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1 Centre for Biomolecular Sciences, School of Biology, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK
2 Division of Gene Regulation and Expression, College of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK
Correspondence
Peter J. Coote
pjc5{at}st-andrews.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Crucially, VMA2 is a multicopy suppressor of both the sorbic acid-sensitive phenotype and the impaired vacuolar-acidification defect of dbf2 cells, confirming a functional interaction between Dbf2p and Vma2p. The yeast V-ATPase is therefore involved in mediating sorbic acid stress tolerance, and we have shown a novel and unexpected role for the cell cycle-regulated protein kinase Dbf2p in promoting V-ATPase function.
Present address: Division of Gene Regulation and Expression, College of Life Sciences, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), the requirement for the membrane-bound transporter Azr1p and Aqr1p for optimal adaptation to short-chain monocarboxylic acids (Tenreiro et al., 2000, 2002), and the key role played by the vacuolar H+-ATPase (V-ATPase) in resistance to the organic acid herbicide 2,4-dichlorophenoxyacetic acid (2,4-D; Fernandes et al., 2003). The best-characterized response to acid stress in yeast is that induced by sorbic acid, a weak-acid food preservative. Optimal adaptation to sorbic acid stress requires induction of the ATP-binding cassette (ABC) efflux pump Pdr12p (Piper et al., 1998), a rapid, weak acid-specific response that is essential for counteracting intracellular anion accumulation. War1p, a novel transcription factor of the Zn2Cys6 family, is the stress-activated regulator of PDR12 (Kren et al., 2003). War1p binds constitutively to the PDR12 promoter irrespective of sorbic acid stress, which is believed to activate War1p through phosphorylation (Kren et al., 2003; Schuller et al., 2004). Notably, the signalling pathway that mediates War1p phosphorylation has not yet been identified.
One of the principal objectives of this study was to identify key regulatory proteins involved in the perception and signalling of weak-acid stress. Screening of the yeast haploid deletion library revealed that cells lacking the well-studied serine-threonine protein kinase Dbf2p, best known for its role in cell cycle regulation (see Yeong et al., 2002), are hypersensitive to sorbic acid. However, the presence or absence of Dbf2p did not affect expression of Pdr12p, suggesting that the kinase mediates sorbic acid tolerance via a novel mechanism.
Here we show that Dbf2p is required for phosphorylation of both the V-ATPase catalytic subunit Vma1p and the non-catalytic subunit Vma2p. V-ATPases are large, complex enzymes responsible for the acidification of many internal compartments in eukaryotic cells (reviewed by Arata et al., 2002; Nishi & Forgac, 2002). In yeast, the V-ATPase acidifies the vacuole by driving the translocation of protons into the lumen. This acidification is crucial for many cellular processes, including endocytosis, targeting of newly synthesized lysosomal enzymes, cytoplasmic pH homeostasis, protein processing and the coupled transport of small molecules. Both Vma1p and Vma2p, and thus a functional V-ATPase, are required for optimal adaptation to sorbic acid. Measurement of V-ATPase-mediated vacuolar acidification revealed that like vma1 and vma2 mutants, dbf2 and kinase-inactive dbf2 strains were also impaired in vacuolar acidification. Overexpression of VMA2 rescued both the sorbic acid sensitivity and the impaired vacuolar acidification of dbf2 cells, implying a functional interaction between the two proteins. Our findings therefore provide strong evidence for a previously unknown role for Dbf2p in maintaining optimal V-ATPase function.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Yeast strains were grown at 30 °C in standard rich medium (YPD) at pH 4.5. All yeast strains are listed in Table 1, and those constructed in this study were prepared in the BY4741a background (Research Genetics). Yeast strains containing tandem affinity purification (TAP) tags were from Open Biosystems, and the myc13-DBF2 strain was prepared by PCR-mediated genomic manipulation (Longtine et al., 1998). VMA2 and VPH1 were chromosomally tagged at the 3' end with GFP–HIS1 cassettes using the method of Sheff & Thorn (2004) and verified by diagnostic PCR.
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), using YPD (pH 4.5) supplemented with 1.5 mM sorbic acid. In liquid culture, growth was monitored in a 24-well microplate (Greiner Bio-One) by measuring OD600 using a PowerWave XS Universal Microplate Spectrophotometer (BioTek Instruments), taking automated readings every 30 min. All treatments were performed in triplicate. Phenotypic testing was performed by diluting overnight cultures of yeast strains to OD600 0.1 and making 10-fold serial dilutions, then spotting 5 µl samples onto appropriate agar plates.
Site-directed mutagenesis and plasmid constructs.
Routine recombinant DNA methodology was performed as described elsewhere (Sambrook & Russell, 2001). The pRS vector series was used for gene complementation and overexpression experiments (Sikorski & Hieter, 1989). Details of constructed vector sequences and oligonucleotides used are available on request. The Stratagene QuikChange method was applied to mutate residue 305 of Dbf2p, carried by pRS313-DBF2, from asparagine (AAC) to alanine (GCT). This created pRS313-DBF2(N305A), which encoded a kinase-inactive version of Dbf2p, as described elsewhere (Mah et al., 2001).
Phosphoproteome analysis and MS.
Phosphoproteome analysis was carried out as previously described (Makrantoni et al., 2005). Briefly, affinity capture of phosphoproteins was performed using the PhosphoProtein Purification Kit from Qiagen. Approximately 25 mg total yeast protein was loaded onto the proprietary affinity columns, which were washed before the elution of bound phosphoproteins. Fractions containing the enriched phosphoproteins were compared by 2D-PAGE as previously described, and proteins were visualized with SYPRO Ruby protein stain and identified by peptide mass fingerprinting (Lawrence et al., 2004). Only protein spots showing reproducible changes in protein abundance were catalogued. Peptide mass fingerprint data were obtained as described previously, using the Mascot search engine to search for proteins with calculated tryptic peptide masses that matched the measured experimentally derived masses with a mass accuracy of ±0.15 Da (Lawrence et al., 2004).
Western blotting of Pdr12p, Vma1p, Vma2p and Dbf2p.
Proteins were transferred from Criterion gels (Bio-Rad) to nitrocellulose membranes (GE Healthcare) by semi-dry blotting. Membranes were subsequently probed according to standard procedures with mAbs to Vma1p (anti-H+-ATPase 69 kDa subunit 8B1) and Vma2p (anti-H+-ATPase 60 kDa subunit 13D11) obtained from Invitrogen, and a polyclonal antibody to Pdr12p (kind gift of Karl Kuchler, University of Vienna). Comparative 2D-immunoblotting was performed essentially as previously described (Lawrence et al., 2004). Immobilized pH gradient 11 cm strips, pH 4–7, were used with a Multiphor II apparatus for IEF (GE Healthcare), loading 40 µg total cell lysate. For the second dimension, 12.5 % Tris/HCl, 1.00 mm precast 11 % polyacrylamide gels (Bio-Rad) were run in a Criterion cell apparatus (Bio-Rad). For some experiments, 40 µg total lysate was treated with 50 U calf intestinal phosphatase (CIF; Sigma-Aldrich) for 1 h at 30 °C prior to IEF.
Vacuolar acidification, vacuolar staining and protein localization.
Vacuolar acidification was assessed from the accumulation of fluorescent quinacrine, using published procedures in which cells were harvested in late exponential phase and transferred to uptake buffer containing 200 µM quinacrine (Morano & Klionsky, 1994; Roberts et al., 1991). Vacuolar accumulation of quinacrine was monitored within 1 h of staining. Phase-contrast and fluorescence images (excitation 
=490–420 nm, emission =528–538 nm) were captured using an Olympus IX70 DeltaVision microscope (Applied Precision LLC). CellTracker Blue 7-amino-4-chloromethylcoumarin (CMAC; Invitrogen) was used to visualize the yeast vacuole. For staining, 1 ml of culture was harvested at OD600 0.8–1.0 by centrifugation for 30 s at 13 400 r.p.m. The pellet was resuspended in 100 µl 25 µM CellTracker Blue solution and incubated at 30 °C for 45 min. Cells were harvested as before, resuspended in 200 µl YPD (pH 4.5) and then incubated at 37 °C for 30 min. Stained cells (2 µl) were fixed by mixing with 2 µl 1 % low-melting-point (LMP) agarose. Phase-contrast and fluorescence images (excitation =340–360 nm, emission =450–457 nm) were captured. To study localization of GFP-tagged Vph1p and Vma2p, images were acquired as described above for quinacrine fluorescence.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Compared to the parent strain, growth of the dbf2 mutant was severely impaired in the presence of increasing concentrations of sorbic acid both on YPD agar, as shown in Fig. 1(a) and in YPD medium (data not shown). Furthermore, a dbf2 allele encoding a mutant Dbf2p (N305A) lacking kinase activity (‘kinase-inactive’ Dbf2p; Mah et al., 2001) showed sorbic acid hypersensitivity similar to that of the dbf2 mutant, indicating that functional kinase activity was required to mediate tolerance to the weak acid. Fig. 1(b) shows that dbf2-dependent sorbic acid hypersensitivity was rescued by complementation with DBF2, confirming that the effect is specific. DBF20 encodes a functionally redundant homologue of Dbf2p to which it shows 80 % amino acid sequence identity (Toyn et al., 1991). Deletion of DBF20 had no effect on tolerance to sorbic acid stress (data not shown), consistent with the much lower kinase activity associated with Dbf20p in comparison with Dbf2p (Toyn & Johnston, 1994). Western blot analysis of a strain expressing TAP-tagged Dbf2p showed that while Dbf2p was expressed under all growth conditions used in this study, no significant changes in Dbf2p level were detected under sorbic acid stress (data not shown).
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), and concurrent with this study, the transcriptional regulator War1p was shown to be required for PDR12 expression (Kren et al., 2003). To determine whether Dbf2p forms part of the pathway that transduces the sorbic acid stress signal to War1p, Western blot analysis was performed using an anti-Pdr12p polyclonal antibody. However, loss of DBF2 affected neither the basal nor the sorbic acid-induced levels of the Pdr12p anion transporter (data not shown), indicating that the kinase is not involved in the War1p–Pdr12p pathway.
Loss of Dbf2p is associated with reduced phosphorylation of the V-ATPase
We sought to identify changes occurring in the yeast phosphoproteome due to deletion of DBF2 that might reveal targets of the protein kinase that were relevant to its requirement in sorbic acid stress tolerance. Using our recently developed method (Makrantoni et al., 2005), total yeast phosphoproteins were prepared from both the parent and the dbf2 mutant strains, grown in the presence or absence of sorbic acid. We identified Vma2p, the B subunit of the yeast V-ATPase (Liu et al., 1996; Vasilyeva & Forgac, 1996), as one of a small number of proteins whose behaviour differed in phosphoprotein preparations from the two strains when analysed by 2D-PAGE. In the parent strain, two isoforms of Vma2p were identified by peptide mass fingerprinting (Fig. 2a, isoforms 1 and 2) with 15 and 16 % peptide coverage, respectively. Isoform 2 had an approximate molecular mass of 58 kDa and a pI of 5.1, while isoform 1 had a slightly higher molecular mass and a pI of 
4.9. Significantly, Fig. 2(a) shows that phosphoprotein extracts of the dbf2 mutant lacked isoform 1, although in both strains the pattern of Vma2p isoforms was not affected by the presence or absence of sorbic acid. To exclude that the loss of isoform 1 in the dbf2 phosphoprotein extract resulted from changed Vma2p abundance, Western blot analysis was performed using an anti-Vma2p mAb and total protein extracted from the wild-type and the dbf2 mutant. Fig. 2(b) shows that the level of Vma2p was unaffected by exposure to sorbic acid or the loss of DBF2.
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). The absence of a more acidic Vma2p isoform in dbf2 phosphoprotein extracts is therefore consistent with loss of a more phosphorylated Vma2p isoform. To study the potential modification of Vma2p in more detail, Vma2p was detected by immunoblotting following 2D-PAGE separation of phosphoprotein preparations, as shown in Fig. 2(c)
. The preparation from the parent strain revealed two isoforms of Vma2p with slightly different pI (panel 1). The apparent pI of these isoforms shifted dramatically away from the acidic end of the IEF strip following pre-treatment with calf intestinal phosphatase (CIP), confirming that Vma2p is phosphorylated (panel 5). In phosphoprotein preparations from the dbf2 strain, all of the Vma2p isoforms were shifted towards the neutral end of the IEF gel strip relative to the isoforms present in the control samples. However, growth in the presence of sorbic acid did not significantly alter the pI of Vma2p isoforms recovered from either strain. These observations are therefore in accordance with the phosphoproteome analysis of Fig. 2(a) and support the notion that Vma2p shows Dbf2p-dependent phosphorylation that is independent of sorbic acid stress.
We performed similar experiments using a mAb to the catalytic subunit of the V-ATPase (Vma1p), which is known to interact with Vma2p. Using the parental strain, a cluster of four protein spots of similar molecular mass but slightly different pI was observed (Fig. 2d, panel 1), which shifted markedly towards the neutral end of the IEF strip following CIP pre-treatment (panel 5), again indicative of phosphorylation. In the dbf2 strain, all these Vma1p isoforms shifted toward the neutral end of the IEF gel strip, indicative of Dbf2p-dependent phosphorylation. As with Vma2p, growth in the presence of sorbic acid did not significantly alter the pI of the Vma1p isoforms in either the parent or the dbf2 strain. Western blot analysis of Vma1p showed that neither loss of DBF2 nor exposure to sorbic acid affected the overall Vma1p level in the cell (data not shown). In summary, our findings show that both Vma1p and Vma2p are phosphorylated and that phosphorylation requires Dbf2p.
To provide further evidence that Vma1p and Vma2p are phosphoproteins, total soluble protein was prepared from cells grown in the presence or absence of sorbic acid, and phosphoproteins were recovered by phosphoprotein affinity chromatography. Fig. 2(e) shows immunoblots of both the flow-through (F; non-phosphorylated proteins) and the eluate (E; phosphorylated proteins) fractions using anti-Vma1p and -Vma2p mAbs. In the parent strain, both Vma1p and Vma2p were detected in the eluate fraction, providing further evidence that both proteins are phosphorylated. However, Vma1p and Vma2p prepared from dbf2 cells were predominantly detected in the flow-through fraction, confirming that the proteins show reduced levels of phosphorylation in the absence of functional Dbf2p.
In a previous study, neither Vma1p nor Vma2p was identified as a direct substrate for Dbf2p (Mah et al., 2005). We re-examined this by attempting to phosphorylate Vma2p in vitro, as described elsewhere (Toyn & Johnston, 1994), using Dbf2p purified from yeast as either a TAP-tag or myc13-tagged fusion protein, together with TAP–Vma2p isolated from yeast as substrate. While both Dbf2p preparations efficiently phosphorylated calf thymus histone H1, neither was able to promote direct phosphorylation of Vma2p (data not shown). Thus, Dbf2p-dependent phosphorylation of Vma2p may not be direct.
V-ATPase function is required for sorbic acid stress tolerance and is dependent on functional Dbf2p kinase
Since loss of Dbf2p function confers sorbic acid hypersensitivity, and Vma1p and Vma2p both show Dbf2p-dependent phosphorylation, we next examined whether the V-ATPase is involved in sorbic acid stress tolerance. Compared to the parent strain, growth of vma1 and vma2 mutant strains was significantly impaired at pH 4.5 in the presence of 1.5 mM sorbic acid both on YPD agar as shown in Fig. 3(a) and in YPD medium (data not shown). In each case, the defect was rescued by complementation with the corresponding gene; see Fig. 3(b). The V-ATPase is inhibited with high specificity by bafilomycin A1, both in vitro and in vivo (Bowman et al., 2004; Drose & Altendorf, 1997). Fig. 3(c) shows that treatment with 10 µM bafilomycin A1 resulted in slight growth inhibition compared to the untreated control, while 1 mM sorbic acid at pH 4.5 resulted in marked growth inhibition. However, the combination of 10 µM bafilomycin together with 1 mM sorbic acid resulted in greater growth inhibition than that induced by exposure to sorbic acid alone, implying that a fully functional V-ATPase is required for optimal adaptation to sorbic acid stress.
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, vma2, dbf2 and kinase-inactive dbf2 mutants grown in the presence or absence of sorbic acid. Quinacrine is a weakly basic dye that accumulates in acidic compartments in response to proton gradients and is routinely used to assess the state of vacuolar acidification in yeast (Umemoto et al., 1990; Weisman et al., 1987). Earlier studies have shown that deletion of VMA1, encoding the V-ATPase catalytic subunit, causes inactivation of the V-ATPase and loss of vacuolar acidification (Morano & Klionsky, 1994). Cells of the parent strain displayed strongly localized vacuolar fluorescence indicative of a normal acidified vacuole, whereas quinacrine accumulation was entirely absent in the vma1 strain, indicating that the vacuole was not acidified (Fig. 4). Similarly, vacuolar fluorescence was almost undetectable in vma2 cells. Significantly, dbf2 cells showed a dramatic reduction of quinacrine fluorescence (Fig. 4), although slight residual fluorescence indicated that the dbf2 vacuolar-acidification defect, although profound, was not as complete as that in vma1 cells. The kinase-inactive dbf2(N305A) mutant displayed a similar vacuolar-acidification defect (Fig. 4), but growth in the presence of sorbic acid did not affect vacuolar acidification in any of the strains tested. These findings indicate that the kinase activity of Dbf2p is required for optimal vacuolar acidification and therefore proper V-ATPase function, demonstrate that a fully functional V-ATPase is required for adaptation to sorbic acid stress, and imply that Dbf2p-dependent phosphorylation of the V-ATPase might be the mechanism by which Dbf2p promotes sorbic acid stress tolerance.
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cells was due to incorrect localization of the V-ATPase, we constructed control and dbf2 strains expressing either VMA2–GFP (as a V1 domain marker) or VPH1–GFP (as a V0 domain marker). Using the blue fluorescent CellTracker dye to visualize the vacuoles, in both the parent and dbf2 mutant cells, Vma2–GFP and Vph1–GFP were both clearly localized normally to the vacuolar membrane in cells grown in the presence (data not shown) or absence of sorbic acid (Fig. 5). Since assembly of the V1 subcomplex and its attachment to the V0 subcomplex require all of the V1 polypeptides (except Vma13p) and all of the V0 polypeptides (Forgac, 1999; Kane et al., 1992; Tomashek et al., 1997), we conclude that loss of Dbf2p kinase is unlikely to affect the localization or assembly of either V-ATPase subcomplex.
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). We therefore explored the functional relationship between Dbf2p and the V-ATPase by examining the effect of multiple extra copies of VMA1, VMA2 and DBF2 on sorbic acid sensitivity in wild-type and dbf2 strains. As expected, high-copy VMA1, VMA2 and DBF2 rescued the sorbic acid hypersensitivity of the respective vma1, vma2 and dbf2 mutant strains, although enhanced sorbic acid tolerance was not induced (data not shown). While the sorbic acid hypersensitivity of the dbf2 mutant was unaffected by multicopy VMA1, multicopy VMA2 rescued both the sorbic acid hypersensitivity and the vacuolar-acidification defect of the dbf2 strain in the presence of sorbic acid (Fig. 6a, b, respectively). In the reciprocal experiments, high-copy DBF2 did not rescue the sorbic acid hypersensitivity of either vma1 or vma2 cells (data not shown). These results provide further evidence for a functional interaction between the Dbf2p kinase and the V-ATPase that mediates resistance to sorbic acid stress, and suggest that high-copy VMA2 may lead to enhanced V-ATPase function in the presence of sorbic acid in the dbf2 mutant.
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and stv1 mutants, which possess functionally redundant polypeptides (Manolson et al., 1994), V-ATPase mutants fail to grow on YPD media buffered to pH 7.5 or supplemented with 100 mM CaCl2 or 4 mM ZnCl2, or on YEP medium with glycerol as the sole carbon source (reviewed by Anraku et al., 1989). Despite displaying reduced vacuolar acidification, dbf2 cells grew normally under all these conditions (Fig. 7). This phenotype is similar to that displayed by the vph1 mutant, and implies that there is some residual V-ATPase function present in dbf2 cells, a conclusion supported by our observation that quinacrine accumulation, and thus vacuolar acidification mediated by the V-ATPase, is reduced but not completely abolished in dbf2 cells (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Role of the V-ATPase in sorbic acid stress tolerance
We found that loss of VMA1 or VMA2, or exposure to the specific V-ATPase inhibitor bafilomycin A1, induced hypersensitivity to sorbic acid. These observations are supported by identification of other V-ATPase genes (VMA4, VMA5, VMA7, VMA8, VMA13 and VMA22) as mediators of sorbic acid tolerance in screens of the yeast deletion mutant collection (Mollapour et al., 2004; Schuller et al., 2004), several of which we independently identified in our own, similar screen (data not shown). A functional V-ATPase is therefore required for optimal adaptation to sorbic acid stress, and available evidence supports the hypothesis that V-ATPase function is required to mediate tolerance to a range of growth-inhibitory compounds in yeast. For example, the yeast V-ATPase plays a crucial role in mediating tolerance to the weak-acid herbicide 2,4-D (Fernandes et al., 2003), and yeast mutants lacking a functional V-ATPase show multidrug sensitivity (Parsons et al., 2004). When sorbic acid diffuses into the cell it dissociates into H+ and sorbate, leading to intracellular acidification. A functional V-ATPase may therefore assist the plasma membrane ATPase Pma1p in removing protons from the cytoplasm to balance the extrusion of sorbate anions via the Pdr1p ABC transporter. In fact, loss of V-ATPase function is also known to cause reduced Pma1p activity (Fernandes et al., 2003), probably by reduced targeting of the ABC transporter to the plasma membrane (Perzov et al., 2000), and so changes in V-ATPase activity may also impact on Pma1p function. Alternatively, if sorbic acid led to dissipation of the vacuolar membrane H+ gradient this might be counteracted by V-ATPase activity. Loss of the vacuolar H+ gradient would disrupt the ability of the vacuole to maintain the homeostasis of many essential nutrients and ions, thus inhibiting growth.
Dbf2p kinase and sorbic acid stress
Dbf2p is a multifunctional, cell cycle-regulated protein kinase that regulates cell division, transcription and the stress response (Lee et al., 1999; Liu et al., 1997a; Toyn & Johnston, 1994). As part of the mitotic exit network (MEN), Dbf2p plays a crucial role in exit from mitosis (reviewed by Yeong et al., 2002). Within the MEN, Dbf2p is activated following phosphorylation by Cdc15p and its activity is dependent on the Dbf2p-associated protein Mob1p (Mah et al., 2001). Dbf2p is also believed to influence gene expression via interaction with the CCR4-NOT complex (Liu et al., 1997a), a general transcriptional regulator (reviewed by Collart, 2003) that affects the expression of genes including those that encode the stress-activated transcription factors Msn2p and Msn4p (Lenssen et al., 2002). Outside its role within the MEN, the mechanisms by which Dbf2p is regulated remain undefined. In contrast to dbf2 strains, temperature-sensitive cdc15 mutants do not show sorbic acid hypersensitivity even when grown very close to their restrictive temperature (data not shown), so we consider it likely that the classic cell-cycle regulators of Dbf2p activity are not required for this function of the kinase. Deletion of DBF2 results in other stress-sensitive phenotypes (Liu et al., 1997b), and although DBF2 expression is induced in response to stresses such as salt and heat, we observed no significant change in the level of Dbf2p during growth in the presence of sorbic acid. This conclusion is supported by two independent transcriptional profiling studies of yeast exposed to sorbic acid stress, neither of which detected upregulation of DBF2 (de Nobel et al., 2001; Schuller et al., 2004). Loss of DBF2 also did not affect the increased expression of the ABC-transporter Pdr12p observed during sorbic acid stress (Piper et al., 1998). Thus, although we cannot exclude the possibility that Dbf2p regulates Pdr12p activity, Dbf2p-dependent sorbic acid tolerance is probably independent of Pdr12p-mediated sorbate anion efflux (Holyoak et al., 1999).
DBF2 and V-ATPase function
Our discovery of Dbf2p-dependent phosphorylation of the V-ATPase subunits Vma1p and Vma2p prompted us to ask whether the Dbf2p kinase influences V-ATPase function. Normal V-ATPase activity results in ATP hydrolysis-dependent vacuolar acidification that is lost in V-ATPase mutants (reviewed by Anraku et al., 1992) and that can be monitored by vacuolar accumulation of quinacrine (Umemoto et al., 1990; Weisman et al., 1987). Since both dbf2 and ‘kinase-inactive’ dbf2 mutants showed a drastic reduction in vacuolar quinacrine accumulation (and thus vacuolar acidification), our results indicate that Dbf2p kinase activity is required for proper V-ATPase function. Compelling support for this hypothesis comes from our finding of VMA2 as a multicopy suppressor of both the sorbic acid-sensitive phenotype and the vacuolar-acidification defect of dbf2 cells. Since dbf2 cells show apparently normal assembly and localization of the V-ATPase, it is possible that elevated expression of Vma2p partially restores the level of phosphorylated Vma2p generated by a Dbf2p-related kinase (Dbf20p, for example). dbf2 and V-ATPase mutants both show glycogen hyperaccumulation (Wilson et al., 2002), providing an additional link between Dbf2p and V-ATPase function.
The S. cerevisiae V-ATPase is composed of 13 subunits organized into two functional domains, a peripheral V1 domain responsible for ATP hydrolysis and an integral V0 domain responsible for proton translocation (Arata et al., 2002). Since deletion of any of the V-ATPase subunit genes leads to complete loss of enzyme assembly and activity (see Forgac, 1999), the seemingly normal localization in the vacuolar membrane of key V1 and V0 domain proteins in the dbf2 mutant implies that the apparent loss of V-ATPase function is not due to disrupted V-ATPase assembly. Thus, Dbf2p is more likely to be a regulator of V-ATPase activity than of synthesis or assembly. Since we did not observe the classic Vma– phenotype in dbf2 cells, despite their vacuolar-acidification defect, dbf2 cells may have residual V-ATPase activity. This situation is reminiscent of the vph1 V-ATPase mutant, in which the presence of a functional homologue (Stv1p; Manolson et al., 1994) presumably confers residual function, and is consistent with slight residual vacuolar accumulation of quinacrine in Dbf2p-deficient cells. It is also possible that Dbf20p, which is redundant with Dbf2p for at least some functions (Toyn & Johnston, 1994), supports a low level of V-ATPase function in dbf2 mutant cells.
Since the phosphorylation state of Vma1p or Vma2p in either wild-type or Dbf2p-deficient cells was independent of sorbic acid stress, Dbf2p-dependent V-ATPase activity may not be regulated by sorbic acid. If Dbf2p is not part of a sorbic acid signalling pathway then Dbf2p may simply promote V-ATPase activity in a constitutive manner such that when sorbic acid is present, V-ATPase function is already optimal. Given that earlier studies have failed to identify Vma1p and Vma2p as Dbf2p substrates (Mah et al., 2005), and that we were unable to detect direct phosphorylation of Vma2p by Dbf2p in vitro, other as-yet unidentified protein(s) acting downstream of Dbf2p may carry out this function. A number of protein kinases have been identified amongst the potential substrates of Dbf2p (Mah et al., 2005; Ptacek et al., 2005), although none of these has yet been associated with V-ATPase function. However, we cannot exclude the possibility that Dbf2p phosphorylates either Vma1p or Vma2p in vivo: Dbf2p, Vma1p and Vma2p exist as components of larger complexes and their interacting proteins may be required to aid phosphorylation. Further work is required to identify the actual protein kinase(s) that directly phosphorylate Vma1p and Vma2p, and to demonstrate the significance of this phosphorylation for V-ATPase function. Other potential Dbf2p substrates include two vacuolar amino acid transporters (Avt3p and Avt4p; Ptacek et al., 2005) along with additional proteins involved in vacuolar function (Mah et al., 2005), providing further evidence for a functional link between Dbf2p and the yeast vacuole.
In summary, we have shown that both Dbf2p kinase and the V-ATPase are required for sorbic acid stress tolerance and have provided evidence that Dbf2p is required for proper V-ATPase function. VATB1 (encoding the human kidney isoform of the V-ATPase B subunit) shares 78 % identity with its yeast homologue Vma2p, and mutations in VATB1 cause distal renal tubular acidosis, a condition characterized by impaired renal acid secretion resulting in metabolic acidosis and sensorineural hearing loss (Karet et al., 1999). It will be interesting to determine whether, as we have shown in yeast, the human homologue of yeast Dbf2p (NDR1) influences the function of human V-ATPases.
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ACKNOWLEDGEMENTS |
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Edited by: D. J. Jamieson
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 1 June 2007;
revised 22 August 2007;
accepted 24 August 2007.
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), with 1 mM sorbic acid (
), with 10 mM bafilomycin A1 (
) and with a combination of 1 mM sorbic acid and 10 µM bafilomycin A1 (
).
