Regulation of polypeptide chain initiation and activity of initiation factor ...

Eur. J. Biochem. 157,39-47 (1986) 8 FEBS 1986

Regulation of polypeptide chain initiation and activity of initiation factor eIF-2 in Chinese-hamster-ovary cell mutants containing temperature-sensitive aminoacyl-tRNA synthetases Sara A. AUSTIN', Jeffrey W. POLLARD', Rosemary JAGUS3and Michael J. CLEMENS' Cancer Research Campaign Mammalian Protein Synthesis and Interferon Research Group, Department of Biochemistry, St George's Hospital Medical School, London Medical Research Council Group in Human Genetic Disease, Department of Biochemistry, King's College, University of London University of Pittsburgh School of Medicine, Pittsburgh (Received January 3,1986) - EJB 86 0004 The regulation of polypeptide chain initiation has been investigated in extracts from a number of wellcharacterized Chinese hamster ovary (CHO) cell mutants containing different temperature-sensitive aminoacyltRNA synthetases. These cells exhibit a large decline in the rate of initiation when cultures are shifted from the permissive temperature of 34°C to the non-permissive temperature of 39.5 "C. During a brief incubation with [35S]Met-tRNAy" or [35S]methionine,formation of initiation complexes on native 40s ribosomal subunits and 80s ribosomes is severely impaired in extracts from the mutant cell lines exposed to 39.5"C. Wild-type cells exposed to 39.5"C do not show any inhibition of protein synthesis or initiation complex formation. Inhibition of formation of 40s initiation complexes in the extracts from mutant cells, incubated at the non-permissive temperature, is shown to be independent of possible changes in mRNA binding or the rate of polypeptide chain elongation and is not due to any decrease in the total amount of initiation factor eIF-2 present. However, assays of eIF-2 . GTP * Met-tRNAy ternary complex formation in postribosomal supernatants from the temperaturesensitive mutants reveal a marked defect in the activity of eIF-2 after exposure of the cells to 39.5"C and addition of exogenous eIF-2 to cell-free protein-synthesizing systems from cells incubated at 34 "C and 39.5 "C eliminates the difference in activity between them. The activity of the initiation factor itself is not directly temperaturesensitive in the mutant CHO cells. The results suggest that the activity of aminoacyl-tRNA synthetases can affect the ability of eIF-2 to bind Met-tRNAy" and form 40s initiation complexes in intact cells, indicating a regulatory link between polypeptide chain elongation and chain initiation. During the acute regulation of protein synthesis in mammalian cells, rapid and reversible changes in translational activity are brought about by mechanisms acting mainly at the level of polypeptide chain inititation [l-51. In mouse Ehrlich ascites tumour cells or HeLa cells, deprived of a single essential amino acid, there is a rapid decline in the rate of protein synthesis, together with disaggregation of polysomes [6-101 and a large decrease in the level of Met-tRNAy"'containing initiation complexes on native 40s ribosomal subunits [S, 91. It has been suggested that the effects of amino acid starvation are brought about by the accumulation of uncharged tRNA species corresponding to the missing amino acid [I1 - 131 but no molecular mechanism involving the known components of the protein-synthetic machinery has been identified to explain this effect. Although direct effects of changes in uncharged tRNA levels on polypeptide chain initiation have not been observed in intact Ehrlich cells or their cell-free systems [14], it is possible that the activity of Correspondence to M . J . Clemens, CRC Group, Department of

Biochemistry, St George's Hospital Medical School, Cranmer Terrace, London England SW17 ORE Abbreviation. CHO cells, Chinese hamster ovary cells. Enzymes. Leucyl-tRNA synthetase (EC 6.1.1.4); arginyl-tRNA synthetasc (EC 6.1.1.19); creatine kinase (EC 2.7.3.2); pyruvate kinase (EC 2.7.1.40); micrococcal nuclease (EC 3.1.31.1).

aminoacyl-tRNA synthetases (aminoacyl-tRNA ligases) themselves may play a regulatory role in mediating the effects of amino acid starvation on the initiation of protein synthesis. In order to test this we have compared the behaviour of wild-type and aminoacyl-tRNA synthetase mutants of Chinese hamster ovary (CHO) cells such as tsH1, Leu-21 and Arg-1. These mutant cells grow and synthesize protein normally in culture at 34 "C, the permissive temperature, but have different temperature-sensitive lesions in either the leucyl-tRNA synthetase (tsH, and Leu-21) or the arginyltRNA synthetase (Arg-1) and are impaired in their ability to charge the cognate tRNAs at non-permissive temperatures. Incubation of the cells at 39.5"C results in a rapid decline in the rate of protein synthesis, which is reversed when the temperature is restored to 34°C [15]. The polysomes become disaggregated at 39.5 "C, indicating a strong lesion in polypeptide chain initiation with relatively less effect on elongation [16]. However, both genetic and biochemical analyses [15-18] have shown that these cells contain a mutation only in a single aminoacyl-tRNA synthetase and the mechanism of the initiation defect has not been analysed in detail. In this paper we describe the use of cell-free extracts from wild-type and temperature-sensitive cells incubated at 34 "C or 39.5"C to examine how the initiation of protein synthesis is regulated in these cells. A preliminary abstract of this work has been published [19].

40 MATERIALS AND METHODS Materials

either used immediately or stored in small aliquots in liquid N2.

~-[~'S]Methioninewas obtained from Amersham International (UK) or from New England Nuclear. Calf liver Analysis of polysome size distributions [35S]Met-tRNA? was prepared as described previously [20]. Polysome size distributions in cell-free extracts were mea~-[~H]Threonine, ~-[~H]isoleucine and ~-[~H]phenylalanine were obtained from Amersham International. '251-protein A sured by centrifugation of l .5 - 3 A260units of extract through was from New England Nuclear. Micrococcal nuclease was 5-ml gradients of 20 - 50% (w/v) sucrose in 0.1 mM sodium from Pharmacia P-L Biochemicals and emetine and cacodylate, pH 6.6, 100 mM KCl, 5 mM magnesium acetate, cycloheximide from Sigma Chemical Co. Purified initiation as described previously [14]. Centrifugation was for 45 min at factor eIF-2 (from mouse Ehrlich ascites tumour cells) was a 200000 x g . The gradients were pumped through a flow cell generous gift from Dr R. Panniers (University of Rochester in a Pye Unicam SP800 recording spectrophotometer and absorbance at 260 nm was recorded. Medical Center, New York). Cell culture The wild-type CHOl cells used were a proline-requiring (Pro-CHO-S) line [21]; tsHl is a temperature-sensitive leucyl-tRNA synthetase mutant selected from a subclone of CHO-S [15]. Arg-1 and Leu-23 are temperature-sensitive arginyl-tRNA and leucyl-tRNA synthetase mutants respectively, selected from a glycine, adenine and thymidine-requiring (GAT-) clone of CHO-S [17, 181. Cell culture was routinely carried out either at 34°C (tsH1, Arg-1 and Leu-21) or 37°C (wild type) in 75-cm2 plastic tissue-culture flasks or in stirred spinner-culture vessels. Growth medium consisted of CI minimal essential medium [22] containing 50 pg/ml asparagine H 2 0 and 8 % (v/v) foetal calf serum.

40s and 80s initiation complexformation in cell extracts

Initiation complex formation was measured by incubation of extracts for 2 min with either [35S]methionine(500 pCi/ml) or [3JS]Met-tRNAy' (approx. 5 x lo6 cpm/ml) under conditions optimal for protein synthesis [9, 141. Incubations contained 25 mM Hepes, pH 7.6, 110 mM KCI, 1 mM magnesium acetate, 6 mM 2-mercaptoethanol, 0.4 mM spermidine, 4% (v/v) glycerol, 1 mM ATP, 0.25 mM GTP, 5 mM creatine phosphate and 180 pg/ml creatine kinase. No unlabelled amino acids were added to those provided by the endogenous pools in the extracts. Cell extracts constituted 60% of the final incubation volume (100 pl). Incubations were carried out at 30 "C unless indicated otherwise. Radioactivity associated with initiation complexes was determined by sucrose density gradient centrifugation and fractionation as Measurement of protein synthesis in intact cells described previously [9, 141. Corrections were made for Cells in the exponential phase of growth (2- 5 x l o 5 background radioactivity by subtracting baseline values (see, cells/ml), growing in magnetically stirred spinner flasks for example, Fig. 1). in temperature-regulated water baths, were either maintained at 34°C or shifted up to 39.5-40°C for 30-45 min in the same growth medium to express the temperature-sensitive Protein synthesis in cell extracts synthetase mutations. In some experiments cells were collected Protein synthesis was assayed in 25-pl incubations by the by centrifugation (360 x g,,, 10 min) and resuspended at the incorporation of [35S]methionine into hot-trichloroaceticsame cell density in CI minimal essential medium minus acid-insoluble products, as described previously [9, 141. Inhistidine with 8% (v/v) dialysed foetal calf serum, and incubations contained components at the same concentrations cubated at 34°C for 30-45 min in order to effect amino acid as described in the previous paragraph. The cell extracts starvation. Towards the end of the incubation period 5 ml constituted 60% of the final volume. cells were removed and incubated at the appropriate temperature with a mixture of [3H]threonine, [3H]isoleucine and [3H]phenylalanine(each at 0.33 pCi/ml) for 15 min. Cells were elF-2 . GTP . Met-tRNAfM't ternary complex formation then processed to establish the relative rate of protein synin postribosomal supernatants thesis as previously described [23, 24). Formation of eIF-2 . GTP * [35S]Met-tRNAye'complexes was measured by incubating postribosomal supernatants with Preparation of cell-free extracts [35S]Met-tRNApe'(approx. 5 x lo6 cpm/ml) at 30°C for the The remainder of the cells from the above incubations times indicated in the figures. The incubations contained were harvested by centrifugation and postmitochondrial cell- 25 mM Hepes, pH 7.6, 100 mM KCl, 1 mM magnesium acefree extracts prepared as described in [9], except that the cells tate, 1 mM ATP, 0.25 mM GTP, 0.5 mM dithiothreitol and were lysed by addition of 0.25% (v/v) Nonidet P40 rather 0.2 mM EDTA. 3 mM phosphoenolpyruvate and 18 pg/ml than by homogenization. In some experiments extracts from pyruvate kinase were added where indicated. Postribosomal tsH1 cells and their wild-type controls were prepared in the supernatant constituted 20% of the incubation volume. 2O-pl presence of 4 mM leucine in order to stabilize the leucyl-tRNA aliquots were removed and placed in 1 ml cold buffer synthetase and attempt to maintain the in vivo activities of containing 20mM Hepes, pH 7.6, 100mM KC1, 2 m M this enzyme. However, this procedure had no effect on the magnesium acetate. The radioactivity bound to eIF-2 was relative abilities of the extracts to form 40s initiation determined by rapid filtration of the samples through individual 2.5-cm nitrocellulose filters or on wells of a Millititer type complexes in vitro. Postribosomal supernatants were prepared from the HA plate (Millipore). The filters were washed twice with the postmitochondrial supernatants by centrifugation at above buffer, dried and subjected to liquid scintillation count130000 x g for 3 h at 4°C. Postribosomal supernatants were ing. Characterization of this assay is described in Results.

41

Direction of Sedimentation 4

1.6

1.3 Control

Histidine - starved

39.5OC A

I

Y

1.8

8 N a

1.4

u

Fraction Number

Fig. 1. Polysome size distributions and initiation complex formation in cell-free extracts from tsH, cells incubated at 34°C or 39.5"C and with or without histidine. Extracts were prepared from tsHl cells incubated at 34°C for 30 rnin in GL minimal essential medium with (A and D) or without (B and E) histidine or from cells incubated at 39.5"C with histidine (C and F). The extracts were either layered directly onto 5-ml 20-50% sucrose gradients and centrifuged for 45 min at 200000 xg., to measure polysome size distributions (A-C) or were first incubated with [35S]methionine(500 pCi/ml) at 30°C for 2 min as described in Materials and Methods. Initiation complex formation (D-F) was then assayed by sucrose gradient centrifugation for 180 min at 200000 x g,, followed by fractionation, as described in Materials and Methods. The positions of native 40s and 60s ribosomal subunits, 80s ribosomes and polysomes arc indicated by arrows. 40s and 80s initiation complexes are indicated by the shaded areas

Analysis of eIF-2 content by immunoblotting of cell extracts

RESULTS

Aliquots of cell extracts were subjected to gel electrophoresis in the presence of sodium dodecyl sulphate and proteins were transferred to nitrocellulose at 60 V for 75 min, as described elsewhere [25]. The nitrocellulose blots were assayed for eIF-2 by binding sheep anti-(eIF-2), followed by rabbit anti-(sheep IgG) and lZ51-proteinA [25]. Radioactivity was detected by autoradiography of the blots.

Formation of initiation complexes in cell-free extracts

Estimations of methionine pool size in cell extracts

1OOyl aliquots of cell extracts were precipitated with icecold 3% (w/v) sulphosalicylic acid and the precipitates removed by centrifugation. The acid-soluble supernatants were analyzed with a Locarte amino acid analyzer and the concentrations of methionine calculated against known standards. Estimation of protein concentrations

The protein concentrations in cell extracts were determined as described previously [9].

When tsHl cells, growing at 34"C, are shifted up to 39.5"C in complete growth medium there is a rapid loss in their ability to charge tRNA with leucine and an 80-90% decline in the rate of protein synthesis [15, 161. Fig. 1 shows the effects of the non-permissive temperature on the distribution of ribosomes in polysomes (Fig. 1A and C), and the levels of [35S]Met-tRNA?"-containing40s and 80s initiation complexes formed on brief incubation of the cell-free extracts prepared from cells exposed to 34°C or 39.5"C (Fig. 1D and F). Exposure to the elevated temperature resulted in polysome disaggregation and large decreases in the formation of 40s and 80s initiation complexes, indicating inhibition of the initiation of protein synthesis at or before the 40s initiation complex stage. The effect of the exposure of the cells to 39.5 "C was observed even though the extracts were assayed at 30 "C. These changes in initiation at the non-permissive temperature are reminiscent of those observed when other cell types are starved for essential amino acids [6 - 101. We therefore also

42 ts H, 34°C

ts H,

39.5"C - 0.8

- 0.6

- 0.4

- 0.2 0 16

I

s c

wt

11 34°C

I

I

wt

1139.5OC

-0

I

Table 1. 4 0 s . Met-tRNA/M" initiation complex formation in extracts from various temperature-sensitive CHO cellmutants incubatedat 34 "C or 39.5 C ; comparison with protein synthesis in intact cells Cells were grown at 34°C as described in Materials and Methods. One half of each culture was shifted up to 39.5"C for 45 min (tsH1 and Leu-21) or 40°C for 3 h (Arg-1) and protein synthesis was monitored in the intact cells during the last 15 min by incorporation of [3H]threonine,isoleucine and phenylalanine in 5-ml aliquots of the cell suspensions. Cell extracts were prepared from the remainder and incubated for 2 min with [3SS]Met-tRNApunder protein synythesis conditions; the labelled extracts were then fractionated by sucrose gradient centrifugation and the radioactivity associated with 40s initiation complexes determined as described in Fig. 1. In order to compare extracts with slightly different ribosome concentrations the results are expressed as counts/min bound to 40s subunits per ,4260 unit of extract

Y

- 0.8

0 u) N

a

Cell line

Temperature of Protein synthesis incubation of cells

n

v)

LD

m

U

1

12

- 0.6

8

- 0.4

tsH,

v

$ i f

Leu-21

0 x

Arg-I

Y

E

-0.2

4 4

"C 34 39.5 34 39.5 34 40

cpm/I05 cells 250 40 360 130 460 350

40s initiation complexes

cpmlA 2 6 0 1200 220 550 190 460 230

0

To test whether the effect of the non-permissive temperature was specific for mutant cell lines, cell-free extracts were Fig. 2. Initiation complex formation in extracts from tsH, and wild- also prepared from wild-type CHO cells incubated at 34°C or type cells incubated at 34°C or 39.5"C.Extracts from tsHl cells and 39.5"C and the levels of initiation complexes formed were wild-type cells, which had been exposed to 34°C or 39.5 "Cfor 30 min, compared to those seen in the tsHl extracts (Fig. 2). In were incubated with [3SS]Met-tRNAp" at 30°C for 2 min. The conditions of incubation and method of analysis were as described in contrast to the tsH, cells there was a small stimulatory effect Fig. 1. 40s initiation complexes (fractions 8 - 14) and 80s initiation of the high temperature on the level of initiation complexes formed in the extracts from the wild-type CHO cells. We have complexes (fractions 4 - 7) are indicated by the shaded areas also investigated the effect of the non-permissive temperature on initiation in another independently derived CHO leucylexamined the effect of histidine starvation on tsHl cells at tRNA synthetase mutant Leu-21, and in Arg-I cells, which 34°C and observed decreases in polysome number (Fig. 1B) have a temperature-sensitive arginyl-tRNA synthetase [18]. and initiation complex formation (Fig. 1E) of a similar Extracts prepared from Leu-21 cells, which have been inmagnitude to the effects of the temperature shift. Protein cubated at 39.5"C, behave similarly to those from tsHl cells synthesis can, therefore, be regulated in these cells by two and exhibit a substantial inhibition of 40s complex formation distinct changes in the external cellular environment, both of (Table 1). This is similar in magnitude to the inhibition of which have similar actions on polypeptide chain initiation. overall protein synthesis at 39.5"C. The Arg-1 cell line is not It is known that incubation at the non-permissive tempera- as sensitive to elevated temperature as tsHl or Leu-21 and ture causes an increase in the rate of protein degradation in after 3 h incubation at 40°C its protein synthetic rate is only CHO cells with temperature-sensitive aminoacyl-tRNA reduced to 75% of that in cells at 34°C. Nevertheless, cellsynthetases [26,27l. This could have the effect of lowering the free extracts prepared from Arg-1 cells incubated at 40°C for specific activity of the radioactive methionine by dilution with 3 h also show a 50% defect in initiation complex formation an increased endogenous methionine pool in the 39.5"C cell (Table 1). These results indicate that the temperature-induced extracts; however, direct amino acid analysis did not reveal lesion in initiation complex formation occurs in several any expansion of the methionine pool (data not shown). Fur- independently isolated mutants containing different thermore, effects of the non-permissive temperature on the temperature-sensitive aminoacyl-tRNA synthetases, but is formation of initiation complexes similar to those shown in not observed in wild-type cells with normal synthetase activity. Since Met-tRNAy turns over rapidly in vitro as a result Fig. 1 were also obtained when precharged [35S]MettRNA)le' was used to label the complexes in extracts from of deacylation, it was important to establish whether the tsHI cells (Fig. 2). The amount of radioactivity bound to difference in labelling of 40s initiation complexes (Fig. 2) was initiation complexes was found to be variable from experiment due to a faster rate of decay of the added [35S]Met-tRNAy to experiment and between different pairs of 34°C and 39.5 "C in 39.5 "C extracts relative to 34°C extracts. As shown in Fig. 3 extracts. However, the effect of the temperature shift on the there was no significant difference in the rate of Met-tRNAf formation of initiation complexes was observed consistently deacylation in the two types of extract from tsHl cells (50% with numerous pairs of 34°C and 39.5"C extracts prepared loss of label in 14 min). Therefore the inhibition of labelling of initiation complexes in 39.5"C cell extracts was not due to on many separate occasions. Fraction Number

43

-I

Table 2. DifJrences in 40s complex formation are not due to changes in mRNA binding or polypeptide chain elongation rute in tsHl cell extracts Extracts were prepared from tsHl cells incubated at 34°C or 39.5"C for 45 min, as described in Materials and Methods. In experiment 1 an aliquot of each extract was treated with micrococcal nuclease (300 units/ml) for 5 min at 30°C in the presence of 1 mM CaCI2, conditions which are sufficient to destroy endogenous mRNA, followed by addition of EGTA to 2 mM. Nuclease-treated and untreated extracts were then incubated with [35S]Met-tRNApfor 2 min as described in Materials and Methods. Further aliquots of extracts were also subjected to nuclease treatment after initiation complex formation, to release any polysome-associated 40s complexes. All six samples were then analysed for labelled 40s complexes by sucrose gradient centrifugation, as in Table 1 . In experiment 2 40s initiation complex formation was allowed to occur by incubation for 2 min with [j5S]Met-tRNAp in the presence or absence of emetine (1 mM) or cycloheximide (100 pg/ml). The samples were analysed by sucrose gradient centrifugation. Note that experiments 1 and 2 used different pairs of extracts and different MettRNAy" preparations Expt

40s initiation complexes from cells incubated at

I

I

0

Additions

10

20 Time (mid

34 "C

30

Fig. 3. Rate ofdeacylation o J ' M e t - t R N A P in extractsfrom tsH, cells. Extracts from tsHl cells, exposed to 34°C ( 0 )or 39.5"c (o), werc incubated with [35S]Met-tRNAyetat 30°C. At the times indicated 5-pl samples of the incubations were pipetted on to Whatman no. 1 filter discs. The filters were washed in three changes of ice-cold 5% (w/v) trichloroacetic acid, followed by ethanol and acetone. The filters were then dried and the cold-acid-insoluble radioactivity determined by liquid scintillation counting

39.5"C

cPm/A 260 1.

2.

None Micrococcal nuclease (before incubation) Micrococcal nuclease (after incubation) None Emetinc Cycloheximide

5670 5560

580 240

6840

460

970 2210 21 10

340 860 5 50

an enhanced rate of deacylation of added [35S]Met-tRNAyet in vitro. Influence of mRNA binding and polypeptide chain elongation rate on levels of initiation complexes

The level of 40s initiation complexes measured in vitro represents a balance between the rates of formation of these complexes and their utilization in subsequent steps of the initiation pathway. In theory a more rapid rate of binding of the 40s complexes to the 5' end of mRNA in polysomes at 39.5"C relative to 34°C could lead to a depletion of these complexes in tsH, cells. In order to test whether the latter process might be responsible for the difference between extracts from tsHl cells incubated at 34°C or 39.5"C, we have assayed labelled 40s complexes formed after a brief micrococcal nuclease treatment sufficient to destroy endogenous mRNA (Table 2). There was no effect on the 34°C extract and a slight decrease in 40s complexes formed in the 39.5 "C extract, relative to non-nuclease-treated incubations. We also examined the effect of nuclease treatment performed after 40s complex formation (Table 2). Again no major effects on the yield of the complexes were observed. These results indicate that changes in binding to polysomal mRNA cannot be responsible for the effects of the non-permissive temperature on the level of 40s initiation complexes in tsHl cells. They also show that the majority of 40s. Met-tRNAy" complexes are not associated with polysomal mRNA in the extracts. Similarly, possible differences in the rate of ribosome movement on mRNA during polypeptide chain elongation appear not to determine the relative levels of 40s complexes;

addition of elongation inhibitors had no effect on the difference in 40s complexes between 34°C and 39.5"C extracts, although this did increase the yield of these complexes (Table 2). Amount and activity of initiation.factor eIF-2 in extracts of tsHl cells

Regulation of the activity of initiation factor eIF-2 has been shown to be a major point of control for polypeptide chain initiation in several eukaryotic systems [ 2 , 3, 9, 28301, the best documented example being the effect of haemin deprivation in reticulocytes [31,32]. We have, therefore, investigated whether the defect in initiation complex formation in extracts from temperature-sensitive mutants is associated with impairment of activity of eIF-2. The eIF-2 content of cell extracts was compared by sodium dodecyl sulphate gel electrophoresis and immunoblotting of postmitochondrial supernatants using a polyclonal sheep anti-(eIF-2) serum. The amount of antibody bound was determined by l 2'I-protein A binding and autoradiography. As shown in Fig. 4, the antiserum binds to both the c( and /3 subunits of eIF-2, as well as to a polypeptide species of 82 kDa, which has been identified as a subunit of the guanine nucleotide exchange factor eIF-2B (R. Jagus, unpublished data). In three separate experiments we have failed to find any difference between 34°C and 39.5"C extracts in the amounts of these components, indicating that the initiation

44

50 = =o-

,.--

- o'

-

39.5OC

-0-0-

- - 0 - - - - - --. - - 0 - - - - - -0 20

10

30

Time (mid

Fig. 4. Identification by immunoblotting of eIF-2 in extracts from tsHl cells. Postmitochondrial supernatants from tsHl cells exposed to 34°C (track 1) or 39.5"C for 45min (track 2) were subjected to electrophoresis on a 15% sodium dodecyl sulphate/polyacrylamide gel and transferred to nitrocellulose paper. After blocking non-specific sites with Blotto [52] the blots were probed with sheep anti-(eIF-2) antibodies as described in Materials and Methods. The amounts of bound antibody were quantified by rabbit anti-(sheep IgG) and '"I-protein A binding, followed by autoradiography. The Q and fl subunits of eIF-2 are indicated, together with the relative molecular masses of these bands and of a larger component which reacts with the anti-(eIF-2) serum

defect at the non-permissive temperature is not due to any degradation or loss of eIF-2 protein during cell incubation or fractionation. Under physiological salt conditions a substantial proportion of the eIF-2 activity in mammalian cells is in the cytosol fraction of the cell, rather than being associated with the ribosomes [33]. We have, therefore, measured ternary complex formation between [3SS]Met-tRNAp, GTP and eIF-2 in postribosomal supernatants. Complex formation in this assay is strongly stimulated by a GTP-regenerating system (Fig. 5A) owing to the removal of GDP, which is inhibitory for eIF-2 activity [34]. Complex formation is dependent on added GTP and is greater at low magnesium concentrations (0.21.O mM) than at high magnesium concentrations (data not shown). These properties are all characteristic of eIF-2 [34371 and provide evidence that the Met-tRNAy binding assay is a valid measure of the activity of this initiation factor. Under the conditions used, ternary complex formation was greatly decreased in the 39.5 "C postribosomal supernatants from tsH, cells, relative to the activity of the 34°C preparations (Fig. SA). No such effect was observed when a similar experiment was performed with postribosomal supernatants from

10 20 Time(min1

30

Fig. 5. Ternary complexformation by postribosomal supernatantsfrom tsH, cells. (A) Effect of incubation of cells at 39.5"C. Postribosomal supernatants were prepared from cells incubated at 34°C or 39.5"C for 30 min. Time courses of ["SIMet-tRNAy .eIF-2. GTP complex formation at 30°C were determined as described in Materials and or absence (- - - -) of a GTPMethods, in the presence (-) regenerating system. The results are corrected for differences in protein concentration in the preparations. ( 0 ) Postribosomal supernatant from 34°C tsHl cells; (0)postribosomal supernatant from 39.5"C tsHl cells. (B) Effect of amino acid starvation. Postribosomal supernatants were prepared from cells incubated in complete medium or in the absence of histidine at 34°C for 30 min. Time courses of [3SS]Met-tRNAy eIF-2 . GTP complex formation were determined in the presence of a GTP-regenerating system. ( 0 ) Postribosomal supernatant from fed tsHl cells; (0)postribosomal supernatant from histidine-deprived tsHl cells 3

wild-type cells (data not shown). Because of the similarities between the effects of the non-permissive temperature and amino acid starvation on initiation (Fig. 1) we also examined the effect of starvation on eIF-2 activity in this assay. A large difference was again obtained when ternary complex formation was measured in postribosomal supernatants from control and histidine-deprived cells (Fig. 5 B). If polypeptide chain initiation in extracts from tsH, cells exposed to the non-permissive temperature is limited by the impaired activity of eIF-2, it should be possible to overcome the inhibition by adding back purified eIF-2 in vitro. Fig. 6 shows that this is the case. Protein synthesis in extracts from cells incubated at 39.5 "C is strongly stimulated by exogenous eIF-2, whereas the factor has little effect on the corresponding

45 extracts from cells maintained at 34°C. As a result, the differ- conditions we cannot detect any dominant inhibitor of initiaence in activity between the extracts is virtually eliminated in tion complex formation in tsHl cells incubated at 39.5"C. the presence of added eIF-2. We have investigated the possibility that tsHl cells in- The effect of high temperature in vitro on initiation cubated at 39.5"C may contain a dominant inhibitor of eIF-2 Since initiation activity in the above experiments reflected activity analogous to the haem-controlled repressor of reticulocytes [2, 3, 38, 391. Equal volumes of tsHl 34°C and the behaviour of the cells from which the extracts were pre39.5 "C postribosomal supernatant were mixed and either pared, it was of interest to see whether a defect in initiation assayed immediately or preincubated at 30°C for 15 min. complex formation could also be generated by incubation of Ternary complex formation was then measured after incuba- tsHi cell extracts at 39.5"C in vitro. Extracts from tsHl cells tion for up to 25 min with [35S]Met-tRNAp.The activity of grown at 34°C were preincubated at 39.5"C for various times eIF-2 in the mixed extracts was close to the mean of the values followed by a further incubation with [35S]Met-tRNAye',and shown by the two extracts individually, even after preincuba- the levels of ternary complexes or 40s initiation complexes tion (data not shown). Therefore, at least under these formed were determined. Table 3 shows the results of the two types of experiment, which indicate that no major defect in either ternary complex or 40s initiation complex formation develops under these conditions. The level of 40s initiation complexes formed is, in fact, higher at 39.5"C than at 30°C, presumably reflecting faster rates of reaction at the higher temperature. This stimulatory effect of the higher temperature occurs also in extracts from wild-type CHO cells (Table 3). These findings indicate that initiation in extracts from tsHl cells is not directly temperature-sensitive and that there is no intrinsic defect in eIF-2 in this cell line. The contrast between the behaviour of the cell extracts and the intact cells in response to the non-permissive temperature suggests that expression of the tsHl phenotype at the level of chain initiation requires integrity of cell structure or function, which is lost on preparation of postmitochondrial supernatants for in vitro studies. 0 20 40 60 eIF-2 ( p g l r n l )

Fig. 6. Stimulation of protein synthesis by eIF-2 in cell extract from tsHl cells incubated at the non-permissive temperature. Cell extracts were prepared from tsH, cells incubated for 45 rnin at 34°C ( 0 )and 39.5"C (0)and assayed for protein synthetic activity as described in Materials and Methods. The indicated amounts of purified eIF-2 (from Ehrlich ascites tumour cells) were added at zero time and incorporation of [35S]methionine (100 pCi/ml) into protein in 2 0 4 aliquots was assayed after 60min at 30°C. Approximately equal amounts of the two cell extracts were used (tsH, 34°C: 108 Az,o/ml; tsH, 39.5"C: 113 ,4z60/rnl)

DISCUSSION In mammalian cells polypeptide chain initiation is regulated by the availability of essential amino acids [4 - 7, 10, 40, 411 and by conditions under which amino acid activation and tRNA charging are inhibited [ l l - 131. In the experiments described here we have shown that amino acid deprivation and impairment of aminoacyl-tRNA synthetase activity have very similar effects on the control of polypeptide chain initia-

Table 3. The effect of incubation at 393°C on formation of initiation complexes in extracts from wild-type and tsHl cells grown at 34°C Extracts were prepared from wild-type or tsHl cells grown at 34°C. For measurement of ternary complex formation, postribosomal supernatants were obtained by high-speed centrifugation (Materials and Methods) and preincubated at 39.5"C for the times shown. [35S]MettRNAr (3 x lo5 cpm) and the other required components were then added and the extent of ternary complex formation was assayed after a further 30 rnin at 395°C as described in Materials and Methods. For measurement of 40s initiation complex formation, cell extracts were preincubated at either 30°C or 39.5 "C for 2 min. [35S]Met-tRNAv(6 x lo5 cprn) was then added and the incubations continued for a further 2 min at the same temperatures. The conditions of incubation and method of analysis on sucrose gradients were as described in Fig. 1 and Table 1 Type of extract

Wild type 34 "C

Ternary complex formation preincubation at 39.5"C

[eIF-2. GTP . MettRNAp]

temperature of incubation

40s initiation complexes

min

CPm 4400 5450 6200 5850 9550 10050 7900 8550

"C

CPdA 2 6 0 4100

0 10 20 30

tsHl 34°C

40s initiation complex formation

0 10 20 30

30 39.5

30 39.5

5150

5700

8350

46 tion in the same cell type (Figs 1 and 5) and that this control is mediated by changes in eIF-2 activity (Figs 5 and 6). However, there is no obvious relationship between tRNA charging per se and initiation since changes in the rate of polypeptide chain elongation, designed to alter the demand for aminoacyltRNAs and hence the ratio of charged: uncharged tRNAs, do not affect the control of 40s complex formation by amino acid supply [14]. This finding suggests that changes in amino acid supply and aminoacyl-tRNA synthetase activity regulate initiation by another mechanism, not directly related to the level of tRNA charging. Our observation that, in cell-free systems from the synthetase mutants incubated at 39.5-4O"C, the defect in initiation is expressed at the level of 40s initiation complex formation confirms an earlier brief report from Vaughan's laboratory [42]. Our data indicate that this defect is due to a decrease in activity but not total amount of initiation factor eIF-2. Formally it is possible that the temperature shift changes the distribution of eIF-2 between ribosomes and supernatant, resulting in decreased ternary-complex-forming activity in the latter (Fig. 5). However, this cannot explain the lower 40s complex formation in unfractionated cell extracts (Figs 1 and 2) or the reversal of protein synthesis inhibition by exogenous eIF-2 (Fig. 6). Heating to 39.5"C of an extract from tsH1 cells grown at 34°C does not cause any large reduction in the level of initiation complexes formed. This shows that there is no temperature-sensitive lesion in eIF-2 activity itself in tsH, cells, a conclusion which is consistent with the genetic evidence that the tsHl mutation belongs to a single complementation group identified as the leucyl-tRNA synthetase. The mechanism by which eIF-2 is inactivated could involve changes in its state of phosphorylation and modification of its ability to exchange guanine nucleotides between successive rounds of chain initiation [31, 32, 341. We have previously been unable to observe any difference in the ability of extracts from fully fed cells and amino-acid-starved cells to phosphorylate eIF-2 in vitro [29,43] and mixing experiments have failed to reveal any dominant soluble inhibitor of ternary complex formation such as an eIF-2 protein kinase. The possibility of an inhibitory protein kinase cannot be completely excluded, however. An alternative mechanism could involve inactivation of the factor eIF-2B, responsible for GDP/GTP exchange and recycling of eIF-2 between successive rounds of protein synthesis [31, 34, 371. Such a phenomenon could also lead to inactivation of endogenous eIF-2, but without any change in the phosphorylation state of the latter. These possibilities are currently under investigation in our laboratory. A possible regulatory mechanism by which aminoacyltRNA synthetases could influence other components of the protein synthetic machinery may be through the reversible phosphorylation of the synthetases. Damuni et al. [44] have presented indirect evidence for changes in the phosphorylation state and activity of several synthetases from rat liver in response to hormonal and nutritional influences and at least one enzyme has been shown to be phosphorylated in intact CHO cells [45]. Although the significance of these findings is not yet clear, the existence of many synthetases in the form of large, multienzyme complexes [46-481, perhaps associated with other protein synthesis factors 1491would be compatible with regulatory links between these enzymes and factors involved in polypeptide chain initiation. The sensitivity of protein synthesis to inhibition at non-permissive temperatures in CHO cells with temperature-sensitive synthetases has been shown to be associated with a difference in the physical state

of these enzymes [50, 511 although recently Mirande et al. (531 were unable to observe this in tsHl cells. In conclusion, the observations described here demonstrate a role for components of the polypeptide chain elongation machinery in the regulation of polypeptide chain initiation, mediated by changes in the activity of initiation factor eIF-2. Further investigations of the mechanisms involved should yield novel information about the translational control of protein synthesis in eukaryotic cells. This work was performed with the skillful technical assistance of Sigrid Burridge, Vivienne Tilleray and Margaret Seagrave. We thank Dr R. Panniers for the generous gift of initiation factor eIF-2. We are grateful to Drs Jenny Pain and Angela Galpine for discussions, Barbara Bashford for the drawings and Vivienne Marvel1 for preparation of the manuscript. The work was supported by grants from the Cancer Research Campaign (S.A.A., M.J.C. and J.W.P.) and the Medical Research Council (J.W.P.). M.J.C. holds a Career Development Award from the Cancer Research Campaign.

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