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Originally published In Press as doi:10.1074/jbc.M605592200 on July 19, 2006

J. Biol. Chem., Vol. 281, Issue 37, 27158-27166, September 15, 2006
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Bem1p Is a Positive Regulator of the Homotypic Fusion of Yeast Vacuoles*

Hao Xu and William Wickner1

From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844

Received for publication, June 12, 2006 , and in revised form, July 18, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Docked vacuoles are believed to undergo rapid lipid mixing during hemifusion and then a slow, rate-limiting completion of fusion and mixing of lumenal contents. Previous genomic analysis has suggested that Bem1p, a scaffold protein critical for cell polarity, may support vacuole fusion. We now report that bem1{Delta} strains have fragmented vacuoles (vps class B and C). During in vitro fusion reactions, vacuoles from bem1{Delta} strains showed a strong reduction in the rate of lipid mixing when compared with vacuoles from the BEM1 parent. The reduction in the overall rate of fusion with bem1{Delta} vacuoles was modest, consistent with lipid mixing as a non-rate-limiting step in the pathway. Although the fusion of either BEM1 (wild-type) or bem1{Delta} vacuoles is stimulated by recombinant Bem1p, the lipid mixing of docked bem1{Delta} vacuoles is highly dependent on rBem1p under certain reaction conditions. Bem1p-stimulated lipid mixing is blocked by well characterized fusion inhibitors including lipid ligands and antibodies to Ypt7p, Vps33p, and Vam3p. Although full-length Bem1p is required for maximal stimulation, a truncation mutant comprising the SH3 domains and the Phox homology (PX) domain retains modest stimulatory activity. In contrast to an earlier report (Han, B. K., Bogomolnaya, L. M., Totten, J. M., Blank, H. M., Dangott, L. J., and Polymenis, M. (2005) Genes Dev. 19, 2606–2618), we did not find phosphorylation of Bem1p at Ser-72 to be required for Bem1p-stimulated fusion. Taken together, Bem1p is a positive regulator of lipid mixing during vacuole hemifusion and fusion.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Membrane fusion in eukaryotic cells is highly regulated. At the core of the fusion machinery are Rab GTPases and their effectors (1), SNAREs2 (SNAP receptors) (2) and regulatory lipids (3–5). Additional fusion regulators include Sec18p/N-ethylmaleimide sensitive fusion factor (NSF) (6, 7), Sec17p/{alpha}-soluble NSF attachment protein (SNAP) (8, 9), SM proteins (10), calmodulin (11), synaptotagmin (12, 13), kinases (20), and phosphatases (14–16), Rho GTPases and actin (17), and V0 (the integral membrane domain of the vacuolar H+-ATPase) (18, 19).

We study membrane fusion using the vacuole of Saccharomyces cerevisiae. Our in vitro fusion assay employs two populations of vacuoles that, upon lipid and content mixing, generate alkaline phosphatase activity (21). Using this assay, we have established that vacuole fusion progresses through three stages: priming, docking, and lipid and aqueous compartment mixing (22). Priming entails a Sec17p/18p- and ATP-mediated liberation of the SNAREs Vam3p, Vti1p, Nyv1p, and Vam7p from the cis-SNARE complex (where all SNAREs are on the same membrane) (22–24). Since isolated vacuoles also have unpaired Vam3p, Vti1p, and Nyv1p, the requirement for ATP and Sec17p/18p can be bypassed if the soluble SNARE Vam7p is added to the reaction (25). Docking requires functional Rab-GTPase Ypt7p and the HOPS complex. HOPS is a heterohexameric complex that has direct affinities for phosphoinositides, Ypt7p, and SNAREs (26, 27). It activates Ypt7p through nucleotide exchange (28) and participates in trans-SNARE complex assembly on apposed membranes (29). Docked vacuoles form a ring-shaped membrane microdomain called the vertex that surrounds the apposed membranes from each organelle (30). Fusion catalysts, including regulatory lipids (sterol, diacylglycerol, and phosphoinositides) (5) and proteins such as Ypt7p, HOPS, and the SNAREs (31), become concentrated at this ring-shaped microdomain. Fusion occurs around the vertex ring, yielding internalized membranes within the larger fused vacuole (31). Fusion proceeds through rapid lipid mixing followed by a slower, rate-limiting content mixing.3

To identify the proteins that catalyze each step of the fusion cascade, we used a genomic approach to identify genes that regulate vacuole size and copy number in growing cells (32). One candidate is Bem1p, a multidomain protein required for cell polarization during budding and mating. Among its many binding partners (33, 34), Bem1p interacts with GTP-Cdc42p via its N-terminal SH3 domains (35) and Cdc24p (a GDP-GTP exchange factor for Cdc42p) via its C-terminal PB1 domain (36, 37). The central PX domain of Bem1p may interact with PI(3)P (38, 39) and with specific proteins (40). In addition, Bem1p can associate with actin (41). Since Cdc42p (42, 43), actin (44), and PI(3)P (5) are implicated in vacuole fusion, we decided to examine whether Bem1p regulates fusion directly. Besides its reported presence at the bud site and bud tip, we found that Bem1p is associated with vacuoles. Vacuoles from bem1{Delta} strains show a dramatic reduction in the rate of lipid mixing. Although the overall fusion of either BEM1 (wild-type) or bem1{Delta} vacuoles is stimulated by rBem1p, the subreaction of lipid mixing of docked bem1{Delta} vacuoles is most highly dependent on Bem1p and requires added rBem1p under certain reaction conditions. We propose that Bem1p regulates lipid mixing during homotypic vacuole fusion.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strain Construction—S. cerevisiae strains BY4742, BY4742 bem1{Delta}::kanMX6, BY4742 pep4{Delta}::kanMX6, and BY4742 pho8{Delta}::kanMX6 were from Research Genetics. PEP4 or PHO8 was deleted from BY4742 bem1{Delta}::kanMX6 by transformation with a PCR product amplified from pRS405 (45) to generate BY4742 bem1{Delta}::kanMX6 pep4{Delta}::LEU2 or BY4742 bem1{Delta}::kanMX6 pho8{Delta}::LEU2. BEM1 was deleted from BJ3505 and DKY6281 (21) by transformation with a PCR product amplified from pUG72 (46) to generate BJ3505 bem1{Delta}::URA3 and DKY6281 bem1{Delta}::URA3.

BEM1 cDNA was amplified from plasmid pDLB1185 (35) using 5'-CGCAGATCTGCTGAAAAACTTCAAACTCTC-3' and 5'-CGGAATTCAGTGGTGGTGATGGTGGTGAATATCGTGAACGGAAATTTTC-3'. The cDNA was inserted into pMBP-Parallel1 (47) between the BamHI and EcoRI sites to generate pMBP-Bem1-His6, which was sequenced and transformed into Escherichia coli Rossetta (DE3) (Novagen).

BEM1 truncation mutants were generated in a similar fashion except that 5'-CGCAGATCTGCTGAAAAACTTCAAACTCTC-3' and 5'-CGGAATTCAGTGGTGGTGATGGTGGTGCAGCTTTGCGCTTTTGTTCTGAGG-3' were used for "1-277," 5'-CGCAGATCTGCTGAAAAACTTCAAACTCTC-3' and 5'-CGGAATTCAGTGGTGGTGATGGTGGTGTTCTCTGTCGAAACCATTATTC-3' for "1–407," 5'-CGCAGATCTGTTTGAAAGAGACGAAAATCAAAAC-3' and 5'-CGGAATTCAGTGGTGGTGATGGTGGTGAATATCGTGAACGGAAATTTTC-3' were used for "408–551," and 5'-CGCAGATCTGGTTGACGGTGAATTATTAGTG-3' and 5'-CGGAATTCAGTGGTGGTGATGGTGGTGAATATCGTGAACGGAAATTTTC-3' were used for "277–551." BEM1 mutants S72A and S72D were created using QuikChange mutagenesis (Stratagene) (48) with overnight DpnI digestion. Oligonucleotides (Invitrogen) corresponding to the mutation sites were 5'-GACATAATTCTAAAGATATTACTGCTCCAGAGAAAGTTATAAAAGCC-3' and 5'-GGCTTTTATAACTTTCTCTGGAGCAGTAATATCTTTAGAATTATGTC-3' for S72A and 5'-GACATAATTCTAAAGATATTACTGATCCAGAGAAAGTTATAAAAGCC-3' and 5'-GGCTTTTATAACTTTCTCTGGATCAGTAATATCTTTAGAATTATGTC-3' for S72D. pMBP-Bem1-His6 was the template for initial primer extension. For expression in yeast, the BEM1 gene was amplified from S. cerevisiae S288C genomic DNA (Invitrogen) using 5'-CGCAGATCTAATGTCGTTATATTTTCAATC-3' and 5'-CGGAATTCTCGAGTGTAAAATCTTTCATATAATTC-3' and then subcloned into centromeric plasmid pRS413 (between BamHI and EcoI sites) (45).

Proteins and Antibodies—MBP-Bem1-His6 and mutant Bem1 proteins were purified using their C-terminal His6 tag. The MBP tag is important to maintain protein solubility. Rossetta (DE3)/pMBP-Bem1-His6 cultures (1L) in terrific broth (49) at 37 °C were induced at A600 = 2.5 with 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside at 25 °C for 6 h. Cells were collected by centrifugation (5,000 x g, 23 °C, 5 min), suspended in 25 ml of PBS, pH 7.4 (49), 5 mM dithiothreitol, 200 µM phenylmethylsulfonyl fluoride, 5 mM benzamidine, 0.62 µg/ml leupeptin, 4 µg/ml pepstatin A, and 24.4 µg/ml Pefabloc SC, and a 10-ml aliquot was disrupted by French press at 4 °C. The lysate was cleared by centrifugation (30 min, 100,000 x g, Beckman 60Ti rotor). The supernatant was incubated with 2 ml of nickel-nitrilotriacetic acid-agarose resin (Qiagen) in 2x PBS, 10 mM imidazole, 8 mM beta-mercaptoethanol, 2 mM dithiothreitol, 80 µM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 0.25 µg/ml leupeptin, 1.6 µg/ml pepstatin A, and 9.8 µg/ml Pefabloc SC. Following 1 h at 4 °C, the agarose beads were washed twice with 20 ml of wash buffer (2x PBS, 20 mM imidazole, 10 mM beta-mercaptoethanol, 250 mM NaCl), twice with 20 ml of wash buffer plus an additional 250 mM NaCl, and three times with 20 ml of wash buffer. Recombinant Bem1p was eluted by 5 ml of 250 mM imidazole in 2x PBS. MBP-Fapp1(PH)N-K, defective for phosphatidylinositol 4-phosphate binding (50), was a gift from C. Stroupe.

Antibodies to GST-Bem1p (a gift from A. Merz) were purified from rabbit serum. Serum (5 ml) was mixed at 4 °C for 4 h with 50 µl of 0.5 M of EDTA, 50 µl of proteinase inhibitor mixture (62 µg/ml leupeptin, 0.4 mg/ml pepstatin A, and 2.44 mg/ml Pefabloc SC) and 250 µl of 20x PBS, and added to a nitrocellulose strip (Bio-Rad) bearing 200 µg of MBP-Bem1 for overnight incubation at 4 °C. Following 5 washes (10 ml) with PBS, the blot was incubated with 800 µl of 0.2 M glycine (pH 2.3) for 3 min at room temperature and the eluate was neutralized with 200 µl of 1 M Tris-Cl, pH 8.0. Affinity-purified anti-Bem1p (0.2 µg/ml) was used in immunoblot analysis.

Other antibodies for immunoblot analysis were described previously (42, 44) except anti-Vps1p IgG (purified by protein A-Sepharose; 10 µg/ml), anti-Cof1p (affinity-purified from rCof1p cross-linked to Affi-Gel 15 agarose; 1 µg/ml), and anti-Pfy1p (affinity-purified from rPfy1p cross-linked to Affi-Gel 15 agarose; 1 µg/ml). Fusion inhibitors were used at the following concentrations: anti-Sec17p IgG (51), 57 µg/ml; anti-Sec18p IgG (51), 70 µg/ml; anti-Ypt7p peptide antibody (42), 33 µg/ml; anti-Vps33p IgG (27), 42 µg/ml; anti-Vam3p IgG (52), 67 µg/ml; MARCKS effector domain, 10 µM (25).

Lipid Mixing Assay—Vacuoles were isolated (21) with 20 mM PIPES-KOH, pH 6.8. Purified vacuoles (160 µg) were incubated with 0.12 mM octadecyl rhodamine B chloride (R18; Molecular Probes) on ice for 15 min. Labeled vacuoles were separated from the unbound dye and used to measure lipid mixing as described.3 Standard (+ATP) fusion assays (30 µl) contained 20 mM PIPES-KOH, pH 6.8, 200 mM sorbitol, 125 mM KCl, 6 mM MgCl2, 1 mM ATP, 1 mg/ml creatine kinase, 29 mM creatine phosphate, 10 mM coenzyme A, 2.9 µg/ml IB2, 0.67 µg of R18-labeled vacuoles, and 5.33 µg of unlabeled vacuoles. Bypass fusion assays (–ATP) contained 20 mM PIPES-KOH, pH 6.8, 200 mM sorbitol, 125 mM KCl, 5 mM MgCl2, 20 mM glucose, 0.61 mg/ml hexokinase (1 unit; Sigma), 13 µg/ml rVam7p (prepared according to A. Merz),4 10 µM coenzyme A, 2.9 µg/ml IB2, and vacuoles. Aliquots (25 µl) of the reaction mixture were transferred to a 384-well plate (round bottom, non-binding surface; Corning, NY), and fluorescent signals were measured every minute and processed as described.3

ALP Maturation Assay—Vacuoles from pep4{Delta} and pho8{Delta} strains (3 µg each) were mixed in 30 µl under either the standard condition (+ATP) or the bypass conditions (–ATP) as above. Following incubation on ice or at 27 °C for 80 min, alkaline phosphatase activity was assayed at 30 °C for 5 min (21).

Docking Assay—Docking reactions with 6 µg of DKY6281 vacuoles were performed in a volume of 30 µl of 20 mM PIPES-KOH, pH 6.8, 200 mM sorbitol, 100 mM KCl, 0.5 mM MgCl2, 0.3 mM ATP, 0.07 mg/ml creatine kinase, 13.3 mM creatine phosphate, 0.7 µg/ml Sec18p, 20 µM coenzyme A, 14.3 µg/ml IB2, and 0.13 µM Cy3-rBem1p. After 30 min at 27 °C, reactions were transferred to ice, mixed with 1 µl of 16 µM MDY-64, and 50 µl of 4% agarose and mounted on slides.

MBP-Bem1-His6 was labeled with Cy3 maleimide (Amersham Biosciences) according to the manufacturer's instructions. Unlabeled dye was quenched by addition of 10 mM mercaptoethanol and 10 mM cysteine and separated from the labeled protein using a PD-10 column (Amersham Biosciences).

Microscopy—Fluorescence microscopy methods were as described (26). To visualize FM4–64 or Cy3, Cy5, or MDY4–64 fluorophores, we used TRITC/Dil, Cy5, or Endow green fluorescent protein filter sets (Chroma Technologies).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Yeast vacuoles undergo constant fusion and fission during cell growth. An imbalance between the fusion rate and the fission rate will cause abnormal vacuole morphology (vam, fragmented vacuole morphology) (32, 53). Deletion of the BEM1 gene from the BY4742 chromosome causes type B vacuole fragmentation (Fig. 1B), as reported (32). Transforming BY4742 (Fig. 1A) or BY4742 bem1{Delta} (Fig. 1B) cells with vector alone does not change their vacuole morphologies. However, transforming BY4742 bem1{Delta} with pRS413-BEM1 restored normal vacuole morphology (compare Fig. 1D with Fig. 1B). To determine whether the vam phenotype is strain-specific, we deleted BEM1 from DKY6281 and BJ3505. DKY6281 bem1{Delta} vacuoles exhibit a type B fragmentation phenotype (Fig. 1F), whereas BJ3505 bem1{Delta} vacuoles exhibit a more severe fragmentation (type C; Fig. 1H). This is supported by quantification of the number of vacuole lobes in random fields of yeast cells. For example, of 97 DKY6281 cells, 91.8% had less than five vacuole lobes, 7.2% had five or more vacuole lobes, and 1% had highly fragmented vacuoles. Of 96 DKY6281 bem1{Delta} cells, only 38.5% had less than five vacuole lobes, whereas 46.9% had five or more vacuole lobes, and 14.6% had highly fragmented vacuoles. Thus, BEM1 is an authentic VAM gene.

Bem1p Does Not Affect the Vacuolar Distribution of Known Fusion Catalysts—Bem1p must associate with the vacuoles to directly regulate fusion. We therefore assayed BEM1 and bem1{Delta} vacuoles for their content of Bem1p by immunoblot with affinity-purified anti-Bem1p antibody (Fig. 2A). The antibody recognized a doublet close to the predicted molecular mass of Bem1p (62 KDa) from wild-type vacuoles but not from bem1{Delta} vacuoles. The protein band with slower mobility may represent the phosphorylated form of Bem1p (54). When compared with proteins such as Pho8p and Vam3p (Fig. 2B), which are predominantly localized to the vacuole, Bem1p is not as highly enriched in the vacuole preparation (Fig. 2B). This is not surprising since Bem1p cooperates with Cdc42p at the cell surface to regulate polarized growth (36).


Figure 1
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  		FIGURE 1.
Vacuoles in bem1{Delta} strains are fragmented. BY4742 cells transformed with vector pRS413 (A) or pRS413-BEM1 (C), BY4742 bem1{Delta} transformed with pRS413 (B) or pRS413-BEM1 (D), DKY6281 (E), DKY6281 bem1{Delta} (F), BJ3505 (G), and BJ3505 bem1{Delta} (H) were grown at 30 °C to stationary phase and stained with 3 µM FM4–64 for 1 h before microscopy. Scale bar in G, 10 µM.

 


Figure 2
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  		FIGURE 2.
Proteins on vacuoles from BEM1 and bem1{Delta} strains. BY4742 pep4{Delta} and BY4742 pep4{Delta} bem1{Delta} strains were grown at 30 °C to log phase in YPD medium. Spheroplasts and vacuoles were prepared as described under "Experimental Procedures." Vacuolar proteins (10 µg, or 3 µg for Cdc42p and Rho1p) or spheroplast proteins (50 µg, or 15 µg for Cdc42p) were subject to SDS-PAGE and immunoblot (A and B). A routine docking assay with 6 µg of DKY6281 vacuoles and 0.13 µM Cy3-rBem1p in 30 µl was performed at 27 °C for 30 min. Vacuoles were stained with 5 µM MDY-64 at the end of the reaction. Images were acquired using Endow green fluorescent protein (C) and TRITC/Dil filter sets (D). E, merged image of C and D. Scale bar in E, 5 µM.

 
To determine whether BEM1 deletion causes trafficking defects that affect fusion, we compared the vacuolar level of known fusion catalysts on BEM1 and bem1{Delta} vacuoles by immunoblot (Fig. 2A). Both BEM1 and bem1{Delta} vacuoles have comparable amounts of SNAREs, Ypt7p, HOPS, most other reported fusion regulators, and Pho8p (alkaline phosphatase), suggesting that Bem1p does not critically regulate trafficking to the vacuole. Vacuoles from bem1{Delta} strains have more Cdc42p than normal (Fig. 2A), probably due to the up-regulation of Cdc42p in bem1{Delta} cells (Fig. 2B).

To examine whether Bem1p has a direct affinity for docked vacuoles, we labeled recombinant Bem1p with Cy3 and added the fluorescent protein to a standard docking assay. rBem1p (Fig. 2, D and E) associates with vacuoles at punctate structures.

Bem1p Stimulates Lipid and Content Mixing in Vitro—Since vacuoles from a bem1{Delta} strain have normal levels of most proteins, they are suitable for study of the roles of Bem1p in fusion. We isolated BEM1 and bem1{Delta} vacuoles and examined their fusion activities through the content-mixing assay ("Experimental Procedures"). We performed the assay under either the standard reaction condition (with ATP) or the bypass condition (with no ATP but with added rVamp7) (25). Vacuoles from BEM1 and bem1{Delta} strains exhibit comparable fusion activities under either condition (Fig. 3, A and B). Since vacuoles from BEM1 and bem1{Delta} strains have similar amounts of maturable pro-ALP (data not shown), Bem1p is not essential for fusion in vitro. However, when recombinant Bem1p is added to the reaction, it stimulates the fusion of vacuoles from either BEM1 or bem1{Delta} strains (Fig. 3, A and B). A slightly more potent stimulation is observed for vacuoles from a bem1{Delta} strain.

To understand why deletion of the BEM1 gene causes striking vacuole fragmentation in vivo but little or no fusion defect in vitro, we postulated that Bem1p might regulate a stage of the fusion pathway that is not rate-limiting in vitro but that may be rate-limiting in vivo. Recent studies3 indicate that vacuole fusion proceeds from a very rapid lipid mixing step to a slow and rate-limiting content mixing step. We therefore examined the effect of Bem1p on lipid mixing. We monitor the rate of lipid mixing by an R18 dequenching assay. In this assay, rhodamine with an 18-carbon acyl chain (R18) is bound to vacuoles at a level that self-quenches the rhodamine fluorescence. The mixing of the lipid phase of these vacuoles with that of a docked neighbor vacuole, which is Rab-, HOPS-, and SNARE-regulated and thus on the authentic fusion pathway,3 dilutes the self-quenched R18 and thus causes a large increase in its fluorescence. BEM1 and bem1{Delta} vacuoles labeled with R18 were reisolated and mixed with unlabeled vacuoles. Lipid mixing assays were performed under standard or bypass conditions and were supplemented with rBem1p and anti-Vam3p where indicated. The fluorescent signal was recorded by a plate reader every minute (Fig. 3, C–F). The initial dequenching rate was calculated ("Experimental Procedures") and is presented in Fig. 3, G and H. There is a remarkable difference in the Vam3p-dependent dequenching rates between vacuoles from either BEM1 or bem1{Delta} strains, whether under regular or bypass fusion conditions. This contrasts with the results from the content mixing assay, where little difference was seen for these same vacuoles. The rate of lipid mixing of vacuoles from a bem1{Delta} strain is strongly dependent on added Bem1p under the bypass condition.

Bem1p-dependent Lipid Mixing Is Sensitive to Established Fusion Inhibitors—Under the bypass fusion condition, the Bem1p-dependent R18 dequenching rate is sensitive to anti-Ypt7p, anti-Vps33p (Vps33p is a subunit of the HOPS complex), anti-Vam3p, and MARCKS effector domain (MED; a ligand for phosphatidylinositol 4,5-bisphosphate (Fig. 4)), showing that Bem1p stimulates the Rab-, HOPS-, and SNARE-dependent fusion pathway. The signal is not sensitive to priming inhibitors such as anti-Sec17p and anti-Sec18p (Fig. 4), consistent with our observation that added rVam7p bypasses the need for ATP-dependent priming (25). MBP-Fapp1(PH)N-K, a control for the effects of the MBP tag on MBP-Bem1p, did not affect the rate of lipid dequenching.

Full-length Bem1p Is Required for Maximal Activity—Bem1p is composed of two N-terminal SH3 domains, a central PX domain and a C-terminal PB1 domain. To examine the importance of each domain, we generated deletion mutants and purified the encoded proteins (Fig. 5A). In the dequenching assay, 1.75 µM each Bem1-derived protein was used for comparison (Fig. 5B). The full-length Bem1p exhibits maximum stimulatory effect. A protein (277–551) lacking the two SH3 domains has no stimulatory activity, whereas a protein (1–407) with only the PB1 domain removed maintains modest activity. A protein consisting of only the PB1 domain is inert in the dequenching assay, but the SH3 domains alone show very weak activity. The results from the dequenching assay are mirrored in the content-mixing assay (Fig. 5C). Bem1p or related derivatives (up to 1.75 µM) were added to bypass fusion reactions. Only full-length Bem1p and 1–407 exhibited stimulatory effects. Taken together, the full-length Bem1p and its SH2 and PX domains are crucial for Bem1p activity in vitro. Of course, we cannot rule out the possibility that the mutants could be misfolded. Bem1p did not interfere with the reporting system since it has no effect when added at the end of a fusion reaction (Fig. 5C, compare lanes 1 and 2).


Figure 3
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  		FIGURE 3.
Bem1p stimulates lipid mixing and content mixing. ALP maturation assays were performed for 80 min by mixing BY4742 pep4{Delta} and BY4742 pho8{Delta} vacuoles or BY4742 pep4{Delta} bem1{Delta} and BY4742 pho8{Delta} bem1{Delta} vacuoles under either the standard fusion condition (A) or the bypass condition (B). Four independent experiments were used to generate the mean value for the "no inhibitor" (no inhib.) condition (the ice value was treated as a background and subtracted). By setting their respective no inhibitor values to 100%, the values for other conditions were adjusted relative to the no inhibitor value. Standard deviations were calculated using Excel. To measure the rate of lipid mixing, BY4742 and BY4742 bem1{Delta} vacuoles were labeled with R18 on ice for 15 min and reisolated by step-gradient flotation. Dequenching assays were performed by mixing 1 part labeled vacuoles with 8 parts unlabeled vacuoles (6 µg total). Fluorescent signals were recorded every minute using a Gemini XPS microplate reader. The lipid mixing signal at each time point was standardized by subtracting the signal at 0 min and then dividing by the maximum dequenching signal seen upon Triton X-100 addition and is expressed as a percentage of this maximum (C–F). The initial rates of dequenching are presented in G and H. The mean dequenching rate for the no inhibitor condition was averaged from four independent experiments. The dequenching rates for other conditions were derived as above.

 


Figure 4
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  		FIGURE 4.
Bem1p-dependent dequenching is blocked by fusion inhibitors. Vacuoles from bem1{Delta} cells were labeled with R18 and mixed with unlabeled vacuoles under the bypass condition (no ATP plus Vam7p). Dequenching rates and standard deviations were calculated from three independent experiments as described in the legend for Fig. 3. MED, MARCKS effector domain.

 
Bem1p Activity Does Not Require Ser-72 Phosphorylation—It has been reported that Cln3p-dependent phosphorylation of Bem1p at Ser-72 is required to promote fusion (54). In this study, the replacement of this serine by aspartic acid, which mimics phosphorylation, supported fusion, whereas the S72A mutant, which could not undergo phosphorylation, had no fusion activity. Because the fusion activities in this report required cytosol, it is not clear whether these Bem1p mutant proteins act directly on the vacuolar fusion machinery or through recruitment of cytosolic factors. We purified these two Bem1p mutant proteins (Fig. 5A) and compared their activity with the wild-type Bem1p in a cytosol-free vacuole fusion reaction (Fig. 6). Under the bypass fusion condition, which eliminates the possibility of Bem1p phosphorylation during the assay, wild-type Bem1p and its S72D and S72A derivatives stimulate fusion in a comparable fashion. Similar results were seen for the standard (+ATP) fusion condition. Interestingly, supplementing the reaction with cytosol did not change the behavior of Bem1p derivatives (data not shown). Thus, phosphorylation at Ser-72 does not appear to be required for Bem1p activity in our assay.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We find that Bem1p stimulates the lipid mixing step of vacuole fusion. Lipid mixing takes place upon merger of the two apposed leaflets of the docked organelles (hemifusion), which can occur well before the merger of distal leaflets and aqueous content mixing. SNARE-dependent hemifusion structures have been implicated in proteoliposome fusion (55, 56), engineered cell fusion (57), and vacuole fusion.3 For vacuole fusion, the rapid hemifusion step is followed by a slow rate-limiting step of content mixing. This notion is supported by our finding that bem1{Delta} vacuoles exhibit a significant reduction of the rate of lipid mixing but not of fusion when compared with BEM1 vacuoles. Bypass reaction conditions permit rapid, Bem1p-dependent lipid mixing, indicating that Bem1p can act at, or prior to, the lipid mixing step. Because no homologs for Bem1p have been discovered in higher organisms, it is possible that the role of Bem1p in fusion is unique to the yeast vacuole.

How might Bem1p regulate lipid mixing during vacuole fusion? As a multidomain protein, Bem1p has the capacity to bind several proteins and lipids. The small GTPase Cdc42p and actin are two Bem1p binding partners that have already been implicated in fusion. Although it is unclear whether these interactions take place on the vacuole or how they might regulate lipid mixing, there is more Cdc42p on bem1{Delta} vacuoles, suggesting that the functional relationship between Bem1p and Cdc42p may extend to the vacuole. Moreover, deletion of the Cdc42p binding domain of Bem1p abolishes Bem1p activity (Fig. 5, B and C). Bem1p may also act on proteins that are critical for lipid mixing via unidentified interactions. The PX domain of Vam7p allows Vam7p interaction with the HOPS complex (26); both these proteins regulate important steps that are required for lipid mixing. It is conceivable that the PX domain of Bem1p might regulate lipid mixing by regulating SNARE-HOPS interaction. It is also possible that Bem1p regulates lipid mixing via direct interaction with PI(3)P, an important regulatory lipid in the fusion reaction. Addressing these possibilities may require generating Bem1p derivatives with point mutations that abolish individual intermolecule interactions.


Figure 5
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  		FIGURE 5.
Full-length recombinant Bem1p is required maximal activity. MBP-Bem1-His6 and mutant Bem1 proteins were purified from Ni2+-agarose beads. Eluted proteins (3 µg) were subjected to SDS-PAGE followed by Coomassie Blue staining (A). WT, wild type. Lipid mixing assays had 1.75 µM Bem1 proteins (B). Fusion assays used BY4742 pep4{Delta} bem1{Delta} and BY4742 pho8{Delta} bem1{Delta} vacuoles under the bypass condition (C). Data from three independent experiments were processed as in the legend for Fig. 3.

 


Figure 6
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  		FIGURE 6.
Phosphorylation at Ser-72 is not essential for Bem1p activity. Mutations at Ser-72 were created using PCR based QuikChange mutagenesis ("Experimental Procedures"). MBP-S72A-His6 and MBP-S72D-His6 were purified by the same method as wild-type (WT) MBP-Bem1-His6. The bypass fusion assay was performed using BY4742 pep4{Delta} bem1{Delta} and BY4742 pho8{Delta} bem1{Delta} vacuoles.

 
Recent studies of p47phox suggest that the function of its PX domain can be masked by an intramolecular interaction with its SH3 domain (58). Phosphorylation of the SH3 domain exposes the PX domain by causing a transition from a close to an open conformation. Interestingly, Bem1p also appears to be activated by phosphorylation at Ser-72 in the first SH3 domain (54). However, in our hands, S72D, which mimics the phosphorylated form, exhibits similar in vitro activity to S72A or the wild type. This discrepancy may arise from the fact that fusion is cytosol-dependent in the studies of Han et al. (54) but not in our assays. Bem1p may stimulate fusion via different pathways under these two conditions.


   FOOTNOTES
 
* This work was supported by a grant from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry, Dartmouth Medical School, 7200 Vail Bldg., Hanover, NH 03755-3844. Tel.: 603-650-1701; Fax: 603-650-1353; E-mail: Bill.Wickner{at}Dartmouth.edu.

2 The abbreviations used are: SNARE, SNAP receptors; SNAP, soluble NSF attachment protein; NSF, N-ethylmaleimide sensitive fusion factor; MBP, maltose binding-protein; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; TRITC, tetramethylrhodamine isothiocyanate; PI(3)P, phosphatidylinositol 3-phosphate; r, recombinant; HOPS, homotypic fusion and vacuole protein sorting complex; px, Phox homology domain; ALP, alkaline phosphatase; MARCKS, myristoylated alanine-rich C kinase substrate. Back

3 Y. Jun and W. Wickner, manuscript in preparation. Back

4 A. Merz, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Drs. D. Lew, C. Stroupe, and A. Merz for strains and reagents and Y. Jun for sharing unpublished data.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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