Abstract
The degradation of damaged proteins is an important vital function especially during aging and stress. The ubiquitin proteasome system is one of the major cellular machineries for protein degradation. Health and longevity are associated with high proteasome activity. To demonstrate such a role in aging of Podospora anserina, we first analyzed the transcript and protein abundance of selected proteasome components in wild-type cultures of different age. No significant differences were observed. Next, in order to increase the overall proteasome abundance we generated strains overexpressing the catalytic proteasome subunits PaPRE2 and PaPRE3. Although transcript levels were strongly increased, no substantial effect on the abundance of the corresponding proteins was observed. Finally, the analysis of the P. anserina strains expressing the sequence coding for the CL1 degron fused to the Gfp gene revealed no evidence for degradation of the GFP-CL1 fusion protein by the proteasome. Instead, our results demonstrate the degradation of the CL1-degron sequence via autophagy, indicating that basal autophagy appears to be a very effective protein quality control pathway in P. anserina.
Highlights
The degradation of proteins, in particular of those that are damaged or are present in excess, is an important vital function of biological systems and is implicated in several cellular processes such as cell cycle control, proliferation, differentiation, apoptosis and protein quality control1
Regulation of proteasome components during aging and oxidative stress In order to address the role of the ubiquitin proteasome system (UPS) on aging of P. anserina, we investigated the expression of the genes coding for proteolytic subunits PaPre2 (β5) and PaPre3 (β1) and of the proteasome assembly factor PaUmp1
A widely used degron is CL1 derived from S. cerevisiae and consisting of 15 hydrophobic amino acids. This sequence was successfully used to detect proteasome activity in a wide range of organisms including yeast47, fly35, mouse36, rat37,38 and human cell cultures39–44, our experiments did not reveal a clear degradation of the whole GFP-CL1 fusion protein as it would be expected for degradation by the proteasome
Summary
The degradation of proteins, in particular of those that are damaged or are present in excess, is an important vital function of biological systems and is implicated in several cellular processes such as cell cycle control, proliferation, differentiation, apoptosis and protein quality control. There are two major pathways involved in protein degradation: autophagy and degradation by the ubiquitin proteasome system (UPS). The UPS consists of a large number of different ubiquitin ligases that act jointly with the proteasome, a multi-protein complex with proteolytic activities. The 26S proteasome consists of two subcomplexes, the catalytic 20S core particle and the 19S regulatory particle. The 19S regulatory particle conveys the identification, deubiquitination, unfolding and transport of the substrate into the proteolytic chamber. The core particle is responsible for the degradation of the target proteins. It is composed of four stacked rings, which enclose the proteolytic chamber. The assembled β-subunits are framed by rings of seven α-subunits, blocking the entrance to the proteolytic chamber, if no regulatory particle is bound (reviewed in:13)
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