Fig 1.
Dbp5 is associated with polysomes and both ribosomal subunits.
(A-B) Wild type yeast lysates separated in sucrose-density gradients were fractionated while measuring the absorbance at 254 nm (A254nm) that results in the displayed profile (A). The corresponding separated protein fractions are shown in Western blot analyses with antibodies against Dbp5 and the ribosomal protein Rps3 (B). The percentage of the total cellular amount of Dbp5 in the non-ribosomal, 40S, 60S, 80S and polysomal fractions is indicated. (C-D) Western blot analyses of Rpl25-GFP immunoprecipitations from gradient fractions of the free ribosomal subunits show co-precipitation of Dbp5. Wild type yeast lysates were separated in sucrose-density gradients and upon fractionation, the free ribosomal subunits containing fractions (red framed area) were subjected to co-immunoprecipitation experiments. The correct fractions were chosen according the flow through photometry profiles displayed in (C). Detection of Mex67 served as positive control and the mitochondrial protein Aco1 served as a negative control.
Fig 2.
Mutants of DBP5 are defective in the nuclear export of both pre-ribosomal subunits.
Fluorescence in situ hybridization experiments with probes against the 25S rRNA (A), the 18S rRNA (B) and poly(A)+RNA (C) reveal a nuclear accumulation of the fluorescent signal in different temperature-sensitive dbp5/rat8 mutants and in a mutant of the karyopherin Xpo1/Crm1 (xpo1-1), which served as positive control, upon 1 h temperature shifts to their indicated non-permissive temperatures. The lower panels of (A) and (B) display a magnification of a representative cell, where the FITC channel was merged with the DNA signal. All single channels and merged pictures are shown in S2 Fig. (WT = wild type). (D) Statistical analyses of (A-C). The upper panel indicates the amount of cells with nuclear accumulation (error bars represent the standard deviation). The lower panel shows the average intensities of the nuclear signal of at least 10 cells relative to the whole cell and compared to wild type. Error bars represent the standard deviation and p-values were calculated by an unpaired students t-test (*** = p<0.001 and ** = p<0.01).
Fig 3.
Mutants of DBP5 show no unique defects in the biogenesis of ribosomal subunits.
(A) The steady state levels of the different rRNAs and their precursors in the dbp5 mutants rat8-2 and rat8-7 are comparable with those in the export mutants rat7-1 and xpo1-1. Northern blot analyses with DIG-labeled RNA probes targeting the indicated rRNA species and U2 snRNAs (loading control) are shown. Total RNA of the different strains was extracted upon 1 h shifts to their restrictive temperatures and 1 μg was separated by formaldehyde/MOPS-agarose gel electrophoresis. (B) The total amount of 40S and 60S subunits is not altered in dbp5 mutants. Flow through photometry (A254nm = absorbance at 254 nm) profiles of ultra-centrifuged sucrose gradients from rat8-2 and rat8-7 cells shifted to their indicated non-permissive temperatures and treated with 100 mM EDTA reveal no significant change of their peak sizes and the 60S to 40S subunit ratio in comparison to the wild type. In contrast, rpl10(G161D) served as a positive control of the assay and shows a decreased 60S peak.
Fig 4.
Genetic and physical interactions of Dbp5 and ribosomal export factors.
(A) Dbp5 genetically interacts with the established ribosomal transport factors Nmd3 and Mtr2. The indicated strains were spotted in serial dilutions onto plates either selecting for a covering the respective wild type gene containing plasmid (selective media) or for the loss of this plasmid (FOA = 5-Fluoroorotic Acid). The strains rat8-3 and rat8-2 in combination with nmd3-2 or mtr2-33, which has a specific export defect for ribosomal subunits, show synthetic growth defects after three days at 30°C. (B) Physical interactions of Dbp5 with Nmd3, Xpo1 and Rio2. Western blot analyses show co-precipitated Nmd3-myc in the Dbp5-GFP immunoprecipitation (left panel) and co-precipitated Dbp5 in the Xpo1-GFP and Rio2-GFP pull-downs (right panel). The DBP5-GFP expressing strain and the wild type (WT) were transformed with an NMD3-13xmyc containing plasmid. Detection of Hem15 served as a negative control and of Rps3 as a positive control.
Fig 5.
Dbp5 does not accompany ribosomal particles through the NPC, but seems to be required at its cytoplasmic side.
(A) Dbp5 does not travel with the pre-ribosomal subunits from the nucleus to the cytoplasm. Fluorescence microscopy images of GFP-tagged Dbp5 show a cytoplasmic distribution in wild type, mtr2-33 and nmd3-2 cells upon shift to 37°C for 1 h. In contrast, GFP-Dbp5 accumulates in the nucleus of shifted xpo1-1 cells. (B) rat7-1 cells show mild ribosomal export defects. Fluorescence in situ hybridization experiments with probes against the 25S rRNA, 18S rRNA and poly(A)+RNA are shown in rat7-1 and wild type cells upon shifts for 1 h to 37°C. Statistical analyses were performed as shown in Fig 2D. (C) The association of Nmd3-myc with GFP-Dbp5 is not increased in the drg1-18 strain compared to wild type (WT). Western blot analyses of an immunoprecipitation experiment with GFP-Dbp5 and co-precipitated Nmd3-myc are shown upon 1 h shifts to 37°C. Detection of Hem15 served as non-binding control.
Fig 6.
The ATPase activity of Dbp5 is dispensable for nuclear export of pre-ribosomal particles.
(A-B) Dominant-negative ATPase-deficient dbp5 mutants do not affect ribosomal transport. Fluorescence in situ hybridization experiments with wild type cells overexpressing DBP5, dbp5-R369G and dbp5-R426Q after 1.5 h galactose induction with probes against the 25S rRNA (A) and the 18S rRNA (B) reveal wild typical distribution of the fluorescent signals in contrast to the nuclear accumulations of the poly(A)+RNA. (C) The cytoplasmic filament-bound ATPase-deficient mutant dbp5-R426Q partially rescues the ribosomal transport defects of rat8-2. Fluorescence in situ hybridization experiments with Cy3-labled probes against 25S rRNAs are shown in rat8-2 cells with an empty vector (p) or overexpressing dbp5-R369G, dbp5-R426Q, dbp5E240Q, dbp5K144Q and DBP5 after 1 h galactose induction. All cells were shifted for the last 30 min of galactose induction to 37°C. (D) Statistical analyses of (C) show the average enrichment of the nuclear signal compared to rat8-2 cells. The nuclear signals of at least 10 cells were set into relation with the whole cell and the signal obtained in wild type. The resulting ratios were compared to rat8-2. Error bars represent the standard deviation and p-values were calculated by an unpaired students t-test (*** = p<0.001 and * = p<0.05).
Fig 7.
Dbp5 does not displace Mex67 from exported ribosomal particles.
(A) Sucrose-density fractionation experiments reveal that Mex67 is associated with polysome-containing fractions from wild type and rat8-2 cells and (B) remains equally ribosome bound upon RNase A treatment. The upper panel shows profiles of wild type and rat8-2 cells upon flow through photometry (A254nm) from ultra-centrifuged sucrose gradients. Before loading, the cells were shifted for 1 h to 37°C and half of the resulting lysates were treated with RNase A. The bottom panel shows the corresponding separated protein fractions in Western blot analyses with direct antibodies against Mex67 and the ribosomal protein Rps3. The mRNA-binding protein GFP-Cbp80 was expressed with a galactose-inducible promoter, subsequently detected with an anti-GFP antibody to serve as a positive control. The ratios of all proteins from the first fractions (non-ribosomal+40S+60S) and the last fractions (80S+polysomal fractions) are indicated. (C-D) Mex67 is RNA-independently associated with polysomes in wild type cells. Western blot analyses of Rpl25-GFP immunoprecipitations from polysomal gradient fractions show co-precipitation of Mex67. Wild type yeast lysates were separated in sucrose-density gradients that were subsequently fractionated. The polysomes-containing fractions (red framed area) were subjected to co-immunoprecipitation experiments that were treated with RNase A. The correct fractions were chosen according the flow through photometry profile displayed in (C). Detection of Rps3 and Dbp5 served as positive controls. Aco1 was detected as non-binding control and the mRNA-binding protein Pab1 as control for the RNase A treatment. (E) The association of Mex67 with ribosomal proteins is unchanged in the rat8-2 strain. Western blot analyses of a co-immunoprecipitation experiment with Mex67-GFP and Rps3 or Rpl35 in wild type and rat8-2 cells, which were shifted for 1 h to 37°C, are shown upon RNase A treatment. Hem15 served as a negative control.
Fig 8.
Dbp5 interacts with Mex67 in vivo and in vitro.
(A) Dbp5 interacts mainly RNA-dependently with Mex67 in vivo. Western blot analyses of GFP-Dbp5 immunoprecipitations show strong co-precipitation of Mex67 without RNase A treatment, while addition of RNase A leads to a weaker, but reproducible interaction with Mex67. Detection of Hem15 served as non-binding control. (B) Direct interaction of recombinant GST-Dbp5 and Mex67, which is increased by ATP addition. Western blot analyses of Glutathione Sepharose pull-downs with GST-Dbp5 or GST and the purified heterodimer His-Mtr2 and Mex67 are shown in the presence or absence of 1 mM ATP and 0.2 mg/ml RNase A. The corresponding Coomassie-stained gel is depicted in S8D Fig.