Sequencing of the Saccharomyces cerevisiae genome has revealed that this organism has no less than 55 putative transcription factors of the Zn(II)2Cys6 zinc cluster family. Functions have now been assigned to several of these, involving processes ranging from primary and secondary metabolism, drug resistance to meiotic development (MacPherson et al., 2006). War1 is the zinc cluster family member found to be involved in resistance to moderately lipophilic weak organic acids, notably the carboxylate food preservatives sorbate, benzoate and propionate (Kren et al., 2003; Schüller et al., 2004). When activated in cells exposed to weak acid stress War1 directs a strong induction of the PDR12 gene, causing the Pdr12 ABC transporter to accumulate to high levels in the yeast plasma membrane (Piper et al., 2001). Pdr12 confers weak acid resistance by acting as a carboxylate anion efflux pump (Hatzixanthis et al., 2003; Mollapour et al., 2008; Piper et al., 1998). Whilst mostly studied from the standpoint of its ability to confer resistance to food preservatives, Pdr12 has a normal physiological role in facilitating the export of certain aromatic and branched-chain organic acids that are toxic side products of amino acid catabolism (Hazelwood et al., 2006).
War1 is nuclear in localization and bound constitutively, through its N-terminal Zn(II)2Cys6 domains, to 5′-CCG-N(23)-CGG-3′ repeat sequences of the PDR12 promoter (Kren et al., 2003). Gain-of-function mutations have been found to cause a still higher affinity for this promoter and to map to the central region of War1 (Gregori et al., 2008). No evidence has yet emerged for any upstream component being involved in the signalling weak acid stress to War1, indicating that this transcription factor may be activated through the direct binding of the weak acid anion (Hatzixanthis et al., 2003; Schüller et al., 2004).
Many yeast Zn(II)2Cys6 transcription factors undergo dramatic phosphorylation changes upon activation. However, in most cases it is not known whether these phosphorylations are required for the activation or are the result of it (Leverentz and Reece, 2006). Gal4, the best-studied Zn(II)2Cys6 transcription factor, is phosphorylated on multiple sites, its phosphorylation by Srb10 on Ser699 being required for full induction of its transcriptional activity (Rohde et al., 2000). War1 also becomes extensively hyperphosphorylated in response to sorbic acid (Gregori et al., 2008; Kren et al., 2003), yet so far no protein kinase has been found essential for its activation. Recently these phosphorylation sites on War1 were analysed by semiquantitative mass spectrometry and found to be downstream of the zinc finger domains but upstream of the activation domain (AD) (Frohner et al., 2010). Remarkably, while the mutation of these phosphoresidues affected War1 hyperphosphorylation and the kinetics of the adaptive response to weak acid stress, it had little effect on the activation of War1 (Frohner et al., 2010). This analysis did not detect any phosphoryations within the War1 AD. We nevertheless decided to investigate the effects of mutating the four potential sites of phosphorylation within this domain. We also screened the collection of non-essential protein kinase mutants for altered War1 activity. Here we show that alanine mutation of two of serines in the War1 AD severely compromises War1 activation, these mutations having dramatically opposite effects on the hyperphosphorylation of War1. However, in the absence of corroborative mass spectrometry data it remains uncertain whether these are true sites of phosphorylation, or merely serine hydroxyl groups that are required for the correct structuring and functioning of the War1 AD.
Materials and methods
Construction of War1 expression vectors
Mutations were introduced into a TOPO cloning kit (Invitrogen) cloned WAR1 gene by site-directed mutagenesis (primer sequences available on request) and confirmed by dye-terminator sequencing. They were then ligated into the LEU2 vector YEp81 (Mollapour and Piper, 2007), so as to give vectors for MET25 promoter regulated expression of these mutant forms of War1.
Yeast strains and culture
A war1∆kanMX4 mutant version of strain BJ2168 (MATaura3–52 leu2-Δ1 trp1-Δ63 prb1–1122 prc-1–407 pep4–3) was constructed, then cotransformed with the above LEU2 vectors for wild-type or mutant War1 expression, also a previously described URA3 episomal centromeric vector that contains a LacZ reporter gene under control of the −681 to −1 region of the PDR12 promoter (Hatzixanthis et al., 2003). Transformants were grown on standard defined (SD) (Adams et al., 1997) minus leucine, uracil and methionine 2% glucose medium, adjusted to pH 4.5 prior to autoclaving, (Mollapour and Piper, 2007), so as to ensure plasmid maintenance and the expression of the plasmid-borne WAR1 gene, then treated with 1 mm sorbate for the indicated time.
Homozygous diploid strains of BY4743 genetic background deleted for non-essential protein kinase genes were from EUROSCARF (http://web.uni-frankfurt.de/fb15/mikro/euroscarf/). These were transformed with the above URA3 vector for PDR12 promoter–lacZ expression, then grown on pH 4.5 SD minus uracil 2% glucose medium (Adams et al., 1997), then treated with 1 mm sorbate for the indicated time.
Measurements of PDR12 promoter activity; also Pdr12 and War1 levels
PDR12 promoter–lacZ fusion expression (mean and standard deviation of eight assays) was determined as previously described (Hatzixanthis et al., 2003). Pdr12 and War1 were visualized on immunoblots using anti-Pdr12 (Piper et al., 1998) and anti-War1 (Gregori et al., 2007; Kren et al., 2003) antisera and the ECL chemiluminescence detection system (GE Healthcare, UK).
Mutations in the War1 AD affect Pdr12 level and Pdr12 promoter activity
Earlier detailed mass spectrometry analysis did not identify any phosphorylation of the War1 AD (Frohner et al., 2010). It is possible therefore that either the phosphorylation of this domain does not occur or that it is extremely transient. There are four potential sites of serine/threonine phosphorylation in this AD region (Figure 1a). Each was mutated to alanine, and these mutant forms then inserted under MET25 promoter control, together with a PDR12 promoter–LacZ reporter gene vector, into strain BJ2168 war1∆. Two of these War1 mutations (S923A and S930A) substantially reduced both basal and sorbate-induced PDR12 promoter–LacZ activity (Figure 1b). Consistent with them abrogating the WAR1 gene rescue of the sorbate sensitivity of war1 mutant cells (Figure 1c), they depleted both the basal and the sorbate-induced levels of Pdr12 (Figure 1d). In contrast War1 mutations T919A and T937A did not affect Pdr12 level, or cause sorbate sensitivity (Figures 1c, d).
Western blot analysis of War1 revealed that these S923A and S930A mutations were not resulting in the destabilization of War1. Instead, they exerted opposing effects on War1 gel mobility (Figure 1d). S923A prevented the sorbate-induced up-shift of War1 on gels, symptomatic of hyperphosphorylation. In contrast, the S930A mutant form of War1 formed a single slow-migrating band, even when isolated from unstressed cells (Figure 1d), indicating this mutant form to be constitutively hyperphosphorylated even in the absence of weak acid stress.
We also investigated the effects of mutating the four potential sites of serine/threonine phosphorylation in this AD region (Figure 1a) to phosphomimic residues (Figure 2). Neither T919E, S930D or S923D prevented Pdr12 induction by sorbate, unlike T937E, which appeared to result in an appreciable Pdr12 expression, even in the absence of sorbate stress (Figure 2).
Screening of the non-essential protein kinase deletion mutants for altered War1 activity
War1 is thought to be the sole activator of the PDR12 gene promoter (Kren et al., 2003; Schüller et al., 2004). Therefore, in seeking a protein kinase that might be a regulator of this transcription factor, we screened strains deleted for non-essential protein kinase genes (109 GO-slim ontology; SGD; http://www.yeastgenome.org/) for altered expression of a PDR12 promoter–LacZ fusion reporter gene. Basal and sorbate-induced LacZ expression was measured in homozygous gene deletion mutants of BY4743 genetic background. Almost all of the strains exhibited wild-type levels of PDR12 promoter–LacZ activity (data not shown). We chose for further analysis the six strains where the basal or the sorbate-induced LacZ activity deviated > 30% from that in the BY4743 wild-type (Figure 3a). These included one mutant (dbf2∆) that had been identified in two earlier studies as exhibiting a reduced tolerance of sorbate stress (Makrantoni et al., 2007; Mollapour et al., 2004).
The Pdr12 and War1 levels of these six deletion strains were analysed on immunoblots. This indicated that none of these six mutant strains were defective in the induction of Pdr12 by sorbate (Figure 3b). However one mutant (mrk1∆) exhibited an elevated Pdr12 level in the absence of sorbate stress (Figure 3b). This may reflect an increased Pdr12 protein stability in this mutant, since it was not reflected in any increase in PDR12 promoter–LacZ expression levels (Figure 3a), and Mrk1 has been implicated in regulation of the E3 ubiquitin ligase involved in endocytosis of plasma membrane proteins, Rsp5 (see Discussion).
War1 undergoes dramatic changes in phosphorylation in response to weak organic acid stress (Gregori et al., 2008; Kren et al., 2003). In all six of these protein kinase deletion strains, War1 exhibited the normal sorbate-induced shifts to these slower mobility, hyperphosphorylated forms (Figure 3b). Therefore, our screen did not identify a protein kinase mutant that was defective in either Pdr12 induction or War1 hyperphosphorylation in response to sorbate. It suggests, therefore, that these events either require one of the few essential protein kinases of yeast or are able to be catalysed by more than one non-essential protein kinase (see Discussion).
The induction of the Pdr12 transporter in response to weak acid stress is not due to any increased WAR1 gene transcription (Kren et al., 2003), but rather to an activation of the War1 transcription factor constitutively bound to the promoter of the PDR12 gene. Commensurate with this induction, War1 also becomes hyperphosphorylated – an event that is reflected in a retarded gel mobility (Figures 1, 3). There is an appreciable basal phosphorylation of War1 even in unstressed cells, yet this phosphorylation is markedly increased still further with the War1 activation caused by sorbate stress (Frohner et al., 2010). However, mutation of the phosphorylation sites that are involved in this process has revealed that the hyperphosphorylation of War1 is not essential for the activation process(Frohner et al., 2010), even though a loss-of-function War1 variant does not show the characteristic gel upshift following weak acid stress (Gregori et al., 2007; Kren et al., 2003).
In this study we attempted to identify protein kinases that affect War1 activity. Our screen of the non-essential protein kinase mutants did not identify a kinase that was essential for either Pdr12 induction or War1 hyperphosphorylation, in response to sorbate stress. Others have recently reported a similar finding (Frohner et al., 2010). It would appear, therefore, that either these events require one of the few essential protein kinases of yeast or that there is appreciable redundancy amongst the non-essential kinases that can perform them. Our screen identified the mrk1∆ mutant as having elevated levels of Pdr12 in the absence of sorbate stress (Figure 3b). This might reflect a compromised Pdr12 turnover by endocytosis in this mutant, since MRK1 is one of the four genes for glycogen synthase kinase 3 (GSK-3) homologues (the others being MCK1, RIM11 and YOL128c). GSK-3 is a known regulator of Rsp5 (Andoh et al., 2000), the single essential ubiquitin ligase catalysing an attachment of non-conventional ubiquitin chains, linked through ubiquitin Lys-63 to plasma membrane proteins in S. cerevisiae. This Rsp5 modification is required for efficient endocytosis of plasma membrane proteins and the sorting of many endocytic and multivesicular body cargoes (Belgareh-Touze et al., 2008).
Many Zn(II)2Cys6 transcription factors migrate as two, or several, distinct bands in their inactive state, only to then shift to higher molecular mass forms as they undergo activation (Leverentz and Reece, 2006). Of the Zn(II)2Cys6 transcription factors of yeast, Gal4 has been the most extensively studied. Whilst its DNA recognition is well understood, even for this exhaustively investigated Gal4 a number of uncertainties still remain as to the precise role of this phosphorylation and how the AD interacts with the transcription initiation machinery. Moreover, in the case of War1 the phosphorylations that generate this hyperphosphorylated state are not critical for the activation process (Frohner et al., 2010). Here we undertook to analyse the potential sites of serine and threonine phosphorylation in the War1 AD. We show that alanine mutation of two such sites (S923 and S930) severely abrogates War1 activation and dramatically affects War1 hyperphosphorylation (Figure 1d). These serine residues are therefore critical for these events. However, no phosphorylation at these two sites has been detected (Frohner et al., 2010; Stark et al., 2010), raising the tantalizing issue of whether these are indeed authentic sites of phosphorylation or merely serines whose side-chain hydroxyl groups are necessary for the proper structuring and functioning of the War1 AD.
We are indebted to Karl Kuchler for extensive discussions and the gifts of materials. Supported by BBSRC Grant No. 31/D17868.