5318 Nucleic Acids Research, 2010, Vol. 38, No. 16 RESULTS Reduction of Rrn3p-levels in growth-arrested yeast cells is dependent on the proteasome We have previously reported that down-regulation of rRNA synthesis in stationary yeast cells coincides with a lower recovery of Pol I–Rrn3p complexes (24). A clear reduction of Pol I–Rrn3p complexes was also observed by us (data not shown) and others when pre-rRNA syn- thesis was down-regulated by the protein synthesis inhibi- tor cycloheximide, by nutrient starvation, or rapamycin, an inhibitor of TOR-kinases which mimics conditions of nutrient starvation (32) (11,12,33,34). This reduction of Pol I–Rrn3p complexes in yeast cor- relates with decreasing amounts of Rrn3p in whole cell extracts. When exponentially growing yeast strains were either treated with cycloheximide or rapamycin, or depleted in an essential amino acid, Rrn3p levels were rapidly reduced (Figure 1A). After 120 min, Rrn3p-levels dropped below 20% of the initial amount in all three conditions. A reduced level of Rrn3p is also detectable if Pol II-dependent transcription is inhibited i.e. when a temperature-sensitive RNA polymerase II mutant (rpb1-1) is shifted to the restrictive temperature, or if cells are in stationary phase (data not shown). In contrast, Pol I levels remain rather stable in growth arrested cells (Figure 1A, lower panel, data not shown). This demonstrates that inactivation of both, TOR pathway, and protein synthesis results in a reduction of the Rrn3p protein level. The rapid down-regulation of Rrn3p in growth ar- rested cells depends on the proteolytic activity of the pro- teasome. In a temperature-sensitive mutant which is defective in CIM3/SUG1, a gene coding for an essential subunit of the regulatory 19S subunit of the proteasome (35,36), Rrn3p is stable upon amino-acid depletion at the restrictive temperature (Figure 1B). In contrast, deletion of vacuolar proteases like PEP4 does not aﬀect the reduction in Rrn3p levels after growth arrest (data not shown). Figure 1. Rrn3p levels are reduced upon TOR inactivation and proteasome-dependent degradation. (A) Rrn3p is degraded upon TOR inactivation and inhibition of translation. Yeast strain RRN3-Prot.A expressing a chromosomally Prot.A-tagged Rrn3p was grown in YPD at 30 C to mid-log phase (OD600 & 0.4; t = 0 min), before the cells were either treated with 200 ng/ml of rapamycin or with 100 mg/ml of cycloheximide, or starved in SDC-Leu (aa-depletion), respectively. At the time points indicated cells were collected and lysed. Same amounts of WCE (20 mg) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p and the Pol I speciﬁc subunit A43, respectively. (B) Rrn3p-degradation depends on proteasome activity. The proteasome ts-mutant strain (cim3-1, TOY 652) expressing a chromosomally TAP-tagged Rrn3p or the isogenic CIM3 WT strain (WT, TOY 651) were grown to mid-log phase in YPD at 24 C (t = 0 min), before the cells were starved at 37 C in SDC-Leu medium (-Leu). At the time points indicated cells were collected and lysed. Same amounts of WCE (30 mg) were analysed by western blotting using antibodies directed against the TAP-tag of Rrn3p and the Pol I subunit A135, respectively. (C) Rrn3p is ubiquitylated. Yeast strain pNOP1-RRN3-Prot.A, expressing Prot.A-tagged Rrn3p from a plasmid was grown in YPD at 30 C to midlog phase, before half of the cells were treated with 200 ng/ml of rapamycin for 10 min. Cells of rapamycin treated and untreated cultures were collected and lysed. Same amounts of WCEs (6 mg) were incubated with either recombinant GST-Dsk2p, or recombinant GST immobilized on 50 ml of glutathione sepharose. After washing, proteins bound to the beads were eluted with SDS sample buﬀer. Same amounts (1%) (50 mg) of input (IN) and ﬂow through (FT), 0.5% of the wash steps (washes) and 50% of the eluate (E) were analysed by western blotting using antibodies directed against the Prot.A-tag of Rrn3p.