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The eIF3 interactome reveals the translasome, a supercomplexlinking protein synthesis and degradation machineries

Zhe Sha†, Laurence M. Brill¶, Rodrigo Cabrera†, Oded Kleifeld§, Judith S. Scheliga¶, MichaelH. Glickman§, Eric C. Chang†,*, and Dieter A. Wolf¶,*†1 Baylor Plaza, Molecular and Cellular Biology Department, Lester and Sue Smith Breast Center,Baylor College of Medicine, Houston, TX 77030¶Burnham Institute for Medical Research, Signal Transduction Program, NCI Cancer CenterProteomics Facility, 10901 North Torrey Pines Road, La Jolla, CA 92037§Department of Biology, Technion - Israel Institute of Technology, 32000 Haifa Israel

SummaryeIF3 promotes translation initiation, but relatively little is known about its full range of activities inthe cell. Here, we employed affinity purification and highly sensitive LC-MS/MS to decipher thefission yeast eIF3 interactome, which was found to contain 230 proteins. eIF3 assembles into a largesupercomplex, the translasome, which contains elongation factors, tRNA-synthetases, 40S and 60Sribosomal proteins, chaperones, and the proteasome. eIF3 also associates with ribosome biogenesisfactors and the importins-β Kap123p and Sal3p. Our genetic data indicated that the binding to bothimportins-β is essential for cell growth, and photobleaching experiments revealed a critical role forSal3p in the nuclear import of one of the translasome constituents, the proteasome. Our data revealthe breadth of the eIF3 interactome and suggest that factors involved in translation initiation,ribosome biogenesis, translation elongation, quality control, and transport are physically linked tofacilitate efficient protein synthesis.

IntroductionThe recruitment of the 40S ribosomal subunit to the mRNA start codon is thought to be therate-limiting step in eukaryotic translation. This process requires the assembly of aribonucleoprotein complex, which joins mRNAs with some 30 different polypeptides referredto as eukaryotic initiation factors (eIFs) (Hershey and Merrick, 2000). 40S ribosomes associatewith the eIF2·GTP/Met-tRNA ternary complex, the multisubunit eIF3 complex, and severalother eIFs to form the 43S pre-initiation complex. This complex then binds to a second proteinassembly organized around eIF4G, resulting in the 43S initiation complex. eIF4G interactswith both the cap-binding protein eIF4E and the poly-A binding protein, thus presumablycircularizing the mRNA. eIF4G also recruits the eIF4A helicase assisting the 43S complex inscanning along the mRNA. Once the start codon is identified, the 43S complex is convertedinto the 48S initiation complex, which forms a stable interaction with the initiator AUG. At

© 2009 Elsevier Inc. All rights reserved.*Corresponding authors: Eric Chang: [email protected], 713-798-3519 (p)/1642 (f), Dieter Wolf: [email protected], 858-646-3117(p)/3149 (f).Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptMol Cell. Author manuscript; available in PMC 2010 October 9.

Published in final edited form as:Mol Cell. 2009 October 9; 36(1): 141–152. doi:10.1016/j.molcel.2009.09.026.

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this point, eIF2-bound GTP is hydrolyzed, leading to dissociation of eIFs, thus allowing the60S ribosomal subunit to join for productive protein synthesis.

eIF3 is the most complex translation initiation factor and plays several important roles thatwere revealed by in vitro reconstitution experiments (Dong and Zhang, 2006; Hinnebusch,2006). First, eIF3 binds to the 40S ribosome and facilitates loading of the eIF2·GTP/Met-tRNAternary complex to form the 43S pre-initiation complex. Subsequently, eIF3 assists in recruitingmRNAs to the 43S complex, presumably involving the RNA recognition motifs found in someof its subunits. Lastly, eIF3 binding to the 40S ribosome prevents the joining of the 60S subunituntil the start codon is identified, eIF2-bound GTP is hydrolyzed by eIF5, and all eIFs arereleased. While these discreet reaction steps were deciphered extensively in vitro, it remainedunclear how they are coordinated in vivo in order to ensure efficient translation.

Whereas human eIF3 consists of 13 subunits, consecutively named eIF3a – m (Damoc et al.,2007; Unbehaun et al., 2004; Zhou et al., 2008), budding yeast contains only five stochiometricsubunits, which are orthologs of human eIF3a, b, c, g, and eIF3i, and the substoichiometriceIF3j. These subunits may constitute a core complex, as all are essential for viability (Asanoet al., 1997; Phan et al., 1998). In the fission yeast, Schizosaccharomyces pombe, eIF3 containsthe same five core subunits, in addition to the non-core subunits eIF3d, e, f, g, h, i, and m(Akiyoshi et al., 2001; Bandyopadhyay et al., 2002; Burks et al., 2001; Crane et al., 2000;Dunand-Sauthier et al., 2002; Ray et al., 2008; Zhou et al., 2005). Two distinct eIF3 complexeswere identified in fission yeast that contain an overlapping set of core subunits but aredistinguished by the presence of the related eIF3e and eIF3m proteins (Zhou et al., 2005). TheeIF3m containing complex appears to mediate the translation of the bulk of cellular mRNAs,whereas the eIF3e containing complex associates with a far more restricted set of mRNAs.Distinct eIF3 complexes may therefore contribute to mRNA specificity of translation.

eIF3 also has functions that are apparently independent of its role in translation initiation. Forexample, some eIF3 subunits interact with the 26S proteasome (Dunand-Sauthier et al.,2002; Hoareau Alves et al., 2002; Paz-Aviram et al., 2008; Yen et al., 2003b). The significanceof this interaction was revealed in S. pombe, where deletion of the non-essential eIF3d/Moe1pand eIF3e/Yin6p confers a series of cellular phenotypes that indicate a defect in proteasomalprotein degradation (Yen et al., 2003b). This defect was pinpointed to a role of these eIF3subunits in the nuclear accumulation and assembly of the 26S proteasome. However, themolecular mechanisms underlying eIF3-directed proteasome localization and assemblyremained unknown.

To clarify this issue and to further elucidate the functions of eIF3, we performed a highsensitivity mass spectrometry analysis of eIF3 complexes purified from S. pombe. This eIF3interactome suggests an extensive repertoire of eIF3 roles in protein synthesis and degradationthus establishing a molecular link between these processes.

Results and DiscussionPurification of eIF3 complexes

To affinity purify eIF3 complexes, two S. pombe strains were used that encode fully functionaleIF3e or eIF3m modified at the endogenous genomic loci with protein A epitope tags precededby a cleavage site for tobacco etch virus (TEV) protease (Zhou et al., 2005). Total cell lysatewas absorbed to IgG coupled magnetic beads, and retained proteins were eluted by cleavagewith TEV protease (Fig. 1A). Mock purifications of cell lysate devoid of any epitope-taggedprotein were included as specificity controls. The eluates of a representative purification serieswere resolved by SDS-PAGE alongside fractions taken at various steps of the purificationprocedure. Approximately 30 – 40% of the TEV cleaved material was released from the beads

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into the supernatant as judged by Coomassie staining (Fig. 1B, compare lanes “TE” and “SE”).The gel also revealed an overlapping but not identical pattern of bands in the eIF3e and eIF3mcomplexes as previously described (Zhou et al., 2005). eIF3e is known to bind the proteasome(Yen et al., 2003b). To confirm the integrity of the purifications, we examined a 19S proteasomesubunit, Rpn1p, and found that it co-purified with both the eIF3e and eIF3m bait (Fig. 1C).

Purifications of the eIF3e and eIF3m complexes were performed in triplicate. For the thirdseries of purifications, cell lysates were treated with RNase A prior to affinity capture, in orderto disrupt protein interactions that were mediated by RNA (Supplementary Fig. 1). Ninesamples (triplicates of mock, eIF3e, eIF3m) were digested with trypsin and analyzed by liquidchromatography-tandem mass spectrometry (LC-MS/MS) on a high-sensitivity LTQ OrbitrapXL mass spectrometer. Each sample was analyzed 3 – 4 times to yield a total of 33 independentLC-MS/MS runs. The resulting mass spectra were searched against the S. pombe proteindatabase using SEQUEST, and search results were subjected to probability-based filteringusing ProteinProphet (Trans-Proteomic Pipeline; Institute for Systems Biology, Seattle, WA)for a false discovery rate of protein identification of ≤ 0.02. Altogether, we identified 3876unique proteins, which equal 77.1% of the predicted S. pombe proteome. The majority of theseproteins appear to be low abundance background, because they were represented by peptidesthat were detected in only a few runs, and because they had very low spectrum counts (< 3).The spectrum count of a protein is the cumulative number of times peptides defining that proteinwere selected for MS/MS scans. Spectrum counts therefore provide a semi-quantitativemeasure of relative protein abundance within and between samples (Liu et al., 2004). To obtaina high confidence set of specific and reproducible eIF3 interacting proteins, the eIF3interactome, we subjected our entire dataset to background subtraction using the mockpurifications and filtering based on spectrum counts (Supplementary Methods).

Composition of S. pombe eIF3 complexesThe high confidence eIF3 interactome comprised 230 proteins, which were consistentlyidentified in all six independent purifications (Fig. 2A). As expected, the 10 known subunitsof S. pombe eIF3 were the most abundant proteins retrieved (Fig. 2A, B). In addition, a novelprotein homologous to human eIF3j (SPAC3A12.13c) was identified as a substoichiometriceIF3 subunit, a finding that is consistent with the observation that eIF3j undergoes regulatedcycles of association and dissociation with holo-eIF3 in human cells (Miyamoto et al., 2005).Interestingly, we found two phosphorylation sites in eIF3j, which might be involved in thisregulation (Supplementary Table 1). In summary, S. pombe eIF3 contains orthologs of allhuman subunits except eIF3k and eIF3l, which are absent from the S. pombe genome.

Next, we performed a comparative quantification of subunits identified in purifications ofeither the eIF3e or the eIF3m bait proteins. Since the masses of individual eIF3 subunits varybetween 30.5 kDa (eIF3j) and 107 kDa (eIF3a), we adjusted spectrum counts to molecularweights and, for better comparison between samples, normalized the numbers to eIF3a(Supplementary Data File 1). The adjustments clarified the suggestion from raw spectrumcounts that eIF3d and eIF3e are substoichiometric components of the complex purified viaeIF3m (Fig. 2B, C). In contrast, the eIF3e bait co-purified a complex containing roughlystoichiometric amounts of all subunits, except eIF3j (Fig. 2C).

These data extend and refine those of our previous study, employing lower sensitivity massspectrometry, which suggested two distinct eIF3 complexes in S. pombe that are distinguishedby the presence or absence of eIF3d and eIF3e (Zhou et al., 2005). Since neither eIF3d noreIF3e are essential for global mRNA translation and cell viability (Akiyoshi et al., 2001;Bandyopadhyay et al., 2002; Bandyopadhyay et al., 2000; Chen et al., 1999; Crane et al.,2000; Yen and Chang, 2000; Zhou et al., 2005), the bulk of protein synthesis under normalgrowth conditions may be executed by a “global” eIF3 complex lacking these subunits, which

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is represented by the complex purified via the eIF3m bait (Fig. 2D). Consistent with thisobservation is our previous demonstration that this global eIF3 binds the majority of cellularmRNAs (Zhou et al., 2005). Under certain conditions, eIF3d and eIF3e may join the globalcomplex to modulate its mRNA specificity. For example, these factors may facilitate therecruitment of mRNAs that are translated under conditions of cellular stress to which eif3d andeif3e mutants are sensitive (Akiyoshi et al., 2001; Bandyopadhyay et al., 2000; Crane et al.,2000; Yen and Chang, 2000).

Since an eIF3 complex containing apparently stoichiometric amounts of eIF3d and eIF3e canbe readily purified from unstressed S. pombe cells (Fig. 1, Fig. 2) as well as from otherorganisms, these proteins may also carry out important, albeit non-essential, functions undernormal growth conditions. The critical target mRNAs of complexes containing eIF3d and eIF3ein stressed and unstressed cells are currently unknown. Nevertheless, recent mass spectrometryanalysis of the intact 13-subunit human eIF3 complex suggested that eIF3d and eIF3e areperipheral subunits that may undergo dynamic exchange (Zhou et al., 2008), although thesignals that can trigger such exchange remain to be identified.

The eIF3 interactome reveals a supercomplex linking protein synthesis and degradationmachineries

The eIF3 interactome also contained other initiation factors of the 43S initiation complex,including eIF2, eIF5A and B, eIF4A and eIF4G (Fig. 2A, 3A, Supplementary Table 2). Severalother eIFs, including eIF1A, eIF2B, eIF5, and the cap binding protein eIF4E1 were alsoidentified, although they did not pass the strict spectrum count thresholds in some of the sixindividual datasets (Supplementary Data File 1). Since none of these interactions was affectedby RNase treatment of the cell lysate (Fig. 3A), S. pombe eIF3 appears to assemble into a RNA-independent complex with other initiation factors of the 43S complex, although we cannotentirely exclude the possibility that short remnants of mRNA resistant to complete RNasedigestion contribute to the interactions.

eIF3 also associated with translation elongation factors, multiple tRNA-synthetases as well asribosomal proteins (Fig. 2A). Remarkably, eEF1A, eEF2, and eEF3 were represented moreabundantly in the eIF3 complex than other 43S subunits (Fig. 3B, Supplementary Table 2). Inaddition, we found eight tRNA-synthetases enriched in the eIF3 complex (Fig. 3C,Supplementary Table 2). These enzymes may constitute the S. pombe homolog of themultisynthetase complex (MSC) of mammalian cells (Dang, 1986). Moreover, we identifiedtwenty four 40S ribosomal subunits and forty four 60S ribosomal proteins (Fig.2A,Supplementary Table 2). Consistent with association of eIF3 being physiologicallyrelevant, a previously published high-copy suppressor screen resulted in the isolation of manyribosomal proteins as suppressors of the growth defect of eif3eΔ cells (Supplementary Fig. 2and (Sha et al., 2007)).

The finding that eIF3 forms stable associations with proteins involved in translation elongationis seemingly at odds with its role in initiation that was established in biochemical reconstitutionexperiments. Most notably, stable binding of eIF3 to 40S ribosomes was shown to inhibit thejoining of the 60S subunit (Kolupaeva et al., 2005). Only upon the action of the eIF5B GTPaseon the 48S complex, can 60S join the 40S subunit thereby releasing eIF3 (Unbehaun et al.,2004). Both of these observations are seemingly inconsistent with the interaction of eIF3 and60S ribosomal proteins revealed here. The eIF3 interactome indicates that the multiplebiochemical functions of eIF3 may be coordinated in a complex manner in vivo. For example,it is possible that eIF3 is only temporarily released upon 40S and 60S subunit joining followedby rebinding to the 80S complex, potentially promoted by protein post-translationalmodifications. Although our data do not prove that eIF3 associates with actively translating80S ribosomes, a recent report provided experimental evidence for this phenomenon in budding

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yeast (Szamecz et al., 2008). eIF3 may subsequently orchestrate the recruitment of factors suchas the MSC and eEFs, which are required for efficient elongation.

The eIF3 interactome also contained 10 out of 17 subunits of the 19S proteasome regulatoryparticle (Fig. 2A, 3D, Supplementary Table 2). The remaining 7 subunits were also identifiedin some of the purifications, but did not pass abundance-based filtering in all of the six datasets(Fig. 3D). We also identified 6 subunits of the 20S proteasome, albeit at low levels(Supplementary Data File 1). This may be due to the fact that our purifications were performedin the absence of ATP, a condition that is likely to cause the 20S subunits to dissociate fromthe 19S subunits. These data are consistent with the previous demonstration that S. pombeeIF3e interacts with a subunit of the 19S lid (Yen et al., 2003b). Likewise, eIF3-proteasomeinteractions were reported in human cells and in plants (Dunand-Sauthier et al., 2002;HoareauAlves et al., 2002;Paz-Aviram et al., 2008). Molecular chaperones of the HSP70/40 family andthe CCT complex, which are known to mediate co-translational protein folding (Albanese etal., 2006;Fedorov and Baldwin, 1997), were also enriched in the eIF3 complex (Fig. 2A,Supplementary Table 2). Lastly, we identified the E2 ubiquitin-conjugating enzyme Ubc4p.

Proteasome recruitment to elongating ribosomes may mediate co-translational degradation ofproteins that cannot be properly folded by chaperones. In order to prevent proteotoxicity, thistight spatial linkage may facilitate the rapid removal of the 30 – 50% of newly synthesizedproteins that are co-translationally degraded by the proteasome (Schubert et al., 2000; Turnerand Varshavsky, 2000). Ubc4p was previously shown to be involved in the ubiquitylation andproteasomal targeting of misfolded nascent proteins (Chuang and Madura, 2005; Seufert andJentsch, 1990).

The protein interactions revealed here suggest that eIF3 organizes a series of protein complexesthat coordinately perform diverse steps in mRNA translation. These interactions may establishcytoplasmic “translation factories” akin to the nuclear transcription factories that coordinatethe synthesis and downstream processing of pre-mRNA (Calvo and Manley; Jackson, 2005;Pandit et al., 2008). Likewise, eIF3 appears to coordinate translation initiation, elongation, andquality control through forming an RNA-independent supercomplex with eIFs, MSC, eEFs,40S and 60S ribosomes, and the proteasome, which we named the “Translasome”.

Notably, we have obtained independent evidence for the translasome upon biochemicalpurification of the budding yeast proteasome by column chromatography and native gelelectrophoresis. Analysis of the 26S proteasome holoenzyme (Glickman et al., 1998) by LC-MS/MS revealed co-purification of the five eIF3 core subunits, twenty three 40S and thirtyeight 60S ribosomal proteins, eEF1, and glutamyl-tRNA synthetase (Supplementary Table 3).Similar interactomes were recently encountered upon cross-linking of proteasome interactingproteins of budding yeast (Guerrero et al., 2008) and during purification of the human 26Sproteasome (A. Kisselev, personal communication). These data strongly suggest that the coretranslasome is conserved across three eukaryotic species and is not an artefact of affinitypurification of eIF3.

eIF3 interaction with ribosome biogenesis factorsThe eIF3 interactome also contained 22 proteins involved in ribosome biogenesis, many ofwhich are components of the small subunit (SSU) processosome (Dragon et al., 2002) (Fig.2A, Supplementary Table 2). These included U3 snoRNP subunits as well as several subunitsof the U3 protein complex required for 35S pre-rRNA transcription (tUTP,) and the UTP-Band UTP-C complexes (Henras et al., 2008) (Fig. 3E). In addition, two proteins involved inmodification of pre-rRNA were identified; Fib1p, which mediates 2'- hydroxyl methylation ofribose, and Gar1p, which catalyzes pseudouridylation (Fig. 3E). Furthermore, 7 helicasesinvolved in the maturation of pre-40S and pre-60S ribosomes as well as Rrp12p and

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SPBC16H5.08c, two factors mediating their nuclear export were identified (Zemp and Kutay,2007) (Fig. 3E).

These findings suggest that eIF3 is also involved in various steps of ribosome biogenesis.Notably, ribosome biogenesis factors were previously found in eIF3 preparations of buddingyeast obtained in systematic protein interaction studies (Gavin et al., 2006; Krogan et al.,2006). These included U3 snoRNP and UTP-C components as well as the nuclear export factorRli1p. In addition, budding yeast eIF3j/Hcr1p has dual roles in translation initiation and 20Spre-rRNA processing (Valasek et al., 2001).

Additional InteractorsMetabolic Enzymes—Beside proteins involved in protein synthesis, the eIF3 complex alsoconsistently co-purified 42 metabolic enzymes belonging to the KEGG pathways centralcarbon, carbohydrate, amino acids, fatty acids and lipids, nucleotides, and vitamins andcofactors (Fig. 2A, Supplementary Table 2). This diverse set of functions and the fact that 81%of these proteins are among the 10% of the most abundant proteins in S. pombe ((Schmidt etal., 2007) and data not shown) implies that they may have co-purified as nascent polypeptides.On the other hand, two of the most abundant interactors, the redundant glyceraldehyde 3-phosphate dehydrogenases (GAPDH), Gpd3p and Tdh1p, are multifunctional enzymesassociated with a wide variety of glycolysis-independent functions, including membranefusion, phosphotransferase activity, nitric oxide sensing, and nuclear RNA export among others(Sirover, 2005). It is thus possible that GAPDHs are genuine translasome components.

Actin cytoskeleton—The eIF3 purifications also contained substantial amounts of actin andthe actin regulators Hob3p and Cpc2p/Rack1 (Fig. 2A, Supplementary Table 2). The latter wasalso isolated as an eIF3d/Moe1p binding protein in a previously reported yeast two-hybridscreen (Chen et al. 2000). Interactions of the protein synthesis machinery with cytoskeletalcomponents have been recognized for decades (reviewed in (Hovland et al., 1996)).Ribosomes, eIF2, eIF4A and B, and eIF3 form cytochalasin D sensitive physical interactionswith the cytoskeleton in HeLa cells (Howe and Hershey, 1984). Likewise, eEF2 and eEF1Aare high affinity F-actin binding proteins (Bektas et al., 1994;Yang et al., 1990). The integrityof the filamentous actin network is critical to normal protein synthesis in mammalian cells(Stapulionis et al., 1997), presumably because it locally organizes components of thetranslation machinery (Liu et al., 1996). For example, human eIF3a interacts with actin duringlocalization to the ER membrane (Pincheira et al., 2001). Interactions with actin may thereforebe involved in localizing the translasome to particular cellular compartments.

Trafficking and transport — physiologically relevant interaction of eIF3 with importins-βKap123p and Sal3p

eIF3 co-purified with several proteins involved in intracellular vesicle trafficking (Fig. 2A,Supplementary Data File 1) and cellular transport mechanisms, most notably membranetransport, mitochondrial transport, and nuclear transport (Fig. 4A). Importin-β molecules arecritical for nuclear transport because they can, either independently or via importins-α, bindthe cargo as well as the nuclear pore complex. There are 13 known importins-β in the S. pombegenome (http://www.sanger.ac.uk/Projects/S_pombe/; (Chook and Blobel, 2001;Chua et al.,2002)); however, only two of them, Kap123p and Sal3p, which are orthologs of budding yeastKap123p and Kap121p/Pse1p, are components of the eIF3 interactome. We assessed thephysiological relevance of the eIF3–importin-β interactions in a series of genetic experimentsin eif3eΔ cells, which, unlike deletion mutants of most other eif3 genes, are viable. We firstgenerated eif3eΔ/+ kap123Δ/+ and eif3eΔ/+ sal3Δ/+ diploid strains and induced them tosporulate. Tetrad analysis showed that kap123Δ eif3eΔ haploids divided only a few times aftergermination, indicating synthetic lethality (Fig. 4B). Although the sal3Δ eif3eΔ double mutant

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http://www.sanger.ac.uk/Projects/S_pombe/

was viable, it grew more slowly than the individual single mutants, a growth defect that couldbe readily detected at elevated temperature (Fig. 4C). We selected three other non-essentialimportins-β that do not bind eIF3e (Kap111p, Kap114p, and Kap104p, (Chen et al., 2004)) butfound no genetic interactions with eif3e (data not shown). Since eIF3 associates with bothKap123p and Sal3p, we tested for genetic interaction between these two genes. Indeed, sal3Δkap123Δ double mutants grew very slowly (Fig. 4D). These data indicate that the associationof eIF3 with Kap123p and Sal3p is critical for normal cell growth.

eIF3e cooperates with Sal3p in proteasome nuclear localization and functionProteasomes concentrate in the nucleus and at the nuclear membrane (Wilkinson et al.,1998). We have previously shown that eIF3e is required for proper proteasome accumulationin the nucleus (Yen et al., 2003b). As a consequence, eif3e mutants, like proteasome mutants,are hypersensitive to canavanine, an arginine analog, whose incorporation into nascent proteinstriggers their removal through proteasomal degradation. To assess the significance of thephysical and genetic interactions between eIF3e and Sal3p, we determined their possiblecooperation in regulating proteasome function. As shown in Fig 5A, cells deleted for sal3, butnot those deleted for kap111, which encodes an importin-β that did not co-purify with eIF3,were highly sensitive to canavanine (8 mg/L). A lower concentration of canavanine (4 mg/L)that only mildly affected the sal3Δ single mutant, still severely impaired the growth of thesal3Δ eif3eΔ double mutant. Taken together, these findings suggested that eIF3e cooperateswith Sal3p in proper proteasome function, possibly by regulating its nuclear localization.

To test this possibility, the 19S lid subunit Rpn7p was tagged with GFP by homologousrecombination resulting in a fusion protein, which was previously shown to be fully functionaland integrated into the 26S proteasome (Sha et al., 2007). Rpn7p-GFP expressed from theendogenous promoter is thus suitable for monitoring the subcellular localization of the entireproteasome. In wildtype cells optically scanned across the mid-section by confocal microscopy,the Rpn7p-GFP signal was most highly concentrated at the nuclear membrane (Fig 5B). Rpn7p-GFP was also readily detectable in the nucleoplasm, but excluded from an area that is presumedto be the nucleolus. The phenotypes of sal3Δ eif3eΔ cells appear to be pleiotropic and can bedevided into two groups. Approximately 70% of sal3Δ eif3eΔ cells resembled eif3eΔ singlemutants, in which Rpn7p-GFP was nuclear, but not concentrated at the nuclear membrane (Fig.5B; (Yen et al., 2003b)). The nuclei of these cells were deformed, a phenotype that can alsobe observed in eif3eΔ cells when maintained at low temperature. The remaining ~30% ofsal3Δ eif3eΔ cells had normal nuclear morphology, but contained very little Rpn7p-GFP inthe nucleoplasm (Fig. 5B). Cells with this deficiency were undetectable either in wildtype orthe single mutants (Fig. 5B and data not shown). In summary, a significant proportion ofsal3Δ eif3eΔ cells appear severely deficient in localizing proteasomes to the nucleus.

To directly assess the efficiency of proteasome nuclear accumulation, we photobleached theentire nucleus and then measured the reappearance of nuclear Rpn7p-GFP over time byquantifying the relative abundance of the GFP signal in the nucleus versus the cytoplasm. WhileRpn7p-GFP nuclear accumulation was only slightly inhibited in the sal3Δ single mutant, itwas substantially reduced in sal3Δ eif3eΔ cells belonging to the fraction of 30% that had verylow nuclear Rpn7p-GFP (Fig. 5C). Inefficient accumulation in the nucleus would be expectedto impair nuclear functions of the proteasome such as its role in DNA double strand breakrepair (Krogan et al., 2004;Tatebe and Yanagida, 2000). Indeed, sal3Δ eif3eΔ cells were farmore sensitive to phleomycin, a chemical that induces DNA double-strand breaks, than eitherof the single mutants (Fig. 5D).

These results demonstrate that proper nuclear localization of the proteasome (Wilkinson et al.,1998) requires eIF3e in addition to Sal3p. The Sal3p-eIF3e cooperation may reflect the twostep mechanism of proteasome nuclear accumulation, which requires (i.) passage through the

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nuclear pore, and (ii.) anchorage to the nuclear membrane by Cut8p (Takeda and Yanagida,2005; Yen et al., 2003a). Sal3p appears to mainly regulate passage through the nuclearmembrane, since the kinetics of Rpn7p-GFP accumulation in the nucleus was slower insal3Δ cells than in wild type cells (Fig. 5C). eIF3e may primarily control nuclear retention, aswe have previously shown that cut8Δ is synthetically growth deficient with eif3eΔ (Yen et al.,2003b).

As with sal3Δ cells, kap123Δ cells were hypersensitive to both canavanine (Fig 5A) andphleomycin (Fig. 5E), suggesting that Kap123p may play a role in proteasome nuclear importthat is partially redundant with Sal3p. The lethality of kap123Δ eif3eΔ cells suggests that eIF3may interact with Kap123p to mediate nuclear trafficking of additional cargos, such asribosomes and ribosome biogenesis factors, whose nuclear functions may be essential for cellviability.

PerspectiveOur eIF3 interactome data suggest that protein complexes that are necessary for proteinsynthesis and degradation can form a supercomplex, which we named the translasome (Fig.6). While surfaces of the individual protein complexes may mediate their interactions, the datafrom this and other studies suggest that the actin cytoskeleton may provide additional physicalsupport. The translasome is proposed to spatially coordinate distinct steps of protein synthesisthus contributing to the efficiency of mRNA translation. The integration of proteasomes intranslasomes could ensure translational fidelity by enabling the timely removal of abnormalnascent proteins.

Although the bulk of eIF3 and other translasome components are cytoplasmic at steady state,our data suggest that translasomes are dynamically localized within the cell because theyassociate with nuclear ribosome biogenesis proteins and with importins-β. In support of thepossibility that eIF3 shuttles between the cytoplasm and the nucleus is the observation thateIF3e concentrates in the nuclei of mammalian cells in a cell cycle-dependent manner (Watkinsand Norbury, 2004). The nuclear trafficking of eIF3e is in part mediated by a conserved leucine-rich NES at its N-terminus (Guo and Sen, 2000). Likewise, point mutations in the PCI domainlead to nuclear accumulation of eIF3e in HeLa cells (Sha et al., 2007). In S. pombe, eIF3e isreadily detected in the nucleus, when eif3d/moe1 is deleted; conversely, eIF3d becomesnuclear, when eif3e is missing (Yen and Chang, 2000). Finally, budding yeast eIF3a, anessential eIF3 core component, was identified as an import cargo of Sal3p/Kap121p andKap123p (Leslie et al., 2004).

These considerations raise the intriguing question of what role nuclear transit of eIF3 may play.Our findings suggest that one of these functions is to promote the proper nuclear accumulationof the proteasome. Whereas Sal3p and Kap123p mediate its transport, nuclear eIF3 appears tobe involved in tethering the proteasome to the nuclear membrane through Cut8p (Yen et al.,2003b). Several lines of evidence suggest that eIF3 then disassociates from the proteasomebefore it can move on to its second nuclear function, which our interaction data suggest is inribosome biogenesis. Firstly, eIF3 subunits do not assume the same nuclear rim association asthe proteasome, arguing against the maintenance of stable eIF3-proteasome interactions in thenucleus. Secondly, the proteasome is apparently excluded from nucleoli (Fig. 5B) into whicheIF3 would have to move in order to assist in ribosome biogenesis. We surmise that eIF3 mayserve as an assembly platform for 90S pre-ribosomal particles in the nucleolus and facilitatenucleoplasmic pre-40S and pre-60S ribosome maturation. A mild 40S biogenesis defect wasrecently described in a budding yeast eIF3a partial loss-of-function mutant (Szamecz et al.,2008). Since eIF3 is directly bound to mature 40S ribosomes in the cytoplasm, it may be co-exported from the nucleus together with pre-40S particles, thus resulting in a short nuclear

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transit time. Consistent with this possibility is the presence of ribosome export factors in thepurified eIF3 complexes.

Although we have no direct evidence for the proposition that eIF3 and the proteasometranslocate into the nucleus as part of the entire translasome, we note that both Sal3p andKap123p were previously shown to regulate the nuclear import of many ribosomal proteins(Leslie et al., 2004; Rout et al., 1997). The nuclear pore allows passage of molecules up to ~10MDa in size (Gorlich and Kutay, 1999), and would therefore be able to accommodate thetranslasome with its estimated size of 7–8 MDa. If nuclear translocation of a fraction of intacttranslasomes occurred, this could explain the low levels of translation factors that have beenobserved in nuclei and at sites of transcription (Brogna et al., 2002; Iborra et al., 2004a; Lundand Dahlberg, 1998). It might even enable a modest level of nuclear translation and nonsense-mediated mRNA decay (Buhler et al., 2002; Iborra et al., 2001), although the widespreadoccurrence of these processes in nuclei remains a matter of debate (Dahlberg and Lund,2004; Dahlberg et al., 2003; Iborra et al., 2004b), and references therein).

Experimental ProceduresYeast strains and methods

Cells were grown in either yeast extract medium (YEAU) or minimal medium (MM) withappropriate supplements (Chen et al., 1999). All experiments were with cells pre-cultured toearly logarithmic phase (2–5 × 106 cells/ml). For growth experiments on plates, cells wereserially diluted 1:5. To test sensitivity to DNA damage, phleomycin stock solution (5 mg/ml,Sigma) was prepared in DMSO, and controls included DMSO lacking phleomycin.

All importins-β mutants used in this study were kindly provided by D. Balasundaram (Chenet al., 2004), except as described below. To measure Rpn7p localization and import in sal3Δor sal3Δ eif3eΔ background, strains SAL3U (sal3Δ) and Y6AR7GFP (yin6/eif3eΔ rpn7-gfp,(Sha et al., 2007)) were crossed to generate sal3Δ/+ eif3eΔ/+ rpn7-gfp diploid cells. Thesecells were then induced to sporulate, and tetrad dissection was performed to isolate rpn7-gfp,sal3Δ rpn7-gfp, eif3e/yin6Δ rpn7-gfp, and sal3Δ eif3eΔ rpn7-gfp cells, and these strains werenamed N7G, SAL3UN7G, Y6AN7G, and S3UY6AN7G. Strain KAP123C was generated bya PCR-based gene deletion method using ClonNat as the selectable marker (Gregan et al.,2006).

Affinity purification of eIF3 complexeseif3e-proA and eif3m-proA cells (strains C648 and C617/1), and their WT parental cells (strainDS448/1) were described previously (Zhou et al. 2005). The affinity purification procedureswere as described with minor modifications (Zhou et al. 2005). Briefly, cells were collectedfrom 2 L cultures at OD595 = 0.6 and disrupted with glass beads in 5 ml lysis buffer (50 mMTris-HCl pH 7.4, 140 mM NaCl, 0.5% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml pepstatin,and 1mM phenylmethylsulfonylfluoride). Crude lysates were centrifuged twice at 14,000 rpmfor 20 min at 4°C, and 100 mg cleared protein in a volume of 5 ml was mixed with 300 µlDyna beads (Dynal Biotech) coupled to rabbit IgG (Jackson Immunochemicals). Mockpurifications were performed in parallel with lysate from untagged cells. Beads were collectedand washed 4 times in 5 ml lysis buffer. Beads were cleaved overnight with 300 U TEV protease(Invitrogen). Protein aliquots were separated by SDS-PAGE, and visualized by CoomassieBlue staining or by Western blotting with anti-Rpn1 antibody (1:1,000, (Yen and Chang,2000)). To quantify protein levels in Western blots, fluorescently conjugated secondaryantibodies were used, and the signals were measured by the Odyssey infrared imaging system(Li-COR Biosciences).

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LC-MS/MSProteins from TEV eluates were reduced, alkylated, and digested with trypsin using standardprocedures (see Supplementary Methods for details). Peptides were desalted with a C18cartridge (Waters). Peptides were dried, re-suspended in 0.1% trifluoroacetic acid/2.0%acetonitrile, and stored at 4 °C until LC-MS/MS analysis. The analyses used a Paradigm HPLC/autosampler (Michrom Bioresources, Inc.) and an LTQ OrbitrapXL mass spectrometer(Thermo Fisher Scientific). A C18 analytical column and an ADVANCE source (Michrom)were used. The RP HPLC gradient (solvent A = 0.1% formic acid; solvent B = 100%acetonitrile) consisted of 2% B to 5% B from 0 to 2.0 min and 5% B to 35 % B from 2.1 to120.0 min. The MS/MS method was top-4, data-dependent; precursors were scanned in theOrbitrap and MS/MS scans were in the ion trap. Dynamic exclusion was enabled. Data wassearched against an S. pombe protein database using Sorcerer™-SEQUEST® (SageNResearch). Static alkylation of Cys, and differential Met oxidation, Lys ubiquitination, and Ser,Thr and Tyr phosphorylation were specified. QTools, which are in-house developed visualbasic macros for automated spectral count analysis, were used to compute spectral counts ofthe proteins (Liu et al., 2004).

LC-MS/MS analyses of the budding yeast 26S proteasome were performed in a similar manner,and are described in Supplementary Methods.

Confocal microscopyRpn7p-GFP localization was visualized by confocal microscopy as described in theSupplementary Methods section.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank D. Balasundaram (Singapore University) for providing materials, M. Petroski for reviewing the manuscript,and K. Motamedchaboki and A. Iranli for bioinformatics support. We also thank Mike Mancini and his staff at theIntegrated Microscopy Core at the Dan Duncan Cancer Center for technical assistance. This work was funded by NSFgrant 0920229 and NIH grant GM059780 to D.A.W. and by NIH grants CA90464 and CA107187 to E.C.C; ZS wassupported by a pre-doctoral fellowship from the DOD (BC030443). LMB is funded through the NIH Center Grants5 P30 CA30199-28 and 5 P30 NS057096. We also thank Glen and Judy Smith for their generous support.

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Fig. 1. Purification of eIF3 binding proteins from fission yeast(A) Flow chart showing the procedure for affinity protein purification of eIF3 complexes fromeIF3e-ProA, eIF3m-ProA, or untagged parental cells (strains C648, C617/1, and DS448/1,respectively).(B) Protein samples collected at various steps throughout the purification as indicated in (A)were separated by SDS PAGE and stained with Coomassie Blue.(C) The same samples as in (B) were analyzed by immunoblot using an antibody against Rpn1p.Asterisks mark eIF3e-ProA and eIF3m-ProA, whose protein A (ProA) tags were recognizedby the secondary antibody. The positions of the IgG heavy chain and the cleaved ProA tag aremarked by arrows.

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Fig. 2. Summary of the eIF3 interactome and distinct eIF3 complexes(A) Intensity map of the spectrum counts of the 230 proteins that were identified, using LC-MS/MS, as specific and reproducible interactors with the eIF3e-ProA and eIF3m-ProA baitproteins. The identified proteins were grouped manually into protein complexes and pathways.Individual proteins are listed in Supplementary Table 2 and Supplementary Data File 1. Datafrom three independent experiments are shown (indicated as #1, #2, #3). In experiment #3, thecell lysate was treated with RNAse prior to affinity capture. The overall signal intensity appearshigher in experiments #2 and #3 because the data represent the spectrum count sums of 4consecutive mass spectrometry runs, whereas samples of experiment #1 were run only 3 times.(B, C) Relative abundance of all 11 S. pombe eIF3 subunits. The left panel (B) shows anintensity map based on raw spectrum counts. The right panel (C) shows the same data afteradjustment to the molecular weight of the subunits and normalization to eIF3a. Onlyexperiments #2 and #3 are shown because their spectral count intensities are directlycomparable (see (A)). (D) Models of fission yeast eIF3 complexes with proposed functions in

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global and mRNA-specific translation. The topology of the complexes is based on results fromtop-down mass spectrometry and was adapted from Zhou et al. (Zhou et al., 2008).

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Fig. 3. Protein components of the translasomeSpectrum count intensity maps of (A) eIFs, (B) eEFs, (C) tRNA synthetases, (D) 19Sproteasome, (E) ribosome biogenesis proteins from purifications 2 and 3 (#2 and #3,respectively).

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Fig. 4. Genetic interactions between kap123Δ and sal3Δ with eif3eΔ(A) Spectrum count intensity map of eIF3 interacting proteins involved in cellular transport.(B) Strain YIN6A (yin6/eif3eΔ) was crossed with strain KAP123U (kap123Δ) (Chen et al2004) and the diploid cells were induced to sporulate to form tetrads. A tetra type tetrad wasdissected to show spore viability of the indicated genotypes (left). A microcolony derived froma kap123Δ eif3eΔ spore was scraped off the plate and visualized by DIC microscopy (right).(C) Cells with the indicated genotypes were obtained from a tetra type tetrad produced byeif3eΔ/+sal3Δ/+ diploid cells, which were obtained by crossing strains YIN6A and SAL3U(Chen et al 2004). These cells were spotted onto YEAU plates and grown at the indicatedtemperatures.(D) Cells with the indicated genotypes were obtained from a tetra type tetrad produced bysal3Δ/+kap123Δ/+ diploid cells, which were generated by crossing strains SAL3U andKAP123C. The cells were spotted onto YEAU plates and grown at 30°C.

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Fig. 5. Efficient nuclear accumulation of the proteasome requires eIF3e and Sal3p(A) The indicated strains were serially diluted and spotted on minimal media plates with orwithout canavanine and grown at 32°C.(B) Cells carrying Rpn7p-GFP were optically sectioned through the middle of the cell bodyby confocal microscopy. Representative confocal images are shown. While Rpn7p-GFP canbe readily seen in the nucleoplasm (except for an area that is presumed to be the nucleolus) ofwild type cells as well as the single mutant cells, there was very little Rpn7p-GFP signal insidethe nucleus of ~30% of sal3Δ eif3eΔ cells (example marked by an arrow).(C) Individual nuclei of the indicated cells were photobleached with a laser (marked by dottedgreen circles), and re-accumulation of nuclear Rpn7p-GFP was recorded in 1 minute intervalsover 60 minutes. To quantify nuclear accumulation, the nucleus of an unbleached cell(arrowhead) was measured to correct for spontaneous photobleaching. The relative intensities(RI) corrected for spontaneous photobleaching at each time point after photobleaching weremeasured, and the ratio of nuclear vs. cytoplasmic RI (RIN/C) were plotted over time. The datain the graph represent values averaged from measuring 10 cells with error bars (SEM).Unpaired student t tests confirmed that the difference between wildtype and sal3 Δ eif3eΔ cellswas highly significant at 38/60 time points with p ≤ 0.01, and at 52/60 time points with p ≤0.05 (Supplementary Table 4). Scale bar = 5 µm.(D) Cells were obtained as described in Fig 4C, spotted onto YEAU plates with or withoutphleomycin, and grown at 32°C.

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(E) Wildtype, sal3Δ, and kap123Δ cells (strains MBY1270, SAL3U, and KAP123U,respectively, Chen et al 2004) were spotted onto YEAU plates with or without phleomycin,and grown at 30°C.

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Fig. 6. Model of the eIF3-associated translasomeMSC = multisynthetase complex; light green circles represent eEFs1, 2, and 3; initiation factorsare in dark blue, eIF3 is in light blue. An actin filament is depicted in purple, and a nascentpolypeptide chain as black balls.

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