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The requirement for Cdc48/p97 in nuclear protein quality control degradation depends on the substrate and correlates with substrate insolubility.

ABSTRACT: Cdc48, known as p97 or valosin-containing protein (VCP) in mammals, is an abundant AAA-ATPase that is essential for many ubiquitin-dependent processes. One well-documented role for Cdc48 is in facilitating the delivery of ubiquitylated misfolded endoplasmic reticulum proteins to the proteasome for degradation. By contrast, the role for Cdc48 in misfolded protein degradation in the nucleus is unknown. In the budding yeast Saccharomyces cerevisiae, degradation of misfolded proteins in the nucleus is primarily mediated by the nuclear-localized ubiquitin-protein ligase San1, which ubiquitylates misfolded nuclear proteins for proteasomal degradation. Here, we find that, although Cdc48 is involved in the degradation of some San1 substrates, it is not universally required. The difference in the requirement for Cdc48 correlates with the insolubility of the San1 substrate. The more insoluble the substrate, the more its degradation requires Cdc48. Expression of Cdc48-dependent San1 substrates in mutant cdc48 cells results in increased substrate insolubility, larger inclusion formation and reduced cell viability. Substrate ubiquitylation is increased in mutant cdc48 cells, suggesting that Cdc48 functions downstream of San1. Taken together, we propose that Cdc48 acts, in part, to maintain the solubility or reverse the aggregation of insoluble misfolded proteins prior to their proteasomal degradation.

SUBMITTER: Gallagher PS

PROVIDER: S-EPMC4004975 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Maintaining mitochondrial function is essential for neuronal survival and offers protection against neurodegeneration. Ubiquitin-mediated, proteasome-dependent protein degradation in the form of outer mitochondrial membrane associated degradation (OMMAD) was shown to play roles in maintenance of mitochondria on the level of proteostasis, but also mitophagy and cell death. Recently, the AAA-ATPase p97/VCP/Cdc48 was recognized as part of OMMAD acting as retrotranslocase of ubiquitinated mitochondrial proteins for proteasomal degradation. Thus, p97 likely plays a major role in mitochondrial maintenance. Support for this notion comes from mitochondrial dysfunction associated with amyotrophic lateral sclerosis and hereditary inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD) caused by p97 mutation. Using SH-SY5Y cells stably expressing p97 or dominant-negative p97(QQ) treated with mitochondrial toxins rotenone, 6-OHDA, or A?-peptide as model for neuronal cells suffering from mitochondrial dysfunction, we found mitochondrial fragmentation under normal and stress conditions was significantly increased upon inactivation of p97. Furthermore, inactivation of p97 resulted in loss of mitochondrial membrane potential and increased production of reactive oxygen species (ROS). Under additional stress conditions, loss of mitochondrial membrane potential and increased ROS production was even more pronounced. Loss of mitochondrial fidelity upon inactivation of p97 was likely due to disturbed maintenance of mitochondrial proteostasis as the employed treatments neither induced mitophagy nor cell death. This was supported by the accumulation of oxidatively-damaged proteins on mitochondria in response to p97 inactivation. Dysfunction of p97 under normal and stress conditions in neuron-like cells severely impacts mitochondrial function, thus supporting for the first time a role for p97 as a major component of mitochondrial proteostasis.

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The requirement for Cdc48/p97 in nuclear protein quality control degradation depends on the substrate and correlates with substrate insolubility.

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