Cytoplasmic redox imbalance in the thioredoxin system activates Hsf1 and results in hyperaccumulation of the sequestrase Hsp42 with misfolded proteins.

Cells employ multiple systems to maintain homeostasis when experiencing environmental stress. For example, the folding of nascent polypeptides is exquisitely sensitive to proteotoxic stressors including heat, pH, and oxidative stress, and is safeguarded by a network of protein chaperones that concentrate potentially toxic misfolded proteins into transient assemblies to promote folding or degradation. The redox environment itself is buffered by both cytosolic and organellar thioredoxin and glutathione pathways. How these systems are linked is poorly understood. Here, we determine that specific disruption of the cytosolic thioredoxin system resulted in constitutive activation of the heat shock response in Saccharomyces cerevisiae and accumulation of the sequestrase Hsp42 into an exaggerated and persistent juxtanuclear quality control (JUNQ) compartment. Terminally misfolded proteins also accumulated in this compartment in thioredoxin reductase (TRR1)-deficient cells, despite apparently normal formation and dissolution of transient cytoplasmic quality control (CytoQ) bodies during heat shock. Notably, cells lacking TRR1 and HSP42 exhibited severe synthetic slow growth exacerbated by oxidative stress, signifying a critical role for Hsp42 under redox-challenged conditions. Finally, we demonstrated that Hsp42 localization patterns in trr1∆ cells mimic those observed in chronically aging and glucose-starved cells, linking nutrient depletion and redox imbalance with management of misfolded proteins via a process of long-term sequestration.

Yeast transcription factor Msn2 binds to G4 DNA.

Sequences capable of forming quadruplex or G4 DNA are prevalent in the promoter regions. The transformation from canonical to non-canonical secondary structure apparently regulates transcription of a number of human genes. In the budding yeast Saccharomyces cerevisiae, we identified 37 genes with a G4 motif in the promoters including 20 genes that contain stress response element (STRE) overlapping a G4 motif. STRE is the binding site of stress response regulators Msn2 and Msn4, transcription factors belonging to the C2H2 zinc-finger protein family. We show here that Msn2 binds directly to the G4 DNA structure through its zinc-finger domain with a dissociation constant similar to that of STRE-binding and that, in a stress condition, Msn2 is enriched at G4 DNA-forming loci in the yeast genome. For a large fraction of genes with G4/STRE-containing promoters, treating with G4-ligands led to significant elevations in transcription levels. Such transcriptional elevation was greatly diminished in a msn2Δ msn4Δ background and was partly muted when the G4 motif was disrupted. Taken together, our data suggest that G4 DNA could be an alternative binding site of Msn2 in addition to STRE, and that G4 DNA formation could be an important element of transcriptional regulation in yeast.

Pan2-Pan3 complex, together with Ccr4-Not complex, has a role in the cell growth on non-fermentable carbon sources.

There are two major deadenylase complexes, Ccr4-Not and Pan2-Pan3, which shorten the 3' poly(A) tail of mRNA and are conserved from yeast to human. We have previously shown that the Ccr4-mediated deadenylation plays the important role in gene expression regulation in the yeast stationary phase cell. In order to further understand the role of deadenylases in different growth condition, in this study we investigated the effect of deletion of both deadenylases on the cell in non-fermentable carbon containing media. We found that both ccr4Δ and ccr4Δ pan2Δ mutants showed similar growth defect in YPD media: when switched to media containing non-fermentable source (Glycerol-Lactate) only the ccr4Δ grew while the ccr4Δ pan2Δ did not. Ccr4, Pan2, and Pan3 were phosphorylated in GlyLac medium, suggesting that the activities of Ccr4, Pan2, and Pan3 may be regulated by phosphorylation in response to change of carbon sources. To get insights how Ccr4 and Pan2 function in the cell growth in media containing non-fermentable source only, we isolated multicopy suppressors for the growth defect on YPGlyLac media of the ccr4Δ pan2Δ mutant and identified two genes, STM1 and REX2, which encode a ribosome-associated protein and a 3'-5' RNA exonuclease, respectively. Our results suggest that the Pan2-Pan3 complex, together with the Ccr4-Not complex, has important roles in the growth on non-fermentable carbon sources.

Pbp1 mediates the aberrant expression of genes involved in growth defect of ccr4∆ and pop2∆ mutants in yeast Saccharomyces cerevisiae.

CCR4 and POP2 genes encode the catalytic subunit of the Ccr4-Not complex involved in shortening mRNA poly(A) tail in Saccharomyces cerevisiae. The ccr4Δ and pop2∆ mutants exhibit pleiotropic phenotypes such as slow and temperature-sensitive growth, aberrant expression of glucose repression genes and abnormal cell wall synthesis. We previously found that the growth defect of the ccr4Δ and pop2∆ mutants is suppressed by deletion of the PBP1 gene, which encodes poly(A)-binding protein (Pab1)-binding protein 1. In this study, we investigated the functional relationship between Ccr4/Pop2 and Pbp1 by measuring changes in gene expression in ccr4Δ and pop2∆ single mutants and ccr4Δ pbp1∆ and pop2∆ pbp1∆ double mutants. We found that expression of HSP12, HSP26, PIR3, FUS1 and GPH1 was increased in ccr4Δ and pop2∆ single mutants. The pbp1∆ mutation not only restored the growth defect but also reduced the increased expression of those genes found in the ccr4Δ and pop2∆ mutants. Over-expression of PBP1 in the ccr4Δ mutant further increased the expression of HSP12, HSP26, PIR3 and FUS1 and exacerbated the cell growth. These results suggest that the aberrant expression of a subset of genes, which is facilitated by Pbp1, contributes to the pleiotropic phenotypes of the ccr4Δ and pop2∆ mutants.

Regulation of LRG1 expression by RNA-binding protein Puf5 in the budding yeast cell wall integrity pathway.

The PUF RNA-binding protein Puf5 is involved in regulation of the cell wall integrity (CWI) pathway in yeast. Puf5 negatively regulates expression of LRG1 mRNA, encoding for a GTPase-activating protein for Rho1 small GTPase. Here, we further analyzed the effect of Puf5 on LRG1 expression, together with Ccr4 and Pop2 deadenylases, Dhh1 decapping activator, and other PUF proteins. We found that the growth defect of puf5∆ mutant was enhanced by ccr4∆ mutation, which was partially suppressed by LRG1 deletion. Consistently, Lrg1 protein level was much more up-regulated in ccr4Δ puf5Δ double mutant than in each single mutant. Interestingly, LRG1 poly(A) tail length was longer in ccr4∆ mutant but not in puf5∆ mutant. Thus, Puf5 regulates LRG1 expression independently from Ccr4, although Puf5 recruits the Ccr4-Not deadenylase complex for mRNA destabilization. Unexpectedly, puf6Δ mutation suppressed the growth defect caused by ccr4Δ puf5∆ mutation. Loss of Rpl43a and Rpl43b ribosomal proteins, the previously identified Puf6 interactors, also suppressed the growth defect of ccr4Δ puf5Δ mutant. Our results suggest that Puf5 functions in the CWI pathway by regulating LRG1 expression in a deadenylase-independent manner, and that Puf6 is involved in the Ccr4- and Puf5-mediated regulation of cell growth through association with Rpl43.

Cytoplasmic deadenylase Ccr4 is required for translational repression of LRG1 mRNA in the stationary phase.

Ccr4 is a major cytoplasmic deadenylase involved in mRNA poly(A) tail shortening in Saccharomyces cerevisiae. We have previously shown that Ccr4 negatively regulates expression of LRG1 mRNA encoding a GTPase-activating protein for the small GTPase Rho1, a component of cell wall integrity pathway, and deletion of LRG1 suppresses the temperature-sensitive growth defect of the ccr4Δ mutant. We have also shown that the slow growth of the ccr4Δ mutant is suppressed by deletion of another gene, PBP1, encoding a poly(A)-binding protein (Pab1)-binding protein 1; however, the underlying mechanism still remains unknown. In this study, we investigated how ccr4Δ and pbp1Δ mutations influence on the length of poly(A) tail and LRG1 mRNA and protein levels during long-term cultivation. In the log-phase ccr4Δ mutant cells, LRG1 poly(A) tail was longer and LRG1 mRNA level was higher than those in the log-phase wild-type (WT) cells. Unexpectedly, Lrg1 protein level in the ccr4Δ mutant cells was comparable with that in WT. In the stationary-phase ccr4Δ mutant cells, LRG1 poly(A) tail length was still longer and LRG1 mRNA level was still higher than those in WT cells. In contrast to the log phase, Lrg1 protein level in the stationary-phase ccr4Δ mutant cells was maintained much higher than that in the stationary-phase WT cells. Consistently, active translating ribosomes still remained abundant in the stationary-phase ccr4Δ mutant cells, whereas they were strongly decreased in the stationary-phase WT cells. Loss of PBP1 reduced the LRG1 poly(A) tail length as well as LRG1 mRNA and protein levels in the stationary-phase ccr4Δ mutant cells. Our results suggest that Ccr4 regulates not only LRG1 mRNA level through poly(A) shortening but also the translation of LRG1 mRNA, and that Pbp1 is involved in the Ccr4-mediated regulation of mRNA stability and translation.