HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 21, 18334-18339, May 24, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/21/18334    most recent M201667200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miyoshi, K. Articles by Mizuta, K. Search for Related Content PubMed PubMed Citation Articles by Miyoshi, K. Articles by Mizuta, K. Social Bookmarking                      What's this?

Normal Assembly of 60 S Ribosomal Subunits Is Required for the Signaling in Response to a Secretory Defect in Saccharomyces cerevisiae^*

Keita Miyoshi^§, Rota Tsujii^§, Hideji Yoshida^¶, Yasushi Maki^¶, Akira Wada^¶, Yasushi Matsui, Akio Toh-e, and Keiko Mizuta^§**

From the  Department of Biological Sciences, Graduate School of Biosphere Sciences and ^§ Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739-8528, Japan, ^¶ Department of Physics, Osaka Medical College, Takatsuki, Osaka 569-0084, Japan, and the  Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received for publication, February 19, 2002, and in revised form, March 8, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A secretory defect leads to transcriptional repression of both ribosomal protein and rRNA genes in yeast. To elucidate the mechanism of the signaling, we previously isolated rrs mutants that were unable to respond to a secretory defect, and we cloned RRS1 encoding a nuclear protein that was required for ribosome biogenesis (Tsuno, A., Miyoshi, K., Tsujii, R., Miyakawa, T., and Mizuta, K. (2000) Mol. Cell. Biol. 20, 2066-2074). We identified duplicated genes encoding ribosomal protein L11, RPL11B as a wild-type allele complementing the rrs2 mutation, and RPL11A in two-hybrid screening using RRS1 as bait. Rpl11p was copurified with Rrs1p in immunoprecipitation analysis. Ultracentrifugation analysis revealed that Rrs1p associated fairly tightly with 60 S preribosomal subunits. These results suggest that signaling in response to a secretory defect requires the normal assembly of 60 S ribosomal subunits including Rrs1p and Rpl11p.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Yeast ribosome consists of 4 rRNAs and 78 ribosomal proteins (RP).¹ The amounts of the components are coordinately regulated corresponding to cell growth mainly at the level of transcription (1, 2). Furthermore, ribosome synthesis appears to be coupled with membrane synthesis; a secretory defect causes specific and significant repression of transcription of both RP and rRNA genes (3-5). To elucidate the molecular mechanism of the regulation in response to a secretory defect, we isolated mutants, named rrs (regulator of ribosome synthesis), in which a secretory defect was unable to cause transcriptional repression of RP genes. RRS1, a previously unidentified gene, was cloned as the wild-type allele complementing one of the mutants. We demonstrated that Rrs1p was essential for growth, localized in the nucleus, and required for ribosome biogenesis, especially for 25 S rRNA maturation and 60 S ribosomal subunit assembly (6). This suggested that the mechanism of the signaling in response to a secretory defect was closely linked to normal regulation of ribosome biogenesis.

By different approaches, other factors in the secretory signaling have been found. We demonstrated that the C-terminal region of Rap1p, a DNA-binding protein, was also essential for the transcriptional repression of both RP and rRNA genes due to a secretory defect (7, 8). Rap1p is involved in both transcriptional activation of many genes including most RP genes and silencing such as the telomere position effect. It is unknown how Rap1p is responsible for the repression due to a secretory defect. Furthermore, it has recently been proposed that plasma-membrane stretch caused by the elevated internal turgor pressure is a trigger of the signaling (5) and that protein kinase C (Pkc1p) is required for the signaling (9). However, mitogen-activated protein kinase cascade of the cell integrity pathway was not required for this response, and components downstream of Pkc1p remain unknown.

In this study, we cloned RPL11B, which encodes ribosomal protein L11 (Rpl11p), as a responsible gene for the rrs2 mutation. We also isolated RPL11A in yeast two-hybrid screening using RRS1 as bait. Rpl11p is encoded by the duplicated genes RPL11A (YPR102c) and RPL11B (YGR085c), formerly named RPL16B and RPL16A (10-12), respectively (for the nomenclature, see Ref. 13 and the Saccharomyces Genome Data base, genome-www.stanford.edu/Saccharomyces/). The RPL11A gene is expressed at twice the level of RPL11B (10). Rpl11Ap and Rpl11Bp differ only at their third amino acid residues, alanine and threonine, respectively (14, 15), and there is no functional distinction between the Rpl11Ap and Rpl11Bp (10). Rpl11p is necessary for the assembly of 60 S ribosomal subunits and is localized near the top surface of the central protuberance, where the 60 S subunit potentially contacts the 40 S subunit (16).

We demonstrate that disruption of either the RPL11A or RPL11B gene attenuates transcriptional repression of both RP and rRNA genes in response to a secretory defect. Rpl11p shows physical interaction with Rrs1p, which may have a central role for the signaling in response to a secretory defect. These results suggest that 60 S ribosomal subunit assembly machinery containing Rpl11p and Rrs1p is essential for the signaling.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Yeast Strains, Media, and a Library-- The yeast strains used in this study are listed in Table I. Deletion-insertion mutations of RPL11A or RPL11B were constructed in diploids, W303 or KM007, as described by Rotenberg et al. (10). Yeast cells were grown in YPD-rich medium, synthetic complete medium containing 2% glucose (SC), or SC dropout medium depending on the plasmid markers (17). Yeast transformation was performed by a lithium acetate procedure (18). A library consisting of partial Sau3A fragments of Saccharomyces cerevisiae genomic DNA inserted into a single-copy yeast vector YCp50 was generously provided by M. D. Rose (19).

Isolation of Mutants-- Mutants were isolated as described previously (6). Briefly, a plasmid (see Fig. 1A) in which the HIS3 gene was driven by the promoter of the ribosomal protein gene RPL28 (formerly called CYH2) was transformed into a temperature-sensitive his3 sly1 strain, which is defective in endoplasmic reticulum-to-Golgi trafficking (3). The cells are phenotypically His⁺ at 25 °C, but at 31 °C, just below the non-permissive temperature for sly1, they grow extremely slowly on SC lacking histidine (SC/His) plates containing 3-aminotriazole because the HIS3 transcription is repressed. The cells were treated with ethyl methanesulfonate to a survival frequency of 25% and grown on SC/His plates containing 3-aminotriazole at 31 °C. A mutant defective in the signaling pathway was expected to grow much faster than the parental strain. Of an estimated 8 × 10⁴ cells plated, 24 colonies that grew rapidly were picked up. Each mutant was tested for its ability to repress transcription of RP genes, RPL28 and RPL3 (formerly named TCM1), in response to a secretory defect.

Cloning of a Mutant Allele-- A mutant allele of the chromosomal gene was isolated by PCR. Total chromosomal DNA was isolated from wild-type and rrs2 mutant cells. DNA fragments including RPL11A or RPL11B were amplified by PCR using two sets of primers (5'-tggaagggaaggtgaaccaagaac and 5'-gtacaataacatagagtttcatcttgg, 5'-tacccttgaaagcccaacatatac and 5'-ttgatgttgtcactgactataccg) and cloned into the pGEM-T vector. The DNA sequence was determined by using two independent clones of respective genes.

Two-hybrid Screening-- pBTM116-RRS1 (20) was introduced into L40 cells (kindly provided by R. Sternglanz), a his3 strain carrying the lexA-HIS3 construct as a reporter gene. This strain was then transformed with the pACT-based S. cerevisiae cDNA library. After 3-7 days of incubation at 25 °C, His⁺ transformants were selected, and the DNA insert of pACT in the cells was characterized.

Two-hybrid Assays of the Rpl11p-Rrs1p Interaction-- The DNA fragments containing the RPL11A or RPL11B open reading frame were amplified by PCR using two sets of primers (5'-tttgaattcctgccaaagctcaaaaccctatgcg and 5'-tttctcgagttatttgtccaaaacatcagcatcg, 5'-tttgaattcctactaaagcccaaaaccctatgc and 5'-tttctcgagattatttatcgagcacatcagcg), and amplified DNA fragments were digested with EcoRI and XhoI. The digested fragments were introduced into pACTII to produce Gal4p activation domain-Rpl11Ap and -Rpl11Bp fusion proteins. Two plasmids for the production of lexA binding domain-fusion proteins and Gal4p activation domain-fusion proteins were cotransformed into L40 strain cells. Leu⁺ Trp⁺ transformants were selected, and serial dilutions of the cell cultures were stamped on SC/Leu, Trp, and His plates containing 5 mM 3-aminotriazole and incubated at 28 °C for 3 days.

Co-immunoprecipitation and Immunoblotting Analysis-- Yeast cells were grown in SC-selective media to a mid-log phase, collected by centrifugation, washed twice with ice-cold immunoprecipitate (IP) buffer (50 mM Tris-HCl, pH7.5, 1 mM EDTA, 10% glycerol, 30 mM NaCl, 0.05% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A), and resuspended in 100 µl of IP buffer. The cells were broken with glass beads by using a vortex mixer at 4 °C. The homogenates were centrifuged twice at 15,000 rpm for 20 min at 4 °C in a microcentrifuge rotor. The supernatants were incubated with the anti-hemagglutinin (anti-HA, Roche Molecular Biochemicals) or anti-Myc (Berkeley Antibody) monoclonal antibodies and protein A-Sepharose beads (Sigma) overnight at 4 °C and then precipitated by centrifugation. The immunoprecipitates were washed five times with IP buffer, fractionated by SDS-PAGE, transferred to Hybond ECL membrane (Amersham Biosciences), and probed separately with the anti-HA or anti-Myc antibodies. Signals were visualized with the ECL detection reagent (Amersham Biosciences) as instructed by the manufacturer.

Northern Blot and [methyl-³H]methionine Pulse-Chase Analyses-- Northern blot and [methyl-³H]methionine pulse-chase analyses were carried out as described previously (3, 6).

Preparation of Ribosome Pellets from Yeast Lysates-- After the addition of cycloheximide at the concentration of 100 µg/ml followed by the addition of crushed ice, yeast cells were harvested and resuspended in complex stabilization buffer (CSB) (21) containing 300 mM sorbitol, 20 mM HEPES-KOH, pH 7.5, 1 mM EGTA, 5 mM MgCl₂, 10 mM KCl, 10% glycerol, 2 mM dithiothreitol, and 100 µg/ml cycloheximide. Glass-bead lysates of the cells were centrifuged at 5,000 rpm for 5 min, and the supernatant was centrifuged again at 15,000 rpm for 20 min at 4 °C in a microcentrifuge rotor. Supernatants (500 µl; 20 A₂₆₀ units) were overlaid on 2 ml of sucrose cushions (CSB containing 500 mM sucrose instead of sorbitol) and centrifuged at 40,000 rpm for 2 h at 4 °C in a Beckman TLA100.3 rotor. Ribosome pellets were resuspended with CSB containing cycloheximide and various concentrations of LiCl, incubated for 30 min at 4 °C, and centrifuged at 40,000 rpm for 2 h at 4 °C in a Beckman TLA100.3 rotor. Ribosome pellets were resuspended in SDS sample buffer.

Polysome Analysis-- Yeast cells were grown in 2 liters of medium to mid-log phase and harvested immediately following the addition of cycloheximide (100 µg/ml) and crashed ice. The pellet was washed twice with buffer A (10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 30 mM MgCl₂, 50 µg of cycloheximide/ml, 200 µg of heparin/ml) and suspended in buffer B (20 mM Tris-HCl (pH 7.6), 100 mM NH₄Ac, 15 mM MgAc₂). After glass-bead lysis of yeast cells, the crude ribosome pellet was prepared by centrifugation at 55,000 rpm for 3 h in a Beckman 90 Ti rotor and resuspended in 0.5 ml of buffer B. Aliquots of the suspension were overlaid on top of 12 ml of 8-45% (w/v) sucrose gradient made in buffer B and centrifuged for 3.5 h at 40,000 rpm at 4 °C in a Beckman SW 40 Ti rotor. The gradients were monitored at 260 nm and fractionated. Fractions containing the 80 S monosomes were pooled and analyzed by the second sucrose density gradient centrifugation under the same conditions as the first centrifugation. For immunoblot analysis, proteins of each fraction were precipitated with 10% trichloroacetic acid, washed with acetone, dried, and resuspended in SDS sample buffer.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Isolation of Yeast Mutants Unable to Respond to a Secretory Defect-- To elucidate the molecular mechanism of the signaling in response to a secretory defect, we obtained rrs (regulator of ribosome synthesis) mutants in which a secretory defect did not cause transcriptional repression of RP genes (6). A plasmid containing the reporter gene, shown in Fig. 1A, was introduced into a his3 sly1 strain, a temperature-sensitive mutant defective in endoplasmic reticulum-to-Golgi trafficking. Mutants were isolated on an SC/His plate containing 3-aminotriazole at the semipermissive temperature of sly1. Previously, we analyzed rrs1 mutant and showed that RRS1 encoded a nuclear protein essential for 25 S rRNA maturation and 60 S ribosomal subunit assembly (6). In this study, rrs2 was chosen for further analysis. In rrs2 sly1 cells, the transcriptional repression of RP genes was significantly attenuated when the cells were transferred to the restrictive temperature (Fig. 1B). Genetic analysis showed that the mutation is recessive; when the mutant cells were crossed to parental sly1 cells of the opposite mating type, RP genes RPL28 and RPL3 were significantly repressed at the restrictive temperature (data not shown).

View larger version (52K): [in this window] [in a new window]   Fig. 1.   Isolation of rrs2 mutant. A, the construct of the reporter gene. The reporter gene consists of the coding sequence of the yeast HIS3 gene fused to the promoter of RPL28. B, Northern analysis of the rrs2 mutant under the secretory defect. Yeast KM003 (sly1 RRS2) and KM201 (sly1 rrs2) cells were grown in YPD medium to log phase (optical density at 600 nm = 0.4-0.6) at 25 °C. Half of the culture was shifted to 36 °C, and after 90 min, the cells were harvested. Total RNA was prepared and separated by gel electrophoresis. Northern blot analysis was carried out using 32P-labeled DNA probes specific for RPL28 and RPL3. Small nucleolar RNA U3 was used as a marker to check equal loading.

rrs2 Has a Mutation in RPL11B-- To isolate a gene complementing the rrs2 mutation, we examined phenotypes of the rrs2 mutant cells and found that rrs2 mutant cells exhibited cycloheximide sensitivity. The cycloheximide sensitivity segregated 2:2 in tetrads from RRS2/rrs2 diploid cells, and every cycloheximide-sensitive clone in three sets of tetrads showed the signaling defect detected by Northern blot analysis (data not shown). By using cycloheximide-sensitive phenotype of rrs2, we succeeded in cloning the RRS2 gene as described below. The rrs2 cells were transformed with a library of yeast genomic DNA constructed in a centromere-based vector, YCp50 (19). Of 1.3 × 10⁴ Ura⁺ transformants, 35 colonies could grow on a YPD plate containing 0.12 µg of cycloheximide/ml. In 11 of the 35 strains, the complementing activity was plasmid-linked. Restriction maps of the plasmid DNAs recovered from these transformants revealed that ten plasmids had an identical 3.8-kb insert (named pRT-1) and one plasmid had an 11.9-kb insert (named pRT-2). We determined a partial DNA sequence of the plasmids. To identify the region of pRT-1 required for complementation, three subclones were constructed, and their complementing activities for the cycloheximide-sensitivity of rrs2 were checked. The complementing activity was fully recovered in the plasmid containing RPL11B. Subcloning of pRT-2 revealed that RPL11A was responsible for the complementing activity. Northern analysis showed that the plasmids containing RPL11A or RPL11B also complemented the signaling defect of rrs2 (data not shown).

To determine the mutated allele in the rrs2 mutant, the RPL11A and RPL11B genes were amplified by PCR using chromosomal DNA prepared from the mutant cells as template. Sequence analysis revealed that RPL11B had only one nucleotide difference (a C-to-T transition) within codon 39, resulting in a stop codon, whereas RPL11A had no difference.

Both the rpl11a- and the rpl11b-null Mutations Affect the Transcriptional Repression of RP Genes in Response to a Secretory Defect-- As the rrs2 mutant has a nonsense mutation in RPL11B, we determined the effect of the rpl11a- and the rpl11b-null mutations (10) on the transcriptional repression of RP genes in response to a secretory defect. Both the rpl11a- and the rpl11b-null mutations attenuated the transcriptional repression of RP genes due to a secretory defect, which was caused by either addition of tunicamycin, an inhibitor of the secretory pathway (Fig. 2A), or shifting sly1 cells to the restrictive temperature (Fig. 2B). The signaling defect in the rpl11a-null mutant was more striking than that in the rpl11b-null mutant, probably reflecting that the RPL11A gene is expressed at twice the level of RPL11B (10). On the other hand, the null mutations of RPS4A or RPS4B (22) that encode a ribosomal protein of the small subunit had little effect on the response to a secretory defect (Fig. 2, A and B), suggesting that the attenuation of transcriptional repression of ribosomal protein genes is specific to a defect in large ribosomal subunits.

View larger version (51K): [in this window] [in a new window]   Fig. 2.   The rpl11 mutation affects the transcriptional repression of both RP and rRNA genes in response to a secretory defect. A and B, Northern analysis of the rpl11 and rps4 mutants when the secretory pathway was blocked. In panel A, yeast strains W303a, KM204 (rpl11a), KM205 (rpl11b), J130A (rps4a), and J130C (rps4b) were grown to log phase (optical density at 600 nm = 0.4-0.6) at 25 °C. Half of the culture was treated with tunicamycin (TM) at a final concentration of 1.0 µg/ml for 4 h at 25 °C, and the cells were harvested. Total RNA was prepared and separated by gel electrophoresis. Northern blot analysis was carried out using 32P-labeled DNA probes specific for RPL7, RPL28, and RPL3. Small nucleolar RNA U3 was used as a marker to check equal loading. WT, wild-type. In panel B, yeast strains KM003 (sly1), KM202 (sly1 rpl11a), KM203 (sly1 rpl11b), KM314 (sly1 rps4a), and KM319 (sly1 rps4b) were grown at 25 °C and shifted to 37 °C for 90 min. Northern analysis was performed as in panel A. C, the rpl11 mutation also affects the transcriptional repression of rDNA in response to a secretory defect. Strains KM003 (sly1), KM202 (sly1 rpl11a), and KM203 (sly1 rpl11b) were grown to log phase (optical density at 600 nm = 1.0) in SC/Met medium at 25 °C. Aliquots from each culture were shifted to 37 °C for 90 min. Each culture was pulsed with [methyl-3H]methionine (10 µCi/ml) for 3 min and then chased with nonradioactive methionine (500 µg/ml). Samples were taken and chilled by pouring onto crushed sterile ice at the time of the addition of cold methionine (t = 0) and after a chase time of 10 min. Total RNA was prepared, and 20 µg of each sample was analyzed by electrophoresis and blotted to Nytran membrane. The membrane was sprayed with En3Hance (PerkinElmer Life Sciences) and exposed to a film for 2 days. The lower gel was blotted and probed for small nucleolar RNA U3.

The rpl11 Mutation Also Affects the Repression of rDNA Transcription in Response to a Secretory Defect-- As a secretory defect causes transcriptional repression of rDNA as well as RP genes (3), we examined whether the rpl11 mutation had any effect on the transcriptional repression of rDNA. We monitored the synthesis and processing of rRNA by [methyl-³H]methionine pulse-chase analysis since newly synthesized precursor rRNA is methylated immediately (23, 24). As shown in Fig. 2C, a secretory defect by shifting the sly1 cells to the restrictive temperature leads to strong repression of rDNA transcription (lanes 3 and 4), consistent with previous data (3). On the other hand, in both the rpl11a and rpl11b strain cells, repression of rDNA transcription due to a secretory defect was significantly attenuated (lanes 7, 8, 11, and 12). These results indicate that Rpl11p depletion affects the signal transduction from a secretory defect to the repression of rDNA transcription as well as that of RP gene transcription.

Rrs1p Shows Physical Interaction with Rpl11p-- Interestingly, RPL11A was also cloned in two-hybrid screening by using RRS1 as bait. Rrs1p, an evolutionarily conserved nuclear protein, is required for pre-rRNA processing and proper assembly of ribosomal subunits in S. cerevisiae (6). RRS1 was originally isolated as the gene responsible for the rrs1-1 mutation, which causes a defect in the secretory response. To identify proteins that physically interact with Rrs1p, we carried out yeast two-hybrid screening on a yeast cDNA library using RRS1 as bait. Among the 21 cDNA clones isolated in this screen, one cDNA encoded most of the sequence of Rpl11Ap. As we reported previously, three clones contained EBP2 cDNA (20). Fig. 3A shows two-hybrid interaction of Rrs1p with Rpl11p in yeast with a positive control of Rrs1p and Ebp2p. HIS3 reporter gene was activated in the presence of Gal4p activation domain-Rpl11Ap fusion protein and lexA binding domain-Rrs1p fusion protein. Rpl11Bp also showed two-hybrid interaction with Rrs1p. The results were reproducible, and the assay using lacZ reporter gene showed similar results (data not shown). The C-terminal truncated protein, rrs1-1, also showed two-hybrid interaction with Rpl11 (data not shown).

View larger version (50K): [in this window] [in a new window]   Fig. 3.   Physical interaction of Rpl11p with Rrs1p. A, two-hybrid interaction. Plasmids expressing Rrs1-fusion protein and Ebp2-fusion protein were used as a positive control. BD, DNA-binding domain; AD, activation domain. B, co-immunoprecipitation of Myc-Rpl11p with HA-Rrs1p. Extracts of strains KM331 (HA-RRS1, EBP2-myc), KM148 (HA-RRS1), and KM330 (EBP2-myc) were immunoprecipitated with anti-HA or anti-Myc monoclonal antibodies. The immunoprecipitates (corresponding to 4.8 µl of cell extract) and the extracts prior to precipitation (input, corresponding to 0.1 µl of cell extract) were analyzed by SDS-PAGE and immunoblotting.

To confirm the interaction between Rrs1p and Rpl11p, we constructed a yeast strain in which both RRS1 and RPL11A on the chromosome were disrupted and complemented with plasmids expressing HA-Rrs1p and Myc-Rpl11Ap. Co-immunoprecipitation analysis confirmed the interaction of Rrs1p with Rpl11Ap as shown in Fig. 3B.

Rrs1p Associates with Ribosomal Particle-- Rrs1p has an important function in ribosome biogenesis, especially in the maturation of 25 S rRNA and assembly of 60 S ribosomal subunits (6). As Rrs1p had two-hybrid interaction with Rpl11p, we examined whether Rrs1p associates with ribosomal particles. Ultracentrifugation was carried out using cell extract from the strain expressing HA-Rrs1p, and ribosome pellet and supernatant fractions were analyzed by Western blotting. As shown in Fig. 4, approximately a half-amount of HA-Rrs1p was detected in the ribosome fraction, and the other half was detected in a soluble fraction in buffer with low ionic strength. To learn the stability of the interaction of Rrs1p with ribosomes, the ribosome fraction was treated with various concentrations of LiCl. Approximately a half-amount of Rrs1p associated with ribosome even at 0.5 M LiCl, and most of Rrs1p dissociated from ribosomes in 1.0 M LiCl. This suggests that the interaction of Rrs1p with a ribosome is rather stable, although it appeared to be less stable as compared with that of ribosomal protein L3. Next, to determine the type of ribosome with which Rrs1p associates, we performed sucrose density gradient centrifugation followed by immunoblot analysis (Fig. 5). In the first density gradient, the centrifugation of crude ribosome, most of the strong signals for HA-Rrs1p were observed in the fractions corresponding to 60 S ribosomal subunits, and weak signals were found in the fractions corresponding to 80 S monosomes and polysomes (Fig. 5B). To clarify whether HA-Rrs1p associates with the lower fractions, a pooled fraction from 80 S fractions of the first gradient was analyzed in the second sedimentation. No signal for HA-Rrs1p was detected (Fig. 5C), indicating that Rrs1p associates with only fractions corresponding to 60 S subunits.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   HA-Rrs1p associates with ribosome. A, yeast KM148 (HA-RRS1) cells were treated with cycloheximide, lysed, and centrifuged through low salt sucrose cushions. Equivalent amounts of ribosome pellet (lane 1) and supernatant (sup; lane 2) were subjected to SDS-PAGE and immunoblot analysis using anti-HA antibodies and anti-ribosomal protein L3 antibodies (kindly provided by J. R. Warner). ppt, precipitate. B, ribosome pellets were treated with increasing concentrations of LiCl and centrifuged again through sucrose cushions containing the same concentrations of LiCl. Ribosome pellets were subjected to SDS-PAGE and immunoblot analysis. Concentrations of LiCl were as follows: 0.01 M (lane 1), 0,1 M (lane 2), 0.25 M (lane 3), 0.5 M (lane 4), and 1.0 M (lane 5).

View larger version (38K): [in this window] [in a new window]   Fig. 5.   HA-Rrs1p cosediments with free 60 S ribosomal subunits. A, polysome profile of ribosomes isolated from KM148 (HA-RRS1) cells and separated using an 8-45% sucrose gradient. B, fractions from the gradient shown in panel A were collected, and proteins were precipitated with 10% trichloroacetic acid and analyzed by SDS-PAGE and immunoblotting. C, fractions containing 80 S monosomes in the first gradient centrifugation shown in panel A were pooled, pelleted, resuspended, and sedimented through the second gradient centrifugation. Each fraction was analyzed by immunoblotting. Ribosomal protein L3 was used as a marker of 60 S ribosomal subunits.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Previously, we isolated rrs mutants in which transcription of RP genes was not repressed in response to a secretory defect (6). We demonstrated that RRS1, the wild-type allele responsible for the rrs1-1 mutation, had an important function in 25 S rRNA maturation and 60 S ribosomal subunit assembly when the secretory pathway functions normally (6). In this study, we have cloned the duplicated genes encoding ribosomal protein L11 not only as functional genes complementing the rrs2 mutation that causes a defect in the secretory response but also in two-hybrid screening using RRS1 as bait. These results suggest that Rrs1p and Rpl11p have a function in the same signaling pathway in response to a secretory defect and that protein-protein interaction of the two proteins may be important in the signaling. The C-terminal truncated form of Rrs1p, rrs1-1p, shows two-hybrid interaction with Rpl11p (data not shown), suggesting that the N-terminal half of Rrs1p is required for the interaction with Rpl11p and that the signal is transduced through the C-terminal half of Rrs1p. In the same two-hybrid screening, we previously identified Ebp2p and demonstrated that Ebp2p had a similar role to Rrs1p in 25 S rRNA maturation and 60 S ribosomal subunit assembly (20). We proposed that ribosome assembly machinery is responsive to the signaling, and in this study, we showed that the rpl11 mutation caused a defect in the signaling and that Rrs1p interacts with Rpl11p, strengthening our model.

Amino acid sequence analysis and immunological analysis revealed that Escherichia coli L5, Bacillus stearothermophilus L5, and Halobacterium cutirubrum L19 are homologues of yeast Rpl11p (16). These three homologues were shown to associate with 5 S rRNA (25-27). Although it is not shown directly that yeast Rpl11p associates with 5 S rRNA, Rpl11p is localized at a position similar to that of its homologue L5 in E. coli ribosomes, near the top surface of the central protuberance of the 60 S subunit (10, 16). Recent analysis by docking of atomic models into a lower resolution cryo-electron microscopy map suggested that Rpl11p was involved in the formation of a bridge with the 40 S subunit via the interaction with Rps13p and that it also interacted with the 5 S rRNA-binding protein Rpl5p and the elbow of the P site-bound tRNA (28).

Our data suggest that approximately a half-amount of Rrs1p associates with ribosomal particles and that the binding is fairly tight. Rrs1p is localized mainly in the nucleolus and also in the nucleoplasm (6). The nucleolus is the site of pre-rRNA synthesis, pre-rRNA processing, and ribosomal subunit assembly. Most ribosomal proteins bind pre-rRNA in the nucleolus, and pre-rRNA processing is tightly linked to ribosomal subunit assembly (29, 30). Early associating ribosomal proteins bind ³⁵S-labeled pre-rRNA to form 90 S preribosome, from which 66 and 43 S preribosomal subunits containing the 27 and 20 S pre-rRNAs, respectively, are formed. The 66 S preribosomal subunit contains late associating ribosomal proteins, and cleavage of 27 S pre-rRNA to 25 S rRNA leads to the 60 S preribosomal subunit. Rrs1p is detected mainly in the 60 S ribosomal subunit fraction in sucrose density gradient ultracentrifugation analysis. Considering that Rrs1p localizes in the nucleus, this suggests that Rrs1p associates with the preribosomal particle. Although HA-tagged Rrs1p also appears to associate with a lower fraction corresponding 80 S monosomes in the first sucrose density gradient ultracentrifugation, the band disappears in the fraction in the second analysis. As 90 S preribosomal particles sediment at nearly the same fractions as 80 S monosomes, and the 60 S ribosomal subunit fraction contains 66 S preribosomal subunits, it seems that Rrs1p associates with 66 S and/or 60 S preribosomal subunits and associates little with the 90 S pre-ribosomal particle if any. As Rrs1p localizes throughout the nucleus, including the nucleolus, Rrs1p might have a role in the transport of large ribosomal subunits from the nucleolus to the nucleoplasm as well as pre-rRNA processing/ribosomal subunit assembly. It has recently been reported that Noc proteins, Noc1, Noc2, and Noc3, are required for the maturation and intranuclear transport of preribosomes (31). The role of Rrs1p on intranuclear transport is now under investigation. Furthermore, we have found a novel nucleolar protein that interacts with Rrs1p,² suggesting that Rrs1p exists as a component of a large complex.

We have presented here that Rrs1p and Rpl11p are essential for the secretory response, but the molecular mechanism of transcriptional repression of both rRNA and RP genes in response to a secretory defect remains to be elucidated. As depletion of Rps4p, a protein of the small ribosomal subunit, had little effect on the secretory response, it is unlikely that a decline of protein synthesis caused by the depletion of Rrs1p or Rpl11p canceled a secretory defect. We propose a model in which the signal from a secretory defect might be transmitted to the large ribosomal subunit assembly machinery including Rrs1p and Rpl11p. However, it is also possible that a decline of 60 S ribosomal subunits affects the signaling in response to a secretory defect. In this case, the yeast cells should sense a very small defect in ribosome synthesis because rpl11b-null mutation could cause a small defect in ribosome synthesis (10). We cannot distinguish between these two possibilities at present because a defect in a component of the ribosomal subunit could affect the assembly of other components. In fact, qsr1-1 and qsr1-24 (32, 33; kindly provided by B. L. Trumpower), both of which have mutations in RPL10, were also defective in the signaling due to a secretory response (data not shown). Although Rpl10p appears to assemble with a large ribosomal subunit at the last step in ribosome biogenesis, rpl10 mutations affect the earlier steps of pre-rRNA processing (34). Nevertheless, we prefer the former model for three reasons. First, RPL11 was isolated in two independent screens. Second, Rrs1p appears to be a key protein in the signaling as the rrs1-1 allele leads to an extremely strong defect in the signaling (6). Third, rrs1-1 cells still have the ability to repress ribosome synthesis by mild heat shock (6) or nitrogen starvation.³ It should be noted that the mechanism of the secretory response is different from that of mild heat shock or nitrogen starvation. According to our model, the mechanism of transcriptional repression in response to a secretory defect is tightly linked to the normal regulatory mechanism that maintains ribosome synthesis. It has recently been proposed that rRNA transcriptional machinery is closely linked to pre-rRNA processing machinery (35). This model supports our idea in which transcriptional repression of rRNA genes is mediated through ribosomal subunit assembly machinery, that is, pre-rRNA processing machinery. In addition, we have found that transcriptional repression of rRNA genes is an early event (8), consistent with our model.

	ACKNOWLEDGEMENTS

We thank T. Miyakawa, E. Tsuchiya, and D. Hirata for valuable discussion, J. R. Warner for anti-Rpl3 antibodies and yeast strains, M. D. Rose for a yeast genomic DNA library, and J. Woolford, R. Sternglanz, and B. L. Trumpower for yeast strains and plasmids.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Biological Sciences, Graduate School of Biosphere Sciences, Hiroshima University, Kagamiyama 1-4-4, Higashi-Hiroshima 739-8528, Japan. Tel.: 81-824-24-7926; Fax: 81-824-24-7926; E-mail: kmizuta@hiroshima-u.ac.jp.

Published, JBC Papers in Press, March 13, 2002, DOI 10.1074/jbc.M201667200

² D. Morita, K. Miyoshi, Y. Matsui, A. Toh-e, H. Shinkawa, T. Miyakawa, and K. Mizuta, unpublished data.

³ K. Miyoshi and K. Mizuta, unpublished data.

	ABBREVIATIONS

The abbreviations used are: RP, ribosomal protein; rrs, regulator of ribosome synthesis; SC, synthetic complete; HA, hemagglutinin.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1.	Warner, J. R. (1999) Trends Biochem. Sci. 24, 437-440[CrossRef][Medline] [Order article via Infotrieve]
2.	Woolford, J. L., Jr., and Warner, J. R. (1991) in The Molecular and Cellular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics (Broach, J. R. , Pringle, J. R. , and Jones, E. W., eds) , pp. 587-626, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
3.	Mizuta, K., and Warner, J. R. (1994) Mol. Cell. Biol. 14, 2493-2502[Abstract/Free Full Text]
4.	Li, B., and Warner, J. R. (1996) J. Biol. Chem. 271, 16813-16819[Abstract/Free Full Text]
5.	Li, Y., Moir, R. D., Sethy-Coraci, I. K., Warner, J. R., and Willis, I. M. (2000) Mol. Cell. Biol. 20, 3843-3851[Abstract/Free Full Text]
6.	Tsuno, A., Miyoshi, K., Tsujii, R., Miyakawa, T., and Mizuta, K. (2000) Mol. Cell. Biol. 20, 2066-2074[Abstract/Free Full Text]
7.	Mizuta, K., Tsujii, R., Warner, J. R., and Nishiyama, M. (1998) Nucleic Acids Res. 26, 1063-1069[Abstract/Free Full Text]
8.	Miyoshi, K., Miyakawa, T., and Mizuta, K. (2001) Nucleic Acids Res. 29, 3297-3303[Abstract/Free Full Text]
9.	Nierras, C. R., and Warner, J. R. (1999) J. Biol. Chem. 274, 13235-13241[Abstract/Free Full Text]
10.	Rotenberg, M. O., Moritz, M., and Woolford, J. L., Jr. (1988) Genes Dev. 2, 160-172[Abstract/Free Full Text]
11.	Moritz, M., Pulaski, B. A., and Woolford, J. L., Jr. (1991) Mol. Cell. Biol. 11, 5681-5692[Abstract/Free Full Text]
12.	Moritz, M., Paulovich, A. G., Tsay, Y.-F., and Woolford, J. L., Jr. (1990) J. Cell Biol. 111, 2261-2274[Abstract/Free Full Text]
13.	Mager, W. H., Planta, R. J., Ballesta, J.-P. G., Lee, J. C., Mizuta, K., Suzuki, K., Warner, J. R., and Woolford, J. L., Jr. (1997) Nucleic Acids Res. 25, 4872-4875[Abstract/Free Full Text]
14.	Leer, R. J., Raamsdonk-Duin, M. M., Mager, W. H., and Planta, R. J. (1984) FEBS Lett. 175, 371-376[CrossRef][Medline] [Order article via Infotrieve]
15.	Teem, J. L., Abovich, N., Kaufer, N. E., Schwindinger, W. F., Warner, J. R., Levy, A., Woolford, J. L., Jr., Leer, R. J., van Raamsdonk-Duin, M. M. L., Mager, W. H., Planta, R. J., Schultz, L., Friesen, J. D., Fried, H., and Rosbash, M. (1984) Nucleic Acids Res. 12, 8295-8312[Abstract/Free Full Text]
16.	Tsay, Y.-F., Shankweiler, G., Lake, J., and Woolford, J. L., Jr. (1994) J. Biol. Chem. 269, 7579-7586[Abstract/Free Full Text]
17.	Kaiser, C., Michaelis, S., and Mitchell, A. (1994) Methods in Yeast Genetics , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
18.	Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163-168[Abstract/Free Full Text]
19.	Rose, M. D., Novick, P., Thomas, J. H., Botstein, D., and Fink, G. R. (1987) Gene (Amst.) 60, 237-243[CrossRef][Medline] [Order article via Infotrieve]
20.	Tsujii, R., Miyoshi, K., Tsuno, A., Matsui, Y., Toh-e, A., Miyakawa, T., and Mizuta, K. (2000) Genes Cells 5, 543-553[Abstract]
21.	Nelson, R. J., Ziegelhoffer, T., Nicolet, C., Werner-Washburne, M., and Craig, E. A. (1992) Cell 71, 97-105[CrossRef][Medline] [Order article via Infotrieve]
22.	Synetos, D., Dabeva, M. D., and Warner, J. R. (1992) J. Biol. Chem. 267, 3008-3013[Abstract/Free Full Text]
23.	Udem, S. A., and Warner, J. R. (1972) J. Mol. Biol. 65, 227-242[CrossRef][Medline] [Order article via Infotrieve]
24.	Chen-Schmeisser, U., and Garrett, R. A. (1977) FEBS Lett. 74, 287-291[CrossRef][Medline] [Order article via Infotrieve]
25.	Horne, J. R., and Erdmann, V. A. (1972) Mol. Gen. Genet. 119, 337-344[Medline] [Order article via Infotrieve]
26.	Smith, N., Matheson, A. T., Yaguchi, M., Willick, G. E., and Nazar, R. N. (1978) J. Cell Biol. 89, 501-509
27.	Spierer, P., and Zimmermann, R. A. (1978) Biochemistry 17, 2474-2479[CrossRef][Medline] [Order article via Infotrieve]
28.	Spahn, C. M. T., Beckmann, R., Eswar, N., Penczek, P. A., Sali, A., Blobel, G., and Frank, J. (2001) Cell 107, 373-386[CrossRef][Medline] [Order article via Infotrieve]
29.	Venema, J., and Tollervey, D. (1999) Annu. Rev. Genet. 33, 261-311[CrossRef][Medline] [Order article via Infotrieve]
30.	Kressler, D., Linder, P., and de la Cruz, J. (1999) Mol. Cell. Biol. 19, 7897-7912[Free Full Text]
31.	Milkereit, P., Gadal, O., Podtelejnikov, A., Trumtel, S., Gas, N., Petfalski, E., Tollervey, D., Mann, M., Hurt, E., and Tschochner, H. (2001) Cell 105, 499-509[CrossRef][Medline] [Order article via Infotrieve]
32.	Thierry, T, Yang, M., Dick, F. A., Schmitt, M. E., and Trumpower, B. L. (1995) J. Biol. Chem. 270, 9961-9970[Abstract/Free Full Text]
33.	Eisinger, D. P., Dick, F. A., and Trumpower, B. L. (1997) Mol. Cell. Biol. 17, 5136-5145[Abstract]
34.	Gadal, O., Strau, D., Kessl, J., Trumpower, B. L., Tollervey, D., and Hurt, E. (2001) Mol. Cell. Biol. 21, 3405-3415[Abstract/Free Full Text]
35.	Fath, S., Milkereit, P., Podtelejnikov, A. V., Bischler, N., Schultz, P., Bier, M., Mann, M., and Tschochner, H. (2000) J. Cell Biol. 149, 575-589[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. Zhang, P. Harnpicharnchai, J. Jakovljevic, L. Tang, Y. Guo, M. Oeffinger, M. P. Rout, S. L. Hiley, T. Hughes, and J. L. Woolford Jr. Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes Genes & Dev., October 15, 2007; 21(20): 2580 - 2592. [Abstract] [Full Text] [PDF]

				F. Bachand, D. H. Lackner, J. Bahler, and P. A. Silver Autoregulation of ribosome biosynthesis by a translational response in fission yeast. Mol. Cell. Biol., March 1, 2006; 26(5): 1731 - 1742. [Abstract] [Full Text] [PDF]

				M. Nariai, T. Tanaka, T. Okada, C. Shirai, C. Horigome, and K. Mizuta Synergistic defect in 60S ribosomal subunit assembly caused by a mutation of Rrs1p, a ribosomal protein L11-binding protein, and 3'-extension of 5S rRNA in Saccharomyces cerevisiae Nucleic Acids Res., August 12, 2005; 33(14): 4553 - 4562. [Abstract] [Full Text] [PDF]

				C. Shirai, T. Takai, M. Nariai, C. Horigome, and K. Mizuta Ebp2p, the Yeast Homolog of Epstein-Barr Virus Nuclear Antigen 1-binding Protein 2, Interacts with Factors of Both the 60 S and the 40 S Ribosomal Subunit Assembly J. Biol. Chem., June 11, 2004; 279(24): 25353 - 25358. [Abstract] [Full Text] [PDF]

				K. Miyoshi, C. Shirai, and K. Mizuta Transcription of genes encoding trans-acting factors required for rRNA maturation/ribosomal subunit assembly is coordinately regulated with ribosomal protein genes and involves Rap1 in Saccharomyces cerevisiae Nucleic Acids Res., April 1, 2003; 31(7): 1969 - 1973. [Abstract] [Full Text] [PDF]

				Y. Zhao, J.-H. Sohn, and J. R. Warner Autoregulation in the Biosynthesis of Ribosomes Mol. Cell. Biol., January 15, 2003; 23(2): 699 - 707. [Abstract] [Full Text] [PDF]

				E. Fossale, V. C. Wheeler, V. Vrbanac, L.-A. Lebel, A. Teed, J. S. Mysore, J. F. Gusella, M. E. MacDonald, and F. Persichetti Identification of a presymptomatic molecular phenotype in Hdh CAG knock-in mice Hum. Mol. Genet., September 15, 2002; 11(19): 2233 - 2241. [Abstract] [Full Text] [PDF]

				D. Morita, K. Miyoshi, Y. Matsui, A. Toh-e, H. Shinkawa, T. Miyakawa, and K. Mizuta Rpf2p, an Evolutionarily Conserved Protein, Interacts with Ribosomal Protein L11 and Is Essential for the Processing of 27 SB Pre-rRNA to 25 S rRNA and the 60 S Ribosomal Subunit Assembly in Saccharomyces cerevisiae J. Biol. Chem., August 2, 2002; 277(32): 28780 - 28786. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 277/21/18334    most recent M201667200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Miyoshi, K. Articles by Mizuta, K. Search for Related Content PubMed PubMed Citation