Figure 1. Most proteins carrying K48-linked ubiquitin chains interact with the Cdc48/UN complex.
(A) S. cerevisiae cells lacking the ABC transporter Pdr5 (BY4741:pdr5Δ) and expressing Npl4-FLAG or Ufd1-FLAG from plasmids were incubated with or without the proteasome inhibitor bortezomib (BTZ). Concentrated cell lysates were incubated with either beads containing FLAG antibodies (Anti-FLAG resin) or biotinylated TUBE recognizing K48-linked ubiquitin chains (BioTUBEK48) and streptavidin beads. Polyubiquitinated proteins were eluted with an excess of trypsin-resistant TUBE (TR-TUBE). The samples were subjected to trypsin digestion, TMT labeling, and analysis by tandem MS.
(B) For each substrate protein detected in BTZ-treated cells, its abundance in the FLAG immunoprecipitation (IP) was divided by its abundance in the K48 ubiquitin pull-down (K48 IP) (Cdc48/K48 ratios). The ratios from Npl4-FLAG and Ufd1-FLAG cells were plotted against each other on a logarithmic scale.
(C) As in (B), but for untreated cells (DMSO instead of BTZ).
(D) As in (B), but comparing data from Npl4-FLAG expressing cells with or without BTZ treatment.
(E) As in (D), but for Ufd1-FLAG expressing cells.
(F) For each detected protein, the Cdc48/K48 ratios were averaged among all tested cell lysates. Proteins between the dotted lines are enriched or depleted by a factor of less than three. The full list of identified proteins is given in Table S1.
See also Figure S1.