5320 Nucleic Acids Research, 2010, Vol. 38, No. 16 analysed whether a cellular redistribution of Rrn3p after nutrient deprivation occurs also in yeast. Immunolocalization experiments conﬁrmed that Rrn3p disappears rapidly after nutrient starvation in wild-type cells, but neither in the N-mutant (Figure 3A), nor in proteasome-deﬁcient cim3-1 cells (Figure 3B). Furthermore, in both mutants the nuclear-cytoplasmic distribution of Rrn3p did not change. The amount of cytoplasmic versus nuclear Rrn3p- N-ProtA was also analysed by western blotting after fractionation of whole cells into nuclei and cytoplasm (Supplementary Figure S2). In accordance with the immunolocalization experiments the ratio between nuclear and cytoplasmic Rrn3p was very similar before and after depletion. From these experiments we conclude that the major population of wild-type yeast Rrn3p does not translocate after nutrient depletion. Maintaining Rrn3p levels upon nutrient depletion preserves the number of Pol I–Rrn3p complexes We asked how nutrient availability inﬂuences formation of Pol I–Rrn3p complexes in wild-type and mutant strains. Many experiments of several groups including ours demonstrated that down-regulation of rDNA transcrip- tion correlates with the dissociation of the Pol I–Rrn3p complex in stationary and growth-arrested cells. We have previously reported that Rrn3p is present in three diﬀerent forms in whole cell extracts of exponentially growing cells (39). The major Rrn3p fraction is monomeric, about 20% are tightly bound to Pol I, whereas the remaining Rrn3p is associated with a high molecular weight complex. To distinguish between the three forms of Rrn3p we per- formed gelﬁltration experiments with whole cell extracts derived from the Rrn3p-Prot.A wild-type and N mutant strain before and after amino-acid depletion (Figure 4A). In nutrient depleted wild-type cells, Rrn3p is signiﬁcantly reduced to a similar extent in all three populations (Figure 4A, panels WT). In contrast, in the N-mutant the amounts of Rrn3p in all fractions before and after nu- trient depletion are comparable (Figure 4A, panels N), suggesting that in this genetic background Pol I–Rrn3p complexes persist upon growth arrest. Similarly, in the cim3-1 mutant strain both, the total amount of Rrn3p and the ratio of complexed versus free Rrn3p did not change signiﬁcantly before and after nutrient depletion (Supplementary Figure S3A, panels cim3-1). These results indicate that maintaining Rrn3p levels upon nutrient depletion preserves the number of Pol I–Rrn3p complexes. To assess the number of Pol I–Rrn3p complexes in the two diﬀerent genetic backgrounds more quantitatively, co-immunoprecipitation experiments under stringent conditions were performed before and after nutrient depletion (Figure 4B). In extracts from nutrient depleted wild-type cells the amount of Rrn3p-Prot.A co-precipitating with HA-tagged Pol I-subunit A43 is strongly reduced, when compared to co- immunoprecipitation experiments with extracts from cells before nutrient depletion (Figure 4B, compare lane 7 with lane 8). In contrast, when the same experiments were carried out with extracts from the N-strain in which Rrn3p levels are maintained after nutrient deple- tion, N-Rrn3p-Prot.A association with Pol I was Figure 3. The subcellular distribution of stabilized yeast Rrn3p does not change upon nutrient starvation. (A) Immunolocalization. pNOP1-RRN3-Prot.A (WT) and pNOP1-RRN3- N-Prot.A ( N) cells either logarithmically growing or after amino-acid depletion were ﬁxed with 4% paraformaldehyde and treated with zymolyase to generate spheroplasts. Anti-protein A antibodies and an Alexa 594-conjugated secondary antibody were used to detect the Prot.A-tagged Rrn3p versions (in red), whereas the DNA was stained with DAPI (in blue). (B) Rrn3p-TAP does not change its subcellular localization if proteasome-dependent degradation is inhibited. The proteasome ts-mutant cim3-1 (TOY 652)(with chromosomally TAP-tagged Rrn3p) and the isogenic WT strain (TOY 651) were grown at 24 C in YPD medium to mid-log phase, before the cells were starved at 37 C in SDC-Leu medium. After 2 h the cells were ﬁxed with 4% paraformaldehyde and treated with zymolyase to generate spheroplasts. TAP-tagged Rrn3p was detected with an a-protein A primary antibody and an Alexa 594-conjugated secondary antibody (in red), while the DNA was stained with DAPI (in blue).