Figure 1. Cotranslational ubiquitination occurs at low levels in vivo.

(A) Nascent chains may be ubiquitinated as they emerge from the ribosome or post-translationally once the protein has been fully synthesized (B) Ribosome-nascent chain complexes (RNCs) contain ubiquitinated material that is released by puromycin. Ultrasedimentation with or without puromycin to release nascent chains separated the supernatant (S) from the ribosome-nascent chain complexes (R), which were subjected to polyUb-affinity isolation (Ubn). Totals and pulldowns were analyzed by SDS-PAGE and ubiquitin immunoblot. (C) Peptidyl-tRNA isolation through CTAB precipitation shows direct ubiquitination of nascent chains. Isolated RNCs were incubated with polyUb-affinity resin or GST-only and the nascent chain-tRNA complexes precipitated with CTAB. Samples run on SDS-PAGE were immunoblotted for ubiquitin. (D) ³⁵S–pulse-labeled nascent chains are ubiquitinated while still ribosome-bound. Cells were pulse-labeled for 1 min with ³⁵S-Met/Cys; fractionated into supernatant (S or Sup), containing newly made, full-length ³⁵S-polypeptides and RNCs (R or Ribo), containing ribosome bound ³⁵S-nascent chains and pulled-down either with GST (Ctl) or polyUb-affinity resin (Ubn). (E) ³⁵S-labeled nascent chains are directly ubiquitinated and can be isolated independently of the ribosome. i) Cells were pulse-labeled and fractionated as before; S: supernatant; R: ribosomal pellet. EDTA was added to the RNCs (R1), which was resuspended and spun again to yield empty ribosomes (R2) and released nascent chains (S2). S2 and R2 were then subject to polyUb-affinity pulldown. Samples were analyzed by SDS-PAGE and autoradiography. ii.) Autoradiogram showing totals of S1/R1 and S2/R2, as well as the polyUb-affinity pulldown of S2 and R2 (Ubn). (F) A small fraction of nascent chains and newly made proteins is cotranslationally ubiquitinated; calculated as the ratio of ³⁵S-labeled nascent chains isolated by Dsk2 over total ³⁵S-labeled nascent chains in the ribosomal or in the supernatant fraction (mean ± SEM, n=59). See also Figure S1.