(A) Ltn1 and Ubr1 contribute to accumulation of Ub conjugates on cdc48-3 ribosomes. Ribosomes from the indicated strains grown at 30°C were isolated from input cell lysates (right panels) by pelleting through sucrose cushions, treated with puromycin, and incubated with UBA resin. The bound fraction (left panels) and inputs were evaluated by SDS-PAGE and immunoblotting with Ub, tubulin (Tub), and Rpl32 antibodies as indicated. The -3 allele of cdc48 was used. (B) The non-stop reporter GFHNS accumulates in Cdc48 pathway mutants. Glass bead/SDS extracts from exponential cultures (grown at 30°C) of the indicated mutants harboring a plasmid that expresses GFHNS were analyzed by SDS-PAGE and IB with anti-Gfp. Tubulin served as the loading control. (C) Cycloheximide chase analysis of cells expressing either the non-stop (GFHNS) or stop codon-containing (GFHStop) reporters. Glass bead/SDS extracts prepared from aliquots harvested at the indicated times from wildtype and mutant cultures were analyzed by SDS-PAGE and IB with anti-Gfp. Samples from WT expressing GFHNS were also evaluated by IB with anti-tubulin to confirm equal loading. Note that extracts prepared from cells expressing GFHStop were loaded at one-fifth the amount of GFHNS. (D) Non-stop protein accumulates in the ubiquitinated state in ufd1 mutants. Glass bead/SDS extracts of wildtype and mutant cells expressing plasmid-borne GFHNS and grown at 37°C for 90 min were evaluated directly (inputs) or immunoprecipitated with anti-Flag antibodies after 10-fold dilution with buffer containing Triton X-100. Bound proteins and inputs were evaluated by SDS-PAGE and IB with anti-Ub and Flag antibodies. UT corresponds to WT cells not expressing GFHNS. (E) Cdc48 and Ufd1 interact selectively with non-stop protein. Lysates prepared from cells expressing Flag-tagged GFHNS (NS) or GFHStop (Stop) reporters as well as cells lacking a plasmid (UT) were immunoprecipitated with anti-Flag antibodies. Total cell extracts (inputs; right panels) and bound proteins (left panels) were evaluated by SDS-PAGE and IB with anti-Cdc48, Ufd1, Flag and Ub antibodies.