Model for Drg1 function in late stages of pre-60S biogenesis. Only factors relevant for this work are shown. Tif6, Rlp24, Nog1, and Arx1 bind to the early pre-60S particles in the nucleus and accompany them into the cytoplasm, where Drg1 associates. Drg1 is required for the release of Nog1 and Rlp24 from pre-60S particles and for the subsequent binding of Rei1. Consequently, Arx1 and Tif6 can be released and recycled back to the nucleus.

Drg1 interacts with late preribosomal particles. Cultures of strains expressing TAP-tagged versions of Nsa3, Rlp24, Nog1, Arx1, Rei1, and Alb1 were grown to late log phase, and the proteins were affinity purified on IgG beads as described in Materials and Methods. Aliquots of the TEV eluates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted. Western blot analyses with polyclonal antibodies directed against Drg1, Arx1, Nog1, Rlp24, and Tif6 are shown below the Coomassie blue-stained gel. A Western blot with a polyclonal antibody directed against the ribosomal protein Rpl16 served as loading control.

Nog1 and Arx1 accumulate in the cytoplasm when Drg1 is depleted. Nog1-TAP (LMA636), Rlp24-TAP (LMA611), Arx1-GFP (LMA606), and Nog2-GFP (LMA607) fusion proteins were produced in strains expressing DRG1 under the control of the GAL1 promoter. Strains were grown in galactose-containing medium and shifted to glucose-containing synthetic medium for 20 h at 30°C to shut down expression of DRG1. The corresponding wild-type strains were grown in glucose-containing synthetic complete medium at 30°C. Thereafter, Nog2-GFP- and Arx1-GFP-expressing strains were examined by epifluorescence microscopy. Nog1-TAP- and Rlp24-TAP-expressing cells were fixed and processed for immune fluorescence using an anti-protein A antibody as described previously (17). Fluorescence and Nomarski images for each strain are shown.

Functional Drg1 is required for correct 27S pre-rRNA processing. Cells from the wild-type strain W303, the drg1-ts mutant FWY111, and the drg1-sup suppressor mutant DTY4 were incubated at 25°C and 37°C for 90 min, pulse-labeled with [¹⁴C]uridine for 2 min, and chased with a 50-fold excess of nonradioactive uridine for 0, 3, 15, and 30 min. RNA was extracted by the hot-phenol method. (A) The RNA was separated on 1.2% formaldehyde agarose gels to follow the processing of high-molecular-weight rRNA precursors. The gel was blotted, and the radioactivity was detected by phosphorimaging. (B) The RNAs of W303 (DRG1) and FWY111 (drg1-ts) were separated on 8% acrylamide-8 M urea gels to detect the formation of low-molecular-weight RNAs. The gel was dried, and radioactivity was detected by exposure of an X-ray film.

Nog1, Rlp24, and Arx1 accumulate in the cytoplasm of the drg1-ts mutant after incubation at the restrictive temperature. Nog1-GFP, Rlp24-TAP, Arx1-TAP, and GFP-Nop4 fusion proteins were expressed in the wild-type strain and the drg1-ts strain. The strains were grown to early log phase at 25°C and incubated for 2 hours at 37°C before they were analyzed by fluorescence microscopy (GFP constructs) or fixed and processed for indirect immune fluorescence and DAPI (4′,6′-diamidino-2-phenylindole) staining (TAP constructs). The indirect immune fluorescence assay was performed according to the method in reference 17 using a polyclonal antibody directed to protein A and a rhodamine-conjugated secondary antibody.

The drg1-ts mutant shows ribosome half-mers in polysome profiles. (A) Schematic representation of protein Drg1. The amino acid changes leading to the thermosensitive phenotype, the suppressor phenotype, and the E617Q variant are marked by arrows. The Walker A and B motifs of the AAA domains D1 and D2 are indicated in the lower part of the diagram. (B) Polysome profiles of the wild-type strain W303, the drg1-ts mutant FWY111, and the drg1-sup suppressor mutant DTY4, incubated for 30 min at 37°C, are shown. Extracts were prepared, and 6.5 A₂₆₀ units of each was loaded on 15 to 50% sucrose gradients as described in Materials and Methods. After ultracentrifugation for 2.5 h at 200,000 × g, polysome profiles were collected. Half-mer polysomes and reduced free 60S subunits observed in the drg1-ts mutant are indicated by filled and open arrows, respectively.

ATP hydrolysis by D2 of Drg1 is required for the release of shuttling proteins. (A) Overexpression of a Drg1 variant containing an E617Q exchange in the Walker B motif of D2 causes accumulation of Nog1 and Rlp24 on late pre-60S particles. Late preribosomal particles were affinity purified from the Arx1-TAP strain after overexpression of a GST fusion of Drg1(E617Q) under the control of the CUP1 promoter. The wild-type GST-Drg1 fusion and the cloning vector served as controls. The strains were grown to early log phase, and expression was induced by addition of 0.5 mM copper sulfate for 3 h. Western blots of the TEV eluates are shown. (B) The Drg1(E617Q) protein exerts a dominant-negative effect on Nog1-GFP localization. The Nog1-GFP strain carrying a plasmid with a GST fusion of Drg1(E617Q) under the control of the CUP1 promoter was grown to early log phase, and the expression of the fusion protein was induced by 0.5 mM copper sulfate for 6 h. Thereafter, cells were inspected by fluorescence microscopy. The expression vector (pYEX4-T1) and the vector expressing the GST fusion with wild-type Drg1 (pAZ7) served as controls.

Drg1 localization. (A) Cell fractionation. Crude extracts from the wild-type strain W303 and from W303 carrying the DRG1 gene on a 2μm plasmid (pGZ252) were fractionated by centrifugation. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted. Drg1, Nop1 (control for nuclear localization), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; control for cytosol) were detected in the resulting fractions by Western blot analysis using specific antibodies. Pellet fractions are marked by P, and supernatant fractions are marked by S. (B) Drg1 in an overexpressing strain visualized by immune gold labeling and electron microscopy. Nuclei and mitochondria are denoted with N and M, respectively. Bar, 0.5 μm. (C) The leptomycin-sensitive crm1 strain expressing GFP-Drg1 or Rpl25-GFP fusions was grown to mid-log phase and treated with 100 ng/ml leptomycin B (+LMB) for 1 h. Then cells were inspected by fluorescence microscopy for the localization of the fusion proteins (upper panels). In the lower panels, the corresponding Nomarski images are shown. An untreated culture of each strain served as control.

Nog1, Rlp24, Arx1, and Tif6 remain associated with pre-60S particles in the drg1-ts mutant and upon depletion of Drg1. (A) Polysome profiles from the temperature-sensitive drg1-ts mutant (ELY118) and the isogenic wild-type strain (ELY1) expressing the Tif6-GFP fusion are shown. The strains were grown at 25°C and shifted to 37°C for 2 h. Extracts were prepared, and 6.5 A₂₆₀ units each was loaded on 15 to 50% sucrose gradients. After ultracentrifugation for 2.5 h at 200,000 × g, polysome profiles were recorded, and fractions of 800 μl were collected. The proteins present in the individual fractions were precipitated with trichloroacetic acid, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted. Western blot analyses with polyclonal antibodies directed against Arx1, GFP, Nog1, Rlp24, and the ribosomal protein Rpl16 are shown. (B) TAP purification of Arx1 containing preribosomal particles after depletion of Drg1. The wild-type strain (LMA397) and the P_GAL1::DRG1 (LMA609) strain expressing Arx1-TAP fusions were grown in galactose-containing synthetic medium. Mid-log cells were transferred to glucose-containing synthetic medium for 19 h to deplete cells of Drg1. Thereafter, TAP purification was performed as described previously (25). Proteins in the eluates were separated by SDS-PAGE and blotted. Western blotting results with antibodies directed against Arx1, Drg1, Rei1, Nog2, Nog1, Rlp24, Tif6, and the ribosomal protein Rpl1 are shown either on TEV eluates or on TAP purifications. (C) The wild-type and drg1-ts strains expressing chromosomally TAP-tagged Arx1 were grown to early log phase and incubated for 60 min at 37°C. After cell lysis, Arx1-containing preribosomal particles were affinity purified on IgG beads and eluted by TEV cleavage. Samples were analyzed by SDS-PAGE and Western blotting. Twice as much eluate was loaded from the wild-type strain to facilitate detection and quantification of Nog1 and Rlp24. (D) Arx1-containing preribosomal particles were TAP purified from the drg1-ts mutant and the isogenic wild-type strain after incubation at 37°C for 1 h. Proteins in the eluates were separated on a 4 to 12% gradient sodium dodecyl sulfate-polyacrylamide gel and stained with Coomassie blue. Prominent proteins present in increased amounts in the eluate from the mutant are indicated by numbers and were identified by mass spectrometry.