The Cdc48 unfoldase prepares well-folded protein substrates for degradation by the 26S proteasome

Abstract

Cdc48/p97 is an essential and highly conserved AAA+ ATPase that uses its protein-unfoldase activity to extract ubiquitinated polypeptides from macromolecular complexes and membranes. This motor has also been implicated in protein-degradation pathways, yet its exact role in acting upstream of the 26S proteasome remains elusive. Ubiquitinated proteins destined for degradation by the proteasome require an unstructured initiation region to engage with the proteasomal translocation machinery, and Cdc48 was proposed to generate these unfolded segments, yet direct evidence has been missing. Here, we used an in vitro reconstituted system to demonstrate the collaboration of Cdc48 and the 26S proteasome from S. cerevisiae in degrading ubiquitinated, well-folded proteins that lack unstructured segments. Our data indicate that a critical role for Cdc48 in the ubiquitin-proteasome system is to create flexible initiation regions in compact substrates that otherwise would be refractory to engagement and degradation by the proteasome.

Fig. 1

Quantitative analyses of model-substrate degradation by the proteasome and unfolding by Cdc48. a Representative fluorescence time courses of three separate experiments showing the proteasomal degradation of K48-GREEN TAIL and the lack of processing for tailless K48-GREEN. b Michaelis–Menten analysis of K48-GREEN TAIL degradation by the 26S proteasome. Shown are the individual rates for three repeats with identical protein samples and the fit. c Michaelis–Menten analysis of K48-RED unfolding by Cdc48 in the presence of Ufd1/Npl4 (Cdc48•UN). Shown are the individual rates for three repeats with identical protein samples and the fit. d Example fluorescence trace for the single-turnover unfolding of K48-RED by Cdc48•UN, fitted to a single exponential decay (red). e Example fluorescence trace for the single turnover unfolding of the K48-GREEN fraction of an activated K48-RED sample, fitted to single exponential decay (light green). f Example fluorescence trace for the single-turnover unfolding of non-activated K48-GREEN by Cdc48•UN, fitted to a single exponential decay (dark green). For d–f, rates were averaged from three measurements (N = 3, repeats with identical protein samples) with standard deviations reported. Missing points at the beginning of each measurement correspond to the dead time for manual mixing and sample transfer. d and e show processing of the same sample of activated mEOS3.2 substrate (red and green) monitored simultaneously at two different wavelengths. Raw data related to this figure is available in Supplementary Data 1

Fig. 2

Cdc48-mediated substrate degradation by the 26S proteasome. a Fluorescence time course for the Cdc48-dependent proteasomal degradation of K48-GREEN (purple; shown is one example trace out of three experimental repeats). The substrate is equilibrated (1), before the addition of Cdc48•UN leads to unfolding (2). After reaching steady state of unfolding and refolding, 26S proteasome was added (3), and a continuous loss of fluorescence signal was observed, indicative of multiple-turnover degradation. No unfolding and therefore no degradation by the proteasome occurred when GREEN substrate without the branched ubiquitin chains was used (black trace). b The rate of K48-GREEN degradation after unfolding by Cdc48•UN correlates with the amount of proteasome added to the coupled reaction. c SDS-PAGE analysis of the endpoints for the Cdc48-coupled degradation assays of Cy5-labeled K48-GREEN. The gel was scanned for Cy5 fluorescence to detect peptide-product formation. Significant peptide formation depended on the presence of Cdc48•UN and the 26S proteasome. The apparent change in banding patterns upon addition of Cdc48•UN (lanes 2, 4, and 6) is likely caused by the large amounts of Cdc48 affecting the migration of ubiquitinated substrate in the gel. See Supplementary Figure 6 for image of entire gel. Raw data related to this figure are available in Supplementary Data 1