5184 Nucleic Acids Research, 2010, Vol. 38, No. 15 we analysed the localization of the 60S reporter L25-eGFP (62) in the diﬀerent strains used in this study. Under per- missive culture conditions (SGal-Ura), L25-eGFP was, as expected for a r-protein, excluded from the vacuole and found predominantly in the cytoplasm in the GAL::RPL35 strain. However, following a shift to non-permissive conditions (SD-Ura) for as short as 3 h, L25-eGFP accumulated in the nucleus in about 80–90% of the ca. 120 GAL::RPL35 cells examined (Figure 5A). In most cells, the ﬂuorescence signal was restricted to the nucleolus, which was detected with the nucleolar marker Nop1-DsRed (62) (see magniﬁcation in Figure 5B). This phenotype is more evident after 9 h depletion where the ﬂuorescence signal was almost lost from the cytoplasm and prominent all over the nucleus without evident restric- tion to the nucleolus (Figure 5A). We did not observe nuclear accumulation of the L25-eGFP reporter in the wild type control strain grown in identical conditions (data not shown). Moreover, no accumulation of nuclear ﬂuorescence was observed upon L35 depletion when we studied the localization of the 40S r-subunit reporter S2-eGFP (59) either in galactose or in glucose medium (Figure 5A). Similar ﬂuorescence microscopy analyses were also per- formed with the Drpl35A and Drpl35B strains. These revealed that both the L25-eGFP and the S2-eGFP report- ers were found in the cytoplasm of all the strains tested. Figure 5. Depletion of L35 leads to nuclear retention of the 60S r-subunit reporter L25-eGFP. (A) RBY175 (GAL::RPL35) cells ex- pressing either L25-eGFP or S2-eGFP were grown in SGal-Ura at 30 C, shifted to SD-Ura for up to 9 h. The GFP signal was analyzed by ﬂuorescence microscopy. (B) Magniﬁed picture of selected GAL::RPL35 cells expressing L25-eGFP and the nucleolar marker Nop1-DsRed, which were depleted for 9 h in SD-Ura. Arrows point to nucleolar ﬂuorescence. However, we observed a mild nuclear retention of the ﬂuorescence signal of the L25-eGFP reporter in the Drpl35A mutant (data not shown). We conclude that both intra-nuclear and nucleo- cytoplasmic transport of pre-60S r-particles is impaired upon defective production of L35. These stalled pre-60S particles most likely contain, besides the stably assembled Rpl25-eGFP, 27SB pre-rRNAs, which are the most obvious pre-rRNAs that accumulated upon deletion of RPL35A and L35 depletion. This impairment is apparent- ly speciﬁc as transport of pre-40S r-particles is unaﬀected upon depletion of L35. L35B assembles in the nucleus Assembly of r-subunits occurs mainly in the nucle(ol)us, although few 60S r-proteins appears to stably load only with cytoplasmic pre-60S r-particles, such as L24 (30,45) or preferentially with them, such as L10 or P0 (38,72–75). No information was available on the course of the incorporation of L35 into pre-60S r-particles, although we suspect it should occur into the nucleus since several bipartite nuclear localization se- quences can be predicted with the cNLS mapper pro- gramme (76). To test whether L35 assembles in the nucleus, we made use of a fully functional L35B-eGFP (Supplementary Figure S5). We ﬁrst monitored the localization of L35B-eGFP in a LMB sensitive strain (64). This sensitivity is due to the presence of the T539C mutation in the exportin Crm1/Xpo1 (77). Treatment of the sensitive strain with LMB impairs export of pre-ribosomal particles and causes their accumulation in the nucleus (62,78). As shown in Figure 6A, the L25-eGFP, as expected for a r-protein, was primarily localized in the cytoplasm in untreated cells and, as reported (62,78), was readily trapped in the nucleus after a treatment with LMB for 3 h. As also shown in Figure 6A, L35B-eGFP localized similarly as L25-eGFP, indicating that it accumulated in the nucleus only after the LMB treatment. Similar nuclear accumulation was observed upon overexpression of the dominant negative nmd3D100 allele (78) in cells expressing either the L25-eGFP or the L35B-eGFP reporter (Figure 6B) but not upon overexpression of a wild type NMD3 allele (data not shown). Nmd3 is the Crm1-dependent adapter for export of pre-ribosomal par- ticles through the nuclear pores (78). The dominant negative Nmd3 100 protein traps pre-60S r-particles in the nucleus (78). Altogether, these data strongly suggest that L35B as- sembles in nucle(ol)ar pre-60S r-particles. L35 associates with earliest pre-60S ribosomal particles To address the timing of the nucle(ol)ar L35 assembly, we aﬃnity puriﬁed L35B-eGFP containing complexes with GFP-Trap beads (see ‘Materials and Methods’ section) and analysed which pre-rRNA species were present in these complexes by northern blot hybridization. As shown in Figure 7, and as expected for a 60S r-protein, there was a signiﬁcant co-puriﬁcation of mature 25S, 5.8S and 5S rRNAs with L35B-eGFP. We believe that the