a Ca²⁺-ATPase that transports Ca²⁺ ions into the vacuole [42]. Although the SNARE-dependent vacuolar Ca²⁺ efflux channel remains undefined, the connections between Ybt1p, Pmc1p and the delayed efflux during fusion suggests that the import and export of Ca²⁺ is linked to the fusion machinery.

Ybt1p and the SNARE machinery

The direct connection between Ybt1p and the fusion machinery remains unknown; however, the link between Ybt1p and Pmc1p suggests that an indirect mechanism is possible. Others have shown that Pmc1p interacts with free Nyv1p, the required R-SNARE in vacuole homotypic fusion [44]. The interaction between Pmc1p and Nyv1p inhibits the transport of Ca²⁺ into the vacuole lumen. After priming, the Q-SNARE complexes compete for Nyv1p binding to form trans-SNARE complexes, thus de-repressing Pmc1p activity and leading to the uptake of extraluminal Ca²⁺. In the absence of Ybt1p we observed the prolonged uptake of Ca²⁺ upon the addition of ATP, suggesting that Pmc1p activity is augmented and that changes in activity might be due to reduced interactions with Nyv1p.

There are several possible mechanisms by which the efflux of Ca²⁺ might contribute to the regulation of fusion. We have found that vacuole fusion strictly depends upon the formation of membrane microdomains that are enriched in the proteins and lipids that regulate fusion [6–8]. Thus any perturbation to the assembly pathway needed for the formation of vertices would undoubtedly alter fusion. Others have found that Ca²⁺ at sub-micromolar concentrations induces clustering of plasma membrane SNAREs [45]. This is thought to occur through the neutralization of negatively charged side chains. It is also likely that Ca²⁺ relieves the electrostatic repulsion between negatively charged phospholipids [46,47]. Interestingly, Zilly et al. [45] also found that elevated Ca²⁺ concentrations (≥10 μM) inhibited SNARE pairing. Although the bulk measurement of Ca²⁺ efflux only measures sub-micromolar concentrations, it is possible that local concentrations of Ca²⁺ at the site of efflux may reach micromolar levels. Thus a delay in Ca²⁺ efflux might allow more trans-SNARE pair formation and enhanced fusion.

Abbreviations used

ABC transporter

ATP-binding cassette transporter

CBP

calmodulin-binding peptide

Cy5

indodicarbocyanine

GDI

guanine-nucleotide-dissociation inhibitor

GFP

green fluorescent protein

GST

glutathione transferase

HOPS

homotypic fusion and vacuole protein sorting

MRP

multidrug resistance-associated protein

MTM1

myotubularin 1

NSF

N-ethylmaleimide-sensitive factor

SB

solubilization buffer

SNAP

soluble NSF-attachment protein

SNARE

SNAP receptor

WT

wild-type