A conserved mitochondrial role for cyclin C in mediating oxidative stress-induced cell death in yeast

Abstract

Cyclin C is a highly conserved component of the Mediator kinase module that regulates transcription through CDK8 stimulation. In addition, the yeast (Cnc1) and human (CCNC) cyclin C exhibit stress-induced mitochondrial translocation to stimulate fission through direct interaction with the dynamin-like GTPase DRP1 (mammals) or Dnm1 (yeast). Gene ablation studies revealed that both Cnc1 and CCNC are required for cell damage-induced regulated cell death (RCD). To determine the relative contributions of cyclin Cs transcriptional and mitochondrial roles in promoting RCD, this study utilized docking simulation algorithms to predict interaction interfaces for CCNC-CDK8 and CCNC-DRP1 heterodimers. As expected, CCNC bound CDK8 through its amino terminal cyclin box domain while DRP1 associated with the second carboxyl cyclin box. Using these predictions, we used site directed mutagenesis on Cnc1 to separate these functions. Importantly, only the DRP1/Dnm1-interaction residues were important for RCD in yeast. Interestingly, although Dnm1 is required for RCD, its fission activity was not. Moreover, Dnm1 is still required for RCD even when Cnc1 is targeted to the mitochondria indicating it is not simply functioning as a tether. Finally, when expressed in yeast, the human CCNC efficiently induced fission and stimulated RCD. Moreover, these functions required predicted DRP1 interaction sites as well. In conclusion, these studies revealed that cyclin C separates its nuclear and mitochondrial activities by utilizing different cyclin box domains. Second, Dnm1-cyclin C interaction, and not transcriptional control, is critical for cyclin C-dependent RCD and this role is conserved from yeast to humans. Author SummaryThe cyclin C protein is found in all eucaryotes and exhibits two conserved functions. First cyclin C activates the CDK8 protein kinase to control transcription of genes involved in the stress response. Second, stress induces cyclin C translocation to the mitochondria where it induces fragmentation by stimulating the fission GTPase DRP1. Mitochondrial fission is an early step in the regulated cell death pathway. Importantly, cyclin C is required for stress-induced cell death raising the question of what the relative contribution of its transcription and mitochondrial roles is. To address this question, we generated mutations in the yeast cyclin C that interfered with its association with CDK8 or DRP1 without affecting the other. We found that the mitochondrial role, but not transcription, was required for cell death in yeast. In addition, when expressed in yeast, the human cyclin C also induced mitochondrial fission and cell death. Finally, although DRP1 is important for regulated cell death, its fission function is not. These results point to a highly conserved role for cyclin C in mediating regulated cell death at the mitochondria. In addition, these results point to a non-enzymatic role for DRP1 in executing the cell death pathway.