For in vivo depletion of L35, the GAL::RPL35 strain was grown in 2% rich galactose medium (YPGal) at 30 C until mid-exponential phase (OD600 = 0.8), and then, har- vested, washed and transferred to YPD. Cell growth was monitored over a period of 18 h, during which the cultures were regularly diluted into fresh YPD medium. As control, the wild-type BY4741 strain was used. At diﬀer- ent time points, samples were collected to perform protein and RNA extractions, and polysome analysis. Sucrose gradient centrifugation Polysome and r-subunit preparations and analyses were performed as previously described (56) using an ISCO UA-6 system with continuous monitoring at A254. Protein extractions and western blotting analyses Total yeast protein extracts were prepared and analysed by western blotting according to standard procedures (55). The rabbit polyclonal anti-L35 (57), anti-L1 (58) and anti-S8 (59) antibodies were gifts from M. Seedorf, F. Lacroute and G. Dieci, respectively. RNA extractions, northern hybridization and primer extension analyses RNA extraction, northern hybridization and primer ex- tension analyses were carried out according to standard procedures (53,60). In all experiments, RNA was extracted from samples corresponding to 10 OD600 units of expo- nentially grown cells. Equal amounts of total RNA was loaded on gels or used for primer extension reactions (60). The sequences of oligonucleotides used for northern hybridization and primer extension analyses have been described previously (61) and are listed in Supplementary Table S2. Phosphorimager analysis was performed in a FLA-5100 imaging system (FujiElm) at the Biology Service (CITIUS) from the University of Seville. Pulse-chase analysis Pulse-chase labeling of pre-rRNA was performed exactly as described previously (56), using 100 mCi of [5,6-3H]uracil (Perkin Elmer; 45–50 Ci/mmol) per 40 OD600 of yeast cells. Total RNA was extracted as above and 20 000 cpm per RNA sample was loaded on gels and analysed as described in (56). Fluorescence microscopy To test pre-ribosomal particle export, the appropriate strains were co-transformed with a pRS316 plasmid har- boring a L25-eGFP reporter (62) or a S2-eGFP reporter (59) and a pRS413 plasmid expressing the nucleolar marker Nop1-DsRed [(62); this work]. Then, several transformants were grown to mid-log phase in selective liquid medium, washed, and resuspended in PBS buﬀer (140 mM NaCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 2.75 mM KCl, pH 7.3). When needed, cells were trans- formed with either the pRS316-GAL-nmd3D100 or the pRS316-GAL-NMD3FL plasmid (gifts from A. Jacobson), which harbour a dominant-negative Nucleic Acids Research, 2010, Vol. 38, No. 15 5179 truncation or a wild-type allele of the NMD3 gene, re- spectively, under the control of an inducible GAL promoter (63). Acquisition was done in a Leica DMR microscope equipped with a DC camera following the in- structions of the manufacturer. Digital images were pro- cessed with Adobe Photoshop 7.0. Leptomycin B (LMB) experiments were carried out as described (64) with slight modiﬁcations (38); brieﬂy, cells from the AJY1539 strain (a gift from A.W. Johnson) were co-transformed with the pUR34-NLS plasmid (gift from M. Corte-Real), which carried the red ﬂuorescent protein DsRed fused to a nuclear localization signal (NLS) (65) and either pRS315-RPL25-eGFP or YCplac111- RPL35B-eGFP. Transfromants were grown at 30 C in the appropriate synthetic 2% glucose (SD) medium to an OD600 of about 0.6. Then, cells from 2 ml culture of selected transformants were concentrated 2-fold in fresh medium and split. LMB (Alexis Biochemicals) in 20% ethanol or 20% ethanol vehicle was added to the cultures at a ﬁnal concentration of 200 ng/ml LMB-0.16% ethanol or 0.16% ethanol, respectively. Cultures were incubated for 1–3 h and then cells were in- spected by ﬂuorescence microscopy as above. Cell morphology was studied under the brightﬁeld microscope with cells ﬁxed with 70% ethanol whose nuclei where stained with 40,6-diamidino-2-phenylindole dihydrochloride (DAPI) at 0.5 mg/ml (54). Aﬃnity puriﬁcation of L35B-eGFP protein GFP-tagged L35B protein was precipitated following a one-step GFP-Trap _A procedure slightly modiﬁed from that suggested in the manufacturer’s instructions (Chromotek). Brieﬂy, 50 ml of GFP-tagged or untagged negative control cells were grown in SD-Ura medium to an OD600 of 0.8, washed with cold water, harvested and concentrated in 500 ml of ice-cold lysis buﬀer (20 mM Tris– HCl, pH 7.5; 1.5 mM MgCl2; 150 mM CH3COOK; 1 mM DTT; 0.2% Triton X-100) containing a protease inhibitor cocktail (Complete, Roche). Cells were disrupted by vigurous shaking with glass beads in a Fastprep -24 (MP Biomedicals) at 4 C and total cell extracts were obtained by centrifugation in a microfuge at the maximun speed (ca. 16100 g) for 15 min at 4 C. To each of the resulting supernatants 50 ml of GFP-Trap _A beads, equilibrated with the same buﬀer, were added and the mixture was incubated for 2 h at 4 C with end-over-end tube rotation. After incubation, the beads were extensively washed three times with 5 ml of the same buﬀer at 4 C and ﬁnally collected. RNA was extracted from the beads and the total cell extracts as exactly described in (66), and the extracted RNA was analysed by northern blotting as above. Flow cytometry Cells grown in logarithmic phase to an OD600 of 0.1–0.3 were harvested, ﬁxed with 70% ethanol and DNA was stained with propidium iodine as previously described (67). Stained cells were analysed using a Becton Dickinson FACScan ﬂow cytometer using CELL