Regulation of Translation in Rabbit Reticulocytes and Mouse L-Cells; Comparison of the Effects of Temperature N E S S L Y CRAIG Department of Biologicul Sciences, University of Maryland Baltimore County, Catonsville, Maryland 21 228
ABSTRACT
Various parameters of protein synthesis were analyzed i n rabbit reticulocytes exposed to various temperatures for u p to five hours. Between 10°C and 40°C total protein synthesis exhibited two different apparent activation energies (36 kcal/mole, 10-24°C; 22 kcal/mole, 2 4 4 O o C ) , a s did protein elongation and release (35 kcal/mole, 10-25°C; 12 kcal/mole, 2540 "C). However, the level of polysomes remained essentially unchanged between 0°C and 42°C which implies that the activation energy for polypep tide initiation is quite similar to that for elongation and is also biphasic. This situation is different from that i n cultured mouse Lcells where the polysome level is dependent on temperatures. Nevertheless, reticulocytes and L-cells appear to be similar i n their temperature dependence of initiation and i n their rate of elongation ( 5 6 amino acidslsecond at 36°C).
While it is clear that protein synthesis in eukaryotic mammalian cells is both different from and similar to protein synthesis in prokaryotic bacterial cells (Lengyel, '74), the situation is less certain for possible differences between protein synthesis in different types of mammalian cells. In general, this is because relatively few types of mammalian cells have been carefully studied - e.g., rat liver, rabbit reticulocytes, and mouse and human tissue culture cells - and more emphasis has been placed on establishing the general principles of translation which are common to all cells. This is a potentially interesting point because there are significant differences between mammalian cells which might be reflected in, or even a product of, differences in the regulation of translation in these cells. For example, cells such as reticulocytes are terminally differentiated to produce a limited number of and p glospecific proteins (essentially bins), whereas tissue-culture cells such as HeLa or L-cells appear to be "non-differentiated" and to synthesize the wide variety of proteins needed for general growth and division. However, a recent series of experiments on the regulation of protein synthesis in mouse L-cells in culture (Craig, '73, '75) has produced results different from what would have been expected on the basis of (Y
J . CELL. PHYSIOL.,87: 157-166
our present understanding of translation in E. coli and in reticulocytes. For example, in L-cells, three inhibitors of RNA synthesis (including actinomycin D and cordycepin) block translation at the level of polypeptide initiation (Craig, '73), whereas the earlier work of Marks et al. ('62) suggested that protein synthesis in rabbit reticulocytes appeared to be completely resistent to actinomycin D. This would imply that there may be an important difference in the mechanism of polypeptide initiation between the two cell types. A second potentially interesting difference between protein synthesis in L-cells and in reticulocytes concerns the temperature dependence of translation. For example, in L-cells, both the level of polysomes and the rate of protein synthesis are a complex function of temperature and the time of exposure to a given temperature which differentially affects the rates of initiation and elongation (Craig, '75). Information about the effect of temperatures on protein synthesis in reticulocytes is more meager relying on the older work of Conconi et al. ('66) who used a relatively narrow range of temperatures and only short incubations (10-15 minutes) in one reported experiment. Their results suggested that in contrast to results in L-cells there Received May 9, '75. Accepted June 30, '75
157
158
NESSLY CRAIG
was a single dependency of translation on temperature and that the polysome levels appeared to be unaffected. Nevertheless, because the L-cell study found that there could be some variability which might also be true in the reticulocyte system, that some temperature effects were demonstrable only after longer incubations, and that it was important to use as wide a temperature range as possible, it seemed important to examine in more detail the effect of temperature on protein synthesis in reticulocytes to establish whether or not the possible differences with L-cells were real. The experiments to be described in this paper show that while some of the differences are real, some are not: reticulocyte polysome levels are temperature independent, but this is because there is a similar and complex temperature dependency of the rates of initiation and elongation in reticulocytes which is not present in L-cells. MATERIALS AND METHODS
Reticulocytes were isolated from anemic rabbits essentially as described by Gilbert and Anderson (‘70). New Zealand white rabbits weighing less than 3 kg were injected with 0.25 ml/kg of 2.5% (w/v) neutralized phenylhydrazine for six days and then bled on the eighth day. At this time, the hematocrit was usually between 16 and 20%. For most experiments, 10-20 ml of ice-cold RWS (“reticulocyte washing solution” - 0.14 M NaCl, 0.05 M KC1, 0.005 M MgC12, adjusted to pH 7.6) containing 2.0 USP unitslml heparin. The cells were washed three times in cold RWS and the buffey coat removed with centrifugation at 900 X g for five minutes at 2°C. The cells were then resuspended in the incubation media and divided into the various experimental portions. For the long term incubation ( > 4 0 minutes) used for polysome analysis the cells were kept fairly dilute, e.g., 0.5-1.0 ml-equivalent of blood in 40 ml media at the appropriate temperature in 125 ml erlenmeyers; for the shortterm labeling experiments the cell concentration was higher, 1 ml-equivalent of blood in 2 ml media in 4 ml “shell vials.” The incubation media was a modified Eagle’s spinner media used for mouse L-cells(Craig, ’73) with 4% decomplemented fetal calf serum (the serum could be omitted without affecting the results) and supplement-
ed with 5% decomplemented anemic rabbit plasma, 10-4 M ferrous ammonium sulfate, 2 X 10-5 M hemin, and 0.005 M HEPES buffer to help maintain pH at 7.5. In labeling experiments the normal concentration of leucine in the media ( ~ 0 . 4 mM) was reduced to 0.04-0.08 mM, and 3H-L-(4,5) leucine (30.7 Cilmmole, New England Nuclear Corp.) at a concentration of 20 pCi/ml was added. The cells were kept in suspension with magnetic stirring and the pH was controlled by flushing the cultures with 5% CO, in air. Polysomes were isolated and analyzed using a modification of the procedure described for mouse L-cell polysomes (Craig, ’73). In brief, aliquots of 2 M O ml ( 0 . 5 1.0 ml-equivalent of blood) were poured onto a slurry of cold RWS and crushed ice (made of RWS), washed three times by centrifugation with ice-cold RWS and then suspended and lysed in 0.8 mi of hypotonic RSB (0.01 M Tris-HC1,pH 7.6,O.Ol M NaC1, 0.0015 M MgC12) for five minutes. The stroma and other fragments were removed by centrifugation at 10,000 g for ten minutes at 3 ” C , and the supernatant layered onto a 12.2 ml 15-45% (w/w) sucrose gradient with a 0.5 ml cushion of 45% sucrose, all in 0.02 M Tris-HC1 pH 7.6, 0.05 M KC1, and 0.002 M MgC12. The gradients were spun in a Spinco SW40 rotor at 40,000 rpm for 75 minutes, and then their absorbance profile at 260 nm was monitored with a Gilford spectrophotometer equipped with a flow cell. The rates of protein synthesis and elongation and termination were determined using a modification of the methods described in Craig (’73, ’75). Aliquots of 0.5 ml(0.25 ml-equivalent of blood) were taken at 4-6 time points from the labeling mixtures described above and added to 10 ml ice cold RWS containing 100 pg/ml cycloheximide and 1.5 mM unlabeled leucine. The cells were washed three times with this solution and then lysed in 1.5 ml cold RSB. The stroma was pelleted at 10,000 g for ten minutes and the supernatant layered over 0.3 ml 20% sucrose in RSB in 2.0 ml tubes which were spun at 48,000 rpm for 90 minutes at 4 ° C in a SpincoType 65 rotor to pellet the ribosomes. The supernatant, as monitored by the hemoglobin color, did not contaminate the ribosomal pellet. Each fraction or aliquot of it
159
TEMPERATURE AND RETICULOCYTE TRANSLATION
w a s solubilized in 0.08 M NaOH with 1.5 was considered to represent the “released” mM leucine, neutralized, and precipitated polypeptides; when the ribosomal fraction’s with 10% trichloroacetic acid. After heat- activity (representing “nascent” polypeping at 85-90°C for 15 minutes, each frac- tides) was included, this constituted the tion was filtered through Whatman GF/C “total” radioactivity. To facilitate comparglass fiber filters. Because there was in- ison between different experiments using tense color quenching of the post-ribosomal different temperatures and cells from difsupernatant aliquots, it was necessary to ferent rabbits, the results were normalized solubilize and bleach each sample. This by making the total incorporated radiow a s done with 0.5 ml 0.1 M NaOH and activity of each experiment at a given time 0.1 ml 30% Hz02 at 50°C for 2 - 4 hours. point (usually the last) equal to one-tenth To permit comparable counting conditions of the time (e.g., 4.0 at 40 minutes) and and to minimize efficiency correction er- then expressing all of the other total and rors, the ribosome and stroma samples were released values in terms of it. also treated in the same way. After the As has been discussed before (Fan and bleached and solubilized samples had been Penman, ’72; Craig, ’73) the lateral time neutralized and cooled, 10 ml of an aque- displacement along the abscissa of the two ous counting scintillation fluid, “Hydro- parallel incorporation curves - one of the mix”(Yorktown Research Corporation), was “total,” the other the “soluble” polypepadded, the samples mixed, dark adapted, tide incorporation -represents one-halfthe a n d counted in a Beckman LSlOOC liquid time to complete and release the average scintillation counter. External standard ra- polypeptide (fig. 4 insert). Because the distios were used to monitor the counting placement was quite small at the higher efficiency and to make corrections when temperatures and difficult to measure acnecessary. The radioactivity of different curately by extrapolating the incorporation time aliquots for a given temperature con- curves to the abscissa, a method based on dition in an experiment was corrected to that of Hunt et al. (‘68) was used. A least the same basis by using the absorbance squares analysis was made of all of the at 260 nm of the solubilized ribosome frac- points used for each “soluble” and “total” tion before the TCA precipitation to calcu- incorporation curve to determine the mean late correction factors. In general, this slope. Then, from the mean difference becorrection was less than 1 0 % . The sum tween the value at any time point for the of the total radioactivity of the post-ribo- “total” and the “soluble” incorporation somal supernatant and the stroma fraction (vertical displacement) and from the mean TABLE 1
Cctlcztlcition of civercige trcinsit cind reletrse t i m e us Slope Temperature OC
40 36 33 30 25 20 18 15 10
Total
0.096-+ 0.003 0.097f 0.002 0.103t 0.003 0.099 -+ 0.003 0.10120.004 0.098s 0.003 0.0982 0.007 0.105f0.004 0.101s 0.004
ci fiinctzon
of tempercititre
1
Soluble
0.092t 0.005 0.095f 0.003 0.092t 0.007 0.099t 0.005 0.099i0.007 0.098? 0.004 0.096% 0.004 0.094t 0.006 0.098c 0.009
Total-soluble 2
0.0162f 0.0009 0.02132 0.0006 0.0248k0.0037 0.0310-+ 0.0011 0.060720.0044 0.1230f 0.0030 0.1751i 0.0054 0.3150& 0.0230 1.0145f0.0260
Transit and release time 3 (seconds)
19.8s 1.2 25.1t 0.8 30.4s 1.2 38.02 1.2 74.32 2.6 150.62 3.6 214.4s 6.4 385.72 35.6 1242.2231.2
The slope k S.E.M. (min-1) determined from a least squares analysis of the normalized values 8-39, S 1 2 experiments per temperature). This would be “(C-B/B-A)” in the insert of figure 4. 2 The mean i S.E.M. of all of the paired total-released time points using the normalized values (n = 8-39, 3-10 experiments per temperature). As diagrammed in the insert of figure 4, this is the mean of all values of “C-B.” There was no sigificant difference between cells exposed to a given temperature for ten minutes or for four hours, and so the results were combined. $ T h e mean t S.E.M. (seconds) calculated by dividing the mean values in column 3 by the mean slope ( = 0 . 0 9 8 min-1) determined from columns 1 and 2, converting to seconds, and multiplying by 2 with the assumption that the nascent chairs are on the average half completed (see text). This is equivalent to “2 (B-A)” i n the figure 4 insert. I
(n
=
160
NESSLY CRAIG
0
30
20
10
TEMPERATURE
(
40
'C. )
Fig. 1 E@ct of temptwitirre on the level of polysomes. Suspensions of reticulocytes were exposed to each temperature for varying lengths of time and their polysomes isolated a n d analyzed a s described in MATERIALS AND METHODS. The fraction of total ribosomal material sedimenting faster than the 4 0 s subunit which was found in polysomes with three or more ribosomes w a s expressed a s a percentage of the relevant value determined for control cells at 36°C i n the same experiment. All plotted points with bars represent means i standard errors. 0 ; "short" times of 10-60 minutes; seven experiments with 4-12 values for each temperature; the percentage for control polysomes was 62.8 i 9.7%. 0;"long" times of 4-5 hours; four experiments, four values for each temperature; control polysome percentage was41.0 i 1.6%
slope of both curves, it was possible to calculate the horizontal displacement (see table 1 for sample calculation). RESULTS AND DISCUSSION
Polysome levels One of the most distinctive features of protein synthesis in rabbit reticulocytes compared to that in mouse L-cells is the relative stability of reticulocyte polysome levels and sizes over the temperature range OO-42" for both short times of 10-60 minutes and for longer times of four to five hours (fig. 1). The only possible exceptions are long incubations at 25°C and incubations at 0°C where the values are 10-20% higher. A typical example of the polysome profiles of incubated reticulocytes is shown in figure 2, where it is clear that not only the relative level is unaffected but also the average size remains the same. As was described in the previous paper (Craig, '75), the corresponding situation in L-cells is significantly different. Here the polysome level is definitely a function of the temperature and time between 0" and 36"C,
reaching a minimum of 30% of the 36°C control levels after four hours of incubation. In addition, 42°C is known to cause a rapid and dramatic decrease in the rate of protein synthesis and in the level of polysomes in both HeLa cells (McCormick and Penman, '68) and L-cells (Schochetman and Perry, '72) due to an inhibition in the initiation process. This is not true for reticulocytes. The relative temperature independence of the reticulocyte polysome level would suggest that the major determinants of the quantity size of reticulocyte polysomes -the rates of polypeptide initiation, elongation, and termination -have roughly similar temperature dependencies in contrast to the situation in L-cells where the dependencies are different (Craig, '75). This hypothesis can be experimentally examined by analyzing the influence of temperature of these rates.
Total protein synthesis The overall rate of protein synthesis as measured by the incorporation of leucine into total acid-precipitable material is di-
161
TEMPERATURE AND RETICULOCYTE TRANSLATION
2. 0
1.6 2. 0
1. 6 c 0 N \o
2.
I
1.2 I
w
E
c
0 N u3
0
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CL 0 v, m
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0.
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Fig. 2 Polysome profiles of cells exposed t o different tempercitztres. Reticulocytes were first incubated a t three different temperatures for specific times, and their polysomes then isolated and displayed in sucrose gradients as described in MATERIALS A N D METHODS. Only the region sedimenting faster than the soluble hemoglobin region which obscures the 40s subunits is shown. A. Incubation at 36°C for 60 minutes. B. Incubation at 5°C for 30 minutes. C. Incubation at 10°C for 45 minutes.
rectly dependent on temperature as is shown in figure 3A. The exact nature of this dependency is somewhat complex, and can be more easily seen in figure 3B where the data has been replotted according to the Arrhenius treatment. Computer analysis of all of the data using a “statistical curvefit” program demonstrated that the curve appropriate to the data is significantly non-linear, and can best be described by a second-degree polynomial equation. However, the data can be approximated
with two linear portions, each statistically linear at the 5% level, and a transition temperature of 24°C. The lines of this approximation are the ones drawn in figure 3B. From these linear regions i t is possible to calculate apparent “activation energies” which would represent a complex summation of the activation energies of the component reactions making up the total process of protein synthesis. Between 24 “C and 40 C the apparent activation energy for total protein synthesis is 21.6 O
162
NESSLY CRAIG
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1.25
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"Ea"= 22 kcal
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2.,
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3.2 3.3 3.4 3.5 3.6 1 i ABSOLUTE TEMPERATURE ( x lo3 )
TEMPERATURE ( Fig. 3 Effect of tempertrture on the rote of p r o t e i n synthesis. Reticulocytes were exposed to each temperature for either 20 minutes or for four hours, concentrated 1 ml-equivalent of blood per 2 ml of media and then labeled at the same temperature with 3H-leucine as described in MATERIALS AND METHODS. In each experiment, the rate of total incorporation into TCA-precipitable material at a specific temperature was compared to the control rate in cells at 36°C to determine the relative rate of synthesis. The data for the 20 minutes and four hours was combined since they were not significantly different. Each plotted value represents the mean of several experiments % standard errors (n = Z 1 6 ) . A. Arithmetic plot. B. Plotted according to the Arrhenius treatment. The apparent activation energies, "Ea," were calculated from the slope of the lines determined by a least squares analysis of the data. See text for more details. i 1.2 kcal (S.E.M., 39 values, 4-16 experiments), whereas between 1 0 ° C and 24°C it is 36.1 i 2.3 kcal (24 values, 2-10 experiments). It was difficult to obtain meaningful values below 10°C because the rate of protein synthesis was quite low. At first glance, this biphasic curve for the temperature dependency of protein synthesis might appear to be different from the results of Conconi et al. ('66). However, this is more apparent than real, since if only data in the temperature range they studied (17 "-40 C) is used, it is possible to fit the data with a linear curve and calculate an activation energy which is exactly the same as the one they O
calculated, i.e., 26.2 Kcal. It is only when a wider temperature range is used and the temperature studies repeated several times to take variability into account that it becomes clear that the total temperature dependency is non-linear. The relationship between protein synthesis and temperature for reticulocytes is similar in several respects to that of mouse L-cells (Craig, '75). In L-cells the temperature dependency is also biphasic over the range 1OoC-36"C, with a transition temperature of 25°C. The apparent "activation energy" for the lower temperatures is basically the same for both cell types (38.8 k 1.9 vs. 36.1 i 2.3 kcal), whereas the
TEMPERATURE AND RETIC ULOCY T E TRANSLATION
163
apparent "activation energy" for the high- insert). The actual normalized mean valer temperatures is somewhat different ues and slopes used in the calculations are (13.6 f 1.0 kcal in L-cells vs. 21.6 2 1.2 shown in table 1. The total transit times kcal for reticulocytes). In the only com- range from 20 seconds at 40°C to 1,242 parable study in bacteria ( E . coli; Gold- seconds at 10°C.These values are plotted stein et al., '64) there also appeared to be arithmetically in figure 5A, and according a biphasic relation between 0" and 37°C to the Arrhenius treatment in figure 5B. for the initial rate of protein synthesis It is clear that the temperature dependency with a break at 25"C,although there was is not linear over this temperature range, only one experimental point above this although i t can be approximated with two temperature and two points below it. The linear regions separated by a transition apparent activation energy for the lower zone centering around 25°C.The apparent temperatures 0"-25"C was 31 kcal, where,- "activation energy" for elongation and reas the value for the higher temperatures lease above 25°C is 12.2 & 1.0 kcal (S.E.M., 95 values, 4-10 experiments), c a n be calculated from their figure as being about 17 kcal. The temperature relation- whereas below 25°C it is 35.0 t 1.1 kcal ship below 10°C was only applicable for (S.E.M., 41 values, 2-3 experitnents). There are several observations which can the first 10-15 minutes since protein synthesis became progressively slower, even- be made concerning these values. The fact tually halting after several hours. As was that below 25°C the apparent activation shown by Friedman et al. ('68) this de- energy for elongation is the same as that cline in rate below 10°C is due to an for total protein synthesis (35.0 f 1.0 vs. inhibition in the rate of polypeptide ini- 36.1 f 2.3 kcal) and that the level of polysomes is also unchanged implies that tiation. the temperature dependency of initiation Ribosome "transit time" is about the same as that for elongation, The rate of polypeptide elongation and and that the major rate limiting step in release, i.e., ribosome "transit time," can total protein synthesis below 25°C could also be determined reasonably directly. As be either (or both) the rate of initiation has been discussed by Fan and Penman or the rate of elongation and release. The ('72) and Craig ('73), among others, if the situation above 25°C is somewhat more incorporation of a n amino acid into the complex. The level of polysomes remains completed and released polypeptides and essentially unchanged (with a possible exinto total cellular protein is measured as ception at 25°C for long-term incubations) a function of time, the two curves will be which suggests that initiation and transit parallel with the displacement between rate are equally affected by temperature. along the time axis representing one-half This would imply that the apparent acthe time needed to translate and release tivation energy for initiation above 25"C, and the average polypeptide (insert, fig. 4). 22 kcal, is quite different. Thus it appears Such an analysis for reticulocytes at dif- possible that neither initiation nor elongaferent temperatures is shown in figure 4. tion are directly rate limiting above 25"C, Because the transit times at the higher but that some other process -e.g., amino temperatures are so short, e.g., d 2 6 sec- acid transport, amino acid activation, etc. onds at 36"C, the actual calculations of - might be an important limiting process. the time displacement of the incorporation These results also demonstrate that much curves were done somewhat more directly of the difference in the temperature depenthan by just extrapolating each curve to dence of protein synthesis and polysome the time axis (abscissa). This procedure level between mouse L-cells and rabbit rewas first used by Hunt et al. ('68) and in- ticulocytes is due to a difference in the volves calculating the lateral time displace- temperature dependency of the rates of ment (B-A in fig. 4 insert) by using the elongation and release. As was shown in actual data of the difference in radioactiv- the previous paper (Craig, '75), elongation ity between the total and released material in L-cells has only a single apparent actiat each time point (C-B in fig. 4 insert) and vation energy of 16 kcal, which is different dividing by the average slope of the paral- from the two activation energies for elonlel incorporation curves (C-B/B-A in fig. 4 gation and release characteristic of reticu-
164
NESSLY CRAIG
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24 36 MINUTES OF INCORPORATION
I
I
48
Fig. 4 Ribosome t r u n s t t t i m e cimtlysis f o r cells exposed to different tempercitztres. The analysis of the rate of polypeptide elongation a n d release in cells exposed to various temperatures for either 20 minutes or for four hours was done a s described in MATERIALS AND METHODS. The results from different experiments with various cell numbers and aliquot sizes a n d different rabbits were normalized by making the total and thus maximum incorporation present at the last sample time equivalent to one-tenth of the time (e.g. 1.6 at 16 minutes, or 4.8 at 48 minutes, depending on how long the incorporation was followed), and then expressing all of the other total and released incorporation values i n terms of it. Because of space limitations, the actual normalized data for only four different temperatures is shown; however the derived transit time values for these and all other temperatures are given in table 1 and shown in figure 5. The insert shows the theoretical basis for this transit time determination; see the text for more details. The solid line represents the total precipitable polypeptide material; the dashed lines, the released polypeptides; 0 , 36OC; X, 2 0 ° C ; 0 , 18°C; 19OC.
.,
~
165
TEMPERATURE AND RETICULOCYTE TRANSLATION
1. 0 A
f
B
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lx
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2 2.c ct c
\
0 A
0 m
I
2. 5
i
3. (
10
20
30
40
3. 2 3.3 3.4 3.5 3.6 1 I ABSOLUTE TEMPERATURE ( x lo3
TEMPERATURE ( OC. ) Fig. 5 Effect of tempercctzire on t h e rcites of p o l y p e p t i d e elongution ctnd termincition. Total average transit and release times i n seconds were determined as described i n MATERIALS A N D METHODS, illustrated in figure 4, and tabulated in table 1. Cells were exposed to each temperature for either 20 minutes or four hours before the labeling began; because the results were not significantly different they were pooled. Each plotted value is the mean of the data from several experiments % standard error (n = 8-39, 3-10 experiments for each temperature). A. The data plotted arithmetically. B. The data, converted to elongation and release rate (no. polypeptides/second/ribosome), plotted according to the Arrhenius treatment. locytes (12 kcal >25"C, 35 kcal
ABSTRACT
Various parameters of protein synthesis were analyzed i n rabbit reticulocytes exposed to various temperatures for u p to five hours. Between 10°C and 40°C total protein synthesis exhibited two different apparent activation energies (36 kcal/mole, 10-24°C; 22 kcal/mole, 2 4 4 O o C ) , a s did protein elongation and release (35 kcal/mole, 10-25°C; 12 kcal/mole, 2540 "C). However, the level of polysomes remained essentially unchanged between 0°C and 42°C which implies that the activation energy for polypep tide initiation is quite similar to that for elongation and is also biphasic. This situation is different from that i n cultured mouse Lcells where the polysome level is dependent on temperatures. Nevertheless, reticulocytes and L-cells appear to be similar i n their temperature dependence of initiation and i n their rate of elongation ( 5 6 amino acidslsecond at 36°C).
While it is clear that protein synthesis in eukaryotic mammalian cells is both different from and similar to protein synthesis in prokaryotic bacterial cells (Lengyel, '74), the situation is less certain for possible differences between protein synthesis in different types of mammalian cells. In general, this is because relatively few types of mammalian cells have been carefully studied - e.g., rat liver, rabbit reticulocytes, and mouse and human tissue culture cells - and more emphasis has been placed on establishing the general principles of translation which are common to all cells. This is a potentially interesting point because there are significant differences between mammalian cells which might be reflected in, or even a product of, differences in the regulation of translation in these cells. For example, cells such as reticulocytes are terminally differentiated to produce a limited number of and p glospecific proteins (essentially bins), whereas tissue-culture cells such as HeLa or L-cells appear to be "non-differentiated" and to synthesize the wide variety of proteins needed for general growth and division. However, a recent series of experiments on the regulation of protein synthesis in mouse L-cells in culture (Craig, '73, '75) has produced results different from what would have been expected on the basis of (Y
J . CELL. PHYSIOL.,87: 157-166
our present understanding of translation in E. coli and in reticulocytes. For example, in L-cells, three inhibitors of RNA synthesis (including actinomycin D and cordycepin) block translation at the level of polypeptide initiation (Craig, '73), whereas the earlier work of Marks et al. ('62) suggested that protein synthesis in rabbit reticulocytes appeared to be completely resistent to actinomycin D. This would imply that there may be an important difference in the mechanism of polypeptide initiation between the two cell types. A second potentially interesting difference between protein synthesis in L-cells and in reticulocytes concerns the temperature dependence of translation. For example, in L-cells, both the level of polysomes and the rate of protein synthesis are a complex function of temperature and the time of exposure to a given temperature which differentially affects the rates of initiation and elongation (Craig, '75). Information about the effect of temperatures on protein synthesis in reticulocytes is more meager relying on the older work of Conconi et al. ('66) who used a relatively narrow range of temperatures and only short incubations (10-15 minutes) in one reported experiment. Their results suggested that in contrast to results in L-cells there Received May 9, '75. Accepted June 30, '75
157
158
NESSLY CRAIG
was a single dependency of translation on temperature and that the polysome levels appeared to be unaffected. Nevertheless, because the L-cell study found that there could be some variability which might also be true in the reticulocyte system, that some temperature effects were demonstrable only after longer incubations, and that it was important to use as wide a temperature range as possible, it seemed important to examine in more detail the effect of temperature on protein synthesis in reticulocytes to establish whether or not the possible differences with L-cells were real. The experiments to be described in this paper show that while some of the differences are real, some are not: reticulocyte polysome levels are temperature independent, but this is because there is a similar and complex temperature dependency of the rates of initiation and elongation in reticulocytes which is not present in L-cells. MATERIALS AND METHODS
Reticulocytes were isolated from anemic rabbits essentially as described by Gilbert and Anderson (‘70). New Zealand white rabbits weighing less than 3 kg were injected with 0.25 ml/kg of 2.5% (w/v) neutralized phenylhydrazine for six days and then bled on the eighth day. At this time, the hematocrit was usually between 16 and 20%. For most experiments, 10-20 ml of ice-cold RWS (“reticulocyte washing solution” - 0.14 M NaCl, 0.05 M KC1, 0.005 M MgC12, adjusted to pH 7.6) containing 2.0 USP unitslml heparin. The cells were washed three times in cold RWS and the buffey coat removed with centrifugation at 900 X g for five minutes at 2°C. The cells were then resuspended in the incubation media and divided into the various experimental portions. For the long term incubation ( > 4 0 minutes) used for polysome analysis the cells were kept fairly dilute, e.g., 0.5-1.0 ml-equivalent of blood in 40 ml media at the appropriate temperature in 125 ml erlenmeyers; for the shortterm labeling experiments the cell concentration was higher, 1 ml-equivalent of blood in 2 ml media in 4 ml “shell vials.” The incubation media was a modified Eagle’s spinner media used for mouse L-cells(Craig, ’73) with 4% decomplemented fetal calf serum (the serum could be omitted without affecting the results) and supplement-
ed with 5% decomplemented anemic rabbit plasma, 10-4 M ferrous ammonium sulfate, 2 X 10-5 M hemin, and 0.005 M HEPES buffer to help maintain pH at 7.5. In labeling experiments the normal concentration of leucine in the media ( ~ 0 . 4 mM) was reduced to 0.04-0.08 mM, and 3H-L-(4,5) leucine (30.7 Cilmmole, New England Nuclear Corp.) at a concentration of 20 pCi/ml was added. The cells were kept in suspension with magnetic stirring and the pH was controlled by flushing the cultures with 5% CO, in air. Polysomes were isolated and analyzed using a modification of the procedure described for mouse L-cell polysomes (Craig, ’73). In brief, aliquots of 2 M O ml ( 0 . 5 1.0 ml-equivalent of blood) were poured onto a slurry of cold RWS and crushed ice (made of RWS), washed three times by centrifugation with ice-cold RWS and then suspended and lysed in 0.8 mi of hypotonic RSB (0.01 M Tris-HC1,pH 7.6,O.Ol M NaC1, 0.0015 M MgC12) for five minutes. The stroma and other fragments were removed by centrifugation at 10,000 g for ten minutes at 3 ” C , and the supernatant layered onto a 12.2 ml 15-45% (w/w) sucrose gradient with a 0.5 ml cushion of 45% sucrose, all in 0.02 M Tris-HC1 pH 7.6, 0.05 M KC1, and 0.002 M MgC12. The gradients were spun in a Spinco SW40 rotor at 40,000 rpm for 75 minutes, and then their absorbance profile at 260 nm was monitored with a Gilford spectrophotometer equipped with a flow cell. The rates of protein synthesis and elongation and termination were determined using a modification of the methods described in Craig (’73, ’75). Aliquots of 0.5 ml(0.25 ml-equivalent of blood) were taken at 4-6 time points from the labeling mixtures described above and added to 10 ml ice cold RWS containing 100 pg/ml cycloheximide and 1.5 mM unlabeled leucine. The cells were washed three times with this solution and then lysed in 1.5 ml cold RSB. The stroma was pelleted at 10,000 g for ten minutes and the supernatant layered over 0.3 ml 20% sucrose in RSB in 2.0 ml tubes which were spun at 48,000 rpm for 90 minutes at 4 ° C in a SpincoType 65 rotor to pellet the ribosomes. The supernatant, as monitored by the hemoglobin color, did not contaminate the ribosomal pellet. Each fraction or aliquot of it
159
TEMPERATURE AND RETICULOCYTE TRANSLATION
w a s solubilized in 0.08 M NaOH with 1.5 was considered to represent the “released” mM leucine, neutralized, and precipitated polypeptides; when the ribosomal fraction’s with 10% trichloroacetic acid. After heat- activity (representing “nascent” polypeping at 85-90°C for 15 minutes, each frac- tides) was included, this constituted the tion was filtered through Whatman GF/C “total” radioactivity. To facilitate comparglass fiber filters. Because there was in- ison between different experiments using tense color quenching of the post-ribosomal different temperatures and cells from difsupernatant aliquots, it was necessary to ferent rabbits, the results were normalized solubilize and bleach each sample. This by making the total incorporated radiow a s done with 0.5 ml 0.1 M NaOH and activity of each experiment at a given time 0.1 ml 30% Hz02 at 50°C for 2 - 4 hours. point (usually the last) equal to one-tenth To permit comparable counting conditions of the time (e.g., 4.0 at 40 minutes) and and to minimize efficiency correction er- then expressing all of the other total and rors, the ribosome and stroma samples were released values in terms of it. also treated in the same way. After the As has been discussed before (Fan and bleached and solubilized samples had been Penman, ’72; Craig, ’73) the lateral time neutralized and cooled, 10 ml of an aque- displacement along the abscissa of the two ous counting scintillation fluid, “Hydro- parallel incorporation curves - one of the mix”(Yorktown Research Corporation), was “total,” the other the “soluble” polypepadded, the samples mixed, dark adapted, tide incorporation -represents one-halfthe a n d counted in a Beckman LSlOOC liquid time to complete and release the average scintillation counter. External standard ra- polypeptide (fig. 4 insert). Because the distios were used to monitor the counting placement was quite small at the higher efficiency and to make corrections when temperatures and difficult to measure acnecessary. The radioactivity of different curately by extrapolating the incorporation time aliquots for a given temperature con- curves to the abscissa, a method based on dition in an experiment was corrected to that of Hunt et al. (‘68) was used. A least the same basis by using the absorbance squares analysis was made of all of the at 260 nm of the solubilized ribosome frac- points used for each “soluble” and “total” tion before the TCA precipitation to calcu- incorporation curve to determine the mean late correction factors. In general, this slope. Then, from the mean difference becorrection was less than 1 0 % . The sum tween the value at any time point for the of the total radioactivity of the post-ribo- “total” and the “soluble” incorporation somal supernatant and the stroma fraction (vertical displacement) and from the mean TABLE 1
Cctlcztlcition of civercige trcinsit cind reletrse t i m e us Slope Temperature OC
40 36 33 30 25 20 18 15 10
Total
0.096-+ 0.003 0.097f 0.002 0.103t 0.003 0.099 -+ 0.003 0.10120.004 0.098s 0.003 0.0982 0.007 0.105f0.004 0.101s 0.004
ci fiinctzon
of tempercititre
1
Soluble
0.092t 0.005 0.095f 0.003 0.092t 0.007 0.099t 0.005 0.099i0.007 0.098? 0.004 0.096% 0.004 0.094t 0.006 0.098c 0.009
Total-soluble 2
0.0162f 0.0009 0.02132 0.0006 0.0248k0.0037 0.0310-+ 0.0011 0.060720.0044 0.1230f 0.0030 0.1751i 0.0054 0.3150& 0.0230 1.0145f0.0260
Transit and release time 3 (seconds)
19.8s 1.2 25.1t 0.8 30.4s 1.2 38.02 1.2 74.32 2.6 150.62 3.6 214.4s 6.4 385.72 35.6 1242.2231.2
The slope k S.E.M. (min-1) determined from a least squares analysis of the normalized values 8-39, S 1 2 experiments per temperature). This would be “(C-B/B-A)” in the insert of figure 4. 2 The mean i S.E.M. of all of the paired total-released time points using the normalized values (n = 8-39, 3-10 experiments per temperature). As diagrammed in the insert of figure 4, this is the mean of all values of “C-B.” There was no sigificant difference between cells exposed to a given temperature for ten minutes or for four hours, and so the results were combined. $ T h e mean t S.E.M. (seconds) calculated by dividing the mean values in column 3 by the mean slope ( = 0 . 0 9 8 min-1) determined from columns 1 and 2, converting to seconds, and multiplying by 2 with the assumption that the nascent chairs are on the average half completed (see text). This is equivalent to “2 (B-A)” i n the figure 4 insert. I
(n
=
160
NESSLY CRAIG
0
30
20
10
TEMPERATURE
(
40
'C. )
Fig. 1 E@ct of temptwitirre on the level of polysomes. Suspensions of reticulocytes were exposed to each temperature for varying lengths of time and their polysomes isolated a n d analyzed a s described in MATERIALS AND METHODS. The fraction of total ribosomal material sedimenting faster than the 4 0 s subunit which was found in polysomes with three or more ribosomes w a s expressed a s a percentage of the relevant value determined for control cells at 36°C i n the same experiment. All plotted points with bars represent means i standard errors. 0 ; "short" times of 10-60 minutes; seven experiments with 4-12 values for each temperature; the percentage for control polysomes was 62.8 i 9.7%. 0;"long" times of 4-5 hours; four experiments, four values for each temperature; control polysome percentage was41.0 i 1.6%
slope of both curves, it was possible to calculate the horizontal displacement (see table 1 for sample calculation). RESULTS AND DISCUSSION
Polysome levels One of the most distinctive features of protein synthesis in rabbit reticulocytes compared to that in mouse L-cells is the relative stability of reticulocyte polysome levels and sizes over the temperature range OO-42" for both short times of 10-60 minutes and for longer times of four to five hours (fig. 1). The only possible exceptions are long incubations at 25°C and incubations at 0°C where the values are 10-20% higher. A typical example of the polysome profiles of incubated reticulocytes is shown in figure 2, where it is clear that not only the relative level is unaffected but also the average size remains the same. As was described in the previous paper (Craig, '75), the corresponding situation in L-cells is significantly different. Here the polysome level is definitely a function of the temperature and time between 0" and 36"C,
reaching a minimum of 30% of the 36°C control levels after four hours of incubation. In addition, 42°C is known to cause a rapid and dramatic decrease in the rate of protein synthesis and in the level of polysomes in both HeLa cells (McCormick and Penman, '68) and L-cells (Schochetman and Perry, '72) due to an inhibition in the initiation process. This is not true for reticulocytes. The relative temperature independence of the reticulocyte polysome level would suggest that the major determinants of the quantity size of reticulocyte polysomes -the rates of polypeptide initiation, elongation, and termination -have roughly similar temperature dependencies in contrast to the situation in L-cells where the dependencies are different (Craig, '75). This hypothesis can be experimentally examined by analyzing the influence of temperature of these rates.
Total protein synthesis The overall rate of protein synthesis as measured by the incorporation of leucine into total acid-precipitable material is di-
161
TEMPERATURE AND RETICULOCYTE TRANSLATION
2. 0
1.6 2. 0
1. 6 c 0 N \o
2.
I
1.2 I
w
E
c
0 N u3
0
1.
z 0.8 a m
I
CL 0 v, m
W
0
1.
z
0.4 a
a m CL 0 v,
0.
m
0
a 0.
TOP
BOTTOM
Fig. 2 Polysome profiles of cells exposed t o different tempercitztres. Reticulocytes were first incubated a t three different temperatures for specific times, and their polysomes then isolated and displayed in sucrose gradients as described in MATERIALS A N D METHODS. Only the region sedimenting faster than the soluble hemoglobin region which obscures the 40s subunits is shown. A. Incubation at 36°C for 60 minutes. B. Incubation at 5°C for 30 minutes. C. Incubation at 10°C for 45 minutes.
rectly dependent on temperature as is shown in figure 3A. The exact nature of this dependency is somewhat complex, and can be more easily seen in figure 3B where the data has been replotted according to the Arrhenius treatment. Computer analysis of all of the data using a “statistical curvefit” program demonstrated that the curve appropriate to the data is significantly non-linear, and can best be described by a second-degree polynomial equation. However, the data can be approximated
with two linear portions, each statistically linear at the 5% level, and a transition temperature of 24°C. The lines of this approximation are the ones drawn in figure 3B. From these linear regions i t is possible to calculate apparent “activation energies” which would represent a complex summation of the activation energies of the component reactions making up the total process of protein synthesis. Between 24 “C and 40 C the apparent activation energy for total protein synthesis is 21.6 O
162
NESSLY CRAIG
\'
1.25
I
B
a
"Ea"= 22 kcal
'0
5'
-
v, v,
g 1.00 >-
v,
z 0 CY
\
?
z
r
I
I
I
0.75
l
\\
R LL
\
0
5
0.50
m W
z
I-
5e 0.25 _I 10
20
30
40 C. 1
2.,
I
I
1
1
3.2 3.3 3.4 3.5 3.6 1 i ABSOLUTE TEMPERATURE ( x lo3 )
TEMPERATURE ( Fig. 3 Effect of tempertrture on the rote of p r o t e i n synthesis. Reticulocytes were exposed to each temperature for either 20 minutes or for four hours, concentrated 1 ml-equivalent of blood per 2 ml of media and then labeled at the same temperature with 3H-leucine as described in MATERIALS AND METHODS. In each experiment, the rate of total incorporation into TCA-precipitable material at a specific temperature was compared to the control rate in cells at 36°C to determine the relative rate of synthesis. The data for the 20 minutes and four hours was combined since they were not significantly different. Each plotted value represents the mean of several experiments % standard errors (n = Z 1 6 ) . A. Arithmetic plot. B. Plotted according to the Arrhenius treatment. The apparent activation energies, "Ea," were calculated from the slope of the lines determined by a least squares analysis of the data. See text for more details. i 1.2 kcal (S.E.M., 39 values, 4-16 experiments), whereas between 1 0 ° C and 24°C it is 36.1 i 2.3 kcal (24 values, 2-10 experiments). It was difficult to obtain meaningful values below 10°C because the rate of protein synthesis was quite low. At first glance, this biphasic curve for the temperature dependency of protein synthesis might appear to be different from the results of Conconi et al. ('66). However, this is more apparent than real, since if only data in the temperature range they studied (17 "-40 C) is used, it is possible to fit the data with a linear curve and calculate an activation energy which is exactly the same as the one they O
calculated, i.e., 26.2 Kcal. It is only when a wider temperature range is used and the temperature studies repeated several times to take variability into account that it becomes clear that the total temperature dependency is non-linear. The relationship between protein synthesis and temperature for reticulocytes is similar in several respects to that of mouse L-cells (Craig, '75). In L-cells the temperature dependency is also biphasic over the range 1OoC-36"C, with a transition temperature of 25°C. The apparent "activation energy" for the lower temperatures is basically the same for both cell types (38.8 k 1.9 vs. 36.1 i 2.3 kcal), whereas the
TEMPERATURE AND RETIC ULOCY T E TRANSLATION
163
apparent "activation energy" for the high- insert). The actual normalized mean valer temperatures is somewhat different ues and slopes used in the calculations are (13.6 f 1.0 kcal in L-cells vs. 21.6 2 1.2 shown in table 1. The total transit times kcal for reticulocytes). In the only com- range from 20 seconds at 40°C to 1,242 parable study in bacteria ( E . coli; Gold- seconds at 10°C.These values are plotted stein et al., '64) there also appeared to be arithmetically in figure 5A, and according a biphasic relation between 0" and 37°C to the Arrhenius treatment in figure 5B. for the initial rate of protein synthesis It is clear that the temperature dependency with a break at 25"C,although there was is not linear over this temperature range, only one experimental point above this although i t can be approximated with two temperature and two points below it. The linear regions separated by a transition apparent activation energy for the lower zone centering around 25°C.The apparent temperatures 0"-25"C was 31 kcal, where,- "activation energy" for elongation and reas the value for the higher temperatures lease above 25°C is 12.2 & 1.0 kcal (S.E.M., 95 values, 4-10 experiments), c a n be calculated from their figure as being about 17 kcal. The temperature relation- whereas below 25°C it is 35.0 t 1.1 kcal ship below 10°C was only applicable for (S.E.M., 41 values, 2-3 experitnents). There are several observations which can the first 10-15 minutes since protein synthesis became progressively slower, even- be made concerning these values. The fact tually halting after several hours. As was that below 25°C the apparent activation shown by Friedman et al. ('68) this de- energy for elongation is the same as that cline in rate below 10°C is due to an for total protein synthesis (35.0 f 1.0 vs. inhibition in the rate of polypeptide ini- 36.1 f 2.3 kcal) and that the level of polysomes is also unchanged implies that tiation. the temperature dependency of initiation Ribosome "transit time" is about the same as that for elongation, The rate of polypeptide elongation and and that the major rate limiting step in release, i.e., ribosome "transit time," can total protein synthesis below 25°C could also be determined reasonably directly. As be either (or both) the rate of initiation has been discussed by Fan and Penman or the rate of elongation and release. The ('72) and Craig ('73), among others, if the situation above 25°C is somewhat more incorporation of a n amino acid into the complex. The level of polysomes remains completed and released polypeptides and essentially unchanged (with a possible exinto total cellular protein is measured as ception at 25°C for long-term incubations) a function of time, the two curves will be which suggests that initiation and transit parallel with the displacement between rate are equally affected by temperature. along the time axis representing one-half This would imply that the apparent acthe time needed to translate and release tivation energy for initiation above 25"C, and the average polypeptide (insert, fig. 4). 22 kcal, is quite different. Thus it appears Such an analysis for reticulocytes at dif- possible that neither initiation nor elongaferent temperatures is shown in figure 4. tion are directly rate limiting above 25"C, Because the transit times at the higher but that some other process -e.g., amino temperatures are so short, e.g., d 2 6 sec- acid transport, amino acid activation, etc. onds at 36"C, the actual calculations of - might be an important limiting process. the time displacement of the incorporation These results also demonstrate that much curves were done somewhat more directly of the difference in the temperature depenthan by just extrapolating each curve to dence of protein synthesis and polysome the time axis (abscissa). This procedure level between mouse L-cells and rabbit rewas first used by Hunt et al. ('68) and in- ticulocytes is due to a difference in the volves calculating the lateral time displace- temperature dependency of the rates of ment (B-A in fig. 4 insert) by using the elongation and release. As was shown in actual data of the difference in radioactiv- the previous paper (Craig, '75), elongation ity between the total and released material in L-cells has only a single apparent actiat each time point (C-B in fig. 4 insert) and vation energy of 16 kcal, which is different dividing by the average slope of the paral- from the two activation energies for elonlel incorporation curves (C-B/B-A in fig. 4 gation and release characteristic of reticu-
164
NESSLY CRAIG
'
w $Transit time
1
"
Released
,
. 0
-
I
1
4
8
1
.
3
I
12
6 16
4.8'
3.6
2.4
/".-' ,, ' ' o@d m0 ,
lao c Released
D
oy
1. 2
0 0 /
n
0
12
I
-
10°C Released
'
0-------
0
--
0'
I
I
I
24 36 MINUTES OF INCORPORATION
I
I
48
Fig. 4 Ribosome t r u n s t t t i m e cimtlysis f o r cells exposed to different tempercitztres. The analysis of the rate of polypeptide elongation a n d release in cells exposed to various temperatures for either 20 minutes or for four hours was done a s described in MATERIALS AND METHODS. The results from different experiments with various cell numbers and aliquot sizes a n d different rabbits were normalized by making the total and thus maximum incorporation present at the last sample time equivalent to one-tenth of the time (e.g. 1.6 at 16 minutes, or 4.8 at 48 minutes, depending on how long the incorporation was followed), and then expressing all of the other total and released incorporation values i n terms of it. Because of space limitations, the actual normalized data for only four different temperatures is shown; however the derived transit time values for these and all other temperatures are given in table 1 and shown in figure 5. The insert shows the theoretical basis for this transit time determination; see the text for more details. The solid line represents the total precipitable polypeptide material; the dashed lines, the released polypeptides; 0 , 36OC; X, 2 0 ° C ; 0 , 18°C; 19OC.
.,
~
165
TEMPERATURE AND RETICULOCYTE TRANSLATION
1. 0 A
f
B
z 1.5 w
lx
k LA
2 2.c ct c
\
0 A
0 m
I
2. 5
i
3. (
10
20
30
40
3. 2 3.3 3.4 3.5 3.6 1 I ABSOLUTE TEMPERATURE ( x lo3
TEMPERATURE ( OC. ) Fig. 5 Effect of tempercctzire on t h e rcites of p o l y p e p t i d e elongution ctnd termincition. Total average transit and release times i n seconds were determined as described i n MATERIALS A N D METHODS, illustrated in figure 4, and tabulated in table 1. Cells were exposed to each temperature for either 20 minutes or four hours before the labeling began; because the results were not significantly different they were pooled. Each plotted value is the mean of the data from several experiments % standard error (n = 8-39, 3-10 experiments for each temperature). A. The data plotted arithmetically. B. The data, converted to elongation and release rate (no. polypeptides/second/ribosome), plotted according to the Arrhenius treatment. locytes (12 kcal >25"C, 35 kcal
