Search

The regular functioning of the nucleolus and nucleus-mitochondria crosstalk are considered unrelated processes, yet cytochrome c(Cc) migrates to the nucleus and even the nucleolus under stress conditions. Nucleolar liquid–liquid phase separation usually serves the cell as a fast, smart mechanism to control the spatial localization and trafficking of nuclear proteins. Actually, the alternative reading frame (ARF), a tumor suppressor protein sequestered by nucleophosmin (NPM) in the nucleoli, is shifted out from NPM upon DNA damage. DNA damage also triggers early translocation of respiratory Ccto nucleus before cytoplasmic caspase activation. Here, we show that Cccan bind to nucleolar NPM by triggering an extended-to-compact conformational change, driving ARF release. Such a NPM–Ccnucleolar interaction can be extended to a general mechanism for DNA damage in which the lysine-rich regions of Cc—rather than the canonical, arginine-rich stretches of membrane-less organelle components—controls the trafficking and availability of nucleolar proteins.

Since the first description of apoptosis four decades ago, great efforts have been made to elucidate, both in vivoand in vitro, the molecular mechanisms involved in its regulation. Although the role of cytochrome cduring apoptosis is well established, relatively little is known about its participation in signaling pathways in vivodue to its essential role during respiration. To obtain a better understanding of the role of cytochrome cin the onset of apoptosis, we used a proteomic approach based on affinity chromatography with cytochrome cas bait in this study. In this approach, novel cytochrome cinteraction partners were identified whose in vivointeraction and cellular localization were facilitated through bimolecular fluorescence complementation. Modeling of the complex interface between cytochrome cand its counterparts indicated the involvement of the surface surrounding the heme crevice of cytochrome c, in agreement with the vast majority of known redox adducts of cytochrome c. However, in contrast to the high turnover rate of the mitochondrial cytochrome credox adducts, those occurring under apoptosis led to the formation of stable nucleo-cytoplasmic ensembles, as inferred mainly from surface plasmon resonance and nuclear magnetic resonance measurements, which permitted us to corroborate the formation of such complexes in vitro. The results obtained suggest that human cytochrome cinteracts with pro-survival, anti-apoptotic proteins following its release into the cytoplasm. Thus, cytochrome cmay interfere with cell survival pathways and unlock apoptosis in order to prevent the spatial and temporal coexistence of antagonist signals.

Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome cfrom mitochondria to the cytosol. To better understand the role of cytochrome cin the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thalianacytochrome cas bait. Using this approach, ten putative new cytochrome cpartners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitrointeraction between cytochrome cand such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thalianacytochrome cinteracts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thalianacytochrome c-targets are closely related to those for Homo sapienscytochrome c(Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280.