Protein synthesis, polyribosomes, and peptide elongation in early development of Strongylocentrotus purpuratus

DEVELOPMENTAL

BIOLOGY

32.32-40 (1981)

Protein Synthesis, Polyribosomes, and Peptide Elongation in Early Development of Strongy/ocentrotus purpuratus’ A. SCOTTGOUSTIN’AND FRED H. WILT Department

of Zoology, University of Califonzia,

Berkeley, California

Received May 15, 1980; accepted in revised fm

94720

July 28, 1980

The absolute rate of protein synthesis in developing embryos of Strm&wentrotvs purpwatw has been measured by lysine incorporation. Protein synthesis rises to about 240 pg hr-’ embryo- i from the two- to eight-cell stage, and then gradually increases to a maximum of over 500 pg hr-’ embryo-i in the blastula. The changes in protein synthesis are accompanied by similar increase in the polyribosomes in the embryo, so that 60-65% of the ribosomes are in polyribosomes by the blastula stage. The data are used to calculate an average peptide elongation rate of 1.8 amino acids ribosome-’ set-‘.

INTRODUCTION

maintained in natural seawater (12-18°C) until use. Gametes were obtained by intracoelomic injection of The synthesis, accumulation, and turnover of proteins 0.5 1M KCl. Eggs were always washed at least three and RNA in sea urchin embryos has been the object of times before use with natural seawater that had been many investigations on the molecular events in early filtered through 0.45 PM HA Millipore filters, which development, a subject recently reviewed by Davidson were previously soaked in boiling water (MPFSW); eggs (1976). Nonetheless, we have not encountered any syswere then dejellied by pouring through a Nitex screen. tematic and quantitative analysis of protein synthesis Excess sperm was always removed from embryo culin early development of Strongylocentrotus purpuratus, tures after fertilization. Embryos were cultured at 14the west coast purple sea urchin. Fry and Gross (1970) 16°C in MPFSW, in one of the three following manners: have examined the “early cleavage” stage of this spe(1) stirred with a 60-rpm clock motor equipped with cies, and Regier and Kafatos (1977) have carried out paddles; (2) by agitation produced by bubbling humimeasurements of protein synthesis in the egg and gastrula. Infante and Nemer (1967) have examined some dified line-compressed air into the seawater at a bubble stages for the level of polyribosomes that are present. rate to achieve moderate saltation (using a 100~~1microcap sharpened at the end as the final vent for air There is now considerable interest in the regulation into the seawater); or (3) culture in a shallow layer of of gene expression in this species, and we have carried out measurements on the absolute rate of protein syn- MPFSW in the bottom of a beaker swirled occasionally thesis and on the proportion of the embryo’s ribosomes by hand (for short, kinetic experiments only). The first present in polyribosomes. The two measurements are two methods resulted in the normal development to the pluteus stage of the majority of embryos, provided that highly correlated, suggesting it is the polyribosome level that determines the general rate of protein syn- antibiotic (50 hg/ml each of streptomycin and penicillin thesis. The data may be used to calculate peptide elon- G, or 100 pg/ml of gentamycin sulfate alone) is included gation rates and to serve as essential information for in MPFSW. The concentration of embryos is determined by countstudies on the expression of specific genes during deing embryos in three aliquots of a 1:20 dilution of the velopment. culture removed with 50-~1 microcaps. MATERIALS

AND METHODS

Labeling of Embryos

Culture of Sea Urchin Embryos

Embryos were labeled at the appropriate stages with L-[4,5-3H]lysine (Schwa&Mann, 40-60 CVmmol). Exact details will be given in the figure legends and the text. In all cases, radioactive label was introduced to the culture in seawater at pH 7.8, regardless of the method of label preparation.

The S. purpuratus used in this work were collected from intertidal sites near Point Arena, California, and 1 Supported by Grant GM-13882 from the National Health. * Recipient of a Regents’ Fellowship of the University

Institutes

of

of California.

32 0012-1606/81/030032-09$02.00/O Copyright All rights

0 1981 by Academic Press. Inc. of reproduction in any form reserved.

GOUSTIN

AND WILT

Protein

Homogenization of Embryos display of polysomes on For experiments involving a sucrose gradient, embryos were washed once with calcium-magnesium-free seawater (CMFSW; Arceci and Gross, 1977) and sterile ice-cold 1.5 M dextrose before homogenization (except in the case of unfertilized eggs, for which 1.0 M dextrose was substituted). Homogenization buffer, designated EGTA medium, contained 0.25 M NaCl, 50 mM Mg(OAc),, 25 mM EGTA (ethylene glycol bis(@-aminoethyl ether) NJ’-tetraacetic acid; Sigma), 25 mM Tris base, and 1% (w/v) in Triton X-100 (Sigma) brought to pH 8.4 with NaOH before the addition of detergent; this buffer was treated with 0.01% diethylpyrocarbonate (DEP) and autoclaved. The washed embryos were first suspended in 510 vol of EGTA medium and an adequate volume of 20 mg/ml bentonite was added to bring the bentonite concentration in the homogenate to 1 mg/ml. The stainlesssteel Dounce-type homogenizer was rinsed with water containing 0.01% DEP just before use. The suspension of homogenization medium, embryos, and bentonite was transferred with a sterile pipet to the ice-cold homogenizer, the ball of the pestle was coated with a microliter of fresh DEP, and homogenization on an ice bath was monitored by phase contrast microscopy. Five to ten strokes were usually sufficient.

Sucrose Density Gradient Centrifugation Polysomes

of

Every material used was treated with a solution of DEP (0.03%, v/v) made up in deionized water (resistance of >16 megohms/cm; Ca’+,
Synthesis in Urchin Embryos

33

eliminate ubiquitous ribonucleases; oven-dried tubes were then siliconized by exposure to one drop of dimethyldichlorosilane (Eastman) made up to 5% (v/v) in carbon tetrachloride (Mallinckrodt, AR), and redried in the oven at 70°C. After centrifugation at lO,OOOg,the pellets were generally free of yellow color (indicating very efficient homogenization), and contained less than 30% of the homogenate RNA. The substantial white pellet is mostly bentonite and fertilization membranes, although in later stages (especially 12 hr) this pellet contained substantial amounts of radioactivity (in [3H]uridine labeling experiments) which proved to be in DNA. Following convention, the supernatant prepared in this manner will be referred to as a postmitochondrial supernatant, although it was not shown to be free of mitochondria or mitochondrial fragments. The gradients were then layered with 0.3-0.6 ml of postmitochondrial supernatant, and centrifuged (at 25°C) in either a Spinco SW 41 rotor, or International SB-283 rotor at 37,000-38,000 rpm for 150-210 min. Gradients were analyzed after being pumped out from the bottom of the centrifuge tube by means of a peristaltic pump. The refractive index of selected fractions was determined, before dilution (1:l) with sterile high-resistance water or 1X KHSB, in preparation for determination of optical density (at both 260 and 280 nm) or acid-precipitable radioactivity [lo%, w/w, trichloroacetic acid (TCA)]. Absorbance due to sucrose (usually less than 0.2 for 1.8 M and 0.09 for 0.45 M) was subtracted out. Sedimentation coefficients were determined by the method of McEwen (1967) using 1.5 g/cm3 as a polysome density, and values for percentage sucrose determined from a standard curve relating refractive index to sucrose molarity (this offers a correction for refractive index contributions of the 0.35 M KC1 buffer). The sedimentation coefficients of ribosomes and polysomes were calculated from an empirical equation relating “-some” number (n) to s value: s = 78YV”.5g(Reisner et al., 1972).

Measurements of the Absolute Rate of Protein Synthesis Embryos were fertilized as described, and cultured to the desired stage. They were then concentrated to 50,000-100,000/m1 in a total volume of 10 ml, generally 80,00O/ml. Labeled lysine was added to 2-20 @/ml of culture. The labelings were performed in a shallow layer in the bottom of a siliconized 400-ml beaker, with periodic swirling. Incorporation was linear up to 60 min under these conditions. Embryos subjected to the conditions described above for up to 60 min will (when diluted back to 20,00O/ml) develop to normal plutei on schedule. Aliquots of 3 ml each were removed at 10,20,

34

DEVELOPMENTALBIOLOGYV0~~~~82,1981

and 30 min, layered over ml ice-cold CMFSW and spun down in a hand centrifuge. The embryos were washed twice more in ice-cold CMFSW (10 ml). The embryo pellet was homogenized as described in 2 ml of 10 mM Tris-HCl, pH 7.4, and quick-frozen at -70°C until use. For total incorporation, aliquots were thawed; protein (Lowry et aZ., 1951) and incorporation were then determined (cpm precipitable with trichloroacetic acid at 1.56 M, or approximately 20%, w/w). Heating to 80°C for 5 min in 1.56 M TCA with subsequent precipitation at 4°C reduced the amount of apparent incorporation by 5-lo%, but did not significantly alter the slope of the line drawn through the lo-, 20-, and 30-min time points. This heating step was eliminated in some experiments. The data were expressed as TCA-precipitable counts per minute per microgram of protein and plotted with that correction. The data were then corrected for counting efficiency (20-33%) and put on a per embryo basis assuming 40 ng protein per egg or embryo of S. purpuratus (Fry and Gross, 1970). The reasons for the selection of L-lysine as a radioactive precursor in these experiments were threefold: (1) pilot experiments with L-leucine resulted in unsatisfactory incorporation curves over the 30-min time course desired, reaching a plateau at lo-20 min. The anomalous behavior of L-leucine is labeling experiments on S. purpuratus has been described by previous workers (Fry and Gross, 1970; Graves, 1973); (2) L-[4,53H]lysine was available at high specific activities; and (3) there was no measurable conversion of label into by-products during 30 min of embryo incubation (virtually all of the 3H-cpm in the tRNA pool assay could be accounted for as displacable by authentic L-lysine).

Measurement of Amino Acid Precursor Pool Specific Radioactivities The specific radioactivity (dpm/pmol) of the embryo lysine pools was determined by a modification of the isotope dilution method of Rubin and Goldstein (1970) on TCA-soluble portions of homogenate. Aliquots of the homogenate were made 1.56 M in TCA, and allowed to sit on ice for 30 min. The acid-soluble supernatants were then extracted with four to six successive portions of 2-3 vol of ethyl ether. This acid-free crude amino acid extract was then lyophilized, dissolved in high-resistance water, and lyophilized again. When dissolved in water once more, these fractions served as 3H-input amino acid in the isotope dilution assay.

Isotope Dilution Assay for L-Lysine The isotope dilution assay developed by Rubin and Goldstein (1970) was adapted for use in the measurement of lysine pool specific radioactivities. Crude ami-

noacyl-tRNA synthetases were prepared from late log phase Escherichia coli strain JC 5422 (a K-12 derivative; from Dr. A. J. Clark, Molecular Biology, UC Berkeley) as described (Kelmers et al., 1965). Crude enzyme (3.4 mg/ml) was stored at -20°C after addition of glycerol to 20% (v/v); aliquots of enzyme are diluted to 1 mg/ ml with 25 mM 2-mercaptoethanol just before use. Transfer RNA was from E. coli K-12 strain (Schwarz/ Mann); its binding capacity was found to be 37-62 pmol of L-lysine per ODzm. The L-[‘4C]lysine standard was the StanStar product from Schwarz/Mann (111 dpm/pmol). The mole standard used was from Beckman, an amino acid analyzer calibration mix containing L-lysine and 19 other amino acids at 2.5 mM (kO.02 mM). The final reaction mixture contained 2 ODBo tRNA, 25 prnol Hepes (Calbiochem) at pH 8.5 (with KOH), 2.5 pmol Mg(OAc)z, 0.05 pmol ATP, 1.25 pmol KCl, 750 pmol of the StanStar [14C]lysine, 50 pg of crude enzyme, 1.25 pmol2-mercaptoethanol (introduced with the crude enzyme), a variable amount of 3H-amino acids extracted from embryos, in a reaction volume of 250 ~1. Although the acylation was 85% complete under these conditions by 5 min at 37”C, incubations were routinely carried out to 30 min. The reaction was stopped by placing the tubes on ice and dividing the tube contents into either three 75-~1 or four 50-~1 aliquots; to which were added TCA to 10% (w/w) containing 2 mM cold L-lysine. The precipitates were collected with suction on Whatman GF/C filters, washed with three rounds of 5 ml each of 10% TCA (2 mM lysine), 5 ml cold 95% ethanol, and finally, 5 ml ethanol/ether (3/l). Filters were dried and counted in a toluene-based scintillant made 4% in NCS (Amersham/Searle). Paper filters (Whatman 540), as suggested by Rubin and Goldstein (1970), were unsatisfactory when used with batchwise washings. The assay is sensitive enough to determine lysine pools in less than 10,000 embryos of S. pzwpuratus (allowing for the use of three different extract concentrations as well as quadruplicate aminoacylation determinations).

Validity of the Lysine Pool Assay The isotope dilution assay requires that tRNA be limiting so that addition of the lysine beyond some point (750 pmol) does not alter the extent of the reaction. This was found to be true for reactions containing a maximum of 2 ODzc,, tRNA (binding capacity of 62 pmol L-lysine per ODz&, even though the standard 750-pmol lysine reaction volumes showed a linear aminoacylation response to added tRNA at least up to 6 ODzo. Aminoacylation was completely dependent on the added tRNA. The strategy of the assay used was to utilize the dim-

GOUSTIN AND WILT

35

Protein Synthesis in Urchin Embryos

satisfactory. Incorporation was linear for at least 60 min (data not shown) and high rates of incorporation were achieved. The absolute rates of total protein synthesis were x calculated using data from the incorporation curves and .J* _/l_j / the measurements of the precursor pool specific activI 77hr 83 hr ity, as explained under Materials and Methods. Figure ,. 72 hr 1 shows representative protein incorporation data from an experiment carried out over the first 8 hr of devel. //I opment, and Fig. 2 shows the measurements of the ly/ sine pool specific activity for these same experiments. i-i Conversion of the protein incorporation data into molar -I I 10 20 30 10 20 10 20 30 10 20 30 accumulations gives the data which are displayed in MINUTES Fig. 3. This figure also presents experimental results FIG. 1. Incorporation of [3H]lysine into total protein in 30-min lafrom other similar experiments encompassing develbeling experiments. S. purpuratus embryos were cultured to the deopment up to the 200-cell stage. The absolute rates rise sired stage and then exposed to 18.5 &i/ml of L-[4,5-3H]lysine. Alifrom a little more than 100 pg protein hr-’ embryo-’ quots were taken at 10,20, and 30 min and homogenized; homogenates (1.1 hr) to about 250 pg hr-’ embryo-’ at 3-4 hr, and provided both the incorporation material and the material for pool assays (Fig. 2). For incorporation data, aliquots of the homogenate then rise again to over 500 pg/hr/embryo, in agreement were used for determination of protein. with the available data of Fry and Gross (1970) who used leucine as a label. It appears that there are two general periods of ininution of ‘*C incorporation into acylated product with successive inputs of 3H-labeled or cold exogenous lysine. crease in rate. The first period probably reflects the Standard curves with O-2500 pmol of added [12C]lysine mobilization of maternal mRNA into polyribosomes were carried out each time determinations were done. (Humphreys, 1971). The explanation for the second rise Since we know the specific activity of the endogenous is not as clear; one tempting possibility is that it reflects the input of newly synthesized mRNAs, which begin to constant input of [14C]lysine, the extent of diminution accumulate with an acceleration in the rate of RNA of 14C-cpmin the acylated product by the introduction of (diluting) 3H-labeled embryo lysine aliquots of un- synthesis seen at the g-to 16-cell transition (Wilt, 1970). known specific activities, allows us to calculate the mole contribution of the 3H-labeled exogenous (embryo) ly- Changes in Polysome Projles sine. Since all of the 3H-cpm in the extract are in lysine, Embryos homogenized in EGTA buffer containing we can use these two pieces of information to arrive at Triton X-100 show practically no polysome degradation, a specific (3H) radioactivity of the embryo lysine pool. as monitored by lack of pulse-labeled amino acids associated with the 80 S monosome peak. Furthermore, Calculation of the Rate of Protein Synthesis 21 hr

35 hr

43 hr

Incorporation of lysine into protein (dpm/embryo containing 40 ng protein) at 10, 20, and 30 min of incorporation is converted to molar incorporation by dividing the accumulated protein radioactivity by the average specific activity of the lysine during the 10 min preceding the time point in question. The lysine content of newly synthesized protein is assumed to be due to contributions to nonhistone proteins (6% lysine) and histones (12% lysine) in varying proportions as determined by Goustin (submitted). The actual percentages of lysine used range from a low of 6% in 1-hr zygotes, to a high of 8% at the 200-cell stage of development.

21 hr

43 hr

35 hr

E I!?I’LT 77 hr

72 hr

L

10

20

30

10

20

30

MINUTES

RESULTS

Absolute Rates of Protein Synthesis The use of high embryo concentrations and low molar concentrations of high specific activity lysine proved

FIG. 2. Measurements of [‘Hllysine pool specific radioactives in embryos from Fig. 1. Portions of the same homogenates as in Fig. 1 (250 ~1) were treated with trichloroacetic acid and the acid-soluble material processed for pool assays as described under Materials and Methods.

36

DEVELOPMENTAL BIOLOGY

VOLUME 82, 1981

when background absorbance due to sucrose is accounted for, the presence of uv absorbing material in the polysome region that is insensitive to RNase (5 pg/ ml, 5”C, 5 min) is virtually nil, and the “polysomal” absorbance removed by RNase all appears in the 80 S monosome peak. Hence, we have monitored the distribution of Azso absorbing material in sucrose density gradients for various developmental stages as a way to determine what fraction of the ribosomes are in polysomes. A typical result for the 200-cell stage is shown in Fig. 4. A prominent 80 S monosome peak is evident, as well as two broad bands of polyribosomes sedimenting with modes of 220 S and 400 S, corresponding to the s (slow) and r (rapid) polysomes first described by Infante and Nemer (1967). Subribosomai peaks at I 5 55-60 S are sometimes obvious (cf. 2-hr embryo, 125 IO Mlllhl,ters I” gradtent Fig. 5). FIG. 4. Absorbance profile of a postmitochondrial supernatant preFigure 5 presents a number of experiments for eggs supernatant was and embryos at various early cleavage stages, as well pared from 200-cell embryos. A postmitochondrial prepared from embryos grown at 15°C to the 200-cell stage (12 hr as the prism stage. It is important to note that there after fertilization), and 0.5 ml of it containing 6 ODzso of material was is virtually no material absorbing at 260 nm in the poly- layered on a 12-ml gradient of sucrose (0.45 to 1.8 Af) made up in 0.35 some region of the unfertilized egg gradient (Fig. 5a). M KCI, 10 m&f Tris, pH 7.41,lO m.Af Mg(OAc)z. The gradient was spun This phenomenon is observed even under conditions in at 37,750 rpm, 170 min, in the SW 41 rotor (Spinco) at 3°C. The which the gradient has been heavily overloaded (over gradient was fractionated using a peristaltic pump; fractions were read for refractive index (closed triangles) and absorbance at 260 nm. 10 ODze, in 0.5 ml loaded on a 12-ml gradient, SW 41) Absorbance due to sucrose was subtracted out and the values for with postmitochondrial supernatant. In this case, the ODzso were plotted (open circles). Sedimentation coefficients (s values) total absorbance at 260 nm (after subtraction of the were calculated using the tables of McEwen (1967). absorbance contributed by the sucrose) in the polysome region of the gradient has been estimated to be less peak, even with the pelleted material scored as polythan 1% of the absorbance of the material in the 80 S somes. In most cases, an absorbance reading was taken of material pelleting to the bottom of the gradient tube during centrifugation. This material was resuspended in NETS buffer (0.1 M NaCl; 10 mM Tris-HCI, pH 7.4; 1 mM EDTA; 0.3% SDS) and read for absorbance at 260 and 280 nm. (Its ODz6,,is represented as fraction P on the gradient profiles.) This material comprised a substantial portion of the total recovered absorbance at 260 nm (especially, Figs. 5e and f). The ratio of absorbance at 260 nm to that at 280 nm (ODzso/ODzso)of this pellet ranges from 1.65 to 1.8’7,and we assume these are sedimented polysomes. These gradient profiles therefore allow us to calculate the fraction of ribosomes that sediment as polysomes. FIG. 3. Changing absolute rates of protein synthesis and levels of An example of this estimation is provided in Fig. 5c for polysomes during development. Measurements of absolute rate of the four-cell stage. The broken line beneath the 80 S protein synthesis were conducted on embryos of S. purpuratus grown peak in this panel separates the area under that peak at 15°C to various stages. The numerals on the top of the figure representing monosomes (the area above the broken indicate the number of cells/embryo. Embryos were labeled with line) from the area under that peak representing the [3H]lysine at 15°C and rates of protein synthesis calculated utilizing measurements of lysine pool specific radioactivities as shown in Figs. tail of the highly absorbing material at the top of the 1 and 2 and measurements mazle at other stages. The fraction of gradient [the area beneath the broken line and to the ribosomes in polysomes at various developmental stages was deterright of the peak). The absorbance of polysomes (all mined from absorbance profiles as shown in Figs. 4 and 5. The curve fractions < 100 S + pellet) is divided by polysome through the points is an attempt to hand draw a reasonable fit to the + monosome absorbance. The results of 13 such deterdata on protein synthesis.

GOUSTIN AND WILT

k P24

,_

P

2

2 4 Mflllters

6

4.5 hr 4

6

8

10

125

6

O” P

2

4 Mllllters

37

Synthesis in Urchin Embryos

Protein

8 10 I” gradlent

12E

L2hr 6

8 10 I” gradlent

12

FIG. 5. Absorbance profiles (260 nm) of postmitochondrial supernatants from embryos of various stages. Postmitochondrial supernatants were prepared from embryos at six developmental stages from unfertilized eggs (a) to prism (f); each was centrifuged and analyzed as explained for Fig. 4.

minations on successive developmental stages have been graphed in Fig. 3, superimposed on a summary of the results on the measurement of absolute rates of protein synthesis. The ordinates for the two sets of data have been chosen so as to indicate a congruence. Further, five stages have been selected for which measurements have been made both for absolute rates of protein synthesis and fraction of ribosomes engaged in protein synthesis (Fig. 6). The data indicate a straight line that extrapolates near the origin. DISCUSSION

The data reconfirm the change in the absolute rate of protein synthesis during the first half-day of sea urchin development, first. described by Berg (1965) in Lytechinus anamnesus. The mobilization of the protein synthetic capacity that occurs at fertilization (Hultin, 1950; Nakano and Monroy, 1958; Epel, 1967) is evidenced

by the mobilization of ribosomes into polysomes (Fig. 3 and Humphreys, 1971) and a rising rate of protein synthesis (Fig. 3). The amount of RNA in a sea urchin embryo remains constant over the first 24 hr of development at about 3 ng per embryo. Furthermore, ribosomal RNA accounts for a constant fraction of the total RNA fromn both unfertilized eggs and embryos (Nemer and Infante, 1967) at about 85% of the total RNA. From these two numbers, and the slope of the line of Fig. 6, we can calculate the rate at which a polysomal ribosome elongates a nascent peptide chain. The calculation assumes only that all protein synthesis occurs on polyribosomes and each polysomal ribosome is an equally efficient growing center for polypeptide chains. The calculation is provided in Table 1. The estimate of 1.8 amino acids per second depends on the accuracy of four sources’upon which some comment isSnseful: (1) the new measurements of absolute protein synthetic rates presented here; (2) the new measurements on the fraction

38

DEVELOPMENTALBIOLOGY

of ribosomes in polysomes; (3) the number of ribosomes per embryo; and (4) the assumption that all protein synthesis takes place on polysomes. This last assumption is made without further justification. The measurements reported here for absolute rates of protein synthesis (122 to 540 ng per embryo per hour) agree well with those of Fry and Gross (1970), who using different amino acid precursors (valine and leucine) and a different method of measurement for pool specific activity (automated amino acid analysis) found values for absolute rates that range from 132 to 805 pg per embryo per hour for S. purpuratus labeled at 15”C, using lower embryo concentrations, but higher molarities of exogenous precursor. The immediate precursor to protein synthesis is the aminoacylated tRNA; it has been argued that measurements should be made of this pool specific activity instead of the free amino acid pool. However, recent measurements on eggs and embryos of S. purpuratus (Regier and Kafatos, 1977) indicate that the pool specific activities of the free pool closely reflect those of the acylated pool (within a factor of 1.5). Similarly, the protein synthetic rates calculated by Regier and Kafatos (1977) for the gastrula of S. purpuratus using an assay for aminoacyl pool specific activity averaged 597 pg per embryo per hour, in remarkable agreement with the values reported here. Several attempts exist in the literature at a quantification of the fraction of ribosomes in polysomes (Mon72 E6oo -”

200~ccl I

.’ L

k

c 500 ol cl

,400 5 L ” 300 :T-: 200 2

0

16-cell

TABLE 1 CALCULATION OF AVERAGE PEPTIDE ELONGATION RATES

(S. purpuratus, 15°C) (1) Total RNA (per embryo)

2.8 ng

(2) Fraction of total RNA that is rRNA*

0.85

(3) Ribosomal RNA (per embryo)

2.4

(4) RNA component of a ribosome’

2.12 X lo6 daltons

(5) Moles of ribosomes per embryo

1.1 fmole

ng

(6) Slope from Fig. 6

790 pg amino acid incorporated per embryo/ hr/l.l fmole of ribosomes

(7) Slope as in (6) on a mole per second basis

2.0 fmole amino acid/ sec/l.l fmole of ribosomes

(8) Taking (5) into account

1.8 mole of amino acid incorporated/see/mole of polysomal ribosomes

’ Three measurements exist for the amount of total RNA in the egg of S. purpuratus. They include (1) the single measurement of Whiteley (1949), who measured phosphate in RNA, estimating total RNA at 3.25 ng per egg; (2) the measurements of Nemer and Infante (1967) who estimated phenol-extractable RNA between 2.56 and 3.08 ng per egg; and (3) the data of Washburn (1971), who set this number at 2.2-3.2 ng per egg. We assume an extinction coefficient for RNA of 40 rg per &o. ’ Nemer and Infante (1967) measure the fraction for both the egg and embryo of S. purpratus to be 85.6%. We use 85% for convenience. ‘This factor assumes 1 mole each of 26 S RNA (1.4 X lo6 daltons), 18 S RNA (0.68 X lo6 daltons), and 5 S RNA (38,000 daltons) per ribosome.

\ 4-cell li.l

\ h&Cell

T 2-cell

5 100 2 I’

a’ Ol

VOLUME82, 1981

IO 20 30 40 50 60 Percent nbosomes in polysomes

FIG. 6. Absolute rates of protein synthesis as a function of percentage ribosomes in polysomes. The data shown in this figure have been chosen from those in Fig. 3; only those stages for which both kinds of measurements (protein synthetic rate and percentage ribosomes in polysomes) existed were utilized. At five stages of development (2-, 4-, 8-, 16-, and 200-cell stages), measurements were made at 15°C of the absolute rate of whole-cell protein synthesis and expressed on a per embryo basis (ordinate). Separate measurements were made (on embryos from the same stages) of the percentage of ribosomes in polysomes (abscissa). Assuming that ribosomes per embryo remain constant at 1.12 fmole per embryo (about 6.75 X lo’), the slope (obtained by linear regression) of this line indicates that each polysomal ribosome is polymerizing amino acids into protein at a rate of 1.8 peptide bonds/set (see Table 1 for calculations).

roy and Tyler, 1963; Infante and Nemer, 1967; Humphreys, 1971). Only Infante and Nemer (1967) report measurements of this fraction in S. purpuratus; they examined several developmental stages up to 24 hr after fertilization. Their data agree with those presented here, with the exception of those for the later stages (10 hr and beyond) and the unfertilized egg. They found that the fraction of ribosomes in polysomes increased to 50% by 10 hr, then fell off markedly after 18 hr (Infante and Nemer, 1967). No such decline was seen in the current work; the difference may lie in our inclusion of Triton X-100 and EGTA in the homogenization buffer. The high background of material sedimenting in the polysomal regions of the unfertilized egg-sucrose gradient (Infante and Nemer, 1967) could be due to aggregates that would dissociate with the high Triton X-100 concentrations utilized in the present work. The thesis of Washburn (1971) includes a large num-

GOUSTIN AND WILT

Protein

ber of independent estimations of RNA content per egg of S. purpuratus. Although she noted a variation between females by as much as 45% in RNA content (range of 2.2 to 3.2 rig/egg), this variation would be minimized in the present experiments by use of multiple batches of eggs on several occasions. Hence, it is unlikely that the estimates for general peptide elongation rate reported here are in error by more than a factor of 2, allowing for an uncertainty of 25% in each of the three measurements enumerated above. The elongation rates for general protein postulated here (1.8 amino acids polymerized per growing center per second) are in agreement with older values existing in the literature. Humphreys (1969) made measurements of the average time a nascent chain remains on polysomes from fertilized eggs of Lytechinus pictus (incubated at 18°C). His data indicate that newly synthesized protein spends an average of 1.16 min as nascent chain on polyribosomes (Humphreys, 1969). We can treat this number as a “half-transit-time” (Fan and Penman, 1970). Assuming an average molecular weight of 30-50,000 daltons for the proteins synthesized by sea urchin embryos, Humphreys’ data argue for elongation rates on the order of 2-3.3 amino acids per second per growing center at 18°C. If we assume a Q10of 3.2 for protein synthesis, these numbers would be 1.4-2.3 amino acids per second if normalized to 15°C. In contrast, recent measurements made on the rates of peptide chain elongation before and after fertilization of S. purpuratus eggs demonstrate slower elongation rates. There is good agreement between the data of Brandis and Raff (1978, 1979) and Hille and Albers (1979) which convincingly demonstrate a 2.5-fold increase in this elongation rate, from 0.13 amino acid per second in the unfertilized egg to 0.33 in the zygote.3 None of the data presented in the present work are sufficient to address the matter of the 2.5-fold increase in the elongation rate at fertilization (Brandis and Raff, 1978). Nevertheless, the elongation rate they have determined for zygotes still falls short of our estimate by more than 5-fold. The reasons for this discrepancy are not clear. The data presented here and those presented by Hille and Albers (1979) and Brandis and Raff (1978, 1979) represent distinctly different experimental approaches to the measurement of peptide elongation rate. The method of Fan and Penman (1970), which compares curves for total incorporation and polysomal incorporation, was used in the experiments of Brandis and Raff (1978,1979) and Hille and Albers (1979). Brandis and Raff (1978) 3 After normalizing the 16.5”C data of Brandis and Raff (1978) and the 12°C data of Hille and Albers (1979) to an intermediate temperature of 15”C, assuming a Qlo of 3.2.

Synthesis

in Urchin Embryos

39

used a second method in addition (Bremer and Yuan, 1968), which involves only a separation of proteins according to size, and a quantitiation of incorporation into various size classes at several time points. The method of Humphreys (1969) represents a third approach, as we have described. Our data represent yet a fourth approach. Each of these indirect methods has its own problems and each contributes to an experimentally difficult determination. A fifth approach has been used, one that represents the only direct measurement of peptide elongation rate in urchin embryos. This method compares the incorporation of histidine into N- and Cterminal cyanogen bromide fragments of histone H2B, and assumes only that protein synthesis is a linear polymerization of amino acids beginning at the N-terminus. For 12-hr embryos labeled at 15”C, this last method has been used (Goustin and Wilt, submitted) to measure the elongation rate of histone H2B and Hl to be 0.7 and 0.84 codon set-‘, respectively. These last values fall in between those of Brandis and Raff (1978, 1979) and Hille and Albers (1979) and the calculation of 1.8 codon set-’ presented in Table 1 of this paper. Although histone peptide elongation rates may not closely reflect those of other protein, the problem of overall peptide elongation rates could clearly benefit from further experimentation. REFERENCES ARCECI, R. J., and GROSS, P. R. 1977. Noncoincidence of histone and DNA synthesis in cleavage cycles of early development. Proc. Nut. Acad. Sci. USA 74(U), 5016-5020. BERG, W. E. 1965. Rates of protein synthesis in whole and half embryos of the sea urchin. Exp. Cell Res. 40, 469-489. BRANDIS, J. W., and RAFF, R. A. 1978. Translation of oogenetic mRNA in sea urchin eggs and early embryos. Demonstration of a change in translation efficiency and following fertilization. Develop. Biol. 67, 99-113. BRANDIS, J. W., and RAFF, R. A. 1979. Elevation of protein synthesis is a complex response to fertilization. Nature (London) 278, 467-

469. BREMER, H., and YUAN, D. 1968. Chain growth rate of messenger RNA in Escherichia coli infected with bacteriophage T4. J. Mol. Biol. 34, 527-540. DAVIDSON, E. H. 1976. “Gene Activity in Early Development,” 2nd ed. Academic Press, New York. EPEL, D. 1967. Protein synthesis in sea urchin eggs: A “late” response to fertilization. Proc. Nut. Acad. Sci. USA 57, 899-906. FAN, H., and PENMAN, S. 1970. Regulation of protein synthesis in mammalian cells. I. Inhibition of protein synthesis at the level of initiation during mitosis. J. Mol. Biol. 50, 655-670. FRY, B. J., and GROSS, P. R. 1970. Patterns and rates of protein synthesis in sea urchin embryos. II. Calculation of the absolute rates. Develop. Biol. 21, 125-146. GOUSTIN, A. S. Two temporal phases for the control of histone gene activity in cleaving sea urchin embryos (S. purpuratus). Submitted for publication. GOUSTIN, A. S., and WILT, F. H. Direct measurement of histone peptide elongation rate in cleaving sea urchin embryos. Submitted for publication.

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