Figure 4. Evidence for stalled nascent chain modification with CAT tails and aggregation in wild type cells.

(A) Stalling Lys-less reporter modification with CAT tails in wild type yeast. All constructs were HA-tagged. Expression of GFP, GFP-R12, GFP K-less ('K-less'), or GFP K-less R12 ('K-less-R12') reporter proteins in the indicated strains, revealed by anti-HA immunoblot. 'R12' is the stalling signal, consisting of 12 suboptimal Arg CGN codons. GFP-R12 expression in ltn1△ cells is used as a control for aggregate formation. Lower panel, shorter exposure to reveal relative steady-state levels of monomeric reporter species. (B) Stalling PtnA-Rz reporter CATylation and aggregation in wild type yeast. All constructs were HA-tagged. Expression of PtnA-STOP-Rz, PtnA-Rz, PtnA-STOP-Rz K-less, and PtnA-Rz K-less reporter proteins in the wild type strain, revealed by anti-HA immunoblot.

DOI: http://dx.doi.org/10.7554/eLife.11794.010

Figure 4—figure supplement 1. Stalling NC reporter aggregation in wild type yeast.

HA-tagged GFP, GFP-R12, GFP K-less ('K-less'), or GFP K-less R12 ('K-less R12') reporters expressed in the MG132-treated wild type strain (or MG132 treated rqc2△ strain as a control, lane 5) were concentrated by anti-HA IP and analyzed by anti-HA immunoblot. Lower panel, the monomeric forms are shown in a shorter exposure to reveal relative steady-state levels of monomeric reporter species, as well as CAT tails in lane 4.

DOI: http://dx.doi.org/10.7554/eLife.11794.011