Journal: Communications Biology

Article Title: Stalled disomes marked by Hel2-dependent ubiquitin chains undergo Ubp2/Ubp3-mediated deubiquitination upon translational run-off

doi: 10.1038/s42003-025-07569-z

Figure Lengend Snippet: a A fraction of Rps20 is polyubiquitinated in unstressed log-phase cells. Lysate from wild type or S20-H 6 cells carrying single K6R (RK), K8R (KR), or double K6R, K8R (RR) mutations. Immunoblots were analyzed with α-Rps20 (left panel, Fig. ) or α-HIS (right panel). The length of ubiquitin chains (ubi 1– 4 ) attached to S20-H 6 is indicated. Background bands above 40 kDa on α-HIS immunoblots are labeled with gray asterisks. b CHX induces polyubiquitination of S20-H 6 in a concentration dependent manner. Log-phase S20-H 6 cells were treated for 10 min with the indicated CHX concentration. c CHX-induced polyubiquitination of S20-H 6 reaches a plateau. S20-H 6 cells were treated with low-dose CHX (collision) or were left untreated (no-collision). d Polyubiquitination of Rps20 in log-phase cells depends on catalytically active Hel2 and on ribosomal protein Asc1. Lysates from log-phase cell cultures of the indicated strains in the S20-H 6 background without treatment (no-collision) or after a 10 min treatment with 1 µg/ml CHX (collision). Untagged wild type served as a control (ctrl). For details see Methods. Immunoblots were decorated with α-HIS, α-Hel2, α-Asc1, and α-Sse1 (loading control). The length of the ubiquitin chain attached to S20-H 6 is indicated; ubi 5+x indicates the smear of polyubiquitinated S20-H 6 species attached to more than 5 ubiquitin residues.

Article Snippet: Antibodies: α-Hel2 (1:2000 (Fig. ), α-Rps20 (1:2000) (Fig. ), α-Ubp6 (1:10,000) (Fig. ), α-Stm1 (1:10,000) (Fig. ), α-Asc1 (1:2000) (Fig. ) , α-Rpl4 (1:10,000) , α-Rps9 (1:5000) , α-Sse1 (1:5000) , α-Pgk1 (1:10,000) are rabbit polyclonal antibodies (Eurogentec, Rospert lab collection). mouse α-HIS (1:1000, BioRad, clone AD1.1.10, MCA1396), α-ubiquitin P4D1 (1:1000, Santa Cruz Biotechnology, sc-8017), α-K63-Ub clone Apu3 (1:1000, Sigma-Aldrich, 05-1308), α-K48-Ub D9D5 (1:1000, Cell Signaling Technology, 8081), mouse α-FLAG clone M2 (1:1000, Sigma, F1804), α-HA (1:2000, Santa Cruz, Y-11, sc-805), α-Luciferase (1:100,000, Sigma L0159), α-rabbit HRP (1:10,000, Pierce, 61-6520) and α-mouse HRP (1:10,000, Santa Cruz, sc-2748).

Techniques: Western Blot, Ubiquitin Proteomics, Labeling, Concentration Assay, Control

Journal: Communications Biology

Article Title: Stalled disomes marked by Hel2-dependent ubiquitin chains undergo Ubp2/Ubp3-mediated deubiquitination upon translational run-off

doi: 10.1038/s42003-025-07569-z

Figure Lengend Snippet: a Rps20 is deubiquitinated upon translational run-off. Glucose-depletion/re-addition experiments were performed with S20-H 6 strains as described in Methods. Untagged wild type served as control (ctrl). S20-H 6 and polyubiquitinated S20-H 6 species were analyzed in lysates by immunoblotting with α-HIS; Sse1 served as loading control. Gray asterisks indicate background bands recognized by α-HIS, brown asterisks indicate a background band, which appeared only in glucose depleted cells (Fig. ). b Deubiquitination of S20-H 6 is blocked by high-dose CHX. S20-H 6 cells were transferred to glucose-free medium, or to glucose-free medium supplemented with high-dose (hd) CHX as described in ( a ). c Ubiquitination pattern of S20-H 6 in strains lacking Ubp2 and/or Ubp3. Log-phase cells of S20-H 6 and ∆ ubp2 , ∆ ubp3 , ∆ ubp2 ∆ ubp3 in the S20-H 6 background were flash-frozen directly ( + glucose) or after 5 min glucose depletion (- glucose). The length of the polyubiquitin chains (ubi 1 to ubi 5+x ) is indicated. d Ubp2 and Ubp3 mediate deubiquitination of Rps20. Shown is a Min to Max plot of Rps20-H 6 mono- and polyubiquitination during log-phase and after glucose depletion. Statistical analysis was performed on the basis of 5 independent experiments as shown in c . S20-H 6 -ubi 1 represents monoubiquitinated Rps20; the sum of S20-H 6 -ubi 2+3+4 bands represent poly-ubi. Higher molecular mass polyubiquitinated S20-H 6 species were excluded from the analysis. p -values represent the comparison between wild type and each mutant strain. e Ubiquitination pattern of S20-H 6 in log-phase and glucose depleted ∆ slh1 , ∆ hel2 , and ∆ ubp2 ∆ ubp3 cells as in c . f Slh1 is required for efficient deubiquitination of tri- and tetra-ubiquitinated Rps20 species. Shown are Min to Max plots of Rps20-H 6 -ubi 1–4 levels during log-phase (left panel) and after glucose depletion (right panel). Statistical analysis was performed on the basis of 3 independent experiments shown in Fig. . For details see Methods. Lines in boxes indicate the mean. n.s.: p > 0.05, * p : < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: Antibodies: α-Hel2 (1:2000 (Fig. ), α-Rps20 (1:2000) (Fig. ), α-Ubp6 (1:10,000) (Fig. ), α-Stm1 (1:10,000) (Fig. ), α-Asc1 (1:2000) (Fig. ) , α-Rpl4 (1:10,000) , α-Rps9 (1:5000) , α-Sse1 (1:5000) , α-Pgk1 (1:10,000) are rabbit polyclonal antibodies (Eurogentec, Rospert lab collection). mouse α-HIS (1:1000, BioRad, clone AD1.1.10, MCA1396), α-ubiquitin P4D1 (1:1000, Santa Cruz Biotechnology, sc-8017), α-K63-Ub clone Apu3 (1:1000, Sigma-Aldrich, 05-1308), α-K48-Ub D9D5 (1:1000, Cell Signaling Technology, 8081), mouse α-FLAG clone M2 (1:1000, Sigma, F1804), α-HA (1:2000, Santa Cruz, Y-11, sc-805), α-Luciferase (1:100,000, Sigma L0159), α-rabbit HRP (1:10,000, Pierce, 61-6520) and α-mouse HRP (1:10,000, Santa Cruz, sc-2748).

Techniques: Control, Western Blot, Ubiquitin Proteomics, Comparison, Mutagenesis