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Hrr25-dependent phosphorylation state regulatesorganization of the pre-40S subunitThorsten Schafer1, Bohumil Maco2, Elisabeth Petfalski3, David Tollervey3, Bettina Bottcher4, Ueli Aebi2

& Ed Hurt1

The formation of eukaryotic ribosomes is a multistep process thattakes place successively in the nucleolar, nucleoplasmic andcytoplasmic compartments1–4. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associatewith numerous non-ribosomal factors before mature 60S and40S subunits are formed5–12. However, most mechanistic detailsof ribosome biogenesis are still unknown. Here we identify amaturation step of the yeast pre-40S subunit that is regulated bythe protein kinase Hrr25 and involves ribosomal protein Rps3. Ahigh salt concentration releases Rps3 from isolated pre-40Sparticles but not from mature 40S subunits. Electron microscopyindicates that pre-40S particles lack a structural landmark presentin mature 40S subunits, the ‘beak’. The beak is formed by theprotrusion of 18S ribosomal RNA helix 33, which is in closevicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associ-ated with pre-40S particles. Hrr25 phosphorylates Rps3 and the40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readilydissociate from the pre-ribosome, whereas subsequent dephos-phorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivodepletion of Hrr25 inhibits growth and leads to the accumulationof immature 40S subunits that contain unstably bound Rps3.

We conclude that the kinase activity of Hrr25 regulates thematuration of 40S ribosomal subunits.To analyse the maturation of pre-40S ribosomal subunits, we

assessed their interactions with non-ribosomal factors. Pre-40Sparticles were isolated by tandem affinity purification (TAP) of theassociated non-ribosomal bait protein Rio2-TAP12 and subjected togel filtration in the presence of a high salt concentration (100mMMgCl2). Under these conditions all associated non-ribosomal factors(namely Rio2, Tsr1, Ltv1, Enp1, Nob1, Hrr25, Dim1 and Dim2) weredissociated from the 40S subunit (Fig. 1a). The ribosomal proteinRps3 was also released, but the bulk of small subunit proteinsincluding Rps8 were not dissociated (Fig. 1a). In contrast, mature40S subunits were not disrupted by 100mMMgCl2; in particular, Rps3remained associated (Supplementary Fig. S1). Gel-filtration chroma-tography of the salt-extracted pre-40S particle further revealed thatEnp1, Ltv1 and Rsp3 were eluted together in intermediate fractions,which is indicative of complex formation (Fig. 1a). The remaining 40Ssynthesis factors (for example Tsr1 and Rio2) were eluted in laterfractions, indicating that they became monomeric.Ltv1 was previously shown to be associated with pre-40 subunits13,

whereas Enp1 is present in both early 90S and late 40S pre-ribosomes12. Consistent with these findings was our observation

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Figure 1 | Ltv1, Enp1 and Rps3 form anextractable pre-ribosomal subcomplex.a–c, Gel-filtration profiles of MgCl2-treatedpre-40S particles. Rio2-TAP (a) or Ltv1-TAP(b, c) purifications were separated underconditions of high (100 mM MgCl2; a, c) or low(10 mM MgCl2; b) salt concentration. Input (L),standard (S) and column fractions (1–17) wereanalysed by SDS–PAGE and Coomassie staining,or by western blotting with anti-CBP (bait),anti-Rps3 and anti-Rps8 antibodies. Band 1, Ltv1;band 2, Enp1; band 3, Rps3; asterisks, TEV(Tobacco Etch Viral protease); diamonds,Hsp70s. A 40S ‘core’ particle is eluted atabout 106 Da. d, Affinity purifications ofLtv1-TAP from lysates at the indicated MgCl2 orNaCl concentrations. Eluates were analysed bySDS–PAGE and Coomassie staining.

1Biochemie-Zentrum der Universitat Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. 2M. E. Muller Institute for Structural Biology, Biozentrum, UniversitatBasel, CH-4056 Basel, Switzerland. 3Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK. 4EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

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that Ltv1-TAP co-precipitated a late pre-40S particle indistinguish-able from the Rio2-TAP particle (Fig. 1b).When the Ltv1 particle wasincubated with 100mM MgCl2, Rps3 and all non-ribosomal factorswere dissociated, and Ltv1, Enp1 and Rps3 were again eluted together(Fig. 1c).To show more directly that Ltv1 and Enp1 form a salt-resistant

complex with Rps3, Ltv1-TAP was affinity-purified under differentsalt conditions. After purification in 100mMMgCl2 or 300mMNaCl(Fig. 1d), most non-ribosomal factors and ribosomal proteins weredissociated from Ltv1-TAP, whereas Enp1 and Rps3 remainedbound. Taken together, these data showed that Ltv1, Enp1 andRps3 exist in a salt-resistant complex that can be released frompre-40S subunits, whereas Rps3 cannot be extracted frommature 40Ssubunits under these conditions.The observation that Rps3 showed differential association with

pre-40S and mature 40S subunits prompted us to compare theirstructures. The eukaryotic small subunit consists of three charac-teristic subdomains referred to as ‘head’, ‘body’ and ‘platform’.Protruding rRNA turns and helices provide additional character-istic features in the 40S subunit structure, termed the ‘beak’,‘shoulder’ and ‘feet’14–16 (see also Fig. 2c). Comparison of theX-ray structure of the prokaryotic 30S subunit17 with cryoelec-tron-microscopy-based reconstruction of the yeast ribosome hasallowed the structure of the mature 40S subunit to be modelled atatomic resolution16.We initially compared negatively stained pre-40S subunits

with mature 40S subunits by electron microscopy (Fig. 2a, andSupplementary Fig. S2a). Both particles exhibit the typical ‘head’,‘platform’ and ‘body’ domains. However, the pre-40S ribosomalparticle lacks the prominent ‘beak’, which is a protrusion ofhelix 33 of the 18S rRNA (Fig. 2a, c). Moreover, additional mass isvisible adjacent to the ‘platform’ of the pre-40S particle, whichcould represent non-ribosomal factors (see also SupplementaryFig. S2a).Next we performed cryoelectron microscopy to further resolve

the structural differences between nascent and mature 40S subunits(Fig. 2b, and Supplementary Fig. S2b). Three-dimensional recon-struction of the pre-40S particle showed the major structural land-marks of the small subunit, but the ‘beak’ was not visible (Fig. 2b, c).However, an elongated structure was revealed in the pre-40S subunit,which emerged from the head region and wrapped around in aclockwise orientation. It is possible that this structure is the pro-genitor of the genuine ‘beak’ that protrudes from the mature 40Ssubunit. We also observed an additional structure on the oppositeside of the head, which could be a flipped beak (see also Supplemen-tary Fig. S2b and animated rotations). Notably, the prokaryotichomologue of Rps3 (called S3) is located at the base of a structurewithin the 30S subunit, which corresponds to the eukaryotic beak17.Three-dimensional reconstruction of the eukaryotic 40S subunitplaces yeast Rps3 in a similar position16, indicating that it mightdefine the exposed position of eukaryotic helix 33 within the mature40S subunit (see also Fig. 2c).The evidence that Rps3 is not incorporated into the pre-40S

subunit in its final conformation would be consistent with a rolefor Rps3 in 40S biogenesis. Depletion of the essential Rps3 from yeastcells resulted in a strongly reduced export rate of 20S rRNA18 andnuclear accumulation of pre-40S subunits (Supplementary Fig. S3).Moreover, 20S and 35S pre-rRNA were increased and 27SA2 pre-rRNA was markedly decreased in the GAL1::RPS3 depletion mutant(Supplementary Fig. S3).We next addressed how the salt-resistant incorporation of Rsp3

into the 40S subunit and formation of the ‘beak’ structure might beregulated. Two protein kinases, Rio2 and Hrr25, are associated withthe purified pre-40S subunits12, indicating that phosphorylationmight have a function in small subunit maturation. Notably, whenLtv1-TAP was isolated from yeast lysates in the presence of phos-phatase inhibitors, two bands, Ltv1 and Enp1, were shifted and

migrated slower in the SDS-polyacrylamide gel when compared witha preparation performed in the absence of phosphatase inhibitors(Supplementary Fig. S4a). These data indicate that Lvt1 and Enp1 arephosphorylated in vivo.To assess whether these proteins can undergo phosphorylation in

vitro, pre-40S particles were purified without phosphatase inhibitorsto allow the removal of phosphate by endogenous phosphatases.Incubation with ATP at 4 8C induced a shift in the mobility of theLtv1 and Enp1 bands on the SDS-polyacrylamide gel, indicating thatthey were phosphorylated in vitro (Fig. 3a). When the pre-40Ssubunits were incubated with ATP at physiological temperature(30 8C), Ltv1, Enp1 and Rps3 were phosphorylated and were alsodissociated from the pre-ribosomes (Fig. 3b). In contrast, other non-ribosomal factors were not dissociated. The bands corresponding tophosphorylated Ltv1, Enp1 and Rps3 were eluted together inintermediate fractions of the gel-filtration column, indicating thatthey remained as a complex (Fig. 3b, and Supplementary Fig. S4b). Incontrast, incubation of the pre-40S particle at 30 8C in the absence ofATP did not cause dissociation of Ltv1, Enp1 or Rps3 from the pre-40S particle (Fig. 3c, and Supplementary Fig. S4b). These data

Figure 2 | Structural comparison of pre-40S and mature 40S subunits.a, Projection averages (left) and electron-density contour maps (right) ofnegatively stained 40S (left pair) and pre-40S (right pair) subunits. Scalebars, 5 nm. b, Three-dimensional reconstruction of mature and pre-40Sparticles from cryoelectron micrographs. Depicted are orientations from thesolvent (left), intersubunit (centre) and top (right) sides. Scale bar, 10 nm.c, Tertiary structure of yeast 18S rRNA with superimposed Rps3 (blue) asdescribed in www.rcsb.org (Protein Data Bank, 1S1H). Major structurallandmarks of the 40S subunit are depicted. d, Development of the beakstructure within isolated pre-40S particles on phosphorylation andsubsequent dephosphorylation. In 23% of the isolated pre-40S particles(affinity-purified by means of Rio2-TAP) beak formation was observed byelectron microscopy after negative staining. Left, class averages; right,electron-density contour maps. Scale bar, 10 nm.

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indicate that treatment with ATP at physiological temperatureinduced the phosphorylation and subsequent dissociation of Ltv1,Enp1 and Rps3 from the 40S pre-ribosome.We next tested whether phosphorylation of the pre-40S particle

was followed by a dephosphorylation step that triggers further pre-40S subunit maturation. No specific phosphatases that participate inribosome biogenesis have been identified, and ATP-treated pre-40Sparticles were therefore incubated with l-phosphatase. Subunitmaturation was monitored by gel-filtration chromatography andelectron microscopy. Biochemical analysis showed that a significantfraction of Rps3 (about 25%) became associated with the mature 40Ssubunits in a salt-resistant manner after a round of phosphorylation

followed by dephosphorylation (Fig. 3d). Consistent with this wasour EM examination, which revealed that 23% of negatively stainedpre-40S particles showed the characteristic beak structure, afterphosphorylation–dephosphorylation treatment (Fig. 2d). Incontrast, Rps3 was not stably associated with 40S subunits if eitherthe phosphorylation or dephosphorylation step was applied alone(Supplementary Fig. S5a, b).To identify the kinase(s) responsible for phosphorylation of the

pre-40S components, the two candidate kinases, RIO2 and HRR25,were repressed in yeast with the use of the regulated GAL1 promoter(Supplementary Figs S6 and S7). Rio2 has been reported to beinvolved in the processing of 20S pre-rRNA19 and nuclear 40S

Figure 3 | Phosphorylation state induces 40Ssubunit maturation. a, ATP-inducedphosphorylation (4 8C) and dissociation of Ltv1and Enp1 (30 8C) from isolated pre-40S subunits.The indicated eluates of Ltv1-TAP andRio2-TAP preparations were analysed bySDS–PAGE and Coomassie staining or westernblotting (anti-Rps3 antibody). Circles indicate baitproteins; arrows identify ATP-induced band shifts.Ltv1-TAP could not be eluted from IgG-beads ontreatment with ATP at 30 8C. b–d, ATP-dependentdissociation of Ltv1–Enp1–Rps3 complex frompre-40S particles. Incubation of Rio2-TAPpreparations with 1 mM ATP (b) or no ATP (c) for30 min at 30 8C and analysis by gel filtration.d, A phosphorylation–dephosphorylation cycleinduces salt-resistant association of Rps3 withthe pre-40S subunit. Gel-filtration profiles ofRio2-TAP preparations treated with 10 mM (b, c)or 100 mM (d) MgCl2, analysed by SDS–PAGE andCoomassie staining or western blotting (anti-Rps3antibody). Bands are identified as follows in b–d:1, Ltv1; 2, Enp1; 3, Rps3; 4, Rio2; 5, Nob1;6, Hrr25; asterisks, TEV protease.

Figure 4 | Hrr25 kinase regulates 40S subunit maturation. a, Hrr25-dependent phosphorylation of Enp1 and Rps3. Ltv1-TAP preparations fromGAL1::HRR25 cells grown in YPGal (yeast extract, peptone, galactose) orYPGlu (yeast extract, peptone, glucose) were incubated with or without ATPand analysed by SDS–PAGE with Coomassie staining. Upward arrows,phosphorylated Ltv1, Enp1 and Rps3; downward arrows, dephosphorylatedLtv1, Enp1 and Rps3. b, Hrr25-dependent dissociation of Enp1 and Rps3from pre-40S subunits. Gel-filtration profiles are shown of Ltv1-TAP

preparations from GAL1::HRR25 cells grown for 12 h in YPGal (left) orYPGlu (right) and incubated with ATP for 30 min at 30 8C. Fractions wereanalysed by SDS–PAGE and Coomassie staining or western blotting (anti-Rps3 antibody). Band 1, Ltv1; band 2, Enp1. c, 40S subunits from Hrr25-depleted cells contain salt-extractable Rps3. Gel-filtration profiles are shownof 100 mM MgCl2-treated mature 40S subunits isolated from GAL1::HRR25cells grown for 12 h in YPGal (left) or YPGlu (right). Fractions were analysedby SDS–PAGE and western blotting (anti-Rps3 and anti-Rsp8 antibodies).

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subunit export12. The casein kinase I isoform Hrr25 has multipleroles in the cell20 but has not been implicated in ribosome biogenesis.Notably, when pre-40S particles were purified from Hrr25-depletedcells and incubated with ATP, Enp1 and Rps3 were apparently notphosphorylated and not released from the pre-40S subunit (Fig. 4a, b).Ltv1 was partly phosphorylated in Hrr25-depleted pre-ribosomesand was dissociated from the pre-40S particle (Fig. 4a, b). In contrast,pre-40S particles isolated from Rio2-depleted cells and incubatedwith ATP still exhibited phosphorylated Enp1, Rps3 and Ltv1, whichwere dissociated at 30 8C (Supplementary Fig. S7). These datashowed that the phosphorylation and subsequent dissociation ofEnp1 and Rps3 from the 40S pre-ribosome is regulated by the Hrr25protein kinase.To determine whether Hrr25 controls Rps3 assembly in vivo, 40S

subunits were isolated from the Hrr25-depleted cells by sucrose-gradient centrifugation and treated with a high salt concentration.Rps3 was released from the 40S subunit on incubation with 100mMMgCl2, whereas most ribosomal proteins, including Rps8, were notdissociated (Fig. 4c). When 40S subunits were isolated from theGAL1::HRR25 strain grown in galactose (no Hrr25 depletion), Rps3was not extracted by 100mM MgCl2 (Fig. 4c). The Hrr25-depletedcells showed defects in 18S rRNA maturation that closely resembledthose seen in strains genetically depleted of Rps3 (compare Sup-plementary Figs S6b and S3b), strongly supporting their functionalinteraction in vivo. Depletion of Hrr25 also inhibited nuclear exportof the 40S subunit (Supplementary Fig. S6c), as previously reportedfor Rps3 depletion18. Taken together, the data reveal that Hrr25functions at a late step in 40S subunit biogenesis that regulates theassociation of Rps3 with the 40S subunit, both in vitro and in vivo.Thus, we have uncovered a maturation step in 40S subunit

biogenesis during which the binding of ribosomal protein Rps3and the structure of the beak region of the 40S subunit are reorgan-ized. Our data indicate that the beak RNA remains flexible while Rps3is not tightly integrated into the 40S subunit. Hrr25-dependentphosphorylation and subsequent dephosphorylation are requiredfor Rps3 to achieve its final association with the 40S subunit, whichinduces beak formation. We speculate that the incorporation ofnegatively charged phosphate groups into the Rps3, Enp1 and Ltv1proteins weakens their association with the 40S subunit as a result ofelectrostatic repulsion. Subsequent removal of the phosphate groupfrom Rps3 might then allow it to form a more stable association withthe rRNA. The significance of the structural reorganization of thepre-40S particle remains to be determined, but a protruding, rigidbeak might hinder passage through the nuclear pore complex. Thenuclear pre-40S particles might retain a transport-competent struc-ture, without a hindering beak, until transit through the nuclear porecomplex. Mutations in Enp1, Ltv1 and Rps3 cause defects in thenuclear exit of pre-40S subunits, although it is unclear whether theyhave direct functions in export. These proteins all seem to be boundto the head region of the nascent 40S subunit, as is Rps15, anothersmall subunit protein with a role in 40S subunit export21. Thus, thepre-40S head region without an exposed beak might have a keyfunction in recruiting nuclear export factors and initiating nuclearexport.

METHODSPre-40S ribosomal subunits were affinity-purified with TAP-tagged baitproteins Ltv1 or Rio2, as described22. Mature 40S subunits were isolated bysucrose-density centrifugation5 of whole cell lysates or by affinity purificationof translation factor Nip1. Isolated ribosomal particles were analysed bySDS–PAGE and Coomassie staining. Associated non-ribosomal factors wereidentified by matrix-assisted laser desorption ionization–time-of-flight massspectrometry5. Pre-40S and mature 40S subunits were subjected to gel-filtrationchromatography under conditions of low (10mM MgCl2) and high (100mMMgCl2) salt concentration to separate subcomplexes and analyse the associationstate of Rps3 by western analysis23. To compare pre-40S andmature 40S subunitsat the structural level, negative-staining electron microscopy and cryoelectronmicroscopy with three-dimensional reconstruction were performed. An in vitro

assay was developed that allowed us to monitor the dependence of thematuration of pre-40S subunits on phosphorylation (addition of ATP) anddephosphorylation (phosphatase treatment). To analyse the role of HRR25 orRPS3 in vivo, pre-rRNA processing analyses12,24 and a fluorescence-based in vivoassay for nuclear export of ribosomal subunits25,26 were applied. For repression ofHRR25,RPS3 andRIO2 gene expression in yeast, the genes were placed under thecontrol of the GAL1 promoter27. A detailed description of the experimentalprocedures used is provided in Supplementary Information.

Received 22 December 2005; accepted 25 April 2006.

1. Warner, J. R. Nascent ribosomes. Cell 107, 133–-136 (2001).2. Fatica, A. & Tollervey, D. Making ribosomes. Curr. Opin. Cell Biol. 14, 313–-318

(2002).3. Tschochner, H. & Hurt, E. Pre-ribosomes on the road from the nucleolus to the

cytoplasm. Trends Cell Biol. 13, 255–-263 (2003).4. Granneman, S. & Baserga, S. J. Crosstalk in gene expression: coupling and

co-regulation of rDNA transcription, pre-ribosome assembly and pre-rRNAprocessing. Curr. Opin. Cell Biol. 17, 281–-286 (2005).

5. Bassler, J. et al. Identification of a 60S preribosomal particle that is closelylinked to nuclear export. Mol. Cell 8, 517–-529 (2001).

6. Harnpicharnchai, P. et al. Composition and functional characterization of yeast66S ribosome assembly intermediates. Mol. Cell 8, 505–-515 (2001).

7. Saveanu, C. et al. Nog2p, a putative GTPase associated with pre-60S subunitsand required for late 60S maturation steps. EMBO J. 20, 6475–-6484 (2001).

8. Fatica, A., Cronshaw, A. D., Dlakic, M. & Tollervey, D. Ssf1p preventspremature processing of an early pre-60S ribosomal particle. Mol. Cell 9,341–-351 (2002).

9. Dragon, F. et al. A large nucleolar U3 ribonucleoprotein required for 18Sribosomal RNA biogenesis. Nature 417, 967–-970 (2002).

10. Grandi, P. et al. 90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP,and 40S subunit processing factors but predominantly lack 60S synthesisfactors. Mol. Cell 10, 105–-115 (2002).

11. Nissan, T. A., Bassler, J., Petfalski, E., Tollervey, D. & Hurt, E. 60S pre-ribosomeformation viewed from assembly in the nucleolus until export to the cytoplasm.EMBO J. 21, 5539–-5547 (2002).

12. Schafer, T., Strauss, D., Petfalski, E., Tollervey, D. & Hurt, E. The path fromnucleolar 90S to cytoplasmic 40S pre-ribosomes. EMBO J. 22, 1370–-1380(2003).

13. Loar, J. W. et al. Genetic and biochemical interactions among Yar1, Ltv1 andRps3 define novel links between environmental stress and ribosome biogenesisin Saccharomyces cerevisiae. Genetics 168, 1877–-1889 (2004).

14. Frank, J., Verschoor, A. & Boublik, M. Multivariate statistical analysis ofribosome electron micrographs. L and R lateral views of the 40 S subunit fromHeLa cells. J. Mol. Biol. 161, 107–-133 (1982).

15. Frank, J., Verschoor, A. & Boublik, M. Computer averaging of electronmicrographs of 40S ribosomal subunits. Science 214, 1353–-1355 (1981).

16. Spahn, C. M. et al. Structure of the 80S ribosome from Saccharomycescerevisiae–-tRNA–-ribosome and subunit–-subunit interactions. Cell 107,373–-386 (2001).

17. Wimberly, B. T. et al. Structure of the 30S ribosomal subunit. Nature 407,327–-339 (2000).

18. Ferreira-Cerca, S., Poll, G., Gleizes, P. E., Tschochner, H. & Milkereit, P. Roles ofeukaryotic ribosomal proteins in maturation and transport of pre-18S rRNA andribosome function. Mol. Cell 20, 263–-275 (2005).

19. Geerlings, T. H., Faber, A. W., Bister, M. D., Vos, J. C. & Raue, H. A. Rio2p, anevolutionarily conserved, low abundant protein kinase essential for processingof 20S pre-rRNA in Saccharomyces cerevisiae. J. Biol. Chem. 278, 22537–-22545(2003).

20. Ho, Y., Mason, S., Kobayashi, R., Hoekstra, M. & Andrews, B. Role of the caseinkinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF,in the transcriptional response to DNA damage in Saccharomyces cerevisiae.Proc. Natl Acad. Sci. USA 94, 581–-586 (1997).

21. Leger-Silvestre, I. et al. The ribosomal protein Rps15p is required fornuclear exit of the 40S subunit precursors in yeast. EMBO J. 23, 2336–-2347(2004).

22. Rigaut, G. et al. A generic protein purification method for protein complexcharacterization and proteome exploration. Nature Biotechnol. 17, 1030–-1032(1999).

23. Siniossoglou, S. et al. A novel complex of nucleoporins, which includes Sec13pand a Sec13p homolog, is essential for normal nuclear pores. Cell 84, 265–-275(1996).

24. Beltrame, M. & Tollervey, D. Identification and functional analysis of two U3binding sites on yeast pre-ribosomal RNA. EMBO J. 11, 1531–-1542 (1992).

25. Gadal, O. et al. Nuclear export of 60s ribosomal subunits depends on Xpo1pand requires a nuclear export sequence-containing factor, Nmd3p, thatassociates with the large subunit protein Rpl10p. Mol. Cell. Biol. 21, 3405–-3415(2001).

26. Milkereit, P. et al. A Noc complex specifically involved in the formation andnuclear export of ribosomal 40 S subunits. J. Biol. Chem. 278, 4072–-4081(2003).

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27. Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 10,953–-961 (1998).

Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank S. Merker, P. Ihrig, J. Reichert, J. Pfannstiel andJ. Lechner for performing mass spectrometry, and M. Seedorf and G. Diecifor the gift of antibodies. B.B. acknowledges support by a grant from EU-NOE(3D-Repertoire). E.H. is recipient of grants from the DeutscheForschungsgemeinschaft and Fonds der Chemischen Industrie. E.P and D.T.were supported by the Wellcome Trust.

Author Contributions Experiments were designed and data were analysed andinterpreted by T.S. and E.H. Strain constructions, DNA recombinant work,

fluorescence microscopy and biochemical analyses (affinity purification, gelfiltration, sucrose gradient centrifugation and in vitro assays) were performed byT.S. Negative-staining electron microscopy was conducted by B.M. and U.A.,and cryoelectron microscopy and three-dimensional reconstruction by B.B.E.P. and D.T. performed rRNA processing analyses. The manuscript was writtenby T.S. and E.H. All authors discussed the results and commented on themanuscript.

Author Information Three-dimensional reconstructions of mature 40S andpre-40S ribosomal subunits have been deposited in the EMBL-EBI MolecularStructure Database (http://www.ebi.ac.uk/msd/) and can be retrieved underaccession numbers EMD-1211 and EMD-1212. Reprints and permissionsinformation is available at npg.nature.com/reprintsandpermissions. The authorsdeclare no competing financial interests. Correspondence and requests formaterials should be addressed to E.H. (cg5@ix.urz.uni-heidelberg.de).

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