Saccharomyces cerevisiae PIP2 mediating oleic acid induction and peroxisome proliferation is regulated by Adr1p and Pip2p-Oaf1p.

Saccharomyces cerevisiae genes involved in fatty acid degradation contain in their promoters oleate response elements (OREs) and type 1 upstream activation sequences (UAS1s) that bind Pip2p-Oaf1p and Adr1p, respectively. The promoter of the PIP2 gene was found to contain a potential UAS1 that consists of a tandem array of CYCCRR half-sites in an overlapping arrangement with a previously characterized ORE. Electrophoretic mobility shift analysis demonstrated that Adr1p bound to UAS1PIP2, and Northern analysis in combination with a lacZ reporter gene confirmed that Adr1p influenced the transcription of PIP2. Immunoprecipitation showed that, in adr1delta mutant cells grown on oleic acid, Pip2p was less abundant compared with the corresponding wild-type. In addition, the amount of Pip2p-Oaf1p that bound to a target ORE in vitro was reduced in mutant extracts compared with the wild-type. Transcription of the oleic acid-inducible genes SPS19 and CTA1, which rely on both Pip2p-Oaf1p and Adr1p for their regulation, was reduced in adr1delta mutant cells. However, by ectopically restoring levels of Pip2p in adr1delta cells grown on oleic acid medium, transcription of both genes increased 2-fold compared with the control. This partial suppression of the adr1delta mutant phenotype was additionally manifested by moderate utilization of oleic acid. Hence, both the expression as well as the action of the two transcription factors, Adr1p and Pip2p-Oaf1p, are interconnected, which allows for an elaborate control of fatty acid-inducible genes.

Saccharomyces cerevisiae Pip2p-Oaf1p regulates PEX25 transcription through an adenine-less ORE.

The role of the Saccharomyces cerevisiae Pip2p-Oaf1p transcription factor was examined in reference to the regulation of the peroxin gene PEX25 involved in peroxisome proliferation. The PEX25 promoter contains an oleate response element (ORE)-like sequence comprising a CGG palindrome lacking a canonical adenine, which is considered critical for element function and Pip2p-Oaf1p binding. Pex25p levels were higher in wild-type cells grown on oleic acid medium than in those grown on ethanol, but this induction was abolished in cells devoid of Pip2p-Oaf1p. Studies based on lacZ reporter genes and in vitro protein-DNA interactions revealed that the PEX25 ORE could bind Pip2p-Oaf1p and confer activation on a basal promoter. These findings reinforced the central role played by Pip2p-Oaf1p in regulating peroxisome proliferation. We also investigated whether Pip2p-Oaf1p is important for regulating genes encoding peroxins involved in protein import into the peroxisomal matrix. Pip2p-Oaf1p was able to bind efficiently to the PEX5 ORE but not to an ORE-like CGG palindrome in the PEX14 promoter. However, immunoblotting revealed that both Pex5p and Pex14p (as well as Pex7p and Pex13p) were not more abundant in cells grown on oleic acid medium compared with ethanol. These data on a functional, adenine-less, PEX25 ORE and a nonfunctional N13-spaced ORE-like sequence in the PEX14 promoter capable of binding Pip2p-Oaf1p prompts readjustment of the ORE consensus to comprise CGGN3TNA/(R)N8-12CCG.

The peroxisomal transporter gene ANT1 is regulated by a deviant oleate response element (ORE): characterization of the signal for fatty acid induction.

Saccharomyces cerevisiae ANT1/YPR128c encodes the peroxisomal adenine nucleotide transporter that provides ATP for intra-peroxisomal activation of medium-chain fatty acids. A lacZ reporter construct comprising the ANT1 promoter was shown to be comparatively more highly expressed in a wild-type strain grown on oleic acid, a long-chain fatty acid, than in pip2Delta(oaf1)Delta mutant cells that are defective in fatty acid induction. The ANT1 promoter was demonstrated to contain a deviant oleate response element (ORE) that could bind the Pip2p-Oaf1p transcription factor and confer activation on a basal CYC1-lacZ reporter gene. Expression of Ant1p as well as other enzymes whose genes are known to be regulated by a canonical ORE was found to be increased in cells grown on lauric acid, a medium-chain fatty acid. We concluded that the signal for induction does not differentiate between long- and medium-chain fatty acids. This signal was independent of beta-oxidation or the biogenesis of the peroxisomal compartment where this process occurs, since a pox1Delta strain blocked in the first and rate-limiting step of beta-oxidation as well as various pex mutant cells devoid of intact peroxisomes produced sufficient amounts of Pip2p-Oaf1p for binding OREs in vitro and for expressing an ORE-driven reporter gene. The signal's durability was shown to be related to the concentration of fatty acids in the medium, since a pex6Delta strain expressed an ORE-driven reporter gene at high levels for a longer period than did isogenic wild-type cells. Generation of the signal was also independent of protein synthesis, as demonstrated by cycloheximide treatment.

Acute glucose starvation activates the nuclear localization signal of a stress-specific yeast transcription factor.

In yeast, environmental conditions control the transcription factor Msn2, the nuclear accumulation and function of which serve as a sensitive indicator of nutrient availablity and environmental stress load. We show here that the nuclear localization signal (NLS) of Msn2 is a direct target of cAMP-dependent protein kinase (cAPK). Genetic analysis suggests that Msn2-NLS function is inhibited by phosphorylation and activated by dephosphorylation. Msn2-NLS function is unaffected by many stress conditions that normally induce nuclear accumulation of full-length Msn2. The Msn2-NLS phosphorylation status is, however, highly sensitive to carbohydrate fluctuations during fermentative growth. Dephosphorylation occurs in >2 min after glucose withdrawal but the effect is reversed rapidly by refeeding with glucose. This response to glucose depletion is due to changes in cAPK activity rather than an increase in protein phosphatase activity. Surprisingly, the classical glucose-sensing systems are not connected to this rapid response system. Our results further imply that generic stress signals do not cause short-term depressions in cAPK activity. They operate on Msn2 by affecting an Msn5-dependent nuclear export and/or retention mechanism.

Preliminary characterisation of DML1, an essential Saccharomyces cerevisiae gene related to misato of Drosophila melanogaster.

A genetic and cell-biological analysis is provided for Saccharomyces cerevisiae DML1 (YMR211w) encoding a Drosophila melanogaster Misato-like protein. Misato and Dml1p are descendants of an ancestral tubulin-like protein, and exhibit regions with similarity to members of a GTPase family that include eukaryotic tubulin and prokaryotic FtsZ. Deletion of DML1 was lethal to haploid cells; sporulated DML1/dml1Delta heterozygotes from different genetic backgrounds gave rise to no more than two viable spores per tetrad. DAPI staining for DNA in combination with Southern analysis using the mitochondrial genes COX3, 15S_rRNA_2, and COB revealed that a significant portion of the surviving meiotic progeny were [rho(0)] lacking mtDNA. In addition, meiotic transmission of centromeric plasmids also appeared to be impaired. Self-complementation using extra-chromosomal copies of DML1 efficiently restored meiotic inheritance of mtDNA, but improved spore viability ratios only in part. Inheritance of mtDNA could also be restored using misato cDNA. Unscheduled expression of DML1 tethered to the inducible ADH2 promoter altered both mitochondrial dispersion and general cell morphology. We propose that Dml1p and Misato have been co-opted into a role in mtDNA inheritance in yeast, and into a cell division-related mechanism in flies, respectively. Dml1p might additionally function in the partitioning of the mitochondrial organelle itself, or in the segregation of chromosomes, thereby explaining its essential requirement.