Extended Data Fig. 4: K11–Ub linkages are not necessary for proteasomal degradation of all cytoplasmic substrates.
From: Distinct proteostasis circuits cooperate in nuclear and cytoplasmic protein quality control
a–d, WT or UbK11R cells expressing stable Ub-M-GFP (a), the N-end-rule substrate Ub-R-GFP (b), the ubiquitin fusion degradation (UFD) substrate UbG76V–GFP (c) or GFP fused to the artificial degron CL1 (d) from galactose-inducible promoters for 4–6 h at 30 °C were shifted to glucose-containing medium for 1 h at 30 °C or 37 °C to shut off expression. Cells were fixed and imaged by fluorescence microscopy. 300 cells were counted per condition, and the percentage of cells with GFP-positive puncta is shown (mean ± s.e.m. from three biologically independent experiments). There was a statistically significant increase in puncta compared with WT when GFP-CL1 (which contains a short amphipathic CL1 helix that could mimic a partially unfolded protein) was expressed in UbK11R cells, as judged by two-tailed Student’s t-test (P = 0.0127). The differences for all other substrates were not significant (ns, P > 0.05). DUB, deubiquitinating enzyme, which cleaves Ub from Ub-M-GFP or Ub-R-GFP.
