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Vol. 19, Issue 12, 5279-5288, December 2008

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Ribosome-associated Complex Binds to Ribosomes in Close Proximity of Rpl31 at the Exit of the Polypeptide Tunnel in Yeast

Kristin Peisker^*,,, Daniel Braun^*,, Tina Wölfle^*,, Jendrik Hentschel^*,, Ursula Fünfschilling, Gunter Fischer, Albert Sickmann^||, and Sabine Rospert^*,

*Institute of Biochemistry and Molecular Biology, ZBMZ, University of Freiburg, D-79104 Freiburg, Germany; Centre for Biological Signalling Studies (BIOSS), University of Freiburg, D-79104 Freiburg, Germany; Fakultät für Biologie, University of Freiburg, D-79104 Freiburg, Germany; Max Planck Research Unit for Enzymology of Protein Folding, D-06120 Halle/Saale, Germany; ^||Department of Proteomics, Institute for Analytical Sciences, D-44139 Dortmund, Germany

Submitted June 30, 2008; Revised August 19, 2008; Accepted September 24, 2008
Monitoring Editor: Jonathan S. Weissman

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ribosome-associated complex (RAC) consists of the Hsp40 homolog Zuo1 and the Hsp70 homolog Ssz1. The chaperone participates in the biogenesis of newly synthesized polypeptides. Here we have identified yeast Rpl31, a component of the large ribosomal subunit, as a contact point of RAC at the polypeptide tunnel exit. Rpl31 is encoded by RPL31a and RPL31b, two closely related genes. rpl31arpl31b displayed slow growth and sensitivity to low as well as high temperatures. In addition, rpl31arpl31b was highly sensitive toward aminoglycoside antibiotics and suffered from defects in translational fidelity. With the exception of sensitivity at elevated temperature, the phenotype resembled yeast strains lacking one of the RAC subunits or Rpl39, another protein localized at the tunnel exit. Defects of rpl31arpl31bzuo1 did not exceed that of rpl31arpl31b or zuo1. However, the combined deletion of RPL31a, RPL31b, and RPL39 was lethal. Moreover, RPL39 was a multicopy suppressor, whereas overexpression of RAC failed to rescue growth defects of rpl31arpl31b. The findings are consistent with a model in that Rpl31 and Rpl39 independently affect a common ribosome function, whereas Rpl31 and RAC are functionally interdependent. Rpl31, while not essential for binding of RAC to the ribosome, might be involved in proper function of the chaperone complex.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Two Hsp70 family members Ssb1/2 (Ssb1 and Ssb2 differ by only four amino acids) and Ssz1 and one J-domain protein (Zuo1) are abundant components of the translation machinery of Saccharomyces cerevisiae (Raue et al., 2007). The three chaperones are genetically linked and form a functional triad. Lack of either SSB1/2, SSZ1, or ZUO1 results in slow growth, cold sensitivity, and pronounced hypersensitivity against aminoglycosides such as paromomycin (Gautschi et al., 2002; Hundley et al., 2002). Ssz1 and Zuo1 assemble into a stable heterodimeric complex termed RAC (ribosome-associated complex). RAC acts as a cochaperone for Ssb1/2 and stimulates its ATP hydrolysis. The function requires both RAC subunits (Huang et al., 2005; Conz et al., 2007).

RAC is anchored to the ribosome via Zuo1 (Gautschi et al., 2001). The idea is that positioning of RAC on the ribosome is required for its interaction with Ssb1/2 (Yan et al., 1998). However, the function of Ssz1 does not strictly depend on stable interaction with Zuo1 or ribosomes (Conz et al., 2007). How exactly Zuo1 anchors RAC is currently unclear. It was proposed that Zuo1 binds to ribosomes, in part, by interaction with rRNA (Yan et al., 1998). However, purified Zuo1 unspecifically interacts with a variety of nucleic acids. Initially, Zuo1 was identified via its ability to interact with Z-DNA (Zhang et al., 1992), it also interacts tightly with tRNA (Wilhelm et al., 1994) and recently was shown to bind to a small inhibitor RNA (Raychaudhuri et al., 2006). The mouse homolog MIDA1 interacts with DNA that forms small stem loop structures (Inoue et al., 2000). The diversity of nucleic acids that interact with Zuo1 raises the question how targeting to a specific binding site on the ribosome is achieved. An attractive hypothesis is that auxiliary interactions with ribosomal protein components mediate specificity (Yan et al., 1998). To what region of the ribosome Zuo1 binds has not been analyzed. The exit region, which has a higher than average concentration of ribosomal proteins (Klein et al., 2004), would be ideally suited to position RAC close to its partner chaperone Ssb1/2. However, direct evidence for binding close to the polypeptide tunnel exit is missing.

In terms of protein composition the archaeal ribosome is a small-scale model of the eukaryotic one (Lecompte et al., 2002). The crystal structure revealed that six proteins Rpl19/L19e, Rpl17/L22, Rpl25/L23, Rpl26/L24, Rpl35/L29, and Rpl31/L31e, form a rim around the polypeptide tunnel exit (Nissen et al., 2000; Klein et al., 2004). Rpl (ribosomal protein of the large subunit) designates the yeast homologues of ribosomal proteins as in Lecompte et al., (2002); see Figure 1E. Rpl39/L39e, a small protein predominantly localized within the polypeptide tunnel also exposes a small surface at the tunnel exit. This set of proteins is supposed to provide a platform for the interaction of ribosome-associated protein biogenesis factors (RPBs), a set of proteins that reversibly interact with ribosomes and affect nascent polypeptides in multiple ways (Rospert et al., 2005a; Raue et al., 2007). To date, only two of the ribosomal proteins at the tunnel exit, Rpl25/L23 and Rpl35/L29, have been shown to interact with RPBs. At least four RPBs, signal recognition particle (SRP), nascent polypeptide associated complex (NAC), the ER-membrane protein ERj1, and the eubacterial trigger factor, interact with ribosomes via Rpl25/L23. SRP interacts with Rpl25/L23, Rpl35/L29, and rRNA of both ribosomal subunits (Pool et al., 2002; Gu et al., 2003; Ullers et al., 2003; Halic et al., 2004). Trigger factor binds to the ribosome through interactions with Rpl25/L23, Rpl35/L29, and 23S rRNA (Kramer et al., 2002; Blaha et al., 2003; Ferbitz et al., 2004; Baram et al., 2005; Schlünzen et al., 2005). NAC and ERj1 were recently found to interact with Rpl25/L23 (Blau et al., 2005; Wegrzyn et al., 2006). Accordingly, the current idea is that this protein constitutes a general factor binding site. However, evidence was recently presented that Rpl35/L29 is the attachment site for the N-acetyltransferase NatA (Polevoda et al., 2008), which is bound to ribosomes via its subunit Nat1 (Gautschi et al., 2003).

Here we set out to characterize the interaction of RAC with the ribosome in more detail. To that end, we have used a cross-linking approach that allowed us to identify Rpl31 as a ribosomal protein that directly contacts the Zuo1 subunit of RAC. Although Rpl31 was not essential for Zuo1 binding to the ribosome, genetic evidence is consistent with the possibility of functional coupling between Rpl31 and RAC. Moreover, we found that simultaneous deletion of the genes encoding Rpl31 and Rpl39 resulted in synthetic lethality as expected if the two proteins at the tunnel exit were involved in a common function of the ribosome.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Media and Culture Conditions
Strains were grown to log phase on 1% yeast extract, 2% peptone, and 2% dextrose (YPD) or in glucose-containing minimal media. Growth defects were analyzed by spotting 10-fold serial dilutions containing the same number of cells to YPD plates. Plates were supplemented with paromomycin at the concentration specified in the figure legends. Plates were incubated at 30°C to analyze the slow growth phenotype, at 20°C to determine cold sensitivity, and at 37°C to screen for temperature sensitivity. Sensitivity to paromomycin was analyzed in liquid cultures as described (Dresios et al., 2000).

Strains and Plasmids
MH272–3f a/ (ura3/ura3, leu2/leu2, his3/his3, trp1/trp1, ade2/ade2; Heitman et al., 1991) was the parental strain for the mutants used in this study. Strains lacking ZUO1 (IDA1: zuo1) and ZUO1/SSZ1 (IDA12: zuo1ssz1) have been previously described (Gautschi et al., 2001). To generate strains lacking Rpl31a, Rpl31b, and Rpl39 RPL31a, RPL31b, and RPL39 plus 600 base pairs up- and 300 base pairs downstream of the respective orf was cloned into pSP65 (Promega, Madison, WI). In the case of RPL31a an internal 638-base pair StyI fragment was replaced with the TRP1 gene. In addition a SpeI/Tth111I fragment was removed in order to delete the start codon of RPL31a plus the following 19 nucleotides of the first exon. In the case of RPL31b an internal BsaAI/Bpu10I fragment of 586 base pairs within the coding region was exchanged for the ADE2 marker. An internal BamHI/BsaBI fragment of RPL39 was replaced for the HIS3 marker. The resulting disruption constructs were used to generate MH272–3fa rpl31a::TRP1, MH272–3f rpl31b::ADE2 and MH272-3f rpl39::HIS3, respectively. After mating of rpl31a::TRP1 with rpl31b::ADE2, the resulting diploid was sporulated, and tetrad dissection was performed. Deletion of both copies of Rpl31 and of Rpl39 was confirmed via immunoblotting. rpl31arpl31bzuo1 was derived by mating rpl31arpl31b with zuo1 followed by sporulation and tetrad dissection.

For expression in yeast, Rpl31a, Rpl24a, Rpl17a, Rpl39, and Zuo1 plus 300-base pairs up- and downstream of the orf were transferred into pYCPlac33 (CEN, URA3). For overexpression of RAC Zuo1 was cloned into pYEPlac181 (2µ, LEU2) and Ssz1 into pYEPlac195 (2µ, URA3; Gietz and Sugino, 1988). Mutations in pYCPlac33-Zuo1 were generated by QuikChange using the manufacturers protocol (Stratagene, La Jolla, CA). Zuo1-15A contains mutations C(167)A, KEEEKKE(290–296)AAAAAAA, RRK(299–301)AAA, ERE(303–305)AAA, E(313)A, and K(315)A (see Figure 5C). pYCPlac33-Zuo1282-331 was generated by introducing two AfeI sites at the positions corresponding to E(282) and K(331) by QuikChange. Subsequently the construct was cut with AfeI followed by blunt-end ligation. pYCPlac33-Zuo1-15A was introduced into zuo1 and rpl31arpl31bzuo1 deletion strains resulting in strains zuo1 + Zuo1-15A and rpl31arpl31bzuo1 + Zuo1-15A. pYCPlac33-Zuo1282-331 was introduced into zuo1 resulting in strain zuo1 + Zuo1282-331. For in vivo read-through experiments MH272–3f-derived strains were transformed with a high copy number plasmid encoding HSP104 (pRS423: 2µ, HIS3; Christianson et al., 1992) to ensure their [psi^–] status (Rakwalska and Rospert, 2004). The lacZ-luc chimera (see Figure 2D) were expressed from pYEPlac195 (2µ, URA3; Gietz and Sugino, 1988). Construction of the read-through reporters is based on previously published vectors; however, the stop codon context was altered such that the basal level of read-through was low (Bidou et al., 2000; Rakwalska and Rospert, 2004). The two genes were separated by a short in-frame linker containing the TGA stop codon (lacZ-STOP-luc) or GCT encoding alanine (lacZ-luc; see Figure 2D).

Peptide Scan
Peptide libraries were synthesized according to standard protocols (Frank, 1992; Reineke et al., 1996; Kramer and Schneider-Mergener, 1998). The amino acid sequence of Zuo1 was used to generate linear overlapping 15mer peptides shifted by one amino acid. Peptides were C-terminally attached to cellulose via a (β-Ala)₂ spacer. After washing in ddH₂0, cellulose membranes were incubated in buffer A (20 mM HEPES-KOH, pH 7.4, 120 mM KAcetate, 5 mM MgAcetate, 2 mM DTT, 0.5 mM PMSF) for 1 h. Unspecific binding sites were blocked by incubation with 2% milk in buffer A overnight at 25°C. Subsequently, membranes were incubated with high-salt washed yeast ribosomes in buffer A containing 2% milk for 10 h at 24°C. Unbound ribosomes were removed by washes with buffer A and peptide-bound ribosomes were transferred to a nitrocellulose membrane for 10 min at 15 V using a semidry blotting apparatus. Ribosomes were visualized with an antibody directed against ribosomal protein Rpl16.

Cross-linking Experiments
Cross-linking reactions were performed either on ribosomes isolated under low-salt conditions (120 mM KAcetate), on ribosomes isolated under high-salt conditions (800 mM KAcetate) after rebinding of purified RAC or on ribosomes in cell lysates. Note that RAC covers about one-third of ribosomes isolated under low-salt conditions (Raue et al., 2007). RAC is released from ribosomes in the presence of 800 mM KAcetate (Gautschi et al., 2001). When the salt concentration is lowered, RAC can rebind to ribosomes. As purified RAC was added in molar excess to high-salt washed ribosomes, a higher fraction is occupied under these conditions (data not shown). A standard reaction contained 20–60 nM ribosomes in 100–200 µl cross-link buffer (80 mM KAcetate, 20 mM HEPES-KOH, pH 7.4, 2 mM MgAcetate, 2 mM DTT, and 1 mM PMSF). Reactions were supplemented with the amino-reactive cross-linking agent BS³ [bis(sulfosuccinimidyl)suberate; Pierce, Rockford, IL] to a final concentration of 0.8 mM and were incubated at 21°C for 20 min. BS³ cross-linking was stopped by addition of glycyl-glycine to a final concentration of 30 mM followed by incubation for 10 min on ice. Direct interaction between Zuo1 and Rpl31 was tested using the zero-length cross-linker EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, Pierce). In this case, 100 µl reactions in cross-link buffer contained 56 nM ribosomes, which were supplemented with 20 mM EDC. Reactions were incubated at 20°C for 20 min. Cross-link reactions in total cell lysate were performed using cells corresponding to an OD₂₈₀ of 300. Cell lysates in a volume of 100 µl cross-link buffer were supplemented with 6.5 mM EDC. Cross-link reactions were analyzed by SDS-PAGE followed by immunoblotting.

For the identification of Zuo1's cross-link partner immunoprecipitations were performed under denaturating conditions. To this end, high-salt washed ribosomes (20 nM) and purified RAC (40 nM) were preincubated at 21°C for 15 min in cross-link buffer. BS³ was added to a final concentration of 2 mM, and the reaction was incubated for 30 min on ice before cross-linking was terminated by the addition of 15 mM glycyl-glycine. Reactions were precipitated with 5% trichloroacetic acid, and protein pellets were resuspended in immunoprecipitation buffer (200 mM Tris-HCl, pH 7.5, 4% SDS, 10 mM EDTA, 100 µg/ml ovalbumin, 1 mM PMSF, and protease inhibitor mix containing 1.25 µg/ml leupeptin, 0.75 µg/ml antipain, 0.25 µg/ml chymostatin, 0.25 µg/ml elastinal, and 5 µg/ml pepstatin A). In a standard reaction, 30 µl of the samples were diluted into 600 µl TNET buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 25 µg/ml ovalbumin, 1% Triton X-100, 0.5 mM PMSF, and protease inhibitor mix). Protein A Sepharose beads, 30 µl, precoated with antibodies directed against Zuo1 were added, and the reaction was incubated over night at 4°C on a shaker. Aliquots of supernatant and the material bound to protein A Sepharose beads were analyzed by SDS-PAGE followed by Coomassie G-250 staining (Neuhoff et al., 1988). The band corresponding to the Zuo1 cross-link was excised from the gel and analyzed by mass spectrometry.

Ribosome Profiles
Yeast cells at an OD₆₀₀ of 0.4 were harvested in the presence of 100 µg/ml cycloheximide. Preparation of extracts was carried out by glass bead disruption in 20 mM HEPES-KOH, pH 7.4, 100 mM KAcetate, 2 mM MgAcetate, 100 µg/ml cycloheximide, and 0.5 mM DTT as described (Ashe et al., 2000). Of each lysate, 80–120 µl corresponding to 10 A₂₆₀ units were loaded onto a 15–55% linear sucrose gradient. After centrifugation for 2.5 h at 200,000 x g (TH641, Sorvall Instruments, Newton, CT) gradients were fractionated from top to bottom with a density gradient fractionator monitoring A₂₅₄ (Teledyne Isco, Lincoln, NE).

Preparation of Ribosomes and Ribosome-Binding Assay
A 10-l culture of yeast grown on YPD was harvested at an OD₆₀₀ of 2.0. Cells were washed with ice-cold water and were subsequently resuspended in 200 ml sorbitol buffer (1.4 M sorbitol, 50 mM KAcetate, and 10 mM DTT) at 25°C. To generate spheroblasts Zymolyase 20T was directly dissolved in the cell suspension to a final concentration of 2.5 mg/gram of cells, and the mixture was incubated at 30°C for 40 min with gentle shaking. Spheroblasts were harvested by centrifugation at 4°C for 5 min at 3800 x g, were washed three times with a total of 0.9 l of ice-cold sorbitol buffer containing 5 mM DTT and were subsequently resuspended in lysis buffer (20 mM HEPES-KOH, pH 7.4, 120 mM KAcetate, 2 mM MgAcetate, 5 mM DTT, 1 mM PMSF, and 1x protease inhibitor mix). The suspension was homogenized with 20 strokes in a glass homogenizer (Bellco, Vineland, NJ). Ribosomes were isolated by consecutive centrifugation for 15 min at 26,900 x g (SS34, Sorvall), 30 min at 81,000 x g (T647.5, Sorvall), and 2 h at 207,000 x g (TI70.1, Beckman, Fullerton, CA). The resulting pellet was resuspended in 1–2 ml of lysis buffer and is referred to as low-salt washed ribosomes. High-salt washed ribosomes were prepared by adjusting low-salt washed ribosomes to 800 mM KAcetate followed by centrifugation through a cushion containing 25% sucrose and 800 mM KAcetate in lysis buffer at 247,000 x g (TLA100.2, Beckman). The resulting pellet was resuspended in lysis buffer and is referred to as high-salt washed ribosomes. Aliquots of low- and high-salt washed ribosomes were stored at –80°C.

To assess the stability of RAC–ribosome complexes, ribosomes were isolated at increasing salt concentrations as previously described (Gautschi et al., 2001). In brief, aliquots of yeast ribosomes resuspended in lysis buffer were adjusted to the indicated KAcetate concentration in a total of 60 µl. Samples were layered on top of a 90-µl sucrose cushion (25% sucrose, 20 mM HEPES-KOH, pH 7.4, 2 mM MgAcetate, 2 mM DTT, 1 mM PMSF, 1x protease inhibitor mix, and KAcetate as indicated). After centrifugation at 217,000 x g (TLA100.1, Beckmann) for 120 min at 4°C, aliquots of supernatant, ribosomal pellet, and a total corresponding to the amount loaded onto the cushion were analyzed by SDS-PAGE followed by immunoblotting.

Miscellaneous
Purification of RAC was performed as described (Conz et al., 2007). A polyclonal rabbit antibody was generated using as an antigen Rpl31a purified after expression of the N-terminally His-tagged protein in Escherichia coli (pET28a, Novagen, Madison, WI). Total yeast protein for immunoblot analysis and quantification was prepared as described (Yaffe and Schatz, 1984). Immunoblots were developed using ECL. Quantification of ribosomes and RAC was performed as described (Raue et al., 2007). AIDA Image Analyzer (Raytest, Straubenhardt, Germany) was used for the quantification. Preparation of cell extracts, determination of β-galactosidase and luciferase activity, and calculation of the read-through efficiencies was performed as described (Rakwalska and Rospert, 2004).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
RAC Can Be Cross-linked to a Single Ribosomal Protein
To localize RAC on the ribosome and to identify potential ribosomal proteins that interact with RAC we have chosen a cross-linking approach. To that end, purified RAC was allowed to rebind to ribosomal particles stripped of interacting proteins. Subsequently RAC–ribosome complexes were isolated and were treated with the amino-reactive homobifunctional cross-linker BS³. The procedure yielded a prominent cross-link product with molecular mass below 80 kDa that was detectable with Zuo1-specific antibodies (Figure 1A). Immunoprecipitation under denaturing conditions confirmed that Zuo1 was contained in the cross-link product (Figure 1A). To identify the protein with which Zuo1 formed the cross-link, the procedure was scaled up, and the cross-link was excised from a Coomassie-stained gel and analyzed by mass spectrometry (Figure 1B). Analysis revealed two peptides corresponding specifically to either Rpl31a or Rpl31b, two 12.8-kDa proteins, which belong to the large group of ribosomal proteins that exist in two nearly identical copies. Rpl31a and Rpl31b differ in only one amino acid and are referred to as Rpl31. The Rpl31 homolog from archaea is one of the proteins localized at the rim surrounding the exit of the polypeptide tunnel (Ban et al., 2000; Figure 1E). Interaction with Rpl31 would position RAC ideally to act on nascent polypeptides. However, the spacer length of BS³ is 11.4 Å, and therefore Rpl31 and Zuo1 must not be in direct contact to be cross-linked by this reagent. To test for direct interaction, we have used the zero-length cross-linker, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), which couples carboxyl groups to primary amines directly. Zuo1 and Rpl31 were efficiently cross-linked also by EDC, indicating that RAC touches Rpl31 when bound to the ribosome (Figure 1C; compare also Figure 4C). The prominent cross-link detected at a molecular mass of 120 kDa was to the Zuo1 partner Ssz1 as it was detected not only by antibodies directed against Zuo1 but also Ssz1 (Figure 1D).

View larger version (33K): [in this window] [in a new window]   Figure 1. The zuotin subunit of RAC binds to ribosomes in close proximity of Rpl31. (A) Cross-linking of high-salt washed ribosomes after rebinding of purified RAC using the homobifunctional, amino-reactive cross-linker BS3 (+). As a control an aliquot was incubated without addition of BS3 (–). Aliquots with (+) or without (–) BS3-treatment were applied to immunoprecipitation reactions under denaturating conditions using protein A Sepharose beads coated with -Zuo1. Zuo1-X, cross-link detected with -Zuo1; U, material unbound; B, material bound to beads coated with -Zuo1 after immunoprecipitation. Immunoblots were decorated with -Zuo1. (B) Scale-up of the experiment shown in A. Coomassie-stained gel of the material bound to beads coated with -Zuo1. The band labeled as Zuo1-Rpl31 was detected only in the sample treated with BS3. (C) Cross-linking of low-salt washed ribosomes with the zero-length cross-linker EDC (+). A mock reaction without addition of EDC is shown as a control (–). Zuo1-Rpl31 indicates the cross-link between Zuo1 and Rpl31. Immunoblots were developed with -Zuo1 or -Rpl31, as indicated. For details compare Materials and Methods. (D) Cross-linking of cell lysate with (+) or without (–) the cross-linker EDC. Zuo1-Ssz1 indicates the cross-link between Zuo1 and its partner subunit Ssz1 detected with -Zuo1 and -Ssz1. (E) Crystal structure of the large ribosomal subunit of the archaea Haloarcula marismortui (PDB: 1S72 and 1JJ2; Ban et al., 2000; Klein et al., 2001). rRNA in yellow, ribosomal proteins in magenta, ribosomal proteins of the platform around the tunnel exit (E) in violet. The nomenclature of the ribosomal proteins is according to Lecompte (Lecompte et al., 2002). The appendix "e" indicates ribosomal proteins confined to archaea and eukaryotes. H. marismortui proteins in close proximity to the tunnel exit correspond to yeast proteins: L24 is the homolog of Rpl26, L29 of Rpl35, L39e of Rpl39, L23 of Rpl25, L19e of Rpl19, L31e of Rpl31, and L22 of Rpl17. The structural representation was prepared with Pymol (http://pymol.sourceforge.net/).

rpl31arpl31b Is Viable But Suffers from Growth Defects Resembling Defects of zuo1

View larger version (44K): [in this window] [in a new window]   Figure 2. Yeast strains lacking Rpl31 display slow growth, sensitivity to high- and low-growth temperatures and suffer from defects in translational fidelity. (A) Phenotypic comparison of wild type, rpl31a, rpl31b, rpl31arpl31b, and rpl31arpl31b expressing RPL31a from a low copy number plasmid. Haploid yeast strains were grown to early log phase at 30°C on minimal glucose medium. Serial 10-fold dilutions containing the same number of cells were spotted onto YPD plates and were incubated as indicated. (B) Phenotypic comparison of wild type, rpl31arpl31b, zuo1, rpl31arpl31bzuo1, and rpl31arpl31b expressing ZUO1 and SSZ1 from high copy number plasmids. Strains were grown as in A. Paro corresponds to 50 µg/ml paromomycin. Total cell extracts of strains shown in A and B were analyzed by immunoblotting using antibodies specifically recognizing Rpl31, and the RAC subunits, Ssz1 and Zuo1, as indicated. (C) Wild-type, rpl31arpl31b, and zuo1ssz1 strains were inoculated to the same OD600 on YPD or into the same medium containing paromomycin as indicated. Cultures were grown until the control without paromomycin had reached OD600 = 1.0, which was set to 100%. (D) Reporter construct for the determination of the frequency of stop codon read-through in vivo. An in-frame fusion between β-galactosidase and luciferase (lacZ-luc) served to determine the relative enzymatic activities upon equimolar expression of the two enzymes. Read-through efficiency was determined using the lacZ-STOP-luc construct in which the two enzymes were separated by a stop codon (for details compare Results and Materials and Methods). (E) Efficiency of stop codon read-through in wild-type and rpl31arpl31b strains in the presence of increasing concentrations of paromomycin. Enzymatic activities were determined in extracts derived from cells grown to an OD600 of 0.4–1 on YPD or YPD supplemented with paromomycin as indicated. Read-through frequency is expressed as the ratio of luciferase/β-galactosidase activity ([RLU]/[A420]) obtained with the lacZ-STOP-luc construct normalized to the ratio obtained with lacZ-luc as described in Rakwalska and Rospert (2004). Experiments were performed at least in triplicate; error bars, SD.

rpl31arpl31b Displays Defects in Translational Fidelity
Growth of wild type, rpl31arpl31b, and zuo1ssz1 was compared in the presence of increasing concentrations of paromomycin. As a result, rpl31arpl31b and zuo1ssz1 displayed similar sensitivity with half-maximal inhibition at app. 5 µM paromomycin (Figure 2C). Translational fidelity in the absence of Rpl31 was directly tested using an in vivo dual reporter construct in which β-galactosidase and luciferase are separated by a single in-frame stop codon (Figure 2D). If the stop codon is recognized β-galactosidase is produced, however, luciferase is not. If an amino acid is incorporated erroneously at the position of the stop codon, the read-through event results in the expression of a β-galactosidase-luciferase fusion protein. Based on this assay, the basal read-through level of rpl31arpl31b was increased (Figure 2E). Read-through was enhanced further when rpl31arpl31b was grown in the presence of paromomycin, whereas no such effect was detected for the wild-type strain (Figure 2E). The combined data indicate that Rpl31, like Zuo1 (Rakwalska and Rospert, 2004), affects the fidelity of translation.

rpl31arpl31b Displays Defects in the Assembly of Ribosomal Subunits
Ribosome profiles of rpl31arpl31b showed a typical halfmer polysome pattern, indicative of inefficient association of large subunits with small subunit translation initiation complexes (Figure 3A). Consistently, the relative level of free small ribosomal subunits was increased in rpl31arpl31b compared with wild type (Figure 3A). The profiles also revealed that the total concentration of ribosomes in rpl31arpl31b was significantly reduced. To determine the decrease of large ribosomal subunits more precisely the concentration of ribosomal protein Rpl17a/b was determined by quantitative immunoblotting using purified Rpl17a as a standard (Raue et al., 2007; Figure 3B). Rpl17 was reduced to 40% of the wild-type level in rpl31arpl31b (Figure 3, B and C). The combined data indicate that rpl31arpl31b suffers from a deficiency in the concentration of ribosomes and from inefficient subunit assembly.

View larger version (29K): [in this window] [in a new window]   Figure 3. Phenotypic defects of yeast strains lacking Rpl31 can be partly suppressed by high levels of Rpl39. (A) Ribosome profiles of logarithmically growing wild-type and rpl31arpl31b strains. Fractionation of the gradients was monitored at 254 nm. Asterisks indicate the position of halfmer ribosomes. (B) Analysis of large subunit concentration in wild type and rpl31arpl31b. Total cell extract corresponding to 0.6 x 107 cells of logarithmically growing wild type and rpl31arpl31b was separated on 10% TRIS-Tricine gels. Purified Rpl17 was applied to the same gel and was analyzed by immunoblotting using -Rpl17 as a large ribosomal subunit marker. The presence of untagged Rpl17a in the standard is due to the addition of total extract as a carrier (Raue et al., 2007). Note that the purified, His6-tagged standard protein has a slightly higher molecular mass. (C) Quantification of ribosomes in wild type (white) and rpl31arpl31b (gray). Densitometric analysis was performed to determine the range of linearity and to quantify protein concentrations in the total cell extracts. Error bars, SD. (D) Wild type, rpl31arpl31b, rpl31arpl31b, harboring the empty vector, or expressing either Rpl17a, Rpl24a, or Rpl39, rpl39, and rpl39 expressing Rpl31 were grown and analyzed as in Figure 2A. Paro corresponds to 50 µg/ml paromomycin. Total cell extracts were analyzed by immunoblotting using antibodies specifically recognizing Sse1 (as a loading control), Rpl17, Rpl31, Rps9, Rpl24, and Rpl39. (E) Ribosome profiles of logarithmically growing rpl31arpl31b, rpl39, and rpl31arpl31b expressing Rpl39 from a low copy plasmid were performed as described in A.

Zuo1 Touches Rpl31, However, the Interaction Is Not Essential for Association of RAC with Ribosomes
Localization revealed that the bulk of RAC cofractionated with 31-ribosomes on sucrose gradients (Figure 4A). The occupation of 31-ribosomes (RAC bound to 55% of 31-ribosomes) was even higher than that of wild-type ribosomes (RAC bound to 33% of ribosomes; Figure 4B). The data are consistent with the relatively constant concentration of RAC but decreased concentration of ribosomes in rpl31arpl31b compared with wild type (Figures 2B and 3C and data not shown). Cross-linking experiments with 31-ribosomes confirmed that the cross-link of Zuo1 to Rpl31 could no longer be formed (Figure 4C). Moreover, no significant bands corresponding to a molecular mass compatible with a cross-link between Zuo1 (49 kDa) and other core ribosomal proteins (<40 kDa) emerged on 31-ribosomes. This suggests that Zuo1 did not employ an alternative ribosomal protein as a binding site in the absence of Rpl31. We conclude that Rpl31 is not essential for stable interaction of RAC with ribosomes and that growth defects observed in rpl31arpl31b are not related to a general loss of RAC binding to ribosomes.

View larger version (65K): [in this window] [in a new window]   Figure 4. Rpl31 is not essential for binding of RAC to ribosomes. (A) Ribosome profiles were run as described in Figure 3A. After fractionation aliquots were analyzed by immunoblotting using -Ssz1/-Zuo1 (RAC), -Rps9, and -Rpl24 as indicated. One-twentieth of the material loaded onto the sucrose gradient was analyzed as a total (T). (B) Quantification of Zuo1 in wild type and rpl31arpl31b. Ribosomes were isolated under low-salt conditions. Analysis of isolated ribosomes was then performed as described in Figure 3, B and C using purified RAC and Rps9a as a standard for the quantifications. The experiments were performed in triplicate. The occupation of ribosomes with Zuo1 is given in percent (Raue et al., 2007). (C) Zuo1 does not form an alternative cross-link to a ribosomal protein in the absence of Rpl31. Cross-linking with BS3 was performed on low-salt washed ribosomes as described in Materials and Methods. Immunoblots were analyzed with -Zuo1 and with -Rpl17 as a control.

View larger version (46K): [in this window] [in a new window]   Figure 5. A highly charged segment within Zuo1 affects the affinity for ribosomes. (A) Wild type, zuo1, zuo1 expressing Zuo1-15A, or zuo1 expressing Zuo1282-331 were grown as in Figure 2A. paro, 200 µg/ml paromomycin. (B) Scan of a Zuo1 peptide library for segments interacting with ribosomes. Zuo1–15mer peptides on the cellulose membrane are shifted by one amino acid per spot. The cellulose membrane was incubated with high-salt washed ribosomes and subsequently ribosomes were transferred to nitrocellulose and analyzed by immunoblotting using -Rpl16 as a ribosomal marker. Box1 corresponds to amino acids 119–130, box2 to amino acids 243–253, box3 to amino acids 257–269, box4 to amino acids 296–305. (C) Amino acid sequence of Zuo1. Interacting segments identified in B are indicated. Amino acids 282–331 are shaded in gray. Mutations introduced in box4, and adjacent amino acids are shown in the bottom panel. (D) Ribosome profiles of zuo1 expressing Zuo1-15A and of zuo1rpl31arpl31b expressing Zuo1-15A were performed as described in Figure 3A. After fractionation aliquots were analyzed by immunoblotting using -Ssz1/-Zuo1 (RAC), -Rps9, and -Rpl24. (E) Zuo1-15A binding to ribosomes is destabilized in the presence of high concentrations of salt. Salt-dependent release of RAC from ribosomes derived from wild type, rpl31arpl31b, zuo1 + Zuo1-15A, and zuo1rpl31arpl31b + Zuo1-15A. Separation of extracts (tot) into a postribosomal supernatant (S) and a ribosomal pellet (P) was performed at increasing concentrations of KAcetate as indicated (mM KAc). Aliquots were separated on 10% TRIS-Tricine gels and subsequently analyzed by immunoblotting using -Ssz1/-Zuo1 (RAC), and -Rpl17 as a ribosomal marker. Boxed is the concentration of KAcetate that resulted in 50% of RAC release.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Within Zuo1 we have identified a segment of 10 amino acids that showed strong interaction with ribosomes in a peptide scan. This segment localized to the highly charged region, a deletion of which abolishes interaction between Zuo1 and ribosomes (Yan et al., 1998). It was thus quite unexpected that severe mutations within this segment showed only minor effects on Zuo1-ribosome interaction. Because 296–305 was the only segment within Zuo1's charged region that displayed significant binding to ribosomes, it seems possible that important contacts are made by other domains of Zuo1. Recent analysis of protein modules mediating RNA binding also point in this direction. Although such modules are diverse, a general theme is strong enrichment of arginines and lysines combined with a significant underrepresentation of glutamates and aspartates (Weiss and Narayana, 1998; Chen and Varani, 2005; Terribilini et al., 2006). Interestingly, the charged region between amino acids 282 and 331, the deletion of which leads to loss of ribosome binding (Figure 5, A and C) contains 5 arginines and 11 lysines, but also contains 14 glutamates and 1 aspartate. Possibly, loss of binding of Zuo1282-331 reflects structural alterations within Zuo1 rather than loss of its primary binding surface. Independent of the question whether or not the charged region of Zuo1 provides the prime ribosome interaction surface, the mode of Zuo1's interaction seems to differ from RPBs previously characterized. Eubacterial trigger factor and eukaryotic NAC contain well-defined, rather small binding surfaces (Kramer et al., 2002; Wegrzyn et al., 2006) and ERj1, a membrane-bound homolog of Zuo1 also requires only a short and well-defined motif for stable interaction (Dudek et al., 2002, 2005; Blau et al., 2005). Binding of SRP might be more complex in that it involves several domains (Pool et al., 2002; Gu et al., 2003; Ullers et al., 2003; Halic et al., 2004, 2006).

Function of Ribosomal Protein Rpl31
Rpl31 belongs to the eukaryotic ribosomal proteins which are not conserved in eubacteria but possess an archaebacterial homolog. In eubacteria an unrelated protein, L17 occupies the location of Rpl31 at the exit tunnel platform (Harms et al., 2001). It has been suggested that proteins not conserved between eubacteria and archaea/eukaryotes have arrived by convergent evolution with the main purpose to fill the cracks between rRNA helices and stabilize the structure (Klein et al., 2004). However, in the case of Rpl31, which exposes large parts to the exterior of the ribosome (Figure 1E) this seems an unlikely scenario. Possibly, Rpl31 has coevolved to function with RPBs that are restricted to the eukaryotic system. Besides RAC, possible candidates would be the Hsp70 homolog Ssb1/2 or the methionine aminopeptidases for which up to now no binding sites have been identified. However, our analysis suggests that none of the known RPBs was severely affected with respect to ribosome association in the absence of Rpl31 (data not shown). Only in the case of NAC we have observed that a significant fraction was recovered in the cytosol and not attached to ribosomes in a rpl31arpl31b strain. This increase in unbound NAC, however, was due to the shortfall in 31-ribosomes compared with wild-type ribosomes and NAC covered all available 31-ribosomes (data not shown).

It was previously reported that yeast deletion strains lacking either RPL31a or RPL31b are viable (Winzeler et al., 1999; Enyenihi and Saunders, 2003). Deletion of the gene encoding RPL31a, but not RPL31b, increases replicative life span in yeast (Kaeberlein et al., 2005; Steffen et al., 2008). Here we show that the double deletion strain rpl31arpl31b is viable but displays severe growth defects. Rpl31 thus belongs to the group of ribosomal proteins with nonessential functions. To date, at the tunnel platform the only other protein dispensable for the life of yeast is Rpl39 (Dresios et al., 2000). Rpl19 (Song et al., 1996), Rpl17 (Winzeler et al., 1999), and Rpl25 are essential components of the yeast ribosome. In the case of Rpl26 and Rpl35, encoded each by two closely related genes, the phenotypes of the respective double deletion strains have so far not been reported (Dresios et al., 2006). We here report synthetic lethality of rpl31arpl31brpl39. According to the crystal structure of the archaeal ribosome Rpl31 does not directly contact any ribosomal protein but contacts domains III, IV, and VI of the 25S rRNA, whereas Rpl39 contacts rRNA of domains I and III (Ban et al., 2000; Nissen et al., 2000). Interconnection of Rpl31 and Rpl39 via domain III of 25S rRNA may cause, or at least contribute, to the synthetic effect. Such a scenario is supported by the observation that Rpl31 as well as Rpl39 (Dresios et al., 2000) affect translational fidelity. As previously discussed Rpl39 forms part of the polypeptide exit and is thus ideally positioned to allosterically transmit effects along the tunnel to the decoding center (Dresios et al., 2001; Rospert, 2004). How this may function on a molecular level and whether Rpl31 and RAC contribute to this intraribosomal signaling cascade awaits further investigation.

	ACKNOWLEDGMENTS

 
We thank Dr. M. Gautschi and members of the Institute for discussion and critical reading of the manuscript. This work was supported by SFB 746, Forschergruppe 967, and by the Excellence Initiative of the German Federal and State Governments Grant EXC 294 to S.R.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08-06-0661) on October 1, 2008.

Address correspondence to: Sabine Rospert (sabine.rospert{at}biochemie.uni-freiburg.de)

Abbreviations used: NAC, nascent polypeptide associated complex; RAC, ribosome-associated complex; 31-ribosomes, ribosomes isolated from a rpl31arpl31b strain; 39-ribosomes, ribosomes isolated from a rpl39 strain; RPB, ribosome-associated protein biogenesis factors; SRP, signal recognition particle.

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