Rrn3p is constitutively ubiquitylated and is rapidly degraded if protein neosynthesis is blocked To investigate whether Rrn3p-degradation is induced by ubiquitylation we compared ubiquitylation of Rrn3p-Prot.A (WT) before and after inhibition of the TOR pathway. It was previously reported that ubiquitylated proteins can be speciﬁcally enriched by their aﬃnity to the immobilized (poly) ubiquitin-binding protein Dsk2p (37). We generated whole-cell extracts from yeast strains expressing Rrn3p-Prot.A before and after 10 min of rapamycin treatment. Rrn3p-Prot.A-containing extracts were incubated with GST (Figure 1C, lanes 1–7) or GST-Dsk2p (Figure 1C, lanes 8–14) fusion protein immobilized on glutathione sepharose. After washings, the GST-baits and associated proteins were eluted and subjected to western blot analysis. Rrn3p-Prot.A was selectively retained by GST-Dsk2p, but not GST alone (Figure 1C and Supplementary Figure S1A, central panel). We also observe speciﬁc retention of (poly)ubiqui- tin and (poly)ubiquitinated proteins on the GST-Dsk2p beads (Supplementary Figure S1A, lower panel). In contrast, binding of the Pol I subunit A135 to immobilized GST-DSK2p in such an experiment was at background level. (Supplementary Figure S1A, upper panel). Rrn3p-Prot.A co-eluting with GST-Dsk2p migrated with slightly lower mobility in SDS–PAGE than Rrn3p-Prot.A in the input and ﬂow-through fractions (Figure 1C, compare lanes 14 with lanes 8 and 9). Even larger Rrn3p-TAP species, likely representing polyubiquitylated Rrn3p-TAP, could be bound to immobilized GST-Dsk2p from extracts of cells expressing the proteasome deﬁcient cim3-1 mutant and cultured at the restrictive temperature (Supplementary Figure S1B). Interestingly, neither the amount of ubiquitylated Rrn3p-Prot.A bound by Dsk2p nor the extent of polyubiquitylation did increase upon rapamycin treatment (Figure 1C and Supplementary Figure S1B). This suggests that Rrn3p ubiquitylation Nucleic Acids Research, 2010, Vol. 38, No. 16 5319 and proteasome-dependent degradation are not induced upon TOR inactivation. In fact, we observe a strong decrease in RRN3 mRNA levels after 20 min of rapamycin treatment (Supplementary Figure S1C). This is in good agreement with previous transcriptome analyses (38). Thus, the observed decrease of the Rrn3p level is rather due to the inhibition of RRN3 expression and the rapid turnover of the protein. A C-terminal Prot.A-tagged Rrn3p lacking the 17 N-terminal amino acids is stable upon nutrient starvation We found a remarkably increased Rrn3p stability in con- ditions in which TOR is inactive in a strain expressing a C-terminally Prot.A-tagged Rrn3p mutant containing a truncation of the 17 N-terminal amino acids ( N) (Figure 2). Deletion of the N-terminal 17 amino acids is required, but not suﬃcient to inhibit Rrn3p-degradation (data not shown). The C-terminal Prot.A-tag also contrib- utes to Rrn3p- N-Prot.A stability, and accordingly Rrn3p-Prot.A fusion proteins show an increased stability compared to Rrn3p-HA (data not shown). The plasmid-encoded N-mutant fully rescues growth in an rrn3 deletion strain (Figure 2B). Rrn3p- N-Prot.A levels remained stable even after 2 h of amino acid depletion whereas in this condition about 80% of Rrn3p were degraded in the corresponding reference strain expressing plasmid encoded wild-type Rrn3p-Prot.A (Figure 2C). In our following studies, this mutant served as a tool to in- vestigate how the stability of Rrn3p inﬂuences the integ- rity of the transcription machinery and the synthesis of rRNA in response to nutrient starvation. Non-degraded yeast Rrn3p keeps its subcellular localization in growth-arrested cells It has previously been reported that upon TOR- inactivation mammalian Rrn3p/TIFIA translocates from the nucleolus to the cytoplasm (12). Therefore, we Figure 2. N-terminally truncated Rrn3p-Prot.A (Rrn3p- N-Prot.A) is stable upon TOR inactivation. (A) Primary structure of the wild-type protein Rrn3p-Prot.A and the N-terminally truncated version Rrn3p- N-Prot.A. The 16 amino acids deleted in Rrn3p- N-Prot.A are indicated in red. The two fusion proteins are expressed from a centromeric vector under the control of the NOP1 promoter in a strain deleted in the endogenous RRN3 locus. (B) Growth curves of strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3- N-Prot.A ( N) cultured at 30 C in YPD. (C) Rrn3p- N-Prot.A resists degradation. Yeast strains pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3- N-Prot.A ( N) were grown in YPD at 30 C to mid-log phase (t = 0 min) and then starved in SDC-Trp (-Trp). 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 Prot.A-tag of the Rrn3p versions and the Pol I subunit A135, respectively.