Fig. 4: Ribosomal proteins are ubiquitinylated by Hel2 in the presence of damaging agents.

a Western blot analysis of ubiquitinated proteins in total lysates and ribosome fractions in the presence of the indicated compounds. b Release of nascent peptides by puromycin does not affect the ubiquitination pattern of proteins in the ribosome fraction. Right panel is a western analysis of the puromycylation reactions under the indicated conditions. Left panel is a western analysis of ubiquitination of ribosome-enriched fraction in the presence and absence of puromycin upon the addition of 4-NQO. c Western-bot analysis showing that the ubiquitination of ribosomal proteins after MMS addition is completely inhibited when HEL2 is deleted. d Western blot analysis used to assess the relative amount of ubiquitinated ribosomal proteins in the presence of 4-NQO when eIF4E, and hence initiation, is inactivated. Lines compare WT and cdc33-ts cells in the presence of 4-NQO at the restrictive temperature. e Sucrose-gradient fractionation of lysates obtained from endogenous HEL2: 3 × FLAG tag yeast cells in the absence and presence of 4-NQO. The traces follow the absorbance at 254 nm, below the traces is the western blot analysis of the fractions using anti-FLAG antibody. f Heat map showing scaled ubiquitination footprints of ribosome and ubiquitin-derived peptides in response to 4-NQO. The samples were digested with trypsin and enriched for diGly, prior to tandem MS analysis. Two independent biological replicates are shown and the modified lysine residues are indicated to the right of the ribosomal protein name. The peptides are organized based on their origin and subsequently ranked by their response to 4-NQO. g Bar graph showing the relative abundance of ubiquitin (eS31) and different ubiquitin chains. h–k Bar graphs showing the relative abundance of corresponding ribosomal proteins (on the left) and diGly modified peptides (on the right) in the absence and presence of 4-NQO (In all cases n = 2, ±SD). l–m Western blot analysis used to follow the ubiquitination of depicted ribosomal proteins in the presence of the indicated compounds. A C-terminal FLAG tag was added to the chromosomal copy of ribosomal protein genes. Source data are provided as a Source Data file.