Threonine 22 phosphorylation attenuates Hsp90 interaction with cochaperones and affects its chaperone activity.

Heat shock protein 90 (Hsp90) is an essential molecular chaperone whose activity is regulated not only by cochaperones but also by distinct posttranslational modifications. We report here that casein kinase 2 phosphorylates a conserved threonine residue (T22) in α helix-1 of the yeast Hsp90 N-domain both in vitro and in vivo. This α helix participates in a hydrophobic interaction with the catalytic loop in Hsp90's middle domain, helping to stabilize the chaperone's ATPase-competent state. Phosphomimetic mutation of this residue alters Hsp90 ATPase activity and chaperone function and impacts interaction with the cochaperones Aha1 and Cdc37. Overexpression of Aha1 stimulates the ATPase activity, restores cochaperone interactions, and compensates for the functional defects of these Hsp90 mutants.

Swe1Wee1-dependent tyrosine phosphorylation of Hsp90 regulates distinct facets of chaperone function.

Saccharomyces WEE1 (Swe1), the only "true" tyrosine kinase in budding yeast, is an Hsp90 client protein. Here we show that Swe1(Wee1) phosphorylates a conserved tyrosine residue (Y24 in yeast Hsp90 and Y38 in human Hsp90alpha) in the N domain of Hsp90. Phosphorylation is cell-cycle associated and modulates the ability of Hsp90 to chaperone a selected clientele, including v-Src and several other kinases. Nonphosphorylatable mutants have normal ATPase activity, support yeast viability, and productively chaperone the Hsp90 client glucocorticoid receptor. Deletion of SWE1 in yeast increases Hsp90 binding to its inhibitor geldanamycin, and pharmacologic inhibition/silencing of Wee1 sensitizes cancer cells to Hsp90 inhibitor-induced apoptosis. These findings demonstrate that Hsp90 chaperoning of distinct client proteins is differentially regulated by specific posttranslational modification of a unique subcellular pool of the chaperone, and they provide a strategy to increase the cellular potency of Hsp90 inhibitors.

Benzoate and Sorbate Salts: A Systematic Review of the Potential Hazards of These Invaluable Preservatives and the Expanding Spectrum of Clinical Uses for Sodium Benzoate.

Sodium benzoate and potassium sorbate are extremely useful agents for food and beverage preservation, yet concerns remain over their complete safety. Benzoate can react with the ascorbic acid in drinks to produce the carcinogen benzene. A few children develop allergy to this additive while, as a competitive inhibitor of D-amino acid oxidase, benzoate can also influence neurotransmission and cognitive functioning. Model organism and cell culture studies have raised some issues. Benzoate has been found to exert teratogenic and neurotoxic effects on zebrafish embryos. In addition, benzoate and sorbate are reported to cause chromosome aberrations in cultured human lymphocytes; also to be potently mutagenic toward the mitochondrial DNA in aerobic yeast cells. Whether the substantial human consumption of these compounds could significantly increase levels of such damages in man is still unclear. There is no firm evidence that it is a risk factor in type 2 diabetes. The clinical administration of sodium benzoate is of proven benefit for many patients with urea cycle disorders, while recent studies indicate it may also be advantageous in the treatment of multiple sclerosis, schizophrenia, early-stage Alzheimer's disease and Parkinson's disease. Nevertheless, exposure to high amounts of this agent should be approached with caution, especially since it has the potential to generate a shortage of glycine which, in turn, can negatively influence brain neurochemistry. We discuss here how a small fraction of the population might be rendered-either through their genes or a chronic medical condition-particularly susceptible to any adverse effects of sodium benzoate.

Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37.

Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes.

Maximising the yeast chronological lifespan.

When investigating aging it is important to focus on the factors that are needed to attain, and which can be manipulated to extend, the longest lifespans. This has long been appreciated by those workers who use Drosophila or Caenorhabditis elegans as model experimental systems to study aging. Often though it seems it is not a consideration in many studies of yeast chronological aging. In this chapter I summarise how recent work has revealed the preconditioning that is needed for yeast to survive for long periods in stationary phase, therefore for it to exhibit a long chronological life span (CLS). Of critical importance in this regard is the nature of the nutrient limitation that, during the earlier growth phase, had forced the cells to undergo growth arrest. I have attempted to highlight those studies that have focussed on the longest CLSs, as this helps to identify investigations that may be addressing - not just factors that can influence chronological longevity - but those factors that are correlated with the authentic processes of chronological aging. Attempting to maximize long-term stationary survival in yeast should also enhance the potential relevance of this organism as an aging model to those who wrestle with the problems of aging in more complex systems. Finally I also give a personal perspective of how studies on the yeast CLS may still yet provide some important new insights into events that are correlated with aging.

Activity of the yeast zinc-finger transcription factor War1 is lost with alanine mutation of two putative phosphorylation sites in the activation domain.

Saccharomyces cerevisiae acquires its resistance to carboxylate weak organic acids by inducing a plasma membrane ABC transporter, Pdr12. These acids activate a Zn(II)2Cys6 zinc-finger transcription factor, War1, which in turn induces the PDR12 gene. Mutation of the four potential sites of serine/threonine phosphorylation within the War1 activation domain revealed that Pdr12 induction was lost with mutations S923A or S930A, but not with the corresponding phosphomimetic mutations S923D or S930D. However, phosphorylation at these two sites has not been detected by mass spectrometry, so it still remains uncertain whether these are true sites of phosphorylation or merely serines whose side-chain hydroxyls are necessary for the proper structuring of the War1 activation domain. Mutation S923A prevented the sorbate-induced hyperphosphorylation of War1, while S930A caused War1 to be in a constitutively hyperphosphorylated state, irrespective of weak acid stress. Screening of non-essential protein kinase mutants of yeast failed to identify a kinase required for Pdr12 induction, or War1 hyperphosphorylation, in response to sorbate treatment. However, the mrk1∆ mutant was identified as having an elevated Pdr12 level in the absence of sorbate stress.

Features of the Streptomyces hygroscopicus HtpG reveal how partial geldanamycin resistance can arise with mutation to the ATP binding pocket of a eukaryotic Hsp90.

Much attention is focused on the benzoquinone ansamycins as anticancer agents, with several derivatives of the natural product geldanamycin (GdA) now in clinical trials. These drugs are selective inhibitors of Hsp90, a molecular chaperone vital for many of the activities that drive cancer progression. Mutational changes to their interaction site, the extremely conserved ATP binding site of Hsp90, would mostly be predicted to inactivate the chaperone. As a result, drug resistance should not arise readily this way. Nevertheless, Streptomyces hygroscopicus, the actinomycete that produces GdA, has evolved an Hsp90 family protein (HtpG) that lacks GdA binding. It is altered in certain of the highly conserved amino acids making contacts to this antibiotic in crystal structures of GdA bound to eukaryotic forms of Hsp90. Two of these amino acid changes, located on one side of the nucleotide-binding cleft, weakened GdA/Hsp90 binding and conferred partial GdA resistance when inserted into the endogenous Hsp90 of yeast cells. Crystal structures revealed their main effect to be a weakening of interactions with the C-12 methoxy group of the GdA ansamycin ring. This is the first study to demonstrate that partial GdA resistance is possible by mutation within the ATP binding pocket of Hsp90.

Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex.

Hsp90 (heat shock protein of 90 kDa) is a ubiquitous molecular chaperone responsible for the assembly and regulation of many eukaryotic signalling systems and is an emerging target for rational chemotherapy of many cancers. Although the structures of isolated domains of Hsp90 have been determined, the arrangement and ATP-dependent dynamics of these in the full Hsp90 dimer have been elusive and contentious. Here we present the crystal structure of full-length yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1. The structure reveals the complex architecture of the 'closed' state of the Hsp90 chaperone, the extensive interactions between domains and between protein chains, the detailed conformational changes in the amino-terminal domain that accompany ATP binding, and the structural basis for stabilization of the closed state by p23/Sba1. Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the chaperone ATPase cycle.

Hsp90 and phosphorylation of the Slt2(Mpk1) MAP kinase activation loop are essential for catalytic, but not non-catalytic, Slt2-mediated transcription in yeast.

In yeast, the Slt2(Mpk1) stress-activated protein kinase directs the activation of two transcription factors, Rlm1 and Swi4/Swi6, in response to cell wall stress. Rlm1 is activated through a phosphorylation by Slt2, whereas the Swi4/Swi6 activation is noncatalytic and triggered by the binding of phosphorylated forms of both Slt2 and a catalytically inactive pseudokinase (Mlp1). Previous studies have delineated a role for the molecular chaperone Hsp90 in the activation of Slt2, but the involvement of Hsp90 in these events of catalytic versus non-catalytic cell integrity signaling has remained elusive. In cells lacking Mlp1, the Hsp90 inhibitor radicicol was found to inhibit the Slt2-mediated catalytic activation of Rlm1, but not the noncatalytic activation of Swi4/Swi6. Mutation of residues in the TEY motif of the Slt2 activation loop strongly impacted both Hsp90 binding and Rlm1-mediated transcription. In contrast, many of these same mutations had only modest effects on Swi4/6 (Slt2-mediated, non-catalytic) transcription, although one that blocked both the Slt2:Hsp90 interaction and Rlm1-mediated transcription (E191G) triggered a hyperactivation of Swi4/6. Taken together, our results cement the importance of the Slt2 activation loop for both the binding of Hsp90 by Slt2 and the catalytic activation of cell integrity signaling.

Resistance of yeasts to weak organic acid food preservatives.

Carboxylate weak acids are invaluable for large-scale food and beverage preservation. However, in response to safety concerns, there is now desire to reduce the use of these additives. The resistance to these compounds displayed by spoilage yeasts and fungi is a major reason why these preservatives often have to be used in millimolar levels. This chapter summarizes the mechanisms whereby yeasts are rendered resistant to acetate, propionate, sorbate, and benzoate. In baker's yeast (Saccharomyces cerevisiae), resistance to high acetic acid is acquired partly by loss of the plasma membrane aquaglyceroporin that facilitates the passive diffusional entry of undissociated acid into cells (Fps1), and partly through a transcriptional response mediated by the transcription factor Haa1. Other carboxylate preservatives are too large to enter cells through the Fps1 channel but instead penetrate at appreciable rates by passive diffusion across the plasma membrane. In Saccharomyces and Candida albicans though not, it seems, in the Zygosaccharomyces, resistance to the latter acids involves activation of the War1 transcription factor, which in turn generates strong induction of a specific plasma membrane ATP-binding cassette transporter (Pdr12). The latter actively pumps the preservative anion from the cell. Other contributors to weak acid resistance include enzymes that allow preservative degradation, members of the Tpo family of major facilitator superfamily transporters, and changes to the cell envelope that minimize the diffusional entry of the preservative into the cell.

The Fps1p aquaglyceroporin facilitates the use of small aliphatic amides as a nitrogen source by amidase-expressing yeasts.

Saccharomyces cerevisiae acquires a resistance to high, toxic levels of acetic acid by destabilizing Fps1p, the plasma membrane aquaglyceroporin through which this acid - in its undissociated state - enters the cell. In this study, Fps1p loss was shown to confer resistances to acetic acid, acrolein and allyl alcohol, not just in S. cerevisiae but also in the osmotolerant spoilage yeast Zygosaccharomyces rouxii. However, in Z. rouxii, the loss of Fps1p severely compromised the use of acetamide and several other small amides as sources of nitrogen, an indication that these amides enter the cells of this yeast by passive diffusion through the Fps1p pore. Saccharomyces cerevisiae cannot grow on acetamide, but was conferred with an ability to use this and other small amides as nitrogen sources by heterologous expression of a Z. rouxii ORF (ZrAMD1) with protein sequence identity to the amdS-encoded amidase of Aspergillus nidulans. This capacity of ZrAMD1-expressing S. cerevisiae to assimilate amide nitrogen was severely compromised by the loss of Fps1p. ZrAMD1 appears to encode the major amidase of Z. rouxii as a Zramd1Delta deletant mutant had, like the Zrfps1Delta deletant, lost the ability to assimilate small amides as sources of nitrogen.

Hog1 mitogen-activated protein kinase phosphorylation targets the yeast Fps1 aquaglyceroporin for endocytosis, thereby rendering cells resistant to acetic acid.

Aquaporins and aquaglyceroporins form the membrane channels that mediate fluxes of water and small solute molecules into and out of cells. Eukaryotes often use mitogen-activated protein kinase (MAPK) cascades for the intracellular signaling of stress. This study reveals an aquaglyceroporin being destabilized by direct MAPK phosphorylation and also a stress resistance being acquired through this channel loss. Hog1 MAPK is transiently activated in yeast exposed to high, toxic levels of acetic acid. This Hog1 then phosphorylates the plasma membrane aquaglyceroporin, Fps1, a phosphorylation that results in Fps1 becoming ubiquitinated and endocytosed and then degraded in the vacuole. As Fps1 is the membrane channel that facilitates passive diffusional flux of undissociated acetic acid into the cell, this loss downregulates such influx in low-pH cultures, where acetic acid (pKa, 4.75) is substantially undissociated. Consistent with this downregulation of the acid entry generating resistance, sensitivity to acetic acid is seen with diverse mutational defects that abolish endocytic removal of Fps1 from the plasma membrane (loss of Hog1, loss of the soluble domains of Fps1, a T231A S537A double mutation of Fps1 that prevents its in vivo phosphorylation, or mutations generating a general loss of endocytosis of cell surface proteins [doa4Delta and end3Delta]). Remarkably, targetting of Fps1 for degradation may be the major requirement for an active Hog1 in acetic acid resistance, since Hog1 is largely dispensable for such resistance when the cells lack Fps1. Evidence is presented that in unstressed cells, Hog1 exists in physical association with the N-terminal cytosolic domain of Fps1.

The Hsp90/Cdc37p chaperone system is a determinant of molybdate resistance in Saccharomyces cerevisiae.

Saccharomyces cerevisiae lacks enzymes that contain the molybdopterin co-factor and therefore any requirement for molybdenum as a trace mineral supplement. Instead, high molybdate levels are inhibitory to its growth. Low cellular levels of heat shock protein 90 (Hsp90), an essential chaperone, were found to enhance this sensitivity to molybdate. Certain Hsp90 point mutations and co-chaperone protein defects that partially compromise the function of the Hsp90/Cdc37p chaperone system also rendered S. cerevisiae hypersensitive to high molybdate levels. Sensitivity was especially apparent with mutations close to the Hsp90 nucleotide binding site, with the loss of the non-essential co-chaperone Sti1p (the equivalent of mammalian Hop), and with the abolition of residue Ser14 phosphorylation on the essential co-chaperone Cdc37p. While it remains to be proved that these effects reflect direct inhibition of the Hsp90 of the cell by the MoO(4) (2+) oxyanion in vivo; this possibility is suggested by molybdate sensitivity arising with a mutation in the Hsp90 nucleotide binding site that does not generate stress sensitivity or an impaired stress response. Molybdate sensitivity may therefore be a useful phenotype to score when studying mutations in this chaperone system.

Chaperone ligand-discrimination by the TPR-domain protein Tah1.

Tah1 [TPR (tetratricopeptide repeat)-containing protein associated with Hsp (heat-shock protein) 90] has been identified as a TPR-domain protein. TPR-domain proteins are involved in protein-protein interactions and a number have been characterized that interact either with Hsp70 or Hsp90, but a few can bind both chaperones. Independent studies suggest that Tah1 interacts with Hsp90, but whether it can also interact with Hsp70/Ssa1 has not been investigated. Amino-acid-sequence alignments suggest that Tah1 is most similar to the TPR2b domain of Hop (Hsp-organizing protein) which when mutated reduces binding to both Hsp90 and Hsp70. Our alignments suggest that there are three TPR-domain motifs in Tah1, which is consistent with the architecture of the TPR2b domain. In the present study we find that Tah1 is specific for Hsp90, and is able to bind tightly the yeast Hsp90, and the human Hsp90alpha and Hsp90beta proteins, but not the yeast Hsp70 Ssa1 isoform. Tah1 acheives ligand discrimination by favourably binding the methionine residue in the conserved MEEVD motif (Hsp90) and positively discriminating against the first valine residue in the VEEVD motif (Ssa1). In the present study we also show that Tah1 can affect the ATPase activity of Hsp90, in common with some other TPR-domain proteins.

Novel stress responses facilitate Saccharomyces cerevisiae growth in the presence of the monocarboxylate preservatives.

Certain yeasts are relatively resistant to the small number of monocarboxylic acids allowed in food preservation, with the result that these preservatives often have to be used in high concentrations in order to prevent spoilage. When grown at slightly acid pH, Saccharomyces cerevisiae acquires elevated resistance to these acids by means of discrete stress responses. Acquisition of resistance to acetic acid involves loss of Fps1p, the aquaglyceroporin of the plasma membrane that facilitates the passive diffusional entry of this acid into cells. Acetic acid stress transiently activates Hog1p mitogen-activated protein kinase, which then directly phosphorylates Fps1p in order to target this channel for endocytosis and degradation in the vacuole. Other carboxylate preservatives (propionate, sorbate or benzoate) are too large to traverse the Fps1p pore. Instead, being more lipophilic than acetic acid, they enter cells mainly by a process of non-facilitated diffusion across the plasma membrane. Once inside the cell, these acids activate War1p, a transcription factor that induces the gene for the Pdr12p plasma membrane ATP-binding cassette transporter. Pdr12p lowers the intracellular levels of propionate, sorbate or benzoate by catalysing the active efflux of the preservative anion from the cell. Still other mechanisms of weak acid resistance are found in Zygosaccharomyces, including a capacity for the oxidative degradation of sorbic and benzoic acids conferred by a mitochondrial monooxygenase, a system absent in S. cerevisiae.

UCS protein function is partially restored in the Saccharomyces cerevisiae she4 mutant with expression of the human UNC45-GC, but not UNC45-SM.

A dedicated UNC45, Cro1, She4 (UCS) domain-containing protein assists in the Hsp90-mediated folding of the myosin head. Only weak sequence conservation exists between the single UCS protein of simple eukaryotes (She4 in budding yeast) and the two UCS proteins of higher organisms (the general cell and striated muscle UNC45s; UNC45-GC and UNC45-SM, respectively). In vertebrates, UNC45-GC facilitates cytoskeletal functions, whereas the 55% identical UNC45-SM assists assembly of the contractile apparatus of cardiac and skeletal muscles. A Saccharomyces cerevisiae she4Δ mutant, totally lacking any UCS protein, was engineered to express as its sole Hsp90 either the Hsp90α or the Hsp90ß isoforms of human cytosolic Hsp90. A transient induction of the human UNC45-GC, but not UNC45-SM, could rescue the defective endocytosis in these she4Δ cells at 39 °C, irrespective of whether they possessed Hsp90α or Hsp90ß. UNC45-GC-mediated rescue of the localisation of a Myo5-green fluorescent protein (GFP) fusion to cortical patches at 39 °C was more efficient in the yeast containing Hsp90α, though this may relate to more efficient functioning of Hsp90α as compared to Hsp90ß in these strains. Furthermore, inducible expression of UNC45-GC, but not UNC45-SM, could partially rescue survival at a more extreme temperature (45 °C) that normally causes she4Δ mutant yeast cells to lyse. The results indicate that UCS protein function has been most conserved-yeast to man-in the UNC45-GC, not UNC45-SM. This may reflect UNC45-GC being the vertebrate UCS protein that assists formation of the actomyosin complexes needed for cytokinesis, cell morphological change, and organelle trafficking-events also facilitated by the myosins in yeast.

Expressed as the sole Hsp90 of yeast, the alpha and beta isoforms of human Hsp90 differ with regard to their capacities for activation of certain client proteins, whereas only Hsp90beta generates sensitivity to the Hsp90 inhibitor radicicol.

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the activity of many of the most important regulatory proteins of eukaryotic cells (the Hsp90 'clients'). Vertebrates have two isoforms of cytosolic Hsp90, Hsp90alpha and Hsp90beta. Hsp90beta is expressed constitutively to a high level in most tissues and is generally more abundant than Hsp90alpha, whereas Hsp90alpha is stress-inducible and overexpressed in many cancerous cells. Expressed as the sole Hsp90 of yeast, human Hsp90alpha and Hsp90beta are both able to provide essential Hsp90 functions. Activations of certain Hsp90 clients (heat shock transcription factor, v-src) were more efficient with Hsp90alpha, rather than Hsp90beta, present in the yeast. In contrast, activation of certain other clients (glucocorticoid receptor; extracellular signal-regulated kinase-5 mitogen-activated protein kinase) was less affected by the human Hsp90 isoform present in these cells. Remarkably, whereas expression of Hsp90beta as the sole Hsp90 of yeast rendered cells highly sensitive to the Hsp90 inhibitor radicicol, comparable expression of Hsp90alpha did not. This raises the distinct possibility that, also for mammalian systems, alterations to the Hsp90alpha/Hsp90beta ratio (as with heat shock) might be a significant factor affecting cellular susceptibility to Hsp90 inhibitors.

Weak acid and alkali stress regulate phosphatidylinositol bisphosphate synthesis in Saccharomyces cerevisiae.

Weak organic acids are used as food preservatives to inhibit the growth of spoilage yeasts, including Saccharomyces cerevisiae. Long-term adaptation to weak acids requires the increased expression of the ATP-binding cassette transporter Pdr12p, which catalyses the active efflux of the weak acids from the cytosol; however, very little is known about the signalling events immediately following application of weak acid stress. We have investigated the effects of weak acids on two stress-responsive signalling molecules, PtdIns(3,5)P2 and PtdIns(4,5)P2, which in S. cerevisiae are synthesized by Fab1p and Mss4p respectively. At low extracellular pH, benzoic acid, sorbic acid and acetic acid all cause a transient reduction in PtdIns(3,5)P2 accumulation and a more persistent rise in PtdIns(4,5)P2 levels. The increase in PtdIns(4,5)P2 levels is accompanied by a reorganization of the actin cytoskeleton. However, changes in PtdInsP2 levels are independent of weak acid-induced Pdr12p expression. In contrast, changing the extracellular medium to alkaline pH provokes a prolonged and substantial rise in PtdIns(3,5)P2 levels. As PtdIns(3,5)P2 synthesis is required for correct vacuole acidification, it is possible that levels of this molecule are modulated to maintain intracellular pH homoeostasis in response to weak acid and alkali stresses. In conclusion, we have expanded the repertoire of stress responses that affect PtdInsP2 levels to include weak acid and alkali stresses.

Global phenotypic analysis and transcriptional profiling defines the weak acid stress response regulon in Saccharomyces cerevisiae.

Weak organic acids such as sorbate are potent fungistatic agents used in food preservation, but their intracellular targets are poorly understood. We thus searched for potential target genes and signaling components in the yeast genome using contemporary genome-wide functional assays as well as DNA microarray profiling. Phenotypic screening of the EUROSCARF collection revealed the existence of numerous sorbate-sensitive strains. Sorbate hypersensitivity was detected in mutants of the shikimate biosynthesis pathway, strains lacking the PDR12 efflux pump or WAR1, a transcription factor mediating stress induction of PDR12. Using DNA microarrays, we also analyzed the genome-wide response to acute sorbate stress, allowing for the identification of more than 100 genes rapidly induced by weak acid stress. Moreover, a novel War1p- and Msn2p/4p-independent regulon that includes HSP30 was identified. Although induction of the majority of sorbate-induced genes required Msn2p/4p, weak acid tolerance was unaffected by a lack of Msn2p/4p. Ectopic expression of PDR12 from the GAL1-10 promoter fully restored sorbate resistance in a strain lacking War1p, demonstrating that PDR12 is the major target of War1p under sorbic acid stress. Interestingly, comparison of microarray data with results from the phenotypic screening revealed that PDR12 remained as the only gene, which is both stress inducible and required for weak acid resistance. Our results suggest that combining functional assays with transcriptome profiling allows for the identification of key components in large datasets such as those generated by global microarray analysis.

Mutation of the Ser18 phosphorylation site on the sole Saccharomyces cerevisiae UCS protein, She4, can compromise high-temperature survival.

Folding of the myosin head often requires the joint actions of Hsp90 and a dedicated UNC45, Cro1, She4 (UCS) domain-containing cochaperone protein. Relatively weak sequence conservation exists between the single UCS protein of simple eukaryotes (She4 in budding yeast) and the two UCS proteins of higher organisms (the general cell and smooth muscle UNC45s; UNC45-GC and UNC45-SM respectively). In vertebrates, UNC45-GC facilitates cytoskeletal function whereas the 55% identical UNC45-SM assists in the assembly of the contractile apparatus of cardiac and skeletal muscles. UNC45-SM, unlike UNC45-GC, shares with yeast She4 an IDSL sequence motif known to be a site of in vivo serine phosphorylation in yeast. Investigating this further, we found that both a non-phosphorylatable (S18A) and a phosphomimetic (S18E) mutant form of She4 could rescue the type 1 myosin localisation and endocytosis defects of the yeast she4Δ mutant at 39 °C. Nevertheless, at higher temperature (45 °C), only She4 (S18A), not She4(S18E), could substantially rescue the cell lysis defect of she4Δ mutant cells. In the yeast two-hybrid system, the non-phosphorylatable S18A and S251A mutant forms of She4 and UNC45-SM still displayed the stress-enhanced in vivo interaction with Hsp90 seen with the wild-type She4 and UNC45-SM. Such high-temperature enforcement to interaction was though lost with the phosphomimetic mutant forms (She4(S18E) and UNC45-SM (S251E)), an indication that phosphorylation might suppress these increases in She4/Hsp90 and UNC45-SM/Hsp90 interaction with stress.