Bidirectional substrate shuttling between the 26S proteasome and the Cdc48 ATPase promotes protein degradation

Abstract

SUMMARYMost eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex, rather than substrate recruitment.In vivoexperiments confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.

Integrative Omics reveals changes in the cellular landscape of yeast without peroxisomes

“…Among the proteins of decreased abundance were Ubx5, Snf6, and Hsp26. Ubx5 is an adapter of the AAA ATPase Cdc48/p97 (Schuberth et al, 2004) and involved in the degradation of proteins by the proteasome (Li et al, 2023 preprint; Noireterre et al, 2023; Verma et al, 2011). The downregulation of the nuclear protein Snf6, which is involved in transcriptional activation (Estruch and Carlson, 1990; Laurent et al, 1991), generally fits the observation that pex3 cells do not show widespread changes in their transcriptional program.…”