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

Most 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. Experiments in yeast cells confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.

A Berlin-sided retrospective of the origins of metabolic control theory.

Metabolic control theory (MCA) is celebrating its 50th anniversary. The theory introduced quantitative terms that describe the importance of an enzyme for the regulation of the overall flux and of metabolite concentrations. MCA was developed independently by two groups. The Berlin group included Reinhart Heinrich, Tom A. Rapoport and Samuel M. Rapoport, and the Edinburgh group Henrik Kacser and James A. Burns. Here, I provide a brief reminiscence from the perspective of the Berlin group.

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

Most 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 vivo experiments confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.