Figure 2. Ubiquitinated nascent peptides linked to tRNA accumulate on ribosomes in cdc48-3 and ufd1-2 mutants.

(A) Puromycin-dependent binding to UBA resin of Ub conjugates from cdc48-3 ribosomes. Ribosomes were affinity-purified from untagged or wildtype and mutant RPL18BTAP cells grown at 37°C for 90 min and eluted with TEV protease in the presence or absence of puromycin (puro), followed by incubation with UBA resin. Bound fractions were resolved by SDS-PAGE and immunoblotted with antibodies to Ub (top panel) or puromycin (lower panel). (B) Transfer of Ub conjugates to puromycin was RNAse A-sensitive. Ribosomes were affinity-purified from cdc48-3 (UT) or cdc48-3RPL18BTAP cells as described above in the absence (lanes 1, 2 and 3) or presence of 200 μg/ml RNAse A. Following elution with TEV protease, samples from the tagged cells (lane 3) were treated with 200 μg/ml RNAse A at 30°C for 10 min before incubation of all samples with puromycin and binding to UBA resin. The bound fractions were evaluated as in (A). (C) CTAB-precipitable Ub conjugates accumulate on cdc48-3 ribosomes. Ribosomes affinity-purified from the same strains used in panel (A) were treated with RNAse A (or not) and subjected to precipitation (pptn) with CTAB. Precipitates were resolved by SDS-PAGE and immunoblotted with anti-Ub. 48 Refers to cdc48-3. (D) Ubiquitinated newly-synthesized proteins accumulate on ribosomes isolated from cdc48-3 and ufd1-2 mutants. Cells were pulse-labeled for 90 s with ³⁵S methionine and chased with cold methionine and cycloheximide for the indicated times. Ribosomes were affinity-purified, eluted with TEV protease in the presence of puromycin, and loaded onto UBA resin. The bound fraction was evaluated by SDS-PAGE followed by autoradiography. Densitometry indicated that <5% of the high MW material was released from ufd1-2 ribosomes between the 0- and 10-min time points.

DOI: http://dx.doi.org/10.7554/eLife.00308.006

Figure 2—figure supplement 1. Cdc48-Ufd1-Npl4 complex is required to clear ubiquitinated nascent peptides from ribosomes.

(A) SDS-PAGE/Coomassie blue profiles of ribosomes affinity-purified from the indicated strains carrying the RPL18BTAP allele. UT: wildtype cells lacking the RPL18BTAP allele. (B) Ribosomes from the indicated strains grown at 30°C for two generations were pelleted through sucrose cushions and analyzed by immunoblotting (IB) for anti-Rpl32 (bottom panel). Resuspended ribosomes were treated with puromycin in high salt and then incubated with UBA resin. The bound fraction was evaluated by SDS-PAGE and IB with anti-ub (top panel). (C) Ribosomes were affinity-purified from wildtype and mutant strains carrying the RPL18BTAP allele and eluted from the magnetic beads with TEV protease in the presence of puromycin. The eluate was either evaluated directly by IB with anti-TAP (bottom panel) or was incubated with UBA beads. The bound material was fractionated by SDS-PAGE and analyzed by IB with anti-puromycin antibody (top panel). (D) Left panel: Total cell lysates for [³⁵S]-labeled cells were fractionated by SDS-PAGE and subjected to autoradiography to show incorporation of label during the 90 sec pulse. Right panel: Ribosomes were affinity-purified from lysates depicted on left. Ribosomes eluted from the magnetic beads with TEV protease were analyzed by SDS-PAGE and autoradiography to reveal newly-synthesized proteins contained in the preparations. UT: wildtype cells lacking the RPL18BTAP allele.

DOI: http://dx.doi.org/10.7554/eLife.00308.007