Fig. 4: K48-polyubiquitinated Vps10 is also cleaved by Rbd2.
From: K48-ubiquitin-dependent proteases cut-up post-ER proteins
A Proteasome- and Rbd2-dependent degradation products detected by α-RFP immunoblotting of the lumenal domain of RFP–Vps10–NG–AID after SCFTIR1-directed ubiquitination. B Immunoblot analysis of Rbd2-dependent degradation products in pup1-T30A pre3-T20A rbd2Δ cells carrying an empty vector (EV) or plasmid expressing catalytically active or inactive (S124A) Rbd2–GFP. Levels of full-length and Rbd2-dependent degradation products were quantified and graphed. C Immunoblot analysis of post-nuclear supernatant (PNS), soluble (S), and membrane-associated pellet (P) fractions from BY4742 RBD2 and rbd2Δ cells, before and after induction of SCFTIR1-directed ubiquitination of RFP–Vps10–NG–AID. Changes in the amount of soluble RFP-tagged fragments were quantified and graphed. D Top: schematic of the six transmembrane domains that comprise the rhomboid fold of Rbd2. Bottom: predicted AlphaFold structure (A0A816BH90). Catalytic residues are highlighted in yellow. E Micrographs of Rbd2–GFP co-expressed with Sec7–RFP or RFP–Vps10. Scale bar = 10 μm. B, C Bar graphs show mean ± SD, overlaid are grey circles representing values from independent experiments (n) of yeast strains described in Supplementary Tables 1 and 4 (B, n = 3 (S124A Rbd2–GFP), n = 4 (others); C, n = 3). Statistical comparisons are indicated by numbered labels, with asterisks or “ns” as prefix to denote significant differences (p < 0.05) or not significant, respectively. Full statistical outcomes are provided in Supplementary data. B Pairwise comparisons were performed using two-sided unpaired t-tests, with Holm–Šidák correction following a one-way ANOVA. C Pairwise comparisons were performed using two-sided two-sample unpaired Welch’s t-test. Source data are provided as a Source Data files.
