Ubiquitin-like domains can target to the proteasome but proteolysis requires a disordered region (2016)
Figure 7

Figure 7. Degradation of Ubp6 proteins in vivo and in vitro.

(A) Schematic representation of S. cerevisiae Ubp6. (B) In vitro degradation of Ubp6 proteins by purified S. cerevisiae proteasome. Full-length Ubp6 (Ubp6, black open circles) and a UbL deletion mutant (Ubp6ΔN, green open circles) escaped proteasome degradation. Attachment of a Cterminal 95 amino-acid tail derived from S. cerevisiae cytochrome b2 allowed the proteasome to degrade the Ubp6 proteins (Ubp6-95, black solid circles and Ubp6ΔN-95, green solid circles). The graph plots the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m. (C) Degradation of Ubp6 proteins in vivo. The steady-state accumulation of N-terminally HA-tagged Ubp6 and mutants in S. cerevisiae. Ubp6 and Ubp6∆N are stable in yeast and not further stabilized by bortezomib. Attachment of a Su9 tail to the Cterminus lead to the degradation of Ubp6 and Ubp6∆N proteins and the proteins were stabilized by bortezomib treatment. (D) The steady-state accumulation of endogenous Ubp6 and Ubp6 derivatives with short (19 amino acid) or long (54 amino acid) tails at their C-termini. The 19 amino-acid tail destabilized Ubp6 and the 54-amino-acid tail lead to complete depletion of Ubp6. Protein levels were determined by western blotting for the HA-tag, Ubp6 or Scs2 in SDS-PAGE gels of S. cerevisiae protein extracts. The proteasome was inhibited by 100 µM bortezomib as indicated. Protein loading levels in each lane were normalized in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2.

Figure EV1. UbL domains inhibit UbLRad23-GFP-95 degradation

Initial degradation rates of UbLRad23-GFP-95 at different concentrations of purified UbL domains plotted against the competitor concentration and fitted to the equation describing competitive inhibition. The UbL domains are yeast Rad23 (A), Dsk2 (C), Atg8 (D), Hub1 (E), ubiquitin (F), Ddi1 (G), Ubp6 (H), Urm1 (I), Mdy2 (J), Rub1 (K), Smt3 (L) and human hsRad23B (B). Graphs show one representative dataset of at least three independent experiments.

Figure EV2. UbL domain-mediated His3 protein degradation depends on proteasome but not ubiquitination

(A) In the E1 temperature sensitive strain uba1-204, the cellular abundance of UbL-His3-Su9 proteins was similar at the permissive (30 ˚C) and restrictive (37 ˚C) temperatures. In contrast, a His3 fusion protein containing a classic N-end rule degron was stabilized at the restrictive temperature (37 ˚C), showing that its degradation depended on the ubiquitination machinery. Bortezomib stabilized both the UbL-His3-Su9 and N-degron-His3-Su9 proteins. (B) The UbL-His3 proteins without initiation region were stable and not affected by the shift to the restrictive temperature or treatment with bortezomib. Protein levels were determined by western blotting for the HA-tag and for Scs2 in SDS-PAGE gels of S. cerevisiae protein extracts. Proteasome degradation was tested by the addition of the proteasome inhibitor bortezomib. Protein loading levels in each lane were estimated in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2.

Figure EV3. UbL domain-mediated YFP protein degradation depends on proteasome but not ubiquitination

(A) Schematic representation of UbL-YFP constructs. The fluorescent substrates consisted of a UbL domain, followed by a yellow fluorescent protein (YFP) domain and a tail (51 amino acids derived from subunit 9 of the Fo component of the Neurospora crassa ATPase, Su9) or a stop codon at C terminus. dsRed (expression control) and UbL-YFP-Su9 (substrate) were expressed from consecutive constitutive promoters on the same CEN plasmid (YCplac33). (B) Stabilization of UbL-YFP substrates with tails (Su9, grey) or without tails (no tail, red) by proteasome inhibitor (bortezomib) treatment. The extent of recovery is calculated as the fraction of the YFP/RFP ratios of cells expressing each construct after bortezomib treatment divided by the YFP/RFP ratios for cells with the same construct after DMSO treatment. (C) The YFP/RFP ratios for cells expressing UbL-YFP substrates with tails (Su9, grey) or without tails (no tail, red) in the E1 temperature sensitive strain uba1-204 at the permissive temperature (30 ˚C; UbL-YFP-Su9, dark grey; UbL-YFP, dark red) and the restrictive temperature (37 ˚C; UbL-YFP-Su9, light grey; UbL-YFP, light red). The median of YFP/RFP value for each construct was calculated from 10,000 cells collected in one flow cytometry run and was used to indicate the concentration of YFP substrate in yeast cells as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.

Figure EV4. Four UbL domains bind with proteasome via the similar residues as ubiquitin

(A) The proteasome-interacting residues on ubiquitin and the UbL domains of yeast Rad23, Mdy2, Rub1 and Dsk2 that are mutated to glutamic acid residues (3E mutation) in this study. Structural models of UbL domains generated by PyMol (version 0.99). PDB IDs: Dsk2 2BWF; Mdy2 4GOC; Rad23 3M62; Rub1 1BT0; ubiquitin 1UBQ. (B) 3E mutations rescued the growth defect of his3 strains expressing UbL-His3-Su9 constructs under selective conditions (+3-AT, -his). Cells expressing the His3 fusion proteins in late log phase were serially diluted and stamped on selective plates. Plates were incubated at 30 ˚C for 3 days for imaging. (C) 3E mutations stabilize UbL-His3-Su9 proteins in yeast cells. Protein levels were determined by western blotting for the HA-tag and for Scs2 in SDS-PAGE gels of S. cerevisiae protein extracts. Protein loading levels in each lane were estimated in by western blotting for the integral endoplasmic reticulum membrane protein Scs2.