Rosemary Jagus$$, Wun-Ing Huangli, Linnea J. Hansen(( , and Mark A. Wilson$ ...... Nelson. E. M.. Lashbrook. C.. and Hershev. J. W. B.. Acad. Sci. U. S. A. 87 ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 267, No. 22, Issue of August 5, pp. 15530-15536,1992 Printed in U.S.A.
Changes in Rates ofProtein Synthesis and EukaryoticInitiation Factor-4 Inhibitory Activity in Cell-free Translation Systems of Sea Urchin Eggs and Early CleavageStage Embryos* (Received for publication, October 22,1991)
Rosemary Jagus$$, Wun-Ing Huangli,Linnea J. Hansen((,and MarkA. Wilson$ From the $Center of Marine Biotechnology, Baltimore, Maryland 21202
The characteristics of cell-free translation systems prepared from unfertilized eggs and early cleavage stage embryos of the sea urchin, Strongylocentrotus purpuratus, closely reflect the developmentally regulated changes in protein synthesis initiation observed i n vivo. Cell-free translation systems prepared over the first 0-6 h following fertilization show gradually increasing activities, mimicking the changes observed i n uivo. The mechanisms underlying these increases are complex and occur at several levels. One factor contributing to the rise inprotein synthetic rate is the gradual increase in eukaryotic initiation factor(e1F)4 activity. This is correlated with the progressive inactivation of an inhibitorof eIF-4 function, which can be reactivated by in vitro manipulations. The relatively slow activation of eIF-4 follows similar kinetics to the increased utilization of maternal mRNA and ribosomes, in contrast to the rapid rise in maternal mRNA activation, and the increase in eIF-2B activity. This slow release from eIF-4 inhibition following a rapid release from eIF-2B inhibition and increased mRNA availability is reflected in the patternof initiator tRNA binding to the small ribosomal subunit observed in cell-free translation systems. In translation systems from unfertilized eggs, initiator tRNA is unable to interact with thesmall ribosomal subunit, consistent with an initial block in both eIF-2B and eIF-4 activity. In translation systems from 30-min embryos, 4 8 S preinitiation complexes accumulate, reflecting the release from inhibition of mRNA availability and eIF2B activity, but continued low activity of eIF-4. The accumulation of initiator tRNA in 48 S preinitiation complexes disappears gradually in translation systems from later embryos, as eIF-4 is slowly released from inhibition.
Fertilization in invertebrates is often associated with rapid and dramatic activation of the translational machinery, along with increased utilization of maternal mRNA stores (1-5). In sea urchins, such as Strongylocentrotus purpuratus, the in-
* This work was supported by National Institutes of Health Grant GM-33631 (to R. J.) and American Cancer Society Fellowship PF2748 (to L. J. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. J To whom correspondence should be addressed. Tel. 410-7834889;Fax: 410-783-4806. 11 Current address: Dept. of Environmental and Chemical Analysis, Environmental Protection Agency, Baton Rouge, LA 70815. 11 Current address: Toxicology in the Health Effects, Division Office of Pesticides Program, EnvironmentalProtection Agency, Washington, D. C. 20460.
crease in protein synthesis begins several minutes after fertilization, reaching rates 30-50-fold higher than in the unfertilized egg by 2-3 h (4, 5). This activation reflects increased utilization of maternally accumulated mRNA and ribosomes (reviewed in Refs. 6 and 7). Traditionally, these observations have been interpreted as demonstrating that maternal mRNA is “masked” (8, 9) and/or that ribosomes require “activation” (10,ll). However, the observations have proved to be refractory to mechanistic elucidation. The recent establishment of highly active cell-free translation systems from sea urchin eggs and early cleavage stage embryos (12-14) is beginning to allow the elucidation of the mechanisms underlying the regulation of maternal mRNA utilization. The cell-free translation systems used forthe work described above, in contrast to earlier reports ofegg and embryo cell-free translation systems, exhibit several essential features: ( a ) the rate of protein synthesis approaches that observed in uiuo (12, 13); ( b ) the systems are capable of reinitiating on endogenous maternal mRNA (12, 14); and (c) the protein synthetic characteristics of the systems reflect those observed in uiuo (12-14). Cell-free translation systems prepared from unfertilized eggs show very low protein synthetic rates, reflecting those observed in uiuo. Cell-free translation systems prepared from embryos show rates of protein synthesis 15-30-fold higher than those prepared from unfertilized eggs (12-14). Deductions regarding the mechanisms underlying developmental activation of protein synthesis have been made by assessing which components of the protein syntheticmachinery can be added to cell-free translation systems from unfertilized eggs to promote increased protein synthetic activity. Studies using these cell-free systems point to a multifactorial mechanism for the activation of protein synthesis during development (14-17). At least two protein synthesis initiation factors, eIF-2B’ and eIF-4, show reduced activity prior to fertilization, and by inference increased activity following fertilization (14-17). These changes in initiation factor activity”occur in coordination with the “unmasking” of maternal mRNA by a process that remains elusive (reviewed in Ref. 19). The addition of mRNA alone is not sufficient to stimulate the activity of the poorly active cell-free translation systems from eggs, but stimulation canbe achieved by the addition of eIF-2B (15, 17, 20) or eIF-4 (14, 16). Stimulation by these initiation factors is significantly increased by including added mRNA, indicating that neither eIF-2B nor eIF-4 function to “unmask” endogenous mRNA. The nomenclature used for initiation factors is that recommended by the Nomenclature Committee of the International Union of Biochemistry (18).The abbreviations used are: eIF, eukaryotic initiation factor; HEPES, 4-2-(hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid GDP(NH)p, guanyl-5”yl imidodiphosphate.
15530
Previous reports from our laboratory (20) show that eIF2B is activated rapidly followingfertilization, by a mechanism that is linked to the fertilization induced increase in redox potential. In addition, our previous studies have shown that the unfertilized sea urchin egg contains an activity that inhibits eIF-4 function in homologous and heterologous cell-free translation systems (12, 16). The inhibitor of eIF-4 does not prevent interaction of the factor with mRNA. Instead, the inhibitor blocks the initiation sequence after the interaction of eIF-4 with mRNA, after the interaction of mRNA with the small ribosomal subunit, butbefore 60 S subunit binding (12). eIF-4 (previously designated eIF-4F, and cap-binding protein) is a three-subunitprotein synthesis initiation factor involved in the interaction of mRNA with the small ribosomal subunit (21, 22), and viewed by many investigators to be the main factor mediating mRNA/smal~ribosomal subunit interaction (18). eIF-4 performs more than one function during the initiation sequence(reviewed in Refs. 24 and 25): the factor binds to the 5'-cap structure of mRNA, promoting interaction of the small ribosomal subunit with mRNA, as well as participating inthe migration of the small ribosomal subunit to the initiator AUG (24,25). Factor eIF-4 is known to be regulated in response to growth factor and nutrientdeprivation (25,26), heat shock (27-29), during the reduction in protein synthetic activity that occurs during mitosis (30), as well as by viral infection of animal cells (31-33). This paper expands our earlier description of cell-free translation systems from sea urchin eggs and 2-h embryos, by comparing the characteristics of extracts made at various times following fertilization. The resulting extracts possess protein synthetic activities that mimic the gradual rise in translation rates observed in vivo with surprising closeness; the same gradual increases in ribosome loading and maternal mRNA utilization that occur in vivo are reflected inthe activity of these systems. This study also looks at theextent t o which restrictions in eIF-4 activity are reversed after fertilization, as well as the time course over which this occurs, and demonstrates that the gradual rise in eIF-4 activity follows the gradual rise in maternal mRNA and ribosome utilization, and isaccompanied by the reversible inactivation of a specific protein inhibitor of eIF-4 function. In addition, this work compares the activation of eIF-4 with the increases in maternal mRNA activation and the rise in eIF-2B activity, showing that the changes in eIF-4 activity occur much more slowly. These relative changes in eIF-4 and eIF-2B activity are reflected in the pattern of initiator tRNA binding to the small ribosomal subunit. EXPERIMENTAL PROCEDURES
Materials-Soybean trypsin inhibitor, ovomucoid, L-a-lysophosphatidylcboline, spermidine, phosphocreatine were from Sigma. RNasin was from Promega Corp. BPA-1000 was from Toso-Haas, Philadelphia, PA; creatine phosphokinase was from Boehringer Mannheim; and ["S]methionine (800-1200 Cilmmol, translation grade) was from Du Pont-New England Nuclear. Handling of Gametes and Embryos-S. purpuratus were obtained from Pacific Bio-Marine Laboratories, Inc. (Venice, CA). Spawning of gametes, in vitro fertilization, and culturing of eggs and embryos were performed as described (12). t w n preparation of Preparation of Cell-free ~ r a ~ ~ Systems-The cell-free translation systems from sea urchin eggs and embryos is described in detail by Hansen et al. (12) and Huang et al. (16). The method uses the buffering, osmotic, and ionic conditions established by Winkler and Steinbardt (10) to resemble those found in early cleavage stage embryos. Briefly, dejelliedeggs and embryos were lysed in 105 m M K' gluconate, 50 mM HEPES-KOH, pH 7.2,40 mM NaCl, 1 mM Mg(CH,CO&, 10 mM EGTA, 220 mM glycerol, 1 mM spermidine, 1 mM dithiothreitol, 300 units/ml RNasin, 1 mg/ml soybean trypsin inhibitor, 1 mg/ml ovomucoid, 250 pg/ml L-a-lysophosphatidylcholine, homogenized, and centrifuged at 10,000 X g for 10 min. The resulting supernatant was dialyzed against three changes of 105
mM K+ gluconate, 50 mM HEPES-KOH, pH 7.2.40 mM NaC1,l mM Mg(CH,CO&, 1 mM spermidine, and 1mM dithiothreitol for a total of 1.5 h, to ensure that the pH and ionic conditions were known and consistent from batch to batch. Note that the pH of the lysis and diaiysis buffers used, pH 7.2, corresponds to the pH found in the embryo, which is also the optimal pH for translation (17,34,35). The pH of the cell-free translation system prepared from unfertilized eggs is therefore higher than thepH of the corresponding intact eggs. The egg and embryo extracts contained approximately 14 2 1.9 pmol of 80 S eq/100 pl. Rabbit reticulocyte cell-free translation systems were prepared as described (36). Cell-free ~ r a ~ ~ ~ w n - S . ~ u egg r p uand r ~embryo t ~ cell-free translation assays contained approximately 0.85 volumes of egg or embryo extract, the volume being adjusted to give a ribosome content of approximately 12 pmol of 80 S eq/100 pl of assay volume. Other components were added to give: 500 p~ glucose 6-pbosphate, 3 units/ ml of creatine phosphokinase, 20 mM phosphocreatine, 0.75 mM ATP, 0.1 mM GTP, 1.85 mM M g ~ C ~ 3 C O ~150 ) * ,p M amino acids minus methionine, and 0.675 p~ [35S]methionine(375 pCi/ml). The final K+ and free M e concentrations were 150 and 1 mM, respectively. The translation systems were incubated at 18 "C, and 2.5- or 5-pl aliquots were removed a t 30 min for the determination of trichloroacetic acid-precipitable radioactivity. The incorporation of ["Clvaline in the rabbit reticulocyte translation system was assayed as described (36). Calculation of Absolute Rates of Protein Synthesis-The endogenous methionine pools were measured by an isotope dilution method (37): incorporation was determined at two different levels of [36S] methionine and simultaneousequations were generated to allow calculation of the endogenous pool size. The methionine pool size in cell-free translation systems from eggs varied from 0.8 to 1p ~while , the pool size in translation systems from embryos varied from 1.5 to 1.8 p M . Using a value of 1%for an average methionine content of proteins and a value of 40 kDa (360 amino acids) for the average size of proteins synthesized (38), the incorporation data was converted into picograms of protein/embryo-h. The ribosome content of the cell-free translation assays, 12 pmo~/lOOpl, was used to convert the incorporation data to(picomole of protein/pmol of ribosome)/min. Initiator tRNA Binding to the Small Ribosomal Subunit-Conditions for 15-35% sucrose gradient fractionation were essentially as described by Safer et al. (37), except that sucrose solutions were 20 mM HEPESprepared in 150 mMKC1,7.5mMMg(CH,CO,),, KOH, pH 7.5, 10 mM EGTA, and the extracts were adjusted to 10 PM GDP(NH)P and1%Triton X-100 prior to loading. For gradients designed to look at the interaction of initator tRNA with initiation intermediates, the translation systems were incubated with [35S] methionine (375 pCi/ml) prior to loading and centrifuged a t 22,000 rpm for 15 h. This allows the separation of different initiation intermedia~s,although most of the polysomes are pelleted. After centrifugation, the gradients were fractionated into 36 fractions and precipitated with cetyltrimethylammonium bromide. This precipitates RNA and proteins bound to RNA, such as peptidyl tRNA. Consequently, at the top of the gradient, precipitated radioactivity represents precipitated Met-tRNAi and Met-tRNAMet.Further down the gradient, precipitated radioactivity represents Met-tRNAi bound to small ribosomal subunits, and closer to thebottom, it represents a combination of Met-tRNAi bound to functioning 80 S ribosomes and polysomes, as well as peptidyl tRNA. Measurement of Percentage of Actiue Ribosomes and Utilization of mRNA-Cell-free translation systems from unfertilized eggs and early cleavage stage embryos were incubated at 18 'C, in theabsence of radioactive amino acids. After incubation, GDPfNH)P,cycloheximide, and Triton X-100 were added to give concentrations of 10 p ~ , 50 pg/ml, and 1%(v/v), respectively. 900-pl samples were layered over 15-45% sucrose gradients and centrifuged at 13,000 rpm for 18 h. The gradients were fractionated into 36 fractions and assayed for absorbance a t A260 and 5'-3H-labeled poly(U) hybridization, as described (39). 5'-3H-Labeled poly(U) hybridization was converted into picomoles of mRNA using a value of 60 nucleotides as the average poly(A) tract length. The percentage of mRNA in use was calculated asthe percentage of 5"'H-labeled poly(U)-hybridizable material bound to 80 S monosomes and polysomes. This was not calculated for the egg cell-free translation system since 5'-3H-labeled poly(U) hybridi~tionwas barely above background. The percent of ribosomes in polysomes was Calculated asthe percentage of absorbing material in polysomes, compared with tbat in polysomes plus subunits, plus the 80 S peak. Since the 80 S peak contains both inactive ribosome couples plus functioning 80 S ribosomes, the percent of active ribosomes was calculated from the percentage of ribosomes in
15532
Activity eIF-4
Rises Slowly after Fertilization
polysomes adjusted for the 80 S ribosomes bound to mRNA. Assay of Inhibitory Activity-eIF-4 inhibitory activity was assayed by measuring the inhibition of translation in a cell-free translation system from rabbit reticulocytes. Sea urchin extracts were first clarified with BPA-1000, a quaternary amine-linked acrylic polymer, often used for prechromatography polishingto remove cell fragments, acidic impurities, suchas DNA, and polar lipids. Pilot studies showed that clarification of egg or embryo extracts had no deleterious effect on protein synthetic activity, and when used at optimal levels gave a slight stimulation of translation (data not shown). The treatment was therefore considered to have no deleterious effects. Without clarification, inhibitory activity showed batch variability, a less clear pattern of decreased activity with time after fertilization,and a more variable response to rescue by eIF-4. Earlier studies in this system have shown the variable presence of an uncharacterized contaminant, removable by BPA-1000, which prevents the normal interaction of eIF-2B with eIF-2, and could account for the variability in inhibitory activity observed (20).Clarificationwith BPA-1000 decreases the batch variability of inhibitoryactivity, allows a clear pattern of decreased activity with time after fertilization to emerge, and permits the measurement of inhibitory activity which is greater than 90% rescuable by added eIF-4. Immediately prior to assay in the reticulocyte translation system, the extracts from eggs and embryos were drop dialyzed against 25 mM HEPES-KOH, pH 7.2, 25 mMKC1, 0.5 mM MgCl,, using Millipore V series filters (0.025 pm, 13 mm diameter) (Millipore applicationreport AR524) for 30 min to give salt concentrationsequivalent to those of the reticulocyte translation system. When used for eIF-4 inhibitory assays, reticulocyte incubations were supplemented with RNasin (300 units/ml), EGTA (2 mM), and soybean trypsin inhibitor (1mg/ml), to ensure that the inhibitory effects of egg extract did not represent spurious RNase or protease activities. Inclusion of thesesupplementsdid not inhibit protein synthetic activity in the reticulocyte translation system, but actually stimulated translation between 5 and 10% (data not shown). EGTA a t the concentrations used is sufficient to inhibit the Ca2+-dependent protease present in extracts of eggs and early embryos. However, EGTA does not have a sufficiently high affinity for Ca2+to inhibit the Ca2+ dependent step in initiation reported by Kumar et al. (40, 41). Purification of eIF-4-eIF-4, a gift from Dr. William C. Merrick, (Case Western Reserve Medical School), was purified as described (22). RESULTS
Characteristics of Cell-free Translation Systems Prepared at Various Times Following Fertilization-Fig. 1 shows the pro-in tein synthetic activity of cell-free translation systems prepared from unfertilized eggs and embryos between 20 min and 6 h following fertilization. A mean value and standard deviation of [35S]methionineincorporation during a 30-min incubation time is given. The data are from 5 different preparations of extracts made at each time point after fertilization. The pool size, calculated as described under “Experimental Procedures,” along with the incorporation data, were used to calculate the absolute rates of protein synthesis shown in the figure. A rate of 87-92.5 pg/embryo-h was found in the cellfree translation system prepared from 2-h embryos, equivalent to 0.2 pmol of protein/pmol of ribosome/min. This rateis 3639% of that observed in the intact2-h embryo ( 5 ) . Comparison of the activities of cell-free translation systems prepared at various times following fertilization showed a gradual increase in activity, from 4 & 2 pg/embryo-h in cellfree translation systems from unfertilized eggs, plateauing at approximately 100 pg/embryo-h between 3 and 6 h. The rate of translation was almost 10-fold higher, at 39 k 8 pg/embryoh, in extracts prepared within 30 min of fertilization than in those prepared from unfertilized eggs. The ratewas increased by 20-25-fold in extracts prepared 2-6 h after fertilization, reaching 89.8 2 2.5 pg/embryo-h in translation systems from 2-h embryos, and 102 & 4 pg/embryo-h in translationsystems from 6-h embryos. These gradual increases in protein synthetic activity of cell-free translation systems prepared at different times following fertilization mimic the pattern of
0
1 2 3 4 5 6 nme extracts made after fertllbation (h)
FIG. 1. Activity of cell-free translation systems prepared from unfertilized eggs and early cleavage stage embryos. Cellfree translation systems from unfertilized eggs and early cleavage stage embryos were prepared as described, incubated under standard conditions, and assayed for [36S]methionine incorporation into trichloroacetic acid-precipitable radioactivity. The incorporation data was converted into picograms/embryo-h, as described under “Experimental Procedures.” The mean and standard deviations of determinations from 5 separate preparations at each time point are shown. T b allow for variation in recoveries, the volume of extract used in the translation assays was adjusted to give the same level of protein. This strategy also gave the same levels of ribosomes. Incorporation of [35S] methionine into trichloroacetic acid-precipitable material after a 30min incubation is shown here.
TABLE I Ribosome and mRNA utilization in cell-free translation systems prepared from unfertilized eggs and early cleavage stage embryos Cell-free translation systems from unfertilized eggs and early cleavage stage embryos were incubated at 18 “C, in the absence of radioactive amino acids, and 900plwas subjected to sucrose density gradientcentrifugation, as described under“ExperimentalProcedures.’’ The gradients were fractionated and the fractions were used to estimate the percentage of active ribosomes and percentage mRNA used, as described under “Experimental Procedures.” Sea urchin system
% Ribosomes
Egg 0.5-hEmbryo 1-h Embryo 2-h Embryo 3-h Embryo 6-h Embryo
2.5 13.5 19.3 31 7.8 32.5 33
a
polysomes
mRNA in mRNAin 80 S pool polysomes % mRNA % Active used ribosomes pmol % pmol %
