Snf1 dependence of peroxisomal gene expression is mediated by Adr1.

Eukaryotes utilize fatty acids by beta-oxidation, which occurs in the mitochondria and peroxisomes in higher organisms and in the peroxisomes in yeast. The AMP-activated protein kinase regulates this process in mammalian cells, and its homolog Snf1, together with the transcription factors Adr1, Oaf1, and Pip2, regulates peroxisome proliferation and beta-oxidation in yeast. A constitutive allele of Adr1 (Adr1(c)) lacking the glucose- and Snf1-regulated phosphorylation substrate Ser-230 was found to be Snf1-independent for regulation of peroxisomal genes. In addition, it could compensate for and even suppress the requirement for Oaf1 or Pip2 for gene induction. Peroxisomal genes were found to be regulated by oleate in the presence of glucose, as long as Adr1(c) was expressed, suggesting that the Oaf1/Pip2 heterodimer is Snf1-independent. Consistent with this observation, Oaf1 binding to promoters in the presence of oleate was not reduced in a snf1Delta strain. Exploring the mechanism by which Adr1(c) permits Snf1-independent peroxisomal gene induction, we found that strength of promoter binding did not correlate with transcription, suggesting that stable binding is not a prerequisite for enhanced transcription. Instead, enhanced transcriptional activation and suppression of Oaf1, Pip2, and Snf1 by Adr1(c) may be related to the ability of Adr1(c) to suppress the requirement for and enhance the recruitment of transcriptional coactivators in a promoter- and growth medium-dependent manner.

Snf1 controls the activity of adr1 through dephosphorylation of Ser230.

The transcription factors Adr1 and Cat8 act in concert to regulate the expression of numerous yeast genes after the diauxic shift. Their activities are regulated by Snf1, the yeast homolog of the AMP-activated protein kinase of higher eukaryotes. Cat8 is regulated directly by Snf1, but how Snf1 regulates Adr1 is unknown. Mutations in Adr1 that alleviate glucose repression are clustered between amino acids 227 and 239. This region contains a consensus sequence for protein kinase A, RRAS(230)F, and Ser230 is phosphorylated in vitro by both protein kinase A and Ca(++) calmodulin-dependent protein kinase. Using an antiphosphopeptide antibody, we found that the level of Adr1 phosphorylated on Ser230 was highest in glucose-grown cells and decreased in a Snf1-dependent manner when glucose was depleted. A nonphosphorylatable Ser230Ala mutant was no longer Snf1 dependent for activation of Adr1-dependent genes and could suppress Cat8 dependence at genes coregulated by Adr1 and Cat8. Contrary to expectation, neither protein kinase A (PKA) nor Ca(++) calmodulin-dependent protein kinase appeared to have an important role in Ser230 phosphorylation in vivo, and a screen of 102 viable kinase deletion strains failed to identify a candidate kinase. We conclude that either Ser230 is phosphorylated by multiple protein kinases or its kinase is encoded by an essential gene. Using the Ser230Ala mutant, we explain a long-standing observation of synergy between Adr1 constitutive mutants and Snf1 activation and conclude that dephosphorylation of Ser230 via a Snf1-dependent pathway appears to be a major component of Adr1 regulation.

Phenomic and transcriptomic analyses reveal that autophagy plays a major role in desiccation tolerance in Saccharomyces cerevisiae.

Saccharomyces cerevisiae can survive extreme desiccation, but the molecular mechanisms are poorly understood. To define genes involved in desiccation tolerance, two complementary genome-wide approaches, phenomics and transcriptomics, have been used, together with a targeted analysis of gene deletion mutants tested individually for their ability to survive drying. Genome-wide phenotypic analyses carried out on a pooled library of single-gene deletion mutants subjected to three cycles of desiccation and re-growth to post-diauxic phase identified about 650 genes that contributed to strain survival in the drying process. Air-drying desiccation-tolerant post-diauxic phase cells significantly altered transcription in 12% of the yeast genome, activating expression of over 450 genes and down-regulating 330. Autophagy processes were significantly over-represented in both the phenomics study and the genes up-regulated on drying, indicating the importance of the clearance of protein aggregates/damaged organelles and the recycling of nutrients for the survival of desiccation in yeast. Functional carbon source sensing networks governed by the PKA, Tor and Snf1 protein kinase complexes were important for the survival of desiccation, as indicated by phenomics, transcriptomics, and individual analyses of mutant strains. Changes in nitrogen metabolism were evident during the drying process and parts of the environmental stress response were activated, repressing ribosome production and inducing genes for coping with oxidative and osmotic stress.

Intracellular trehalose is neither necessary nor sufficient for desiccation tolerance in yeast.

Trehalose is thought to be important for desiccation tolerance in a number of organisms, including Saccharomyces cerevisiae, but there is limited in vivo evidence to support this hypothesis. In wild-type yeast, the degree of desiccation tolerance has been shown previously to increase in cultures after diauxic shift and also in exponential-phase cultures after exposure to heat stress. Under both these conditions, increased survival of desiccation correlates with elevated intracellular trehalose concentrations. Our data confirm these findings, but we have tested the apparent importance of trehalose using mutant strains with a deleted trehalose-6-phosphate synthase gene (tps1Delta). Although tps1Delta strains do not produce trehalose, they are nevertheless capable of desiccation tolerance, and the degree of tolerance also increases after diauxic shift or heat stress, albeit slightly less than in the wild type. Conversely, when wild-type yeast is subjected to osmotic stress, mid-exponential-phase cultures produce high concentrations of intracellular trehalose but show little improvement in desiccation tolerance. These results show that there is no consistent relationship between intracellular trehalose levels and desiccation tolerance in S. cerevisiae. Trehalose seems to be neither necessary nor sufficient for, although in some strains might quantitatively improve, survival of desiccation, suggesting that other adaptations are more important.