(A) Top panels, diagrams of reporter constructs encoding stalling-prone nascent chains and respective controls. PolyLys-dependent stalling (left): GFP-Flag-HIS3 fusion protein control (K0), its bona fide nonstop (NS) protein derivative, and a derivative fused to 12 lysines (K12). Endonucleolytic mRNA cleavage-dependent stalling (middle): Protein A ZZ domain-Ribozyme-GFP fusion constructs. A self-cleaving ribozyme (Rz) within coding sequence generates a nonstop (NS) mRNA encoding stalled Protein A (PtnA). Controls are constructs with a cleavage-defective ribozyme generating a full-length PtnA-GFP fusion (rz), or with a stop codon preceding the Rz cleavage site (STOP-Rz), such that nascent PtnA is not expected to become stalled in ribosomes. Horizontal lines represent the encoded polypeptides. Arg CGN codon-dependent stalling (right): GFP-R12-RFP (GRR), where R12 is encoded by unpreferred Arg codons. Lower panels, reporter protein expression in a wild type strain (WT; BY4741), a LTN1-deleted strain (ltn1△) or a strain whose endogenous Ltn1 lacks the RING domain (Ltn1 △R). Immunoblots of SDS-boiled cell extracts: anti-Flag, anti-PtnA, or anti-GFP to monitor reporter expression, and anti-Pgk1 as loading control. The migration of CATylated species is indicated. Asterisks indicate bands of unknown identity. (B) Stalling reporter slow-migrating species are pelleted upon high speed centrifugation. The extract of a K12 reporter-expressing ltn1△ strain was pre-cleared by centrifugation at 1000 x g for 5 min and its supernatant (S1) was then subjected to 16,000 x g for 10 min. The resulting supernatant (S16) and pellet (P16) were analyzed by western blot against Flag tag (K12), Rpl3 (a 60S ribosomal protein), Pgk1 (phosphoglycerate kinase 1, a soluble protein) and ubiquitin (high-molecular weight conjugates migrating above 120 kDa are shown). (C) Translational stalling is required for reporter aggregation. NS and K12 reporter protein expression in strains lacking Ltn1 and/or the translational stalling factor, Hel2.