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tagged versions of each of these factors, before or after an 18h shift to glucosecontaining medium, to sucrose gradient fractionation. Then fractions were analyzed by Western blot using antiGFP antibodies to detect GFPtagged Nmd3 or Rlp24, respectively. As shown in supplemental Fig. S7, we could not detect significant changes in the pattern of sedimentation of Nmd3GFP or Rlp24GFP upon L40 depletion, which were still found in the 60 S region of the gradients. This result indicates that these proteins remain bound to high molecular mass complexes, most likely aberrant cytoplasmic pre60 S rparticles, upon L40 depletion. Taken together, our data indicate that Nmd3 and Rlp24 require L40 to be efficiently displaced fromcytoplasmic pre60 S rparticles to recycle back to the nucleolus. Expression of HAL40A Leads to Sordarin Resistance, whereas rpl40a and rpl40b Cells Show Hypersensitivity to This CompoundBoth the GTPaseassociated center and the sarcinricin loop have been proposed to! interact directly with the G domain of eEFs see Refs. 36, 127 and references therein. Moreover, the rstalk Pproteins and L12, which is in close proximity of L40 Fig. 9A, has also been previously identified to interact with eEF2 36. Both eEF2 and rstalk proteins have been described to be targets of members of the sordarin family of antifungal compounds 69, 70, 128, 129. To evaluate38400 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME Downloaded from httpwww.jbc.org by guest on November 11, 2013 287 NUMBER 45 NOVEMBER 2, 2012Role of L40 in Yeast Ribosome Synthesis and FunctionFIGURE 10. Nmd3 and Rlp24 are not properly recycled back to the nucleus upon depletion of L40. The GALRPL40 strain, JDY925 pAS24RPL40A, was transformed with CEN URA3 plasmids that allow expression of GFPtagged versions of either Arx1, Mex67, Mrt4, Nmd3, Rlp24, or Tif6. Cells were grown at 30 C in SGalLeuUra Gal and shifted for 18 h to SDLeuUra 18 h Glc. Then the subcellular localization of these GFPtagged prot! eins was examined by fluorescence microscopy. Arrows point to the nucleolus. Approximately 200 cells were examined for each reporter. About 85 cells expressing GFPtagged Nmd3 or Rlp24 showed full cytoplasmic fluorescence upon L40 depletion. Practically all cells gave the results shown in the pictures for remaining reporters.whether L40 could mediate inhibition by or resistance to sordarin, the single rpl40a and rpl40b strains or the rpl40 null mutant expressing as the sole source of L40 a HAtagged version of L40A were screened for their ability to grow in the presence of different concentrations of the sordarin derivative GM193663. As a control, we used both an isogenic wildtype and rpl35b strain. As shown in Fig. 11, we found that the strain expressing the HAtagged L40A rprotein is highly resistant to sordarin resulting in a more than 30fold increase in the 50 inhibitory concentration IC50 value compared with its isogenic wildtype counterpart. In contrast, the single rpl40! a and specially the rpl40b mutant were clearly much more sensitive to t! his drug than the wildtype strain the IC50 value obtained for the rpl40a and rpl40b mutants was at least 10fold lower than that of the wildtype strain. Interestingly, this sensitivity is not simply related to a lower dosage of an rprotein because the IC50 value of an rpl35b mutant is practically identical to that of the wildtype strain. Moreover, the effect of sordarin seems to be very specific because we could not have observed enhanced sensitivity or resistance to other translation antibiotics anisomycin, cycloheximide, hygromycin B, paromomycin, and neomycin for the above strains when compared with their isogenic wildtype counterpart. Finally, to test if the sordarin resistance of the strain expressing the HAL40A protein is the indirect result of the destabilization of rproteins such as L12, L1, or P0, whose lossoffunction mutations have been reported to induce sordarin resistance 68, 128, we prepared samplesFIGURE 11. Cells expressing the HAtagged L40A are resistant to ! sordarin. Growth inhibition assays were performed in liquid YPD medium with or without different amounts of the sordarin derivative GM193663 in a microtiter format. The relative growth after incubation for 18 h at 30 C in the presence versus the absence of the antibiotic is the mean of four independent experiments. The strains evaluated were as follows W3031A wildtype open circles, JDY925 YCplac33UBHARPL40A HARPL40A closed circles, JDY919 rpl40a open square, JDY922 rpl40b , open triangle, JDY711 rpl35b open rhombus.enriched in preribosomal particles and mature ribosomes containing either wildtype L40 or HAtagged L40A see Experimental Procedures, and we washed them with a high salt soluNOVEMBER 2, 2012 VOLUME 287 NUMBER 45 JOURNAL Downloaded from httpwww.jbc.org by guest on November 11, 2013 OF BIOLOGICAL CHEMISTRY38401Role of L40 in Yeast Ribosome Synthesis and Functiontion 0.5 M NH4Cl. As shown in supplemental Fig. S8, no differences were detected in the purification r! ate of a subset of rproteins, including L12, L1, and P0. Thus, these rp! roteins are stably associated with ribosomes both in wildtype and the HAL40Aexpressing strain. Taken together, these results suggest that L40 specifically and functionally interacts with eEF2 during translation elongation. retained in the nucleolus upon depletion of L40 Fig. 5. Therefore, we must assume that, although prerRNA processing reactions are apparently taking place in a correct way, pre60 S particles from L40depleted cells are not fully competent for nucleocytoplasmic export. We have shown that an HAtagged L40 is predominantly assembled in the cytoplasm see below thus, we are tempted to speculate that wildtype nuclear pre60 S rparticles are devoid of L40, and most likely retention of these particles upon L40 depletion, as well as the abovementioned 23 S prerRNA accumulation, is occurring as a secondary effect due to inefficient recycling of a critical nucleocytoplasmic shuttling factor to the nucleus. A scenario similar to this one has already been demonstrated for! the nuclear shuttling factors Nmd3, Mrt4, and Rlp24, which are recycled to the nucleus upon cytoplasmic assembly of L10 56, P0 61, 104, 135, and L24 136, respectively for further discussion see Refs. 20, 103, 126. Interestingly, our data demonstrate that L40 is an additional rprotein involved in the release of Nmd3 and Rlp24 from cytoplasmic pre60 S rparticles Fig. 10 and supplemental Figs. S6 and S7. How does the depletion of L40 block the release of these factors One possibility is that L40 acts as an effector of the energyconsuming factors involved in the release of Nmd3 and Rlp24 from cytoplasmic pre60 S rparticles. So far, the GTPase Kre35 95 and the AAAATPase Drg1 137 are the factors described to be required for the release of Nmd3 and Rlp24, respectively. Drg1, which is apparently one of the earliest acting factors during the cytoplasmic maturation of 60 S rsubunits, is required for the release of other factors in addition to Rlp24, among them Arx1, Mrt4, Nog1, and ! Tif6 103, 126, 137 however, our results show that the recycling of Arx1! and Tif6 seems not to be affected by the depletion of L40 Fig. 10. Kre35 is apparently one of the latest acting factors in this process 95, 103, and the release of Nmd3 by Kre35 also requires the rprotein L10. Thus, another possibility that could explain the lack of proper release of these factors upon L40 depletion is that assembly of L40 is coordinated to that of L10 indeed, although the structural analyses suggest that Nmd3 and L10 can bind simultaneously to pre60 S rparticles 7, 10, 122, coimmunoprecipitation data are fully consistent with a model in which L10 could be preferentially loaded after Nmd3 release onto cytoplasmic pre60 S particles in wildtype conditions 56. Interestingly, analyses of the 60 S rsubunit structure suggest that Nmd3 and L40 must sequentially associate with pre60 S particles because their binding sites on 60 S rsubunits seem mutually exclusive 7, 10, 122. More puzzling is the role of L40 in the release of Rlp24 from cytoplasmic pre60 S rparticl! es. It has been hypothesized that Rlp24 and L24 successively occupy the same rRNAbinding site during ribosome synthesis 58. If this hypothesis is correct, the binding site of Rlp24 would be placed about 10 nm away from that of L40, and therefore, a wave of conformational changes is required to explain our observation. Surprisingly, whether L10 is required to release Rlp24 from pre60 S rparticles has so far not been addressed. Clearly, understanding the precise mechanism by which L40 participates in the recycling of Nmd3 and Rlp4 will require further work. We have also addressed the course of assembly of L40. Previous data suggested that yeast L40 might associate with preDISCUSSION In this work, we have addressed the role of yeast L40 rprotein in 60 S rsubunit biogenesis and function. L40 was not modeled in the crystal structure of the 50 S rsubunit from Haloarcula marismortui 11, and it has only been recently localized in the structure of eukaryotic 60 S rsubunits from S. c! erevisiae or Tetrahymena thermophila see Fig. 9 7, 10. Two strain syste! ms were used for the phenotypical analysis of L40 rprotein. First, we analyzed single rpl40a and rpl40b null mutants. Our results indicate that both genes contribute to growth and normal accumulation of 60 S rsubunits, with the role of RPL40A being apparently more important than that of RPL40B Fig. 1. Although a functional specificity between many paralogous rproteins has been described 101, 130, 131, we could not find any distinction between L40A and L40B at least for growth and polysome profile analyses under standard laboratory conditions supplemental Figs. S3 and S4. Second, we established a conditional rpl40 null strain by disrupting the RPL40B gene and placing the ORF of RPL40A under the control of a GAL promoter, which allows genetic depletion of the L40 rprotein by transcriptional repression in cells growing with glucose as carbon source. Polysome profile analyses strongly indicate that 60 S rsubunits lacking L40 are defective in subunit joining see below and sugges! t that both rsubunits may have an increased susceptibility to degradation at longer time points of L40 depletion Fig. 2 similar observations were made for the null rpl24 mutant and upon L1 depletion 47, 132, 133. Consistent with the polysome profile results, pulsechase labeling, Northern hybridization, and primer extension analyses in the L40depleted strain indicate that L40 is practically dispensable for prerRNA processing. Thus, we could only observe a modest delay in 27 SB and 7 S prerRNA processing upon depletion of L40 Fig. 3. Consequently, the relative amounts of these precursors slightly increase, although the steadystate levels of mature 25 S and 5.8 S rRNA only decrease at late time points of depletion Fig. 4. In addition, the depletion of L40 leads to a mild delay in early cleavage events of prerRNA processing at the A0, A1, and A2 sites that have subsequent consequences for the levels of 20 S prerRNA and mature 18 S Figs. 3 and 4 this delay has been extensively r! eported for many mutants in genes encoding 60 S rsubunit assembly facto! rs and 60 S rproteins and is likely due to inefficient recycling of transacting factors that cannot efficiently dissociate from aberrant pre60 S rparticles for further discussion see Refs. 23, 134. These data strongly suggest that L40 is not strictly required for the prerRNA processing reactions and that ribosomes lacking L40 are efficiently degraded. This is very unusual because all essential 60 S rproteins studied so far, except perhaps L1 47, 133 and L10 82, play critical roles during ribosome maturation. Strikingly, pre60 S particles are38402 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME Downloaded from httpwww.jbc.org by guest on November 11, 2013 287 NUMBER 45 NOVEMBER 2, 2012Role of L40 in Yeast Ribosome Synthesis and Function60 S particles at a relatively late stage during the course of the 60 S rsubunit assembly 49. Here, we present several lines of evidence that yeast L40 may assemble in the cytoplasm, in complete agreement with the above described role in the release ! of shuttling factors Figs. 6 8 and supplemental Fig. S2. However, we cannot fully exclude the possibility, as also suggested by the presence of predicted NLS sequences within L40, that some assembly could take place in the nucleus in wildtype conditions and that the HA tag could partially interfere with the import of the HAL40 protein. Besides L40, few other rproteins have been described to assemble exclusively L24 and L10 or predominantly P0 in the cytoplasm 56, 58, 61, 103, 104, 135, 136. Further experiments are required to discard the possibility that L40 may also assemble into pre60 S particles within the nucleus. Finally, we have addressed the function of L40 in translation. As stated by Wilson and Nierhaus 138, the assignment of a specific role, in terms of translational activity, to an individually given rprotein is extremely difficult. This is based on the highly cooperative nature of the interactions between rRNA and rproteins and between the rproteins themselves.! Indeed, there are many reports describing that even small changes in t! he primary sequence of an rprotein have significant long distance effects on the rRNA structure 30, 59, 139 141. Our data suggest that L40 is required for the joining of 60 S and 40 S rsubunits and for the translocation process during translation. Both functions are fully consistent with the position of L40 on the mature 60 S rsubunit 7, 10. The rsubunit joining role of L40 is inferred from the analysis of the polysome profiles of rpl40a and rpl40b mutants but especially of those of L40depleted cells. These polysome profiles showed a pronounced decrease in 80 S and polysomes and, more importantly, the accumulation halfmer polysomes without a net reduction in the levels of free 60 S rsubunits, as indicated by a ratio of free 60 S to 40 S rsubunits that is relatively similar to that of wildtype cells Fig. 2. Interestingly, the L10 and L11 rproteins, which are close neighbors of L40 Fig. 9A 7, 10, have been previously involved in rsubunit joining 82, 142. Moreover, a mutationa! l change in the L33 rprotein, identified due to its Gcd phenotype derepression of Gcn4 translation, has been suggested to impede the rsubunit joining reaction 143. Consistent with this finding, our preliminary data suggest that both the rpl40a strain and, to the lesser extent, the rpl40b mutant confer a Gcd phenotype that could be suppressed by overexpression of the translation initiation ternary complex compoMet nents eIF2 and tRNAi .4 Because of the fact that Nmd3 is locked on cytoplasmic pre60 S particles upon L40 depletion see above, further experiments are needed to determine whether L40 has a direct role in the rsubunit joining reaction because the location of Nmd3 within the pre60 S rparticles is apparently incompatible with the joining of 60 S and 40 S rsubunits 122. In agreement with this, it has been shown that Nmd3 does not bind to 80 S ribosomes or polysomes 144. In addition, we hypothesize that L40 participates in the translocation process during translation. T! his is deduced from the selective sordarin hypersensitivity of the rpl4! 0a and rpl40b mutants and the increased resistance to this drug of cells expressing HAtagged L40 as the sole source of L40 Fig. 11. Sordarin derivatives inhibit fungal protein synthesis by stabilizing the eEF2ribosome complex 69, 70, and many sordarinresistant mutations, which allow eEF2 to function in translocation even when bound to a sordarin molecule, are linked to the genes that encode eEF2 and the rstalk P0 and L12 proteins 68 70, 128, 129, 145. Thus, these observations support a role for the rstalk proteins during translation elongation. Our observations that L40 is another target of sordarin strongly suggest that eEF2 and L40 may also interact functionally during the conformational switch that occurs during translation elongation. Whether L40 makes physical contact with eEF2 or whether the HAL40 protein exerts sordarin resistance by affecting the functionality of rstalk proteins or cytoplasmic assembly factors, e.g. the eEF2related GTPase Efl1Ria1 124, 126, 146, rem! ains to be elucidated. In conclusion, we have shown that L40 assembles late into pre60 S subunits and may participate in rsubunit joining and eEF2 function during the translation process. Future indepth analyses will be required to understand the mechanistic details of the role of L40 during cytoplasmic 60 S rsubunit assembly as well as during the initiation and elongation steps of translation.AcknowledgmentsWe are indebted to the colleagues mentioned in the text for their gift of material used in this study. We also thank M. Carballo, L. Navarro, and C. Reyes of the Biology Service CITIUS from the University of Seville for help with the phosphorimager analysis, F. MonjeCasas for valuable advice on immunofluorescence experiments, and J. P. G. 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