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Ribosomal analysis of rapid rates of protein synthesis in the Antarctic sea urchin Sterechinus neumayeri.

Introduction

Marine organisms in Antarctica's Southern Ocean reside in one of the coldest marine environments on Earth, where seawater temperatures are usually subzero for most of the year (Littlepage, 1965; Hunt et al., 2003). Organisms in such "extreme" environments have evolved many unique biochemical adaptations for specific protein-regulated processes, as in Antarctic fish (Chen et al., 1997; Detrich et al., 2000; Fields et al., 2001; Kawall et al., 2002). Generally, for most animals protein synthesis is an ATP-demanding process that accounts for a significant proportion of the metabolic "cost of living," ranging from 19% to 42% of metabolic energy expenditure (e.g., Hawkins et al., 1986; Houlihan, 1991; Podrabsky and Hand, 2000). The highly conserved nature of protein synthesis, and its large metabolic demand for ATP, has resulted in many studies of protein synthesis (Fraser and Rogers, 2007) as a mechanism for regulating metabolic responses to different environmental conditions (e.g., low temperature: Smith and Haschemeyer, 1980; low oxygen: Hand, 1996; low food: Pace and Manahan, 2006, 2007a).

Recent studies have found that rates of protein synthesis in some Antarctic organisms are comparable to those in similar organisms from warmer habitats (Fraser et al., 2002; Pace and Manahan, 2007b). When rates of synthesis were measured in gill lysates of temperate and Antarctic scallops, rates of synthesis at 15 (degrees) C were higher in the Antarctic species (Storch and Portner, 2003). Along with high rates of protein synthesis in the cold, high concentrations of total RNA have also been measured in Antarctic marine invertebrates (Marsh et al., 2001; Robertson et al., 2001; Fraser et al., 2002, 2004). It has been proposed that high rates of protein synthesis in Antarctic species are achieved, in part, by elevated amounts of RNA per cell (Marsh et al., 2001; Fraser and Rogers, 2007). High amounts of RNA might compensate for low activities of RNA synthetic capacities, resulting in high rates of protein synthesis cold environments. Direct measurements to test these hypotheses are, however, still lacking. To our knowledge there are no direct studies of translational rates and biosynthetic capacities of ribosomes isolated from Antarctic organisms.

There is a considerable literature, published over the past decades, on subcellular analyses of ribosomal activities as a direct mechanistic basis for quantifying translation rates in many animal species--e.g., arthropods, echinoderms, mammals, and molluscs (Fan and Penman, 1970; Brandis and Raff, 1978; Goustin and Wilt. 1981; Hershey, 1991; Hofmann and Hand, 1992; Hand, 1996; Frerichs et al., 1998; Larade and Storey, 2002). Such studies have significantly increased our understanding of the activities of protein synthetic machinery during fundamental biological processes such as cell division (Fan and Penman, 1970). Additionally, ribosomal analysis has been applied to the study of metabolically suppressed states (e.g., hibernation: Frerichs et al., 1998) and to anoxia-induced metabolic depression (Hand, 1996; Larade and Storey, 2002). In developmental biology, embryonic stages of sea urchins have been important organisms for studying regulation of DNA, RNA, and protein synthesis (reviewed by Davidson, 1986). Sea urchin embryos have been used extensively to elucidate the role and regulation of protein synthesis in the complex suite of biochemical processes that are initiated soon after fertilization and during early cell division (Epel, 1967; Humphreys, 1969, 1971; Rinaldi and Monroe, 1969; Winkler et al., 1980; Rees et al., 1995; also reviewed in Davidson, 1986). In particular, direct measurement of ribosomal transit times and peptide elongation rates have been the focus of several studies attempting to understand the mechanistic basis of the enormous increase ([greater than or equal to]100-fold) in rates of protein synthesis that occur immediately after fertilization (Berg and Meters, 1979; Hille and Albers, 1979; Goustin and Wilt, 1981, 1982).

In the current study we present an analysis of ribosomal activities related to rates of protein synthesis in developmental stages of the Antarctic sea urchin Sterechinus neumayeri (Meissner, 1900). During development of this species at subzero temperatures, elevated rates of protein synthesis and turnover have been reported; these high rates occur for uniquely low metabolic costs (Marsh et al., 2001; Pace and Manahan, 2007b). These elevated rates measured at--1 (degrees)C are equivalent to those reported for other species of sea urchins that develop at much higher temperatures (15-25 (degrees) C). Elevated rates of mRNA synthesis have also been measured in embryos of the Antarctic sea urchin (Marsh et al., 2001). While amounts of total RNA have been quantified in several Antarctic organisms (reviewed in Fraser and Rogers, 2007), direct measurements of protein synthetic capacity--at the level of ribosomal activity on mRNA transcripts--have not been undertaken to our knowledge for any Antarctic species. The mechanisms remain unknown as to how rates of mRNA synthesis, cellular mRNA amounts, and rates of protein synthesis are related in Antarctic organisms. High rates of protein synthesis in Antarctic organisms might simply be the outcome of elevated amounts of RNA. Alternatively, high protein synthesis rates might be regulated by high ribosomal translational activities (i.e., fast peptide elongation rates in the cold). In this study we report that developing stages of an Antarctic sea urchin possess high peptide elongation rates at subzero seawater temperatures, comparable to rates previously reported for developmental stages of temperate sea urchins. These findings for the Antarctic sea urchin support the hypothesis that fast peptide elongation rates are the basis for high rates of protein synthesis in this species. This result has important implications for understanding the mechanisms of temperature compensation for organisms living in cold environments.

Materials and Methods

Spawning and animal culturing

All seawater used for culturing and for experiments was sterile-filtered (0.2-[micro]m pore-size). Gravid adults of the sea urchin Sterechinus neumayeri were collected from McMurdo Sound, Antarctica. Adult animals were maintained in the Crary Laboratory aquarium at McMurdo Station at near-ambient conditions (--1.0 [degrees]C). Animals were spawned by intracoelomic injection with 0.5 mol [l.sup.-1(1) KC1. All cultures were started from gametes that had a fertilization success of 90% or greater. Fertilized eggs were suspended in 20 1 of seawater and gently stirred. Cultures were kept at the ambient temperature conditions of the seawater in McMurdo Sound (ca.--1.0 [degrees]C). Upon reaching the larval feeding stage, larvae were fed Dunaliella tertiolecta at an algal concentration of 15 cells [mu][1.sup.-1]. Culture water was changed once every 3 days.

Separation of ribosomes in eggs, embryos, and larvae

All reagents were made in diethylpyrocarbonate (DEPC)-treated water and stored in reagent bottles that had been heated for 5 h at 210 [degrees]C to eliminate ribonuclease activity. Homogenized preparations of the specific developmental stages under study were ultracentrifuged for analysis of the distribution of ribosomes in monoribosomes and polyribosomes. Individuals were enumerated and 100,000 were prepared for each analysis. Animals were immediately washed in a Pipes homogenization buffer (0.25 mol [1.sup.-1] NaCl, 5 mmol [l.sup.-1] MgAc, 25 mmol [1.sup.-1] EGTA, 1 mmol [1.sup.-1] DTT, 10 mmol [1.sup.-1] Pipes pH 6.8, 0.1% Triton X100) containing a protease inhibitor cocktail (catalog # P8340, Sigma Chemical Co.). Homogenates were resuspended in a final volume of 3 ml of buffer and loaded into a 7-ml Kontes glass homogenizer. Ten gentle passes of the B-pestle (large clearance) followed by 10 gentle passes with the A-pestle (small clearance) were used to fully homogenize samples. Each homogenate was then immediately centrifuged at 12,000 X g for 15 min at 1 [degrees]C. The post-mitochondrial supernatant was then layered onto a 15%-60% (w/v) discontinuous sucrose gradient prior to ultracentrifugation. This discontinuous gradient was used to separate and quantify peaks associated with monoribosomes and polyribosomes. The gradient consisted of 22 ml of a linear 15%-30% sucrose gradient above 5 ml of a 60% sucrose cushion containing EDTA. Sucrose gradients were made up in Pipes gradient buffer (0.25 mol [1.sup.-1] NaCl, 5 mmol [1.sup.-1] MgAc, 5 mmol [1.sup.-1] EGTA, 1 mmol [1.sup.-1] DTT, 10 mmol [1.sup.-1] Pipes, pH 6.8). Gradients were poured using a 40-ml gradient maker (C.B.S. Scientific Co.) attached to a Labconco autodensiflow gradient fractionator. The 60% sucrose cushion was made in 10 mmol [1.sup.-1] Pipes buffer (pH 6.8) and 10 mmol [1.sup.-1] EDTA. Samples so prepared were ultracentrifuged at 28,000 rpm for 5 h in a SW28 rotor using a Beckman L-80 ultracentrifuge at a temperature of 1 [degrees]C. Under these conditions, monoribosomes (monosomes) migrated to the 15%-30% sucrose layer, while the heavier polyribosomes (polysomes) migrated to the 60% sucrose cushion. Due to the presence of EDTA within the 60% sucrose cushion, all ribosomes dissociated from their respective mRNA transcripts and accumulated as one large polysome peak, independent of the original number of ribosomes associated with each mRNA transcript. Detection of ribosomal material was achieved by pumping gradients from the top of each ultra-centrifuge tube into a spectrophotometer equipped with a 500-[micro]1 flow-cell. Ribosomal peaks were quantified at 260 nm, the UV absorbance used to detect nucleic acids in ribosomes.

Peptide labeling and separation of polyribosome size classes in embryos

Developing stages of the Antarctic sea urchin were further analyzed to determine the range of polysome size classes by quantifying the number of ribosomes attached to each mRNA transcript during translation. Embryos were incubated in 20 ml of filtered seawater containing 10 [micro]Ci of [14.sup]C-labeled amino acid mixture (Perkin Elmer, 50 mCi mmo[l.sup.-1]) for 30 min. After incorporation of isotope, embryos were prepared for ultracentrifugation. Post-mitochondrial fractions were then layered onto a continuous 15%-60% sucrose gradient and ultracentrifuged as described above. Use of this gradient allowed for analysis of the "native" state of the polyribosome size structure and for the determination of the number of ribosomes attached to, and actively translating on, mRNA transcripts. After ultracentrifugation, samples were removed in 1-ml aliquots, starting from the top of the ultracentrifugation tube, and quantified at 260 nm. Additionally, radioactivity in the nascent peptides associated with polyribosome structures was determined in trichloroacetic acid (5% cold TCA) precipitates of each ultracentrifuge fraction. TCA precipitates were collected on GF/C filters (Whatman) and counted in a quench-corrected Beckman scintillation counter.

Determination of ribosomal transit times in embryos and larvae

The amount of time for a ribosome to traverse an mRNA transcript and make a completed peptide (i.e., ribosomal transit time) in the Antarctic sea urchin was measured at--1 [degrees]C. This procedure estimates the average transit time of a ribosome on an mRNA transcript by following the incorporation of radiolabeled amino acids from nascent peptides in the polysome pool to completed peptides in the soluble protein pool (details in Brandis and Raff, 1978, 1979; Hille and Albers, 1979). The polysome pool contains nascent peptides and is detected as the heavier ultracentrifugation fraction because of multiple ribosomes attached to single mRNA transcripts. The soluble protein pool contains completed proteins released from ribosomes that are detected as the lighter ultracentrifugation fractions. Embryos and larvae (250,000) were suspended in 100 ml of seawater containing 30 [micro]Ci of a mixture of 15 different [14.sup]C-labeled L-amino acids (Perkin Elmer, specific activity = 50 mCi mmo[l.sup.-1]). During a time-course of exposure to isotope, at each time point sampled an aliquot was removed (50,000 individuals) and prepared for ultracentrifugation as described above. For this analysis, a discontinuous sucrose gradient (15%-30% linear with a 60% sucrose/EDTA cushion) was employed to quantify the amount of radioactivity associated with polysomes (i.e., radiolabeled nascent peptides still attached to polysomes). The aggregation of polysomes at the gradient-cushion interface at 30% to 60% sucrose allowed for accurate quantification of polysomes, as well as the amount of radioactivity from the [14.sup]C-amino acid mixture that was incorporated into nascent peptides associated with polysomes.

Ribosomal transit time is calculated by quantifying the rate of movement of radioactive amino acids from the nascent peptide pool into the completed peptide pool. Details on these kinetic calculations are given in Fan and Penman (1970); Waterlow et al. (1978); and Hille and Albers (1979). In brief, the analysis is based on (1) measuring the rate at which nascent peptides are released from ribosomes as completed peptides (where the number of ribosomes engaged in protein synthesis is not changing during the analysis) and (2) the total radioactivity in proteins being synthesized (both nascent and completed peptides). Under steady-state conditions, one full transit time after the addition of the radiolabeled amino acids will result in an equal amount of radioactivity in nascent peptides (associated with polysomes) and completed peptides (Waterlow et al., 1978; pp. 33--34). After the first transit time, the radioactivity in the nascent peptides will remain constant. The additional increase in the completed peptide pool for each subsequent transit time will be equivalent to the amount of radioactivity in the nascent peptide pool. For example, in the first transit time there will be equal radio-activity in nascent and completed peptides. In the second transit time there will be 2 times more radioactivity in the completed peptides than in the nascent peptides (which will not change significantly for the remainder of the experiment). After the third transit time, there will be 3 times more radioactivity in the completed peptide pool than in the nascent peptide pool due to an equal amount of new radiolabeled peptides being released at each transit time. By graphing the kinetic change in the total radioactivity of nascent and completed peptides and the radioactivity in the completed peptides, two parallel lines will result (after one transit time has occurred). In such a graphical analysis, after one transit time half of the total radioactivity is from completed peptides. Hence the difference in the x-axis intercepts of completed peptides and total peptides will equal one-half of the ribosomal transit time (i.e., the ribosomal transit time will equal twice the difference in the x-axis intercepts). This analysis gives an estimation of ribosomal transit time that is independent of initiation rate and the number of polysomes (Fan and Penman, 1970; Hille and Albers, 1979). Finally, conversion of ribosomal transit times to rates of peptide elongation was completed in the current study using an average mRNA transcript size of 2000 base pairs for sea urchin embryos (Davidson, 1986, p. 152).

Results

Separation of ribosome classes in eggs, embryos, and larvae

The proportion of ribosomes active in polyribosomes was measured in different developmental stages of the Antarctic sea urchin Sterechinus neumayeri. Sucrose gradient ultra-centrifugation was effective in separating ribosomes not involved in protein synthesis (i.e., present in monoribosomal fraction) from ribosomes that were engaged in synthesis (i.e., in polyribosomal fractions). To quantify the proportion of ribosomes in the monoribosome (monosome) and polyribosome (polysome) fractions, sucrose gradients with an EDTA-containing cushion were used to accumulate all of the different size classes of polysomes (measured as one large peak). In unfertilized eggs, ribosomes were accounted for in the monosome fraction (peak labeled as "monoribosome fraction" in Fig. 1A). The peak labeled "soluble fraction" represents protein that did not enter the sucrose gradient during ultracentrifugation (remained in the homogenization buffer loaded on top of the sucrose gradient). After fertilization there was a large recruitment of ribosomes into polysome structures (peak labeled as "polyribosome fraction" in Fig. 1B). In the data shown in Figure 1B for 5-day-old blastulae, 36.5% of ribosomes were present in the polysome fraction. A further series of analysis on this batch of blastulae (n = 4) resulted in a mean of 45.4% ([+ or -] 1.1%, SEM) of ribosomes in polysomes (Table 1). An additional analysis was conducted on two different batches of blastulae obtained from gametes spawned from different parents (total of three different batches of blastulae). This resulted in a total of 14 ribosomal analyses that gave a mean of 42.3% ([+ or -]1.0%, SEM) of ribosomes present in polysomes (Table 1). In 10-day-old gastrulae, the proportion of ribosomes in polysomes increased to 75% ([+ or -]1.3%) (n = 4 replicates, Table 1). This represents a developmental increase of 1.7-fold in the percent of ribosomes in polysomes in gastrulae compared to blastulae. Later in development, for well-fed larvae (68-days-old), 58.1% ([+ or -]0.6%) of ribosomes were present in the polysome fraction (Table 1).
Table 1

Percent of ribosomes in polysomes for developmental stagesof the
Antarctic sea urchin Sterechinus neumayeri

Developmental stage     Percent ribosomes in
                       polysomes ([+ or -]SE)

Blastula (n = 4)        45.5% ([+ or -]1.1)
Blastula (n = 5)        44.4% ([+ or -]0.4)
Blastula (n = 5)        37.8% ([+ or -]0.8)
Average                 42.3% ([+ or -]1.0)
Gastrula (n = 4)        75.0% ([+ or -]1.3)
Pluteus larva (n = 4)   58.1% ([+ or -]0.6)


The data in Figure 1 show the accumulated total for all polysomes, independent of the number of ribosomes on an mRNA transcript. A more detailed analysis of the different polysome size classes was performed for blastulae (Fig. 2). For this analysis, the sucrose gradient ranged linearly from 15% to 60% and did not employ the use of an EDTA cushion, but rather separated each polysome class by size (ribosome number on a transcript). The 42.3% (Table 1) of ribosomes in polysomes of blastulae were present in polysome structures that ranged from 2 to 8 ribosomes per mRNA transcript (Fig. 2). In addition to defining the size classes of polysomes, information was also obtained on protein synthesis activity as measured by incorporation of [14.sup]C-labeled amino acids. These latter data are given in Figure 2 as the dotted line showing radioactive peaks in eight different polysome fractions. Determination of the cumulative peak areas for radioactivity under the eight peaks labeled on Figure 2 revealed that 93% of the radio-active signal was present in polysomes. These results demonstrate that most of the protein synthetic activity in embryos is associated with polysome structures, with only a relatively small fraction (7%) in monosomes (labeled as peak "1" in Fig. 2) even though the majority of ribosomes are present as monosomes (see large UV peak above radio-active peak "1" in Fig. 2).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Ribosomal transit times and rates of peptide elongation in embryos and larvae

The protocols employed in this study allowed for the reliable separation of polysomes from monosomes and the effective radioactive labeling with amino acids of nascent peptides still attached to polysomes (Fig. 2). The goal in the next series of experiments was to determine ribosomal transit times. The rates at which [14.sup]C-labeled amino acids were incorporated into nascent peptides (i.e., polyribosome fraction, as in Fig. 1B) and into completed peptides (i.e., soluble fraction, Fig. 1) were the quantitative basis for calculating the time required for a ribosome to traverse an mRNA transcript while synthesizing protein (translation). Figure 3 shows the time course for incorporation of radioactive amino acids into the polysome, monosome, and soluble fractions of blastulae. After a 5-min exposure to [14.sup]C-labeled amino acids (Fig. 3A), the incorporation of radioactivity can be seen as a small peak in the polysome fraction. By 17 min (Fig. 3B) more radioactive amino acids have been incorporated into nascent peptides. Only a small amount of radioactivity was present in the soluble fraction, representing completed peptides. This amount of radioactivity was significant, being 6 times above background as determined by liquid scintillation counting (peak maximum = 1254 dpm; background = [approximately equal to] 200 dpm) (graphically, this peak looks small in Fig. 3B due to the scale of the y-axis). After 41 and 71 min (Fig. 3C, D), increases in radioactivity occurred in both the nascent and completed peptide fractions as radioactive proteins were being synthesized by ribosomes and released as completed proteins.

[FIGURE 3 OMITTED]

The kinetic data used to calculate ribosomal transit time are displayed in Figure 4 for experiments spanning therange of developmental stages analyzed in this study (early blastulae [5-day-old] and late-stage larvae [68-day-old]). Radioactive labeling with amino acids of these two pools is separated by a time interval that is the result of the lag in radiolabeled nascent peptides becoming fully labeled, ribosome-dissociated peptides. As referenced in the Materials and Methods section, calculations of ribosomal transit times are based on differences in intercepts of the x-axis for radioactive incorporation into all peptides ([14.sup]C-amino acids in the nascent and completed peptide fractions) and into the completed peptide fraction. This analysis is based on the fact that after one ribosomal transit event there will be equal radioactivity in both the nascent peptide fraction and the completed peptide fraction. After a second ribosomal transit, radioactivity in the nascent peptide fraction will remain constant because ribosomes released completed peptides prior to reinitiating and translating another transcript. This release of completed peptides, following each ribosomal transit event, will result in an increase in radioactive proteins in the soluble fraction. At steady-state radioactive labeling of the polysomes, the rate of incorporation of radioactive amino acids should be similar for the total amount of radioactive amino acids in peptides (the sum of radioactivity in the nascent and completed peptide fractions) and for radioactivity in the completed peptide fraction only. Graphically this results in two parallel lines when plotting the total radioactivity (dpm) in nascent and completed peptides and the radioactivity present in completed peptides (the soluble fraction). In Figure 4A statistical analysis showed that the two lines shown are parallel (ANOVA of combined regressions: df = 1,6; P > 0.05). While this steady-state incorporation is being established, the change in total radioactivity and the radioactivity in the completed peptide fraction will not be similar. Therefore, slope calculations were made on later time points when steady-state incorporation of radioactively labeled amino acids had clearly been achieved (i.e., early data points were excluded from regression analysis in Fig. 4A). Given parallel lines, the lag in movement of radioactivity into the completed protein fraction results in different x-axis intercepts for (i) the rate of radioactive incorporation into the total peptide fraction (nascent and completed) and (ii) the completed peptide fraction. The ribosomal transit time is calculated as 2 times the difference in the x-axes intercepts (i.e., equivalent to one-half the ribosomal transit time: further details on this rationale given in Fan and Penman, 1970; Brandis and Raff, 1978; Waterlow et at., 1978; and Hille and Albers, 1979). For blastulae (Fig. 4A), the x-intercept for total radioactive incorporation (representing nascent peptides and completed peptides) was 40.7 min (see Fig. 4 legend for regression details). The x-intercept for radioactivity in the completed protein fraction was 53.8 min. Hence the average ribosomal transit time for blastulae of the Antarctic sea urchin measured at a temperature of -1 [Degree]C was 26.2 min--that is, 2 times the difference of the x-axis intercepts [(2X (53.8 - 40.7)].

[FIGURE 4 OMITTED]

Figure 4B displays a similar analysis for ribosomal transit lime in larvae (68-day-old), Again, statistical analysis of regressions for the appearance of radioactive amino acids in the total peptide pool and the completed peptide pool, respectively, showed that the slopes were not significantly different (ANOVA of combined regressions: df = 1, 12; P > 0.05). The x-axis intercepts for ribosomal transit times in larvae were 31.5 and 46.78 min for the appearance of radioactive amino acids in the total peptide pool and the completed protein pool, respectively. This resulted in an average ribosomal transit time of 30.6 min.

Rates of peptide elongation can be calculated from the ribosomal transit times displayed in Figure 4. Peptide elongation rates were determined using an average mRNA transcript size of 2000 base pairs (for sea urchins: Davidson, 1986), which is equivalent to 667 codons. For blastulae (Fig. 4A), the peptide elongation rate was 0.42 codons [s.sup.-1] [667 codons [Division Sign] (26.2 min X 60 s [min.sup.-1])]. The peptide elongation rate in larvae (Fig. 4B) was 0.36 codons [s.sup.-1] [667 codons [Division Sign] (30.6 min X 60 s [min.sup.-1])]. Using methods identical to those employed to collect the data illustrated in Figure 4, additional experiments (total n = 7) were conducted on different batches (gametes from different parents) of blastulae, gastrulae, and larvae of S. neumayeri. The average ribosomal transit time was 36.8 min ([+ or -] 7.2, SE) (Table 2). This resulted in an average peptide elongation rate of 0.36 ([+ or -] 0.05, SE) codons [s.sup.-1] (Table 2).
Table 2

Results of regression analysis used to calculate ribosomal transit
times in developmental stages of the Antarctic sea urchin
Sterechinus neumayeri

     Stage          Fraction    x-intercept     [r.sup.2]
                                     (min)

Hatched blastula **  Total            40.67       0.99
                     Soluble          53.80       0.99
Hatched blastula     Total             2.96       0.97
                     Soluble          14.55       0.99
Hatched blastula     Total            -7.20       0.96
                     Soluble           6.69       0.94
Hatched blastula     Total             4.39       0.99
                     Soluble          15.12       0.99
Hatched blastula     Total             7.19       0.99
                     Soluble          38.54       0.99
Gastrula             Total             1.24       0.98
                     Soluble          34.00       0.99
Pluteus larva **     Total            31.50       0.97
                     Soluble          46.78       0.98

                                   Average
                              ([+ or -]SE)

Stage                    Ribosomal         Peptide elongation
                     transit time (min)  rate*(codon [s.sup.1])

Hatched blastula **                26.3                    0.42
Hatched blastula                   23.2                    0.48
Hatched blastula                   27.8                    0.40
Hatched blastula                   21.5                    0.52
Hatched blastula                   62.7                    0.18
Gastrula                           65.6                    0.17
Pluteus larva **                   30.6                    0.36
                                   36.8                    0.36
                                    7.2                    0.05

The ribosomal transit time is calculated as twice the difference
between the x-axis intercepts of total and soluble (14)C-labeled
amino acids incorporation rates (details in text) Regression
analyses based a range of 3-7 data points for all experiments
given below The ribosomal transit time is calculated as twice
the difference between the .x-axis intercepts of total and
soluble (14) C-labeled amino acids incorporation rates
(details in text. Regression analyses based a range of 3-7
data points for all experiments given below.

* Peptide elongation rate calculated using an average
transcript length of 2.0 kbp (= 667 codons).

** Developmental stages for which data are presented in
Figure 4.


These results measuring the ribosomal characteristics of developing stages of S. neumayeri show that, while the rate at which peptides were synthesized by ribosomes did not change significantly during development (Table 2; Fig. 4), the number of ribosomes involved in protein synthesis did vary significantly during development (Table 1, Fig. 1). As will be discussed below, this change in the number of ribosomes engaged in protein synthesis is likely to be a key regulator of whole-animal rates of protein synthesis during development.

Discussion

The goal of this study was to understand the role of translational regulation in maintaining the high rates of protein synthesis known to occur at subzero temperatures in the Antarctic sea urchin Sterechinus neumayeri (Marsh et al., 2001; Pace and Manahan, 2007b). Fractional rates of protein synthesis (i.e., synthesis rate per unit of total protein content) in embryos of the Antarctic sea urchin are about 2% [h.sup.-1] at -1 [degrees]C, in the same range as rates for species of sea urchin from warmer environments (e.g., Arbacia punctulata = 2% [h.sup.-1] at 25 [degrees]C: Fry and Gross. 1970). The observed relationship between high rates of synthesis of mRNA and protein (Marsh et al, 2001), and the presence of large amounts of total RNA in Antarctic species (Fraser and Rogers, 2007), has led to the suggestion that high protein synthesis rates are the result of available synthetic machinery. Specifically, regulation of synthesis rate might be controlled by the presence of high amounts of mRNA that could counteract the assumed low rates of biosynthesis that would occur at low temperatures. Alternatively, rates could be regulated by having high peptide elongation rates from ribosomes translating protein from mRNA transcripts. The latter explanation would require considerable temperature compensation at the level of ribosomal activity. This hypothesis was tested in the current study.

Direct measurements were made of the rate that ribosomes manufacture proteins from mRNA transcripts at cold temperatures. Our results show that the average ribosomal transit time, which represents the amount of time required for a ribosome to traverse an mRNA transcript, was 36.8 [+ or -] 7.2 min for developmental stages of the Antarctic sea urchin at -1[degrees]C (Table 2). From this ribosomal transit analysis, the rate of peptide elongation was 0.36 [+ or -] 0.05 codons [s.sup.-1] (Table 2). This value for the Antarctic sea urchin S. neumayeris is based on the average size of an mRNA transcript in sea urchin embryos (e.g., the average mRNA transcript size in .S. purpuratus is 2000 base pairs: Davidson, 1986). For the Antarctic species, this corresponds to a high rate of translation, comparable to that of sea urchin species developing at temperatures of 15-20 [degrees]C. For instance, Brandis and Raff (1978) measured ribosomal transit times of 12-24 min at 17 [degrees]C for developmental stages of the sea urchin Strongylocentrotus purpuratus. These authors reported a transit time of 25 min for tubulin protein, equivalent to a peptide elongation rate of 0.32 codons [s.sup.-1] for the synthesis of this particular protein. Hille and Albers (1979) measured a ribosomal transit time of 30 min in embryos of S. purpuratus (12[degrees]C), a value similar to the average ribosomal transit time of 36.8 [+ or -] 7.2 min reported here for the Antarctic sea urchin at -1[degrees]C (Table 2). Goustin and Wilt (1981) measured the elongation rates in embryos of .S. purpuratus and reported a rate of 0.33 codons [s.sup.-1] at 15 [degrees]C. This rate, measured at a temperature that is 16 [degrees]C warmer than used for embryos of the Antarctic sea urchin, is remarkably close to the current measurement in S. neumayeri of 0.36 [+ or -] 0.05 codons [s.sup.-1] (Table 2). illustrating high translation rates at -1[degrees]C.

The high rates of peptide elongation in a cold-water Antarctic sea urchin are even more striking when considered in the context of the known relationship between temperature and ribosomal activity. Craig (1975, 1976) studied ribosomal transit times in mammalian cells and showed that rates slowed down with decreasing temperatures. In mouse cells, for instance, transit time was 2 min at 36[degrees]C and increased to 22 min at 10 [degrees]C. In a more comprehensive analysis of temperature sensitivity and ribosomal activity in different organisms and proteins (avian and mammalian cell types, and sea urchin embryos). Goustin and Wilt (1982) constructed an Arrhenius plot showing the relationship between peptide elongation rate and temperature. Their analysis demonstrated a linear relationship from 15 to 41 [degrees]C with a [Q.sub.10] of 3.2. If this [Q.sub.10] value is used to predict the rate of peptide elongation at -1.0 [degrees]C in the Antarctic sea urchin, a rate of 0.05 codons [s.sup.-1] would be expected. The actual measured rate at -1.0 [degrees]C is 7-fold higher than predicted, at 0.36 [+ or -] 0.05 codons [s.sup.-1] (Table 2). Clearly, translational activity at the level of individual ribosomes is "temperature compensated" in the Antarctic sea urchin, and this subcellular process has unique biochemical capacities for protein synthesis in this cold-water species.

The proportion of ribosomes that were actively engaged in protein synthesis (in polysomes) in the Antarctic sea urchin is similar to that reported for other species of sea urchins from warmer environments. Humphreys (1971) found that [greater than]1% of ribosomes were associated with polysomal structures in unfertilized eggs of the temperate sea urchin Lytechinus pictus. After fertilization in this species, ribosomes were immediately recruited to polysomes; in 4-h-old embryos, 20% of ribosomes were in polysome structures. In unfertilized eggs of another temperate sea urchin, S. purpuratus, the percent of ribosomes in polysomes has also been reported to be low, ranging from 0% to 5% (Infante and Nember, 1967; Goustin and Wilt, 1981). By the blastula stage, 50% of ribosomes were in polysomes (Infante and Nemer, 1967). These general patterns of ribosomal recruitment to polysomes reported for temperate species of sea urchins are similar to those we report for the Antarctic sea urchin. Unfertilized eggs of S. neumayeri had no measurable amount of ribosomes in polysomes (monosomes are dominant: Fig. 1A). By the blastula stage (5-d-old, developing at -10 .[degrees]C, an average of 42.3% [+ or -] 1.0% of ribosomes were in polysomes (Table 1; Fig. 1B).

The number of individual ribosomes attached to an mRNA transcript during protein synthesis in the Antarctic sea urchin (Fig. 2) is also consistent with ribosomal distributions in temperate sea urchin embryos. Measurements with high-sensitivity radiochemical analysis (cf. lower sensitivity UV absorption) showed that the maximum discernible number of ribosomes in polysomes of embryos of S. neumayeri was 8 ribosomes per mRNA transcript. This number is similar to results obtained from direct observations with an electron microscope of ribosomes in embryos of the sea urchin L, pictus (Martin and Miller, 1983). In that study, the average number of ribosomes in polysomes for 12-h-old embryos was 7 ribosomes per mRNA transcript. In blastulae of the Antarctic sea urchin, the majority (93%) of the 14C-amino acids synthesized into nascent peptides was found to be associated with polysomal fractions (Fig. 2: data based on integrating the area under the curve showing radioactivity present in the 8 different polysomal fractions detected). This radioactive labeling pattern revealed that the majority of protein synthesis occurred on polysomes in blastulae. Gastrulae had the highest observed proportion of ribosomes in polysomes, at 75% (Table 1). This increase in polysomes is coincidental with measurements of increases in protein synthesis in gastrulae of S. neumayeri (Marsh et al., 2001; Pace and Manahan, 2007b). The increase in the recruitment of ribosomes into polysomes is a common regulatory mechanism for protein synthesis, including dramatic increases in synthesis rates in fertilized sea urchin eggs (Epel, 1967; Jenkins et al., 1978; Goustin and Wilt, 1981). For instance, Regier and Kafatos (1977) found a 113-fold increase in rates of protein synthesis during development from egg to gastrula. The observation that late-staged feeding larvae of S. neumayeri have only 58.1% [+ or -] 0.6% of ribosomes present in the polysome fraction (Table 1) shows that this stage of development has a large reserve of ribosomes somes that could be engaged to further enhance rates of protein synthesis. Even during this larval stage with a relatively high growth (larvae fed at 15 algal cells micro[sup-1], there is still a large, unrealized potential for biosynthetic activity. Regulation of growth at the ribosomal level may be an important factor for the calculated long-life duration times (larvae: Shilling and Manahan, 1994) of these stages of development, despite the capacity for high biosynthetic activities.

The direct measurements of peptide elongation rates from an analysis of ribosomes engaged in translation can be used to calculate a rate of protein synthesis in embryos. This calculation is of value because it provides an independent measurement of protein synthesis rate for direct comparisons with more traditional methods (in vivo labeling with radioactive amino acids of embryos and larvae to measure rates of protein synthesis: Marsh et al., 2001; Pace and Manahan, 2007b). The components and conservative assumptions involved in this calculation of protein synthesis rate are given below and in Table 3. The molecular weight of the ribosomal RNA component of a sea urchin ribosome is 2.12 X 10[sup 6] g mol (Goustin and Wilt, 1981). The amount of total RNA in embryos of S. neumayeri is 115 ng individual [sup-1] (Marsh et al., 2001). Using data from developing stages of sea urchins, we assumed the percentage of total RNA that is rRNA to be 85% (Infante and Nemer, 1967). Therefore, 98.4 ng individual[sup-1] of the total RNA is rRNA (85% X 115 ng individual [sup-1]). Using Avogadro's number in conjunction with the known amount of rRNA and the molecular weight of the RNA in a ribosome, the number of ribosomes in a blastula of S. neumayeri is 2.8 X [10.sup.10] Measurements made in this study of the Antarctic sea urchin show that, of this total number of ribosomes, the percent that was active in blastulae was 42.3% (Table 1), representing 1.2 X [10.sup.10] ribosomes engaged in protein synthesis. The measured peptide elongation rate (translation) was 0.36 codons [s.sup.-1] (Table 2). Multiplying this elongation rate by the number of active ribosomes results in 4.3 X [10.sup.10] amino acid residues being incorporated into peptides per second in a blastula-staged embryo of S. neumayeri. The average molecular weight of amino acids in protein, based on analysis of the amino acid composition of developmental stages of S. neumayeri, is 142.1 ng nmol[sup.-1] (Marsh et al., 2001). Using this value, the mass-rate of protein synthesis is 3.6 ng protein blastula [sup.-1]h [sup.-1] for S. neumayeri at[sup.-1] [degrees]C. A similar set of calculations for gastrulae that have 75% of their ribosomes in polysomes (i.e., 2.1 X [10.sup.10]active ribosomes per individual) yielded a higher protein synthesis rate of 6.5 ng protein embryo [sup.-1]h [sup.-1]. This increase in protein synthesis at gastrulation is consistent with the previous measurements by Marsh et al. (2001) and Pace and Manahan (2007b) where a 2-to 4-fold increase in rates was observed from the blastula to gastrula stage. Importantly, these biosynthetic rates are strikingly similar to rates measured in embryos of echinoderm species from warmer environments (see below). Using the rates determined from direct measurements of ribosomal activity in the current study and a protein content of 203 ng embryo-(1) (Pace and Manahan, 2007b), fractional rates of protein synthesis ranged from 1.8% to 3.2% h-(1). These values are comparable to fractional rates of protein synthesis reported for embryos of warmer-water echinoderms--e.g., Lytechinus pictus (15 [degrees] C) = 0.6% h-(1) (Pace and Manahan, 2006); Stronglyocentrotus purpuratus (16 [degrees] C) = 1.1% h-(1) (Goustin and Wilt, 1981); Arbacia punctulata (25 [degrees] C) = i.9% h-(1) (Fry and Gross, 1970).
Table 3

Calculation of protein synthesis rates in blastulae of Sterechinus
neumayeri utilizing biochemical measurements of RNA amounts and
ribosome activity

Calculation steps  Protein synthesis  Calculated value
                   parameter

(1)                MW                 2.12 X 10 (6) g
                   [sub.ribosome]     mol-(1)

(2)                Total RNA          115 ng
                                      individual-(1)

(3)                mRNA               8.2 ng
                                      individua-(1)'

(4)                rRNA               98.4 ng
                                      individual-(1)

(5)                Number of          2.8 X 10 (1)
                   ribosomes          individual-(1)

(6)                Percent of active  1.2 X 10 (1)
                   ribosomes          individual-(1)
                                      (42.3%)

(7)                Elongation rate    0.36 codons
                                      s-(1)

(8)                Total amino acid   4.3 x 10 [9]
                   incorporation      amino acids s
                   rate               [-1)

(9)                Average molecular  142.1 ng
                   weight of amino    nmol-(1)
                   acid

(10)               Calculated         3.6 ng protein
                   protein synthesis  individual-(1)
                   rate               h-(1)

(11)               In vivo protein    2-6 ng protein
                   synthesis rate     individual-(1)
                                      h-(1)

(1) Goustin and Wilt (1981).

(2), (3), (9) Marsh et al. (2001).

(4) rRNA is 85.6% of total RNA (Infante and Nemer, 1967).

(6), (7) This study.

(11) Marsh et al. (2001); Pace and Manahan (2007b).


What might the molecular biological mechanisms be that allow translational elongation to proceed at such high rates at low temperatures? During adaptation to colder temperatures, amino acid substitutions are proposed to increase the flexibility of the "moving parts" of proteins (enzymes) around active sites, thereby allowing greater access for substrate binding (D' Amico et al., 2002; Hochachka and Somero, 2002; Portner et al., 2007). This mechanism provides the conformational plasticity necessary for high catalytic efficiency in a low-energy environment. The balance between stabilizing interactions (electrostatic interactions, hydrogen bonds, etc.) and flexibility made possible by adaptive changes in protein structure results in kinetic changes (Feller and Gerday, 1997; Fields et al., 2001; Georlette et al., 2004; Johns and Somero, 2004). The increased catalytic efficiency is typically attributed to a reduction in the activation enthalpy of the enzymatic reaction (Georlette et al., 2004). Such cold-adaptation strategies have been observed in enzymes critical to protein synthesis. Studies on Antarctic methanogens (Thomas and Cavicchioli, 2000; Thomas et al., 2001) have shown that elongation factor 2 (EF-2) possesses the attributes of a cold-adapted enzyme. EF-2 is a central enzyme in protein synthesis as it binds and hydrolyzes GTP to cause ribosomal translocation during peptide elongation. Future studies that examine the isolated components of the translational machinery (e.g., ribosomes, ribosomal proteins, aminoacyl tRNA synthetases) of developing embryos of S. neumayeri will greatly increase our understanding of the fundamental principles and mechanisms underlying high rates of protein synthesis at low temperatures.

The data presented here are based on the use of a distinctively different set of protocols from previous measurements of protein synthesis in S. neumayeri and support the conclusion of high biosynthetic capacity during development of this Antarctic sea urchin. This subcellular approach has provided significant new insights into the mechanisms by which these high rates are achieved in the cold. While the presence of high amounts of RNA in cells can be correlated with high rates of protein synthesis, developing stages of the Antarctic sea urchin S. neumayeri possess translational machinery that imparts novel kinetic characteristics, allowing for rapid rates of biosynthesis. These findings contribute to an expanding dataset of unique biochemical attributes for life in the cold and provide new insights into the cellular mechanisms that produce high biosynthetic capacities in animals developing at subzero temperatures.

Acknowledgments

We thank David Ginsburg, Allison Green, and Michael Moore for assistance with culturing embryos and larvae in Antarctica, and Dr. Patricia von Dippe for aspects of the biochemical analysis. We thank Rob Robbins, Howard Tobin, and the staff of the Crary Laboratory at McMurdo Station, Antarctica, and Raytheon Polar Services for support. This work was supported by the National Science Foundation under Grant No. 01-30398.

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DOUGLAS A. PACE(1), ROBERT MAXSON(2), AND DONAL T. MANAHAN(1),*

(1) Department of Biological Sciences, University Park Campus, and (2) Department of Biochemistry and Molecular Biology, Health Sciences Campus, University of Southern California, Los Angeles, California 90089-0371

Received 2 January 2009; accepted 30 September 2009.

* To whom correspondence should be addressed. E-mail: manahan@usc.edu
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