Identification of Ald6p as the target of a class of small-molecule suppressors of FK506 and their use in network dissection.
FK506 inhibits the Ca2+/calmodulin-dependent protein phosphatase calcineurin, which plays a critical role in yeast subjected to salt stress. A chemical genetic screen for small molecules that suppress growth inhibition by high NaCl plus FK506 identified a structurally related class of suppressors of FK506 (SFKs) named SFKs 2-4. To identify possible protein targets for these small molecules, a genome-wide screen of approximately 4,700 haploid yeast deletion strains was undertaken for strains showing resistance to high NaCl plus FK506. This screen yielded a number of genes not previously implicated in salt stress, including ALD6, which encodes an NADP(+)-dependent aldehyde dehydrogenase, and UTR1, which encodes an NAD+ kinase. Transcriptional profiling of yeast treated with SFK2 indicated that the SFKs target the Ald6p pathway. In addition, screening of the deletion strains for hypersensitivity to SFK2 yielded ZWF1, encoding glucose-6-phosphate dehydrogenase, which has been shown to play an overlapping role with Ald6p in NADPH production. Furthermore, the SFKs inhibited the activity of Ald6p in vitro. Having established that the SFKs target Ald6p, they were used as tools to implicate systematically other gene products in the Ald6p pathway, including Utr1p, which may function by supplying Ald6p with its NADP+ cofactor. Furthermore, growth improvement by the SFKs on high NaCl plus FK506 was shown to require GPD1, which encodes an NADH-dependent glycerol-3-phosphate dehydrogenase that is important for the production of glycerol in response to osmotic stress.
Butcher
Rebecca A
RA
Department of Chemistry and Chemical Biology and Howard Hughes Medical Institute, Harvard University, Cambridge, MA 02138, USA.
Schreiber
Stuart L
SL
D000444:Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D020869:Gene Expression Profiling; D016681:Genome, Fungal; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006238:Haploidy; D009249:NADP / Q000378:metabolism; D009997:Osmotic Pressure; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D016559:Tacrolimus / Q000037:antagonists & inhibitors* / Q000494:pharmacology
D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D012965:Sodium Chloride; D009249:NADP; D005954:Glucosephosphate Dehydrogenase; D000445:Aldehyde Oxidoreductases; D000444:Aldehyde Dehydrogenase; C018699:aldehyde dehydrogenase (NAD(P)+); D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase; C487503:UTR1 protein, S cerevisiae; D005990:Glycerol; D016559:Tacrolimus