Ccr4-Not complex subunits Ccr4, Caf1, and Not4 are novel proteolysis factors promoting the degradation of ubiquitin-dependent substrates by the 26S proteasome

Degradation of short-lived and abnormal proteins is essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4, Caf1, and Not4 proteins are known components of the Ccr4-Not multimeric complex. Ccr4 and Caf1 have established roles in transcription, mRNA de-adenylation and RNA degradation etc., while Not4 was shown to have important roles in regulating translation and protein quality control pathways. Here we show that Ccr4, Caf1, and Not4 have a novel function at a post-ubiquitylation step in the UPS pathway by promoting ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4, CAF1, or NOT4 genes in Saccharomyces cerevisiae. Additionally, we show that Ccr4, Caf1, and Not4 bind to cellular ubiquitin conjugates, and that Ccr4 and Caf1 proteins interact with the proteasome. In contrast to Ccr4, Caf1, and Not4, other subunits of the Ccr4-Not complex are dispensable for UFD substrate degradation. From our findings we conclude that the Ccr4-Not complex subunits Ccr4, Caf1, and Not4 have a novel function outside of the canonical Ccr4-Not complex as a factor targeting ubiquitylated substrates for proteasomal degradation. •A novel function of Ccr4-Not complex subunits Ccr4, Caf1, and Not4 in proteolysis.•Post-ubiquitylation targeting function for Ccr4, Caf1, and Not4 is identified.•Ccr4, Caf1, and Not4 co-precipitate cellular ubiquitin conjugates.•Ccr4, Caf1, and Not4 [29] bind 19S regulatory particle complex of 26S proteasome.