Ubiquitin independent degradation of cellular proteins by proteasomes

The molecular basis for coordinated regulation of protein synthesis and degradation is not understood. Here we report that the 20S proteasome endoproteolytically cleaves the translation initiation factors eIF4G, a subunit of eIF4F, and eIF3a, a subunit of eIF3. The cleavage of eIF4G or eIF3a differentially affects the assembly of ribosomal pre-initiation complexes on different cellular and viral mRNAs in an in vitro system containing pure components. Inhibition of proteolytic activity of the 20S proteasome with specific inhibitors prevents cleavage of both factorsin vitro and in vivo, restores assembly of ribosomal complexes in vitro and differentially affects translation of different mRNAs in vivo . These studies demonstrate the importance of the ubiquitin independent degradation activity of proteasomes in regulation of cellular processes and suggest a link between protein synthesis and degradation.;The critical role of the ubiquitin-26S proteasome system in regulation of protein homeostasis in eukaryotes is well established. In contrast, the impact of the ubiquitin-independent proteolytic activity of proteasomes is poorly understood. Through biochemical analysis of mammalian lysates, we find that the 20S proteasome, latent in peptide hydrolysis, specifically cleaves more than 20% of all cellular proteins. Thirty intrinsic proteasome substrates (IPSes) were identified and in vitro studies of their processing revealed that cleavage occurs at disordered regions, generating stable products encompassing structured domains. The mechanism of IPS recognition is remarkably conserved in the eukaryotic kingdom, as mammalian and yeast 20S proteasomes exhibit the same target specificity. Further, 26S proteasomes specifically recognize and cleave IPSes at similar sites, independent of ubiquitination, suggesting that disordered regions likely constitute the universal structural signal for IPS proteolysis by proteasomes. Finally, we show that proteasomes contribute to physiological regulation of IPS levels in living cells and the inactivation of ubiquitin-activating enzyme E1 does not prevent IPS degradation. Collectively, these findings suggest a significant contribution of the ubiquitin-independent proteasome degradation pathway to the regulation of protein homeostasis in eukaryotes.;Further characterization of attributes that facilitate the ubiquitin independent proteasomal degradation of a substrate was performed in order to identify the fundamental features that influence degradation kinetics. We have identified the primary proteasomal cleavage sites within eIF4G, engineered and examined several recombinant eIF4G proteins of variable stability. Proteolytic analysis of deletion mutants of recombinant eIF4G demonstrated that an unstructured region of less than 40aa is sufficient for protection of eIF4G from proteolysis. Thus it appears that the length of an unstructured region contributes significantly to substrate susceptibility. Many IPSes were purified in complex with additional proteins and exhibited markedly slower degradation kinetics compared to that of the pure IPS. The tissue of origin was also found to affect degradation kinetics, as 20S proteasome species from rabbit muscles, spleen, CNS, and reticulocytes exhibited distinct kinetics, whereas 26S species from both muscles and RRL displayed similar kinetics. Together these studies demonstrate that modifications to the substrate or enzyme can affect degradation kinetics, suggesting multiple layers of potential regulation in ubiquitin independent protein degradation.