Eukaryotic (eIF) 5A stimulates synthesis in Saccharomyces cerevisiae

Allen Hendersona and John W. Hersheyb,1

aDepartment of Microbiology and Immunology, University of California, 513 Parnassus Avenue, Room S-472, San Francisco, CA 94143; and bDepartment of Biochemistry and Molecular Medicine, University of California, 4408 Tupper Hall, Davis, CA 95616

Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved February 23, 2011 (received for review June 9, 2010)

Within the field of eukaryotic protein synthesis, one factor re- have normal polysome profiles (11), suggesting that eIF5A is not mained putative for decades: initiation required for general protein synthesis. factor (eIF) 5A. Because eIF5A is an essential protein required for We chose to reexamine the possibility that eIF5A stimulates cell proliferation, and one easily targeted by inhibitors, identifying translation for several reasons. First, the degree to which eIF5A its role in the cell remains important and urgent. Recent reports consistently stimulated met-puro formation did not substantially support early findings that eIF5A stimulates protein synthesis differ from what was observed when depleting UBR5A from and newly assign the factor a role in elongation rather than initia- yeast cells. Second, the primary physiological activity of eIF5A tion. Here we show that eIF5A directly stimulates protein synthesis had remained elusive. Finally, we discovered potential artifacts on native mRNAs, that rapid depletion of eIF5A in vivo immediately in the eIF5A depletion experiments that were a basis for doubting leads to a 2-fold inhibition of protein synthesis, and that both the that eIF5A plays a role in general translation initiation. By immediate and lasting effects of eIF5A depletion are a reduction optimizing the depletion experiments we find that loss of eIF5A in polysome size concomitant with eIF5A depletion. Addition of correlates with a substantial inhibition of protein synthesis in vivo, purified eIF5A to a depleted lysate results in a roughly 2-fold sti- UBR5A depletion results in a pronounced effect on polysome

mulation of protein synthesis in vitro, a result consistent with both distribution, and supplementing depleted lysates with eIF5A BIOCHEMISTRY – older methionyl puromycin synthesis data and more recently stimulates protein synthesis in vitro. published findings. We find that although eIF5A is not required for While this manuscript was being prepared, other laboratories protein synthesis, it stimulates the process by about 2- to 3-fold. reported quantitatively similar effects of eIF5A depletion on Our data, along with other published results, reinforce the conclu- global translation (12, 13). However, although these reports sion that eIF5A stimulates protein synthesis with one important conclude that eIF5A stimulates elongation, the polysome profiles difference: Polysome profiles observed immediately after eIF5A we observe during and after eIF5A depletion are diagnostic for a depletion are diagnostic for a role in initiation. This discrepancy role in initiation. Possible explanations for this discrepancy are is discussed. discussed, as is a recently published structure suggesting that EF-P stimulates only the formation of the first peptide bond (14). hypusine ∣ Results ukaryotic translation initiation factor (eIF) 5A is a small Control Experiments Suggest That eIF5A Acts in Translation. We first – E(16 18 kDa), essential protein that is ubiquitous and highly asked whether previously published data, obtained with strain conserved among eukaryotes and archaea (1, 2). Several crystal UBHY-R, may have been subject to an artifact. Some degrada- structures of archaeal aIF5A have been published within the last tion of UBR5A, the rapidly depleted protein expressed in strain – decade (2 4). The less-conserved C-terminal portion is classified UBHY-R, may have occurred during lysate preparation, inaccu- as an oligonucleotide binding motif (4). The N-terminal portion is rately representing in vivo UBR5A levels. We found this effect to more highly conserved and bears the absolutely unique feature of be negligible, although a striking media-dependent effect was a/eIF5A: a specific, posttranslationally modified lysine known as ϵ observed, indicating that UBR5A depletion in SD glucose is hypusine [N -(4-amino-2-hydroxybutyl)lysine] (5). This residue impaired (Fig. S1). Depletion of UBR5A was previously lies at the tip of an extended loop in the crystal structures, the demonstrated by blotting extracts of cells grown in rich media. sequence of which (STSKTG-hypusine-HGHAK) is universally If UBR5A depletion in minimal media is inefficient, then in vivo conserved in eukaryotes (6). Remarkably, a crystal structure of 35 assays of [ S]methionine incorporation into protein by UBHY-R the prokaryotic eIF5A homolog, called P grown in minimal media could have been misleading. We also (EF-P), shows that both structurally resemble tRNA (7). The essential function of eIF5A remains obscure. The factor surmised that, because the TIF51A coding region was not com- was originally isolated from ribosomal high-salt washes (8, 9), pletely deleted in UBHY-R, reversion to wild type at the TIF51A suggesting a role in translation. Purified eIF5A stimulates a locus by homologous recombination with the plasmid-borne model assay of translation initiation, methionyl–puromycin (met- UBR5A construct was possible, absent selection. Reversion does puro) synthesis (9, 10). Although eIF5A does not affect any step occur during growth in rich media (Fig. S2). Growth in selective prior to 80S initiation complex assembly (10), the factor increases media prior to all experiments prevents contamination with re- met-puro formation by 2- to 3-fold (9, 10). Whether eIF5A sti- mulates translation in vivo was tested by depleting eIF5A in yeast Author contributions: A.H. and J.W.H. designed research; A.H. performed research; A.H. (11). These depletion experiments used a “rapid-depletion” yeast contributed new reagents/analytic tools; A.H. and J.W.H. analyzed data; and A.H. and strain, UBHY-R, engineered to express an unstable form of J.W.H. wrote the paper. eIF5A (UBR5A). By rendering UBR5A expression dependent The authors declare no conflict of interest. on the GAL1 promoter in a null genetic background, eIF5A is This article is a PNAS Direct Submission. rapidly depleted by shifting UBHY-R cells into glucose-contain- 1To whom correspondence should be addressed. E-mail: [email protected]. ing media. UBHY-R cells grown in glucose exhibited only a mild This article contains supporting information online at www.pnas.org/lookup/suppl/ (30%) reduction in the rate of protein synthesis and appeared to doi:10.1073/pnas.1008150108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1008150108 PNAS ∣ April 19, 2011 ∣ vol. 108 ∣ no. 16 ∣ 6415–6419 Downloaded by guest on September 29, 2021 vertants as determined by periodic selection tests and prolonged The effect of growth medium on protein synthesis rates is also growth suppression in glucose-containing media. These results sug- readily seen when polysome profiles are determined. In the case gest that a role for eIF5A in translation may have been masked of YP galactose (Fig. 2C), heavy polysomes are much more pre- by UBHY-R-specific effects. Therefore we asked whether, as ex- dominant compared to those from cells grown in SD galactose pected for a translation factor (9), eIF5A associates with poly- (Fig. 2A), indicative of more efficient translation initiation in rich somes. As since reported by others (15), we found that it does media. Following depletion, polysomes are severely reduced in bind to (Fig. S3), although not strongly. Finally, we rea- YP-glucose medium (Fig. 2D), whereas the effect in SD-glucose soned that because eIF5A is not absolutely required for the medium (Fig. 2B) is more modest, although polysome distribu- formation of met-puro in vitro, a general reduction in translation tion changes similarly in both cases. These observations are activity (expected in cells grown in minimal media; see below) consistent with the protein synthesis rate data (Fig. 1; ref. 11). could mask a potential effect of UBR5A depletion on general The results obtained in YP media indicate that eIF5A stimulates translation. This possibility is addressed in the next section. the rate of protein synthesis by at least twofold. We conclude that the growth conditions used in earlier depletion studies masked The Effect of eIF5A Depletion on Translation Is More Pronounced in important effects of eIF5A depletion on protein synthesis. In Rich Media. The growth medium used to propagate UBHY-R in- this experiment, UBHY-R cells were incubated in glucose-con- fluences both the rate and extent of protein synthesis inhibition taining medium for 5–6 h in order to replicate conditions used 35 upon UBR5A depletion. By comparing the rate of [ S]methio- previously (11). nine incorporation into total protein by UBHY-R cells before and during depletion, we observe a media-dependent effect on eIF5A Directly Stimulates Protein Synthesis in Vitro. The demonstra- protein synthesis (Fig. 1 and Fig. S4). Extended depletion (for tions above that eIF5A depletion reduces global translation in approximately 5 h) inhibits translation initiation when either rich yeast involve polysome profile analysis and in vivo protein synth- [yeast extract peptone (YP)] or minimal [synthetic defined (SD)] esis assays. Both techniques show an effect of eIF5A depletion in media are employed, but the effect in rich media is more rapid intact cells, but the time required to reduce eIF5A levels means and larger. Protein synthesis is inhibited by 2-fold after 1 h of that we cannot rule out possible indirect effects of eIF5A deple- UBR5A depletion in rich media (Fig. 1 and Fig. S4C), whereas tion. The difference between the rates of UBR5A depletion a similar degree of inhibition is not observed until 2–3 h of incu- (Fig. S1) and the reduction of [35S]methionine incorporation bation in minimal media (Fig. S4 A and B). Extended depletion (Fig. 1) also suggests that secondary effects might contribute for approximately 5 h results in a 4-fold inhibition of protein to translational repression. We therefore sought a more direct synthesis in rich media; in minimal media a lower inhibitory effect measure of eIF5A stimulation of protein synthesis and asked (between 2.5- and 3-fold) is observed after similar depletion whether the addition of purified eIF5A to depleted lysates would times. In both minimal and rich media we observe a greater in- stimulate protein synthesis in vitro. hibition of protein synthesis than was initially reported in minimal Toward this end, we prepared translationally active extracts media (about 1.4-fold; see ref. 11); based on our present findings from UBHY-R cells incubated in YP glucose for 2.5 h. The we conclude that this difference arises from media effects and the UBR5A-depleted extracts were then supplemented with purified artifacts described in the previous section. The effects of eIF5A eIF5A(lys or dhp) or eIF5A(hyp) purified from yeast. Protein depletion are seen as early as 1 h of depletion, when protein synthesis on endogenous messages was measured by following synthesis rates already have fallen by 2-fold in YP media. These the incorporation of [35S]methionine into acid-insoluble protein. differences are likely due to more efficient translation initiation It is important to recognize that such stimulation by eIF5A by cells in YP media, although this is difficult to demonstrate by depends on having a sufficient level of methionine in the assay, this method without quantifying intracellular methionine pools. and that the concentration of total methionine dramatically af- fects activity (Fig. S5). The use of recombinant eIF5A expressed in and prepared from bacteria, rather than eIF5A purified from yeast, eliminates possible contaminating yeast proteins that might affect protein synthesis. We also tested whether yeast-derived FLAG-tagged eIF5A(hyp) would differ in activity from the bac- terially derived eIF5A preparations. Although eIF5A(hyp) shows greater stimulatory activity than eIF5A(dhp) during met-puro synthesis, both forms are active and specific; eIF5A(lys) or a homodeoxyhypusine-modified analog of eIF5A shows no stimu- latory activity (16). We demonstrated that protein synthesis in depleted lysates depends on new initiation events by using a cap analog (m7GTP) to sequester eIF4E and block cap-dependent initiation. The result, shown in Fig. 3A, reveals an initial rapid incorporation of [35S]methionine resistant to inhibition, presum- ably due to elongation of nascent proteins on preexisting polysomes. After 10–20 min, protein synthesis in the presence of m7GTP virtually ceases, whereas uninhibited lysates show a near-linear increase in [35S]methionine incorporation. We inter- pret the latter to mean that [35S]methionine incorporation after Fig. 1. The effect of UBR5A depletion on protein synthesis rates is pro- 20 min depends on new initiation events. Thus the slope after nounced when cells are grown in rich media. W303-1A and UBHY-R cells 20 min is a measure of the rate of initiation. The two slopes were grown in YP galactose, then resuspended either in YP glucose (“De- in Fig. 3A differ by a factor of 10, indicating that m7GTP inhibits ” “ ” “ ” pleted UBHY-R and W303-1A/Glucose ) or galactose ( Induced UBHY-R) initiation by 90%. That protein synthesis and new initiation per- as indicated. Duplicate samples were processed independently. [35S]methio- nine incorporation and protein concentrations were determined as described sist in severely depleted lysates is expected, given that polysomes in Materials and Methods. Protein synthesis rates were measured as cpm also persist in depleted cells, albeit reduced in size (Fig. 2), incorporated per μg total protein per minute of labeling, then expressed as and that methionyl-puromycin formation in vitro proceeds in the a percentage of the respective value at time zero. absence of added eIF5A (9, 10).

6416 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1008150108 Henderson and Hershey Downloaded by guest on September 29, 2021 Fig. 2. The effect of UBR5A depletion on polysomes is more pronounced in rich media. UBHY-R cells were cultured in the indicated galactose-containing media (A and C) until reaching mid-log phase. Half of the culture was treated immediately with CHX and used to prepare a nondepleted extract. The other half was resuspended in the indicated media (B and D) with glucose and incubated at 30 °C for 5 (SD) or 6 (YP) h. After glucose incubation, extracts were prepared in the

same manner. Approximately three OD units of each extract were loaded onto 10–60% (SD) or 10–50% (YP) sucrose gradients and centrifuged. Absorbance BIOCHEMISTRY scans at 254 nm were obtained for each gradient. The ratio of polysomes to total ribosomes was calculated by numerical integration and is given for each profile (Upper Right). The profiles are representative of three independent analyses.

The effect of adding various forms of eIF5A to depleted lysates Our data indicate that eIF5A stimulates translation directly. is shown in Fig. 3B. Distinct stimulation of [35S]methionine incor- Depletion of eIF5A from yeast cells results in a rapid decrease poration is apparent and depends upon the presence of deoxyhy- in ribosomal loading on messages (Fig. 2) that is concomitant pusine or hypusine (Fig. 3B and Fig. S6). This specificity was seen with the factor’s disappearance (Fig. S1). Under optimal growth previously with the methionyl–puromycin assay, although unlike conditions, the immediate effect of depleting eIF5A is a twofold our current result, the quantitative activity of eIF5A(dhp) and inhibition of protein synthesis (Fig. 1 and Fig. S4C). Although eIF5A(hyp) then differed. This may reflect a disparity between translation initiation proceeds in the absence of eIF5A (Fig. 1 mammalian and fungal translation (10). The average difference and Fig. S1), supplementing the factor in vitro (Fig. 3B and in slopes we observed, comparing added eIF5A(dhp) or eIF5A Fig. S6) directly restores translation activity on endogenous (hyp) with buffer alone at points after 20 min, was 1.8 ± 0.1 messages to a degree that is surprisingly consistent with multiple among nine experiments and multiple depleted lysates. Therefore met-puro synthesis assay results (stimulation factor: 2.8 ± 0.6) exogenous eIF5A stimulates protein synthesis, likely at the initia- (9, 10). The parity between our in vivo and in vitro data is parti- tion phase (see Discussion), by about 2-fold in this system. A cularly important. When cells are depleted of eIF5A for 2.5 h, slightly larger degree of stimulation of protein synthesis (2.5-fold) protein synthesis rates fall by a factor that is essentially identical is seen if one simply subtracts the amount of [35S]methionine to the degree of stimulation observed when supplementing incorporated in the presence of m7GTP from each of the corre- extracts prepared from these cells. This result suggests that sponding time points to give initiation-dependent protein synth- any immediate secondary effects of depleting eIF5A activity are esis. Also noteworthy is the fact that the rate of [35S]methionine nominal, although such secondary effects may play a role during incorporation during the first 10 min of incubation does not sig- later phases of depletion. nificantly differ between reactions stimulated and not stimulated The question of whether eIF5A plays a role in protein synth- by eIF5A (Fig. 3B), suggesting that eIF5A does not immediately esis has been analyzed in detail by other groups recently (12, 13, affect the elongation rate (see Discussion). We conclude that 17). Their results, and our data, demonstrate that eIF5A plays a eIF5A is not essential for global protein synthesis, but is required direct role in translation. A recent analysis of mutant forms of for optimal translation of a substantial number of endogen- eIF5A also supports this conclusion (18). Therefore, the numer- ous mRNAs. ous pleiotropic phenotypes seen with mutant forms of eIF5A may be due to changes in the translational efficiencies of specific Discussion mRNAs. Our findings lead to three important conclusions. First, eIF5A That eIF5A acts in translation is now well-supported. How- stimulates protein synthesis directly and globally, presumably ever, our data must be reconciled with other reports that indicate on all or most yeast mRNAs, although further study must deter- distinctly different roles of eIF5A in protein synthesis. While this mine its precise mechanism of action. Second, assay optimization work was in preparation, two groups reported data supporting a is required when characterizing eIF activities that have only a role for eIF5A in translational elongation (12, 13); a third group modest effect on translation rates. Third, and related, suboptimal reported a crystal structure, EF-P bound to 70S, that suggests a experimental conditions contributed to the erroneous and widely role for the eIF5A homolog in the formation of the first peptide cited conclusion that eIF5A does not act in general translation bond only (14). Our data agree with the interpretation that eIF5A based on work employing strain UBHY-R. These conclusions acts as an elongation factor, but only with respect to formation of are addressed in detail below. the first peptide bond. The polysome profiles we observe upon

Henderson and Hershey PNAS ∣ April 19, 2011 ∣ vol. 108 ∣ no. 16 ∣ 6417 Downloaded by guest on September 29, 2021 parallel lines, suggesting that elongation rates decreased through- out their experiment. Finally, the dipeptide release experiment required isolation of the substrate, during which time the tRNA in the ribosomal E site may have been lost. EF-P binding to the E site, and by homology eIF5A binding, is incompatible with simultaneous occupancy by stripped tRNA at the same site (14). Met Because E-site occupancy by tRNAi is observed or expected following formation of the first peptide bond and translocation (and by other tRNAs in all subsequent elongation steps), it is difficult to rationalize a general elongation activity for eIF5A after formation of the first peptide bond. It is important to note that robust quantification of eIF5A activity in translation was observed only after optimizing experi- mental conditions in vivo and in vitro. First, artifacts unique to strain UBHY-R were determined (Figs. S1 and S2). Second, we observed a strong effect of UBR5A depletion only after maximiz- ing in vivo protein synthesis (Ref. 11, Figs. 1 and 2 and Fig. S4). Third, efficient protein synthesis on endogenous mRNAs in vitro depends on raising the molar concentration of radioactive methi- onine by cold methionine supplementation (Fig. S5). Although we have not determined the step(s) inhibited by low methionine concentrations in vitro, relieving such inhibition is a sensible and likely required step to take when examining the function of weakly stimulating factors such as eIF5A (9, 10). We favor a model in which the primary physiological activity of eIF5A is to stimulate formation of the first peptide bond. In support of this idea we note the following: (i) eIF5A was initially found not to stimulate elongation on polyU in vitro in the presence of saturating levels of eEF1A and eEF2 (8); (ii) eIF5A was more recently found to stimulate only the formation of the first two peptide bonds on polyU, but not other templates, in the presence of eEF activity (13); (iii) most compelling, the eIF5A homolog EF-P makes contacts with L1 in the large ribosomal subunit that are incompatible with simultaneous stripped tRNA E-site occupancy (14). Considering our experiments showing diminished polysome profiles upon depletion, a failure to stimu- late elongation on preexisting mRNAs in vitro, and the above considerations, we conclude that eIF5A stimulates the formation of the first peptide bond. However, we cannot rule out the possibility that differences in genetic backgrounds, effects of prolonged eIF5A inactivation, or experimental procedures may account for other interpretations of eIF5A activity. Materials and Methods Fig. 3. Although not required for initiation, eIF5A directly stimulates pro- Plasmids and Proteins. Plasmids pYAH-F5A and pYAH-F5A(K51R) were con- tein synthesis in vitro in a hypusine-dependent manner. Translation extracts structed by PCR (19) from pBM-TIF51A (20) and YRp7-5A(K51R) (21). The am- depleted of UBR5A for 2.5 h were prepared and assayed in vitro for protein plified fragment was inserted into the BamH1 and EcoR1 sites of p414Gal1 synthesis as described in Materials and Methods. To identical reaction (22) and used to express FLAG-tagged eIF5A and its K51R mutant form in 7 aliquots was added m GTP to inhibit initiation, or a buffer control (A). In strains YAH2 and YAH60. His6-eIF5A(lys) was expressed in strain BAH6 B, extracts were supplemented with buffer alone, or at five molecules [BL21(DE3) carrying the eIF5A cDNA in pET28c]. To generate eIF5A(lys), per 80S ribosome of eIF5A(lys), eIF5A(dhp), or FLAG-tagged eIF5A(hyp), His6-eIF5A(lys) was purified from strain BAH6 and treated with thrombin the latter protein being purified from yeast. Ribosome concentration in the to remove the tag. Similarly, eIF5A(dhp) was obtained from BAH6 by treating extract was determined by using the molar extinction coefficient of 80S as purified eIF5A(lys) with His6-yDHS (23, 24). Native FLAG-tagged eIF5A [F5A 5 × 107 −1 −1 M cm at 260 nm and by assuming that ribosomes are 80% of (hyp)] was purified from yeast strain YAH2 with an anti-FLAG resin. Details of total RNA (25). Experiments shown are representative of multiple indepen- these procedures are provided in SI Materials and Methods. dent experiments. Extract Preparation and Western Blotting. Cells were propagated at 30 °C in YP eIF5A depletion indicate that eIF5A is required for efficient in- medium containing either 2% glucose or 2% galactose. For depletion studies, itiation; we see no other interpretation that explains a decrease in cells were washed once with sterile room-temperature water containing efficiently loaded mRNAs after depletion of eIF5A. However, 2% glucose, then resuspended in either YP or SD glucose or galactose as the polysome profiles obtained by Dever and coworkers (13) lead indicated. Two lysis methods were employed. The nondenaturing method in- volved glass bead vortexing and was described previously (11). For the dena- to the conclusion that eIF5A promotes translation elongation. It turing method, a similar glass bead procedure was used, except that the is unclear how to reconcile our polysome profiles with theirs. buffer was 40 mM Tris-HCl, pH 6.8, 4% SDS, 4% glycerol, and 0.1 M DTT, However, their other data suggesting a role in elongation are and samples were boiled for 10 min. Lysate proteins were resolved and more difficult to interpret. In particular, some of the results were blotted with rabbit anti-yeast eIF5A (11). UBR5A bands were quantified with obtained after a number of hours of eIF5A depletion, when sec- TotalLab TL100 (Nonlinear Dynamics, version 2006b).

ondary effects of eIF5A depletion likely occur. The ribosome In Vivo Protein Synthesis Measurements. W303-1A and UBHY-R cells (11) were transit time experiments were not done under steady-state con- pulse-labeled with [35S]methionine at various times following the shift from ditions; total and released incorporation data do not generate YP galactose to YP glucose. About three OD595 units of cells (processed in-

6418 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1008150108 Henderson and Hershey Downloaded by guest on September 29, 2021 dependently in duplicate) were washed in water/carbon source, resuspended rotor for approximately 1 h. Polysome profiles were generated by following in 300 μL YP (glucose or galactose), and incubated with 100 μCi Trans-35S- absorbance at 254 nm and fractions collected using an ISCO fractionator. Label (MP Biomedicals) at 30 °C for 15 min with shaking. Labeling was termi- nated by adding 1-mL ice-cold stop solution [1.2 mg∕mL methionine, In Vitro Translation Assays. Cells were grown in galactose media, shifted to 0.1 mg∕mL cycloheximide (CHX)] and incubating on ice briefly. Cells were glucose to deplete eIF5A, and lysed as described in detail in SI Materials collected and snap-frozen in liquid nitrogen, then resuspended in 15% tri- and Methods. Translation assay reactions contained 60 ± 10% extract and chloroacetic acid (TCA) (1 mL), incubated on ice for 15 min, and boiled at eIF5A proteins as indicated, with final reaction conditions of 30 mM Hepes, 95 °C for 15 min to deacylate tRNAs. Denatured protein was pelleted, sus- pH 7.5, 3.8 ± 0.1 mM total MgCl2, 2 mM ATP, 0.5 mM GTP, 0.2 mg∕mL creatine pended in 1 mL acetone at −20 °C, stored 30 min at −20 °C, and centrifuged kinase, 12 mM creatine phosphate, 165 ± 10 mM KOAc/KCl, 100 μM amino again. Precipitated proteins were dissolved in 400 μL 1% SDS, boiled for acids (lacking methionine), 30–40 μM unlabeled methionine, 0.17 μM trans- 10 min, and clarified by centrifugation. The protein content in 50-μL aliquots lation grade [35S]methionine (1175 Ci∕mmol; MP Biomedicals), 1 mM AEBSF was determined with a BCA kit (Pierce) against a bovine serum albumin (added fresh), and 0.3 units∕μL RNAprime (added fresh). Reactions were control. To measure radiolabel incorporation, 25-μL aliquots were counted initiated by adding extract and were performed at 20 ± 2 °C. At the indicated in a Beckman LS 6000 IC scintillation counter. The rate of protein synthesis times, 5-μL aliquots were withdrawn, spotted on Whatman #1 filters, soaked was calculated for each sample as cpm∕μg protein∕ min. in fixative (15% TCA, 20 mM cold methionine), swirled, and stored at 4 °C until assay completion. The fixative was decanted and replaced, boiled Polysome Profile Analysis. Cells were grown as above, brought to 0.1 mg∕mL briefly, and allowed to cool to room temperature. Filters were washed twice CHX and rapidly cooled on ice. Pellets were washed, resuspended in poly- with methanol, air-dried, and then counted in a Beckman LS 6000 IC scintilla- some lysis buffer [20 mM Hepes, pH 7.5, 140 mM KCl, 5 mM MgðOAcÞ2, tion counter. 0.5 mM DTT, 1% Triton X-100, 1 mg∕mL heparin, 100 U∕mL RNAprime (Eppendorf), 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) and ACKNOWLEDGMENTS. We thank Hans Johansson, Myung Hee Park, and complete protease inhibitors (Roche)] at approximately 1-mL buffer per gram Martin Hoyt for helpful discussions. This work was supported by National of cells and lysed by vortexing with glass beads. Lysates were centrifugation Institutes of Health (NIH) Grants GM22135 and GM073723 from the US briefly at 1;000 × g, and three A260 units were loaded onto sucrose gradients Public Health Service. C.A.H. was supported in part by NIH training Grant and centrifuged at 54,000 rpm in a Beckman TL100 centrifuge in a TLS55 GM07377.

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