The biogenesis of the large (60S) ribosomal subunit in eukaryotes involves

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The biogenesis of the large (60S) ribosomal subunit in eukaryotes involves nucleolar, nucleoplasmic, and cytoplasmic steps. redundancy between Reh1 and Rei1, as a double deletion of and results in a synthetic growth defect (22), and overexpression of can partially suppress the pDEQ2 [or (including 300 nucleotides upstream and downstream, to include regulatory regions) from wild-type yeast genomic DNA with the primers DMP001 (5-GGGGGGGGATCCAGCACATCCACTCTCATTCCCGATATTCC) and DMP002 (5-GGGGGGCTCGAGCTTCAGTCTTCAGCAGCTATTTCCTTTGCT) or the primers DMP003 (5-GGGGGGCTCGAGCCGTCCATGCGATATGAGCTGATTC) and DMP004 (5-GGGGGGGTGTCCCAACCCCGTGTCCG). The PCR products were digested with BamHI and XhoI and ligated into the same sites of pRS315 (37). pMP003 (open reading frame using primers DMP005 (5-GGGGGGGGATCCATGAGCTCTACTTTCTTTACATGCAACTG) and DMP006 (5-GGGGGGCTCGAGCTATTGCAACAACTCATCCCTGTAATGTG). The PCR products were digested with BamHI and XhoI and ligated into the same sites of p415 TEF (31). pAJ1004 SAG pontent inhibitor (for 5 min). Cells were washed in ice-cold breaking buffer A (20 mM TrisHCl, pH 7.5, 100 mM NaCl, 30 mM MgCl2, 100 g/ml cycloheximide, 200 g/ml heparin), repelleted by centrifugation as above, resuspended in breaking buffer A (1.5 ml per g of wet cell weight), lysed by glass bead vortexing (10 cycles of 45 s of vortexing followed by 45 s on ice), and cleared by centrifugation (20,000 at 4C for 20 min). Approximately 10 OD260 units of cleared lysate were layered on 11 ml of 7 to 47% (mass/vol) sucrose gradients (containing 50 mM TrisHCl, pH 7.5, 50 mM KCl, 12 mM MgCl2, 1 mM dithiothreitol [DTT]) and centrifuged in an SW41 Ti rotor, Optima L-90K Ultracentrifuge (Beckman Coulter Inc., Fullerton, CA) at 40,000 rpm (200,000 at 4C for 30 min), separated on 5 to 15% polyacrylamide-SDS gels as described above, and transferred to nitrocellulose membranes (Immobilon-P; Millipore, Billerica, MA). The following primary antibodies were used in this study at the indicated dilutions in 3% nonfat milk-TTBS (20 mM SAG pontent inhibitor ITGB2 Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20): mouse monoclonal antibodies anti-FLAG M2 (1:10,000; Sigma, St. Louis, MO) and anti-Rpl3 (1:5,000; J. Warner, Albert Einstein College of Medicine, New York, NY); and rabbit polyclonal antibodies anti-Arx1 (1:1,000; M. Fromont-Racine, Institut Pasteur, Paris, France), anti-Nmd3 (1:5,000; A. Johnson, University of Texas, Austin, TX), anti-Rei1 (1:1,000, M. Fromont-Racine), anti-Rlp24 (1:1,000, M. Fromont-Racine), anti-Rpl10 (1:2,000, B. Trumpower, Dartmouth Medical School, Hanover, NH), and anti-Tif6 (1:1,000, F. Fasiolo, IBMC, Strasbourg, France). Secondary antibodies (goat anti-mouse and goat anti-rabbit antibody-horseradish peroxidase conjugates) were used at 1:20,000 dilutions, and visualization of peroxidase activity was performed with a SuperSignal West Femto chemiluminescence kit (Pierce, Rockford, IL). [35S]methionine pulse-chase analysis. Pulse-chase analysis of 3FLAG-Reh1 or 3FLAG-Rei1 immunoprecipitations was performed essentially as described previously (15). 3FLAG-Reh1 (MP011/pMP004) or 3FLAG-Reh1 (MP002/pMP003) strains were grown in 250 ml of minimal medium lacking leucine and methionine at 30C to mid-log phase (OD600 of 1 1). Cells were pelleted and resuspended in 9 ml of the same medium. l-[35S]methionine (3.5 mCi EXPRE35S35S protein labeling mix; Perkin Elmer, Waltham, MA) was then added. After 5 min, cells were pelleted and resuspended in 9 ml of medium containing 200 g/ml unlabeled methionine. Samples (1.0 ml) were removed to an ice bath upon addition of unlabeled methionine (0 min), and at 5, 10, 20, and 40 min after chase. Pelleted cells were lysed by glass bead vortexing on ice and cleared, and FLAG immunoprecipitation was carried out as described SAG pontent inhibitor above. Immunoprecipitated proteins were separated by SDS gel electrophoresis and transferred to nitrocellulose, and Rpl3 was visualized by Western blotting (as described above). [35S]methionine was subsequently visualized by autoradiography. Fluorescence microscopy. Cells had been transformed having a centromeric plasmid expressing either (pAJ1015) or (pAJ1004). To determine localization of Arx1-GFP in the (MP011/pMP004) or (MP002/pMP003) cells had been separated by sucrose denseness sedimentation (7% to 47% sucrose). Positions of 40S, 60S, and 80S subunits are indicated. 3FLAG-Reh1 or 3FLAG-Rei1 was recognized by Traditional western blotting using anti-FLAG antibody. (B) Nascent subunits run after through Reh1 and Rei1 complexes. Cells including 3FLAG-Reh1 or 3FLAG-Rei1 had been tagged for 5 min with [35S]methionine, accompanied by addition of excess unlabeled methionine as referred to in Strategies and Components. Examples had been eliminated at the changing times indicated after addition of run after. Extracts were prepared, and proteins were immunoprecipitated with anti-FLAG antibody. Immunoprecipitated proteins were analyzed by Western blotting for Rpl3.