Figure 2. Regulation of the ubiquitin independent degron of Rpn4 by NatA and contribution to degradation of ubiquitin-fusion degradation substrates.
(A) Flow cytometry analysis of strains expressing the UbiG76V-tFT reporter. (B) Domain architecture of Rpn4. (C) Degradation of C-terminally TAP-tagged Rpn4 after blocking translation with cycloheximide. Whole-cell extracts were separated by SDS–PAGE followed by immunoblotting with antibodies against protein A and Pgk1 as loading control. (D) Flow cytometry analysis of strains expressing the indicated Rpn4 N-terminal sequences fused to the tFT. mCherry/sfGFP ratios were normalized to the mean mCherry/sfGFP ratio of the wild type strain. (E) Extracted ion chromatograms of Nt-acetylated and unmodified N-terminal peptides derived from full-length Rpn4 variants obtained by label-free mass spectrometry. (F) Half-lives of C-terminally TAP-tagged Rpn4 variants estimated by cycloheximide chase. Mean half-lives and 95% CI of six replicates are plotted together with the half-life of each replicate. p: one-sided unpaired t test. (G) Flow cytometry analysis of strains expressing the UbiG76V-tFT reporter. mCherry/sfGFP ratios were normalized to the mean mCherry/sfGFP ratio of ubr1∆ ufd4∆ cells. AN: Rpn4A2N. p: one-sided unpaired t test.