In response to oxidative stress, cells must choose either to live or to die. Here we show that the E3 ligase SCFGrr1 mediates the destruction of Med13, which releases cyclin C into the cytoplasm and results in cell death. The Med13 SCF degron is most likely primed by the Cdk8 kinase and marked for destruction by the MAPK Slt2.

Many cells experience hypoxia, or low oxygen, and respond by dramatically altering gene expression. In the yeast Saccharomyces cerevisiae, genes that respond are required for many oxygen-dependent cellular processes, such as respiration, biosynthesis, and redox regulation. To more fully characterize the global response to hypoxia, we exposed yeast to hypoxic conditions, extracted RNA at different times, and performed RNA sequencing (RNA-seq) analysis. Time-course statistical analysis revealed hundreds of genes that changed expression by up to 550-fold. The genes responded with varying kinetics suggesting that multiple regulatory pathways are involved. We identified most known oxygen-regulated genes and also uncovered new regulated genes. Reverse transcription-quantitative PCR (RT-qPCR) analysis confirmed that the lysine methyltransferase EFM6 and the recombinase DMC1, both conserved in humans, are indeed oxygen-responsive. Looking more broadly, oxygen-regulated genes participate in expected processes like respiration and lipid metabolism, but also in unexpected processes like amino acid and vitamin metabolism. Using principle component analysis, we discovered that the hypoxic response largely occurs during the first 2 hr and then a new steady-state expression state is achieved. Moreover, we show that the oxygen-dependent genes are not part of the previously described environmental stress response (ESR) consisting of genes that respond to diverse types of stress. While hypoxia appears to cause a transient stress, the hypoxic response is mostly characterized by a transition to a new state of gene expression. In summary, our results reveal that hypoxia causes widespread and complex changes in gene expression to prepare the cell to function with little or no oxygen.

Mitochondria exist in an equilibrium between fragmented and fused states that shifts heavily toward fission in response to cellular damage. Nuclear-to-cytoplasmic cyclin C relocalization is essential for dynamin-related protein 1 (Drp1)–dependent mitochondrial fission in response to oxidative stress. This study finds that cyclin C directly interacts with the Drp1 GTPase domain, increases its affinity to GTP, and stimulates GTPase activity in vitro. In addition, the cyclin C domain that binds Drp1 is contained within the non–Cdk binding second cyclin box domain common to all cyclin family members. This interaction is important, as this domain is sufficient to induce mitochondrial fission when expressed in mouse embryonic fibroblasts in the absence of additional stress signals. Using gel filtration chromatography and negative stain electron microscopy, we found that cyclin C interaction changes the geometry of Drp1 oligomers in vitro. High–molecular weight low–GTPase activity oligomers in the form of short filaments and rings were diminished, while dimers and elongated filaments were observed. Our results support a model in which cyclin C binding stimulates the reduction of low–GTPase activity Drp1 oligomers into dimers capable of producing high–GTPase activity filaments.

Cyclin C-Cdk8 kinase negatively regulates the yeast phagophore gene ATG8. After nitrogen starvation, repression is relieved by ubiquitin proteasome degradation of cyclin C. This is important for survival as degradation occurs prior to cyclin C translocation to the mitochondria where it induces mitochondrial fragmentation and promotes cell death.

Eukaryotic cells, when faced with unfavorable environmental conditions, mount either pro-survival or pro-death programs. The conserved cyclin C-Cdk8 kinase plays a key role in this decision. Both are members of the Cdk8 kinase module that, along with Med12 and Med13, associate with the core Mediator complex of RNA polymerase II. In Saccharomyces cerevisiae, oxidative stress triggers Med13 destruction, which releases cyclin C into the cytoplasm to promote mitochondrial fission and programmed cell death. The SCFGrr1 ubiquitin ligase mediates Med13 degradation dependent on the cell wall integrity pathway, MAPK Slt2. Here we show that the AMP kinase Snf1 activates a second SCFGrr1 responsive degron in Med13. Deletion of Snf1 resulted in nuclear retention of cyclin C and failure to induce mitochondrial fragmentation. This degron was able to confer oxidative-stress-induced destruction when fused to a heterologous protein in a Snf1 dependent manner. Although snf1∆ mutants failed to destroy Med13, deleting the degron did not prevent destruction. These results indicate that the control of Med13 degradation following H2O2 stress is complex, being controlled simultaneously by CWI and MAPK pathways.