Temperature-dependent protein synthesis capacities in Antarctic and temperate (North Sea) fish (Zoarcidae)
Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany
* Author for correspondence at present address: Animal Ecophysiology, Alfred-Wegener-Institut für Polar- und Meeresforschung, Am Handelshafen 12, D-27570 Bremerhaven (e-mail: dstorch{at}awi-bremerhaven.de)
Accepted 5 April 2005
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Summary |
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Key words: RNA translational capacity, RNA:protein ratio, Zoarces viviparus, Pachycara brachycephalum, cold acclimation, cold adaptation
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Introduction |
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Enabling of the protein synthesis machinery to function at very low
`operating temperatures' in vivo has been suggested to be brought
about by elevated tissue RNA:protein ratios (milligrams RNA per gram protein).
Accordingly, this parameter is commonly used as an indirect measure of the
in vivo protein synthesis capacity of a tissue
(Waterlow et al., 1978;
Sugden and Fuller, 1991
;
Houlihan, 1991
). Increased
RNA:protein ratios have been found upon cold acclimation or during winter in
various tissues of fish, and for the last two decades this finding has been
interpreted to reflect cold compensation of RNA translational activities
(kRNA in vivo, defined as grams protein
synthesized in vivo per gram RNA per day, also known as RNA
translational efficiency), at low temperatures
(Goolish et al., 1984
;
Foster et al., 1992
;
Foster et al., 1993
;
Mathers et al., 1993
;
McCarthy and Houlihan, 1997
;
McCarthy et al., 1999
).
Accordingly, it seems very tempting to extrapolate this interpretation to
cold-adapted stenotherms from polar regions. The increase in RNA:protein
ratios in Antarctic species reflected by increased RNA levels in cold
stenotherms from the Southern Ocean
(Whiteley et al., 2001
;
Robertson et al., 2001
;
Marsh et al., 2001
;
Fraser et al., 2002
) has in
fact been suggested to counteract a thermally induced reduction in RNA
translational efficiency in vivo.
These interpretations imply that in vivo translational efficiency
falls in the cold because of a reduction in individual biochemical processes
involved in protein synthesis. Such a decrement has never been demonstrated.
Synthesis and maintenance of higher RNA levels to counteract the negative
effect of cold temperatures on translational activity may further imply higher
costs of protein synthesis in the cold. Fraser et al.
(2002) proposed that
maintaining considerably elevated tissue RNA concentrations causes high
metabolic costs of growth in the cold compared to temperate and tropical
species. However, increased RNA stability may also lead to enhanced RNA levels
at no extra costs and, in the light of energy savings commonly observed in
Antarctic species, the protein synthesis machinery should rather be energy
efficient with a cold adapted RNA translation apparatus that operates at
enhanced catalytic efficiency. In fact, similar in vitro protein
synthesis capacities in gills of the Antarctic scallop Adamussium
colbecki measured at 0°C and in the temperate scallop Aequipecten
opercularis measured at 25°C, were explainable by the ninefold higher
and, thus, cold-compensated RNA translational capacity (kRNA
in vitro, defined as grams protein synthesized in
vitro per gram RNA per day) on top of a twofold elevated RNA content
(Storch et al., 2003
) at
similar energetic costs (Storch and
Pörtner, 2003
). Elevated RNA contents in cold adapted
ectotherms may therefore be the result of enhanced RNA stability resulting
from low RNA turnover rates, and may not reflect enhanced energy costs.
Elevated levels of mRNA and protein synthesis sites, in turn, would support
short diffusion pathways for newly synthesized proteins to their final usage
sites.
In the present study we tested this hypothesis in two confamilial fish
species adapted to different temperature regimes, the Antarctic eelpout P.
brachycephalum and the temperate eelpout Z. viviparus, which are
both members of the cosmopolitan fish family Zoarcidae. We determined the
translational capacity of the protein synthesis machineries using an in
vitro cell-free system isolated from gills and white muscle. The in
vitro amino acid incorporation system is a far more sensitive and
immediate indicator of RNA translational capacity than the in vivo
RNA:protein ratio (Lied et al.,
1985; Houlihan,
1991
). It has the inherent advantage that both the actual protein
synthesis capacities of the tissue (expressed as mg protein synthesized in
vitro per mg fresh mass per day) and, considering the RNA concentration
within the tissue, the translational capacities of the RNA (kRNA
in vitro) can be quantified accurately under optimized
physiological conditions, i.e. at unrestricted energy and amino acid supply in
the assay (Storch and Pörtner,
2003
).
To our knowledge, this is the first such comparison of confamilial fish species from polar as well as temperate waters and should allow access to potential differences between cold acclimation effects and features of evolutionary cold adaptation to Antarctic conditions. If a capacity for thermal acclimation still exists in polar fishes, such an analysis should also reveal to what extent thermal acclimation may occur differently in polar compared to temperate fish. Based on the findings in the pectinids we hypothesize that the protein synthesis apparatus may be permanently cold adapted in the stenotherms, linked to enhanced RNA translational capacities in excess of effects of higher RNA:protein ratios.
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Materials and methods |
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The long-term acclimation experiment was conducted for 10 months between
February and December 2002. Both eelpout species were acclimated to habitat
(control) temperatures and a common temperature of 5°C, which is well
within the thermal tolerance window of both species
(Van Dijk et al., 1999;
Mark et al., 2002
). Control
animals were kept at 0.0±0.5°C for P. brachycephalum and
10±1°C for Z. viviparus. The second group of both species
acclimated at 5.0±0.5°C and 34±1 PSU were held in the same
large basin, separated by a grid. Throughout the 10-month acclimation period,
control animals (0°C for P. brachycephalum and 10°C for
Z. viviparus), warm-acclimated P. brachycephalum (5°C)
and cold-acclimated Z. viviparus (5°C), were fed live sand shrimp
(Crangon crangon) ad libitum once per week. The validity of
this approach is supported by the observation that Antarctic eelpout displayed
positive growth under these conditions. After 10 months in vitro,
experiments were conducted within 2 months in January/February 2003.
Preparation of cell-free translation systems
Prior to tissue dissection, the fish were killed by a sharp blow to the
head and ensuing transection of the spine. The fish were immediately placed on
ice and tissues were quickly excised. Gills were dissected by cutting the
branchial arches at their sites of attachment. Only the soft gill filaments
without arches were used for lysate preparation. White muscle was taken from
the dorsolateral aspect along the backbone after the skin had been removed.
P. brachycephalum, unlike Z. viviparus, exhibits a
subcutaneous thick layer of fat tissue, which had to be removed along with the
skin. White muscle was freeze-clamped in liquid N2 while the gill
lysate was freshly prepared. The cryo-preservation did not affect the protein
synthesis system as has also been shown for lysates prepared from epaxial
muscle of the cod Gadus morhua
(Lied et al., 1982).
The preparation of lysates from P. brachycephalum and Z. viviparus followed the same procedure. Gill filaments of one to two animals were pooled to provide enough tissue for all measurements. Half of the gill tissue was freeze-clamped in liquid N2 for later examination of amino acid composition in tissue protein, and of RNA and protein content. Approximately 150350 mg of gill tissue was transferred to a chilled, hand operated, loosely fitting 2 ml glass homogenizer and homogenized with five strokes in 1 vol. ice-cold extraction buffer containing 30 mmol l1 Hepes, 250 mmol l1 sucrose, 120 mmol l1 potassium chloride, 10 mmol l1 magnesium chloride, 7 mmol l1 2-mercaptoethanol, 20 mmol l1 dithiothreitol (DTT), adjusted to pH 7.1 at 25°C, and supplemented with 107 U ml1 RNasin ribonuclease inhibitor (Promega, GmbH, Mannheim, Germany). The homogenate was immediately transferred to 1.5 ml Eppendorf tubes and centrifuged at 16 000 g for 30 min at 0°C. The freshly prepared post-mitochondrial supernatant was used as the lysate and kept on ice until the start of in vitro translation assays. Samples of the lysates were frozen in liquid nitrogen for later determination of RNA and of free endogenous phenylalanine (Phe). White muscle lysates were prepared in the same way as gill lysates except that muscle tissue (7501250 mg) was ground to a fine, homogeneous powder under liquid nitrogen using a pre-cooled mortar and pestle before homogenization in 0.6 volumes of ice-cold extraction buffer. This preliminary step was essential for a better mechanical pulping of the stringy muscle tissue.
Cell-free in vitro translation assays
Protein synthesis rates were determined in vitro at four different
temperatures (0, 5, 10, 15°C). Buffer composition and osmolarity of the
cell-free system were set to mimic the intracellular fluid of marine fish and
to compensate for dilution of cellular components occurring upon lysis of the
gill or of white muscle tissue. A refinement of assay conditions was achieved
by preliminary experiments with variable Phe, ATP, GTP and phosphocreatine
(PCr) concentrations measured in gill and muscle lysates of the North Sea
eelpout at 10°C. Previous analyses of temperate versus Antarctic scallop
muscle had demonstrated that such optimized conditions are also effective in
studies of the same parameters in polar confamilials (REF). For measurements
of protein synthesis capacity, translation assays were conducted under
optimized physiological conditions. Incorporation of
[2,3,4,5,6-3H]Phe into protein was measured as a function of time
in 11-µl samples from an incubation medium containing 22.5 µl lysate 75
µl1 and final concentrations of 30 mmol
l1 Hepes buffer (sodium salt, adjusted to pH 7.1 at
25°C), 120 mmol l1 potassium chloride, 7 mmol
l1 magnesium chloride, 5 mmol l1 DTT, 0.2
mmol l1 spermidine, all amino acids except Phe at 0.1 mmol
l1, 6 µmol l1 Phe (including 30 µCi
of [2,3,4,5,6-3H]Phe, Amersham; 116 Ci mmol l1; 1
mCi=37 MBq), 52 units RNasin ribonuclease inhibitor (Promega), 1 mmol
l1 ATP, 1 mmol l1 GTP and 30 mmol
l1 PCr and 2.25 U creatine phosphokinase as an ATP
regeneration system to avoid a limitation of energy supply. The contribution
of low molecular mass components from the lysate to the final concentration
was not included in these numbers. Before starting translation with 22.5 µl
lysate the reaction mixture was allowed to equilibrate in a water bath set to
the experimental temperature of 0°C, 5°C, 10°C or 15°C for 5
min. After the addition of lysate, the assay was quickly subdivided into
aliquots of 11 µl and returned to the water bath set at the experimental
temperature. Reactions were run for given time periods and were terminated by
2 µl pancreatic RNAse (25 U ml l1). Subsequently, 11
µl samples were pipetted onto Phe-saturated, semi-wet Whatman GF/C filters
to minimize non-specific adhesion of radioactive Phe to the filter. Filters
were immersed in ice-cold 10% TCA, containing 5 mmol l1 Phe
as carrier, then washed once in 10% and twice in 5% ice-cold TCA. After a
final rinse in 95% ethanol the filters were allowed to dry in air before
dissolving them in 5 ml scintillation cocktail (Packard, 57% tritium counting
efficiency). Radioactivity in the precipitated protein was determined by
liquid scintillation counting. A zero control, which contained RNAse right
from the beginning of the assay to prevent protein synthesis, was used to
correct for background due to non-specific binding of
[2,3,4,5,6-3H]Phe to components of the lysate. Results were
expressed as [2,3,4,5,6-3H]Phe incorporated into trichloroacetic
acid-precipitable protein (as d.p.m. 11 µl1 assay) and
was later converted to µg protein mg1 fresh mass
day1 (see below).
Analytical methods
Total RNA and free endogenous Phe levels in lysates were determined as
previously described (Storch et al.,
2003; Storch and Pörtner,
2003
). For the determination of RNA and protein contents, tissues
were ground to a fine, homogeneous powder under liquid nitrogen using a
pre-cooled mortar and pestle. Two 100 mg sub-samples of the resultant power
were homogenized in ice-cold 0.2 mol l1 perchloric acid
(PCA). Homogenates were centrifuged at 16 000 g for 1 min at
0°C and the remaining precipitate was washed twice in 0.2 mol
l1 PCA. Subsequently, the pellet was resuspended in 0.3 mol
l1 NaOH and incubated for 1 h at 37°C. Samples (15
µl) were taken for the determination of protein levels using a modified
Lowry technique with bovine serum albumin as a standard (Sigma procedure no.
P5656). Protein and DNA were then precipitated from the remaining alkaline
digest by the addition of ice-cold 20% PCA, and after centrifugation the
resultant acid-soluble fraction was removed for the estimation of RNA levels
by ultraviolet absorption at 232 nm and 260 nm
(Storch et al., 2003
). In
addition to animals used for lysate preparation, further control specimens and
those acclimated to 5°C were taken for measurements of RNA and protein
content. Total amino acid composition of tissue protein was determined by
acidic hydrolysis followed by HPLC analysis (Dr Wiertz, Dipl. Chem. Eggert and
Dr Joerissen GmbH, Hamburg, Germany).
Derived parameters and statistics
Protein synthesis rates in gill and white muscle tissues at experimental
temperatures were determined using the initial, linear intercept of
time-dependent [2,3,4,5,6-3H]Phe incorporation curves. Protein
synthesis rates were converted from d.p.m. 11 µl1 assay
min1 via nmol Phe 11 µl assay1
min1 into µg protein mg1 fresh mass
day1. Rate of phenylalanine incorporation (nmol Phe 11 µl
assay1 min1) =
[2,3,4,5,6-3H]Phe incorporated into protein (d.p.m. 11
µl1 assay min1)/specific activity
(d.p.m. nmol1 Phe). The specific radioactivity, expressed as
Bq nmol Phe1 (1 d.p.m.=1/60 Bq), of each assay was
calculated from the amount of added radioactive and non-radioactive Phe in the
assay plus the measured free endogenous Phe of the lysates.
For the conversion from nmol Phe to grams of protein, one approach is to simply assume that the ratio of the incorporated radiolabelled amino acid to any of the 20 other common amino acids is 1:1 and that the average molecular mass of an amino acid is 110. Using these assumptions, each nmol of Phe incorporated into protein is equivalent to 2200 ng of protein. In the present study, however, we were concerned about potential differences in amino acid composition of protein from the polar and temperate species. Therefore, the concentration ratio of each individual amino acid to Phe was measured in protein of white muscle and gills from both species. These data and the molecular masses of the respective amino acids were used to calculate the correct relationship between nmol Phe incorporated and grams of protein synthesized.
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All data are expressed as means ± standard error (± S.E.M.) unless stated otherwise. Numbers (N) of determinations are given in parentheses or figure legends. Prior to analysis, assumptions of normal distribution of the studied variables and homogeneity of variances were tested. If any of the assumptions was violated, the data were transformed by xx, which resulted in significantly improved normality and homogeneity of variances. Statistical differences at the 5% level were tested using analysis of variance (ANOVA) followed by the StudentNewmanKeuls post hoc test.
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Results |
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Despite identical protocols used for both species free endogenous Phe levels in white muscle lysates of warm-acclimated P. brachycephalum (5°C) were significantly reduced compared to control animals (0°C) but were still significantly higher than in lysates prepared from white muscle of cold-acclimated (5°C) or control (10°C) Z. viviparus (Table 2). There was no detectable difference in free endogenous Phe levels in gill lysates, either between species or within species maintained at various temperatures. The free endogenous Phe levels in the lysates were used to correct for the resulting differences in specific radioactivity.
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RNA and protein concentrations as well as RNA:protein ratios, as traditional indicators of the in vivo capacity of protein synthesis, were measured in tissues of control animals, and of warm-acclimated P. brachycephalum and cold-acclimated Z. viviparus, respectively, for an evaluation of effects of long-term acclimation and evolutionary cold adaptation (Fig. 1). RNA levels in white muscle of control P. brachycephalum (0°C: 0.68±0.06 µg RNA mg l1 fresh mass, N=6) and of control Z. viviparus (10°C: 0.78±0.07 µg RNA mg l1 fresh mass, N=6) were similar (Fig. 1A). In warm-acclimated P. brachycephalum (5°C) white muscle RNA was significantly reduced to 0.48±0.02 µg RNA mg1 fresh mass (N=6), whereas cold-acclimated Z. viviparus (5°C) showed a significant elevation of white muscle RNA to 1.37±0.09 µg RNA mg1 fresh mass (N=6). Thus, there was a significant difference in RNA content of white muscle between species at 5°C. RNA:protein ratios of white muscle tissue (Fig. 1C) followed the patterns of RNA content (Fig. 1A) because of equal protein contents (Fig. 1B) in both species. Gill tissues of both eelpout species displayed very similar values and exhibited no significant changes in RNA and protein concentrations and in RNA:protein ratios upon long-term acclimation to 5°C (Fig. 1DF). Gill RNA content in the Antarctic eelpout at 0°C was 2.71±0.10 µg RNA mg1 fresh mass (N=6) and at 5°C was 3.08±0.19 µg RNA mg1 fresh mass (N=6). These values were very similar to 3.19±0.54 µg RNA mg1 fresh mass (N=6) and 2.71±0.29 µg RNA mg1 fresh mass (N=6) determined in gills of cold-acclimated and of control Z. viviparus, respectively.
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In vitro phenylalanine incorporation
Phenylalanine incorporation into protein requires the complete protein
synthetic machinery of the cell. The system used here resembles the buffering,
osmotic and ionic conditions of the tissues. One goal of this study was to
compare protein synthesis capacities in P. brachycephalum and Z.
viviparus acclimated to various temperatures. By using the
post-mitochondrial supernatant in an optimized cell-free system the capacities
of white muscle and gill lysates of both species could be studied under their
in vivo physiological conditions.
Fig. 2 shows the time course
for the incorporation of [2,3,4,5,6-3H]Phe into trichloroacetic
acid-precipitable material at different temperatures by lysates prepared from
gills of P. brachycephalum (Fig.
2). The time course of in vitro translation in lysates
prepared from both tissues were essentially the same principal pattern,
regardless of acclimation temperature and species, as exemplified by the gill
lysate in Fig. 2: a linear
period of incorporation was followed by a progressive reduction of reaction
velocity. The reaction approached completion in an asymptotic manner
thereafter. A higher degree of incorporation of Phe into protein was achieved
at higher temperatures, combined with an earlier relative slowing of the
reaction. Short periods of linear incorporation in lysates are typical when
compared to other non-reticulocyte cell-free systems
(Hofmann and Hand, 1994;
Kim and Swartz, 2000
).
In vitro protein synthesis and RNA translational capacity
Protein synthesis capacities (µg protein mg1 fresh
mass day1) and RNA translational capacities (mg protein
mg1 RNA day1) were calculated from the
initial linear Phe incorporation rates
(Fig. 2) considering Phe
concentrations in the lysates (Table
2), as well as amino acid composition of tissue protein and the
RNA concentration in tissues (Fig.
1A,D) and lysates.
P. brachycephalum (0°C) compared with Z. viviparus (10°C)
In vitro protein synthesis capacities in white muscle
(Fig. 3A,C) and in gill lysates
(Fig. 3B,D) displayed
significant temperature dependence in control P. brachycephalum
maintained at 0°C (filled circles) and in control Z. viviparus
maintained at 10°C (filled squares). Protein synthesis capacities were
significantly higher over the total temperature range in white muscle of
P. brachycephalum (Fig.
3A) than in white muscle of Z. viviparus
(Fig. 3C), although the same
RNA contents were found in white muscle tissues of both species
(Fig. 1A). An exponential
increase in protein synthesis capacities was observed in white muscle with
rising temperature in control P. brachycephalum and in control Z.
viviparus. However, protein synthesis capacities of white muscle followed
different Q10 values in control P. brachycephalum
(Q10=3.8±0.5, N=5) and control Z.
viviparus (Q10=7.0±0.2, N=3)
(Table 3). The same findings
were even more distinct in gill tissues
(Fig. 3B,D) despite equal RNA
contents in the gills of both species (Fig.
1D). In the temperature range 0 to 10°C protein synthesis
capacities were considerably higher in gills of control P.
brachycephalum (Fig. 3B)
with a significantly lower temperature dependency compared to control Z.
viviparus (Fig. 3D)
(Q10=3.0±0.3 vs Q10=10.6±0.8). At
15°C, protein synthesis capacities of P. brachycephalum were
below those of gill tissues of control Z. viviparus. Activation
energies of protein synthesis were lower in both tissues of control P.
brachycephalum (muscle: Ea=85±9 kJ
mol1; gill: Ea=71±6 kJ
mol1) than in tissues of control Z. viviparus
(muscle: Ea=127±2 kJ mol1; gill:
Ea=155±5 kJ mol1).
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The Z. viviparus cell-free system had a very low rate of protein synthesis at 0°C in both tissues (muscle: 2.5± 0.6 µg protein mg1 fresh mass day1; gill: 3.1±0.7 protein mg1 fresh mass day1) when compared to the much higher protein synthesis capacities in tissues of P. brachycephalum at 0°C (muscle: 11.8±2.2 µg protein mg1 fresh mass day1; gill: 19.7±2.7 µg protein mg1 fresh mass day1). These four- to fivefold higher in vitro protein synthesis capacities in tissues of P. brachycephalum can be explained by the four- to fivefold higher RNA translational capacity in both tissues (control P. brachycephalum: white muscle 4.68±0.83 mg protein mg1 RNA day1, N=5; gill 3.09±0.34 mg protein mg1 RNA day1, N=5, vs control Z. viviparus: white muscle 1.16±0.36 mg protein mg1 RNA day1, N=3; gill 0.57± 0 mg protein mg1 RNA day1, N=3). At its respective habitat temperature of 10°C, protein synthesis capacities were twofold higher in both tissues of control Z. viviparus compared to control P. brachycephalum at its habitat temperature of 0°C.
Control P. brachycephalum (0°C) vs warm-acclimated P. brachycephalum (5°C) and control Z. viviparus (10°C) vs cold-acclimated Z. viviparus (5°C)
As expected protein synthesis capacities
(Fig. 3) and RNA translational
capacities (Fig. 4) in lysates
prepared from tissues of 5°C acclimated specimens (unfilled symbols) were
temperature dependent as already specified for the protein synthesis
capacities of control animals (see above). The different modes of data
depiction in Figs 3 and
4 reveal whether higher protein
synthesis capacities in tissues can be explained by higher translational
capacities of the RNA independent of the RNA content within the tissue.
Fig. 4 depicts the effect of
long-term acclimation to 5°C compared to control temperature on the in
vitro RNA translational capacities in white muscle
(Fig. 4A,C) and in gill
(Fig. 4B,D) of both species. In
this long-term acclimation experiment we found significant differences between
the two species:
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(2) A significant reduction in RNA translational capacities was observed in lysates prepared from tissues of warm acclimated P. brachycephalum compared to the `cold' control animals at 0°C (Fig. 4A,B) over the measured temperature range. In contrast, the RNA translational capacities in lysates of tissues of Z. viviparus declined significantly upon cooling (Fig. 4C,D). This means that RNA translational capacities increased in P. brachycephalum at lower temperatures whereas Z. viviparus experienced a decrease in RNA translational capacities upon cold acclimation when measured in the in vitro cell-free system under the same physiological conditions.
In the light of varying RNA contents upon acclimation the effect of RNA translational capacity on protein synthesis in white muscle was especially interesting (Fig. 3A,C). High RNA contents amplify high RNA translational capacities and support high protein synthesis, as observed in white muscle lysates of control P. brachycephalum. This contrasts with low RNA contents and low RNA translational capacity in white muscle lysates of warm-acclimated P. brachycephalum, which result in significantly lower protein synthesis capacities. In contrast, in Z. viviparus similar protein synthesis capacities result at both acclimation temperatures, indicating compensation upon cold acclimation. White muscle lysates of cold-acclimated Z. viviparus display high RNA contents, which counteract low RNA translational capacities and bring protein synthesis capacity to the same level as seen under control conditions.
Warm-acclimated P. brachycephalum (5°C) vs cold-acclimated Z. viviparus (5°C)
The comparison of both species acclimated to the same temperature is
interesting, especially in white muscle because of the large difference in RNA
levels (Fig. 1A) due to warming
of P. brachycephalum versus cooling of Z. viviparus.
Therefore, in Fig. 5A,B we
contrasted the RNA translational capacities and protein synthesis capacities
of white muscle from warm-acclimated P. brachycephalum and
cold-acclimated Z. viviparus. At an acclimation temperature of
5°C, RNA translational capacity in white muscle lysates of P.
brachycephalum was significantly above the RNA translational capacities
found in Z. viviparus in vitro
(Fig. 5A) over the whole range
of assay temperatures. This was also valid for gill tissues. Despite the much
higher RNA contents in white muscle of Z. viviparus at 5°C the
observed significantly higher RNA translational capacity in P.
brachycephalum yielded similar protein synthesis capacities in both
species (Fig. 5B).
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Discussion |
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The optimized cell-free system developed in the present study allows direct
comparison of protein synthesis capacities in white muscle and gills of P.
brachycephalum and of Z. viviparus acclimated to various
temperatures. The gill and white muscle lysates prepared from P.
brachycephalum and from Z. viviparus in the present study had
between 800- and 3000-fold higher protein synthesis rates than earlier in
vitro determinations in Antarctic fish liver (control P.
brachycephalum: muscle 3.5%, gills 11.8% protein synthesized per day,
measured at 0°C; control Z. viviparus: muscle 5.3%, gills 18.7%,
measured at 10°C). The 1000-fold lower in vitro than in
vivo protein synthesis rates in liver of Trematomus bernacchii
(Haschemeyer and Williams,
1982) and the order of magnitude by which the eelpout cell free
systems exceed the activity of the `Trematomus' cell-free system
corroborate the view that, because of improved methodology, values obtained
here reflect the full in vitro capacity of the protein synthesis
machinery. Capacity results above in vivo rates found in temperate
and polar ectotherms emphasise the validity of our approach, despite the
admittedly artificial nature of the in vitro system.
RNA translational capacity and RNA translational efficiency
These two terms should be carefully defined, before comparing in
vitro capacity and in vivo translational efficiency. The in
vitro translational capacity reflects the topmost capacity of the
ribosomes to synthesize protein and can be determined by optimizing the in
vitro assay as described above (kRNA in
vitro indicated by the rate of in vitro protein synthesis
per unit RNA; Storch et al.,
2003). The RNA:protein ratio in freshly collected tissues is
commonly used as an indirect indicator of this capacity in vivo. For
example gills are among the most active tissues with respect to protein
turnover, which is reflected in a high RNA:protein ratio in both eelpout
species at all temperatures when compared to white muscle
(Fig. 1C,F). Muscle naturally
shows very low protein turnover rates associated with high protein retention
times. This was indicated by virtually identical protein concentrations in
white muscle and gills (Fig.
1B,E) at significantly lower RNA contents in muscle
(Fig. 1 A,D). The translational
efficiency, in turn, is the extent to which the RNA translational capacity is
utilized in vivo (kRNA in vivo indicated
by the rate of in vivo protein synthesis per unit RNA;
Waterlow et al., 1978
;
Houlihan, 1991
).
With adequate precaution, we attempt, in the following, to compare the measured in vitro capacities with in vivo protein synthesis rates and translational efficiencies found in literature. In vivo rates of protein synthesis and translational efficiency measured in gills and white muscle of various fish species in relation to their ambient temperature were compiled (Table 4) for comparison with the in vitro data obtained in both zoarcid species acclimated to their ambient temperature and measured in vitro at the same temperature. The in vitro RNA translational capacities obtained in gill and white muscle of both zoarcids exceed the values of in vivo efficiency determined in tissues of other fish species inhabiting various temperature environments and this trend is more distinct in white muscle compared to gill. This global comparison suggests that actual in vivo protein synthesis rates remain far below capacity. For a confirmation of this conclusion, in vivo protein synthesis should certainly be measured in the two zoarcids, however, their sluggish mode of life and low rate of energy turnover compared to other teleosts included in Table 4 supports the validity of this conclusion.
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Furthermore, our data strongly indicate that the protein synthesis machinery is cold-compensated in the Antarctic P. brachycephalum at 0°C. Enhanced capacities of the protein synthesis apparatus especially in the cold resemble high enzyme capacities of aerobic metabolism, which are cold compensated too, despite reduced standard and maximum metabolic rates. Such excess capacities in metabolic and protein synthesis functions may be relevant for rapid adjustment of metabolic and functional equilibria and for full metabolic flexibility in response to external and internal stimuli in the permanent cold. The extremely high capacity of the protein synthesis system in the white muscle and also in the gill of the Antarctic eelpout strongly supports these conclusions.
Temperature acclimation and adaptation of the protein synthesis machinery
The isolated translational machinery and its endogenous mRNA levels are a
snapshot of the cell, which mirrors the previous history of the in
vivo protein synthesis system. Therefore, in vitro protein
synthesis capacities were compared in animals of equal body size that had
experienced long-term acclimation to standard laboratory conditions. This
minimizes the effects of fluctuating environmental parameters such as food
availability, light:dark regimes and temperature, which is especially
important when comparing animals in a latitudinal cline.
Thermal acclimation apparently has an effect on the temperature dependence
and activation energies of the temperate, but not of the Antarctic species.
Arrhenius activation energies are altered in response to the thermal history
in cell-free systems prepared from the eurythermal Z. viviparus. This
is the case for gill and white muscle, and data in the literature also
indicate that this may be a common feature. Significantly reduced activation
energies of protein synthesis rates were found in eel hepatocytes upon cold
acclimation (Jankowsky et al.,
1981). Many enzymes of cold-adapted species display reduced
activation enthalpies to counterbalance the expected decrease in reaction
velocity during cooling (Hochachka and
Somero, 1984
; Marshall,
1997
; D'Amico et al.,
2002
). Protein synthesis requires a variety of enzymes and the
overall activation energy mirrors that of the whole protein synthesis
machinery.
The protein synthesis apparatus from white muscle and gill of the cold adapted P. brachycephalum also displays the expected cold-induced drop in activation energy, when compared to the synthesis apparatus of the respective tissues of temperate Z. viviparus maintained at 10°C. However, the protein synthesis complexes of P. brachycephalum did not exhibit a rise in activation energy during warming. Considering the long-term pattern of evolutionary cold adaptation, the cold-adapted protein synthesis machinery of P. brachycephalum might have lost (some of) the ability to adjust to warmer temperatures. Nonetheless, RNA translational capacity was reduced in the Antarctic species upon warming within the measured temperature range, reflecting a clear capacity for thermal adaptation. This observation is somewhat unexpected for an Antarctic stenotherm but is in line with the apparent well being and net growth of warm acclimated P. brachycephalum during long-term maintenance.
Ectotherms living at high latitudes display cold compensated enzyme
capacities despite reduced metabolic rates. These patterns have been
interpreted to reflect a downward shift of oxygen-limited thermal tolerance
windows (Pörtner, 2002).
P. brachycephalum, naturally adapted to environments with low,
constant annual mean temperatures, have higher RNA translational capacities at
levels of RNA in gills and white muscle that are similar to those in their
confamilial counterparts from temperate habitats. This trend may reflect cold
compensation of enzyme capacities involved in protein synthesis in both high
and low turnover tissues such as gills and white muscles.
The present data also allow a re-examination of whether higher RNA:protein ratios are suitable to compensate for lower RNA translational efficiencies at lower temperatures in general in cold acclimation and adaptation. The RNA concentrations of gill and white muscle did not differ between the species maintained at their habitat temperatures. Similar RNA levels in gills regardless of acclimation temperature and species emphasizes that the RNA:protein ratio does not exclusively reflect cold compensated protein synthesis capacity. This leaves upregulation of RNA translational capacity as a crucial component in the cold compensation process.
However, major changes in RNA:protein ratios were found upon acclimation in
white muscle of both zoarcid species. As already mentioned, protein synthesis
rates of white muscle in fish are known to correlate very well with growth
rates, and react sensibly to temperature changes upon acclimation
(Houlihan et al., 1988;
McCarthy and Houlihan, 1997
;
McCarthy et al., 1999
).
Therefore, the following discussion will be confined to white muscle. A role
for RNA:protein ratio in cold compensation can only be confirmed for
cold-acclimated eurythermal eelpout Z. viviparus, whereas it does not
apply to the cold-adapted Antarctic eelpout P. brachycephalum.
Reduced RNA translational capacities in white muscle of Z. viviparus
were clearly counteracted by elevated RNA:protein ratios resulting in similar
protein synthesis capacities in both acclimation groups of Z.
viviparus.
In contrast to the original hypothesis, cold adapted P. brachycephalum exhibited no increase in RNA:protein ratios compared to temperate eelpout, but displayed increased RNA translational capacities instead. Nonetheless, RNA levels are temperature dependent in the Antarctic eelpout, evidenced by a decrease in RNA levels during warm acclimation. At the same time, warming of the Antarctic eelpout caused a drop in translational capacities in white muscle (Figs 1, 4). Accordingly, both the levels of tissue RNA and of RNA translational capacity are thermally dependent in similar ways in both eelpout species, however, the range of RNA levels seen in Z. viviparus between 5 and 10°C is the same as in the Antarctic species at lower temperatures, between 0 and 5°C. This emphasizes the role of adjustments in translational capacity (per unit RNA) during thermal adaptation.
Changing translational capacities may also be involved in other species. In
different organs of rainbow trout, increased specific elongation rates were
measured upon cold acclimation in cell-free systems
(Simon, 1987). Simon did not
determine RNA levels in the assay, however, he concluded that the increased
specific elongation rates in 4°C-acclimated trout are due to an effective
enhancement of enzymatic elongation factor activities.
We conclude that P. brachycephalum evolved a cold-adapted RNA
translation machinery with reduced Arrhenius activation energies to function
efficiently at low temperatures, however, at lower RNA:protein ratios than
seen in the temperate eelpout when cold acclimated. An increased RNA:protein
ratio to counteract the temperature induced reduction of translational
efficiency does not hold for the highly adapted protein synthesis machineries
of Antarctic fish. This contrasts with observations of increased RNA:protein
ratios in many eurythermal fish (Houlihan,
1991; McCarthy and Houlihan,
1997
). It also contrasts with reduced in vitro
translational capacities as seen in eurythermal Z. viviparus upon
cold acclimation (this study). Increased RNA:protein ratios observed in
cold-adapted invertebrates (Marsh et al.,
2001
; Fraser et al.,
2002
; Storch et al.,
2003
) also contrast with our present observations in Antarctic
fish. As a corollary, further comparative in vitro and in
vivo studies are needed in Antarctic ectotherms to evaluate whether these
patterns and phenomena and the differentiation between fish and invertebrates
reflect unifying mechanisms of cold compensation in protein synthesis
operative in the permanent cold.
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References |
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