Proc. Nati Acad. Sci. USA Vol. 80, pp. 3148-3152, June 1983 Biochemistry

Inhibition of eukaryotic protein chain initiation by vanadate (protein synthesis) RAjINDER SINGH RANU Department of Microbiology and the Graduate Program in Cellular and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523 Communicated by Raj C. Bose, December 30, 1981 ABSTRACT Vanadate inhibits protein chain initiation in - grade) from Baker; cetyltrimethylammonium bromide and bit reticulocyte lysates. The evidence that supports this conclusion poly(uridylic acid) from Sigma; purified 9S globin mRNA from is as follows: (i) the biphasic kinetics of inhibition in which protein Searle (Wycombe, England); and [3S] (1,200 Ci/ synthesis is maintained at the control rate for 1-2 min is followed mmol; 1 Ci = 3.7 X 1010 Bq), [14C]leucine (320 mCi/mmol), by an abrupt decline in the rate of synthesis; (ii) inhibition is as- and [14C]phenylalanine (270 mCi/mmol) from New England sociated with a marked disaggregation of polyribosomes and a Nuclear. Sparsomycin (NSC 59729) was provided by Natural concomitant increase in 80S ; and (iii) vanadate concen- Products Branch, Division of Cancer Treatment, National Can- trations that inhibit protein chain initiation do not inhibit poly- cer Institute. The noncapped satellite tobacco necrosis virus peptide chain elongation or the aminoacylation of tRNA. In par- was Clark of tial reactions of protein chain initiation, vanadate concentrations (STNV) RNA provided by J. (University Illinois, that inhibit protein synthesis have no detectable effect on the for- Urbana, IL). The sources of other reagents have been de- mation of eukaryotic initiation factor eIF-2-promoted ternary scribed (12). The following procedures also have been de- complex with Met-tRNAf and GTP and on the assembly of 40S scribed: preparation of rabbit reticulocyte lysates and protein- ribosomal subunit-Met-tRNAf complexes. On the addition of synthesis reaction mixtures, assay of protein synthesis, the mRNA, the 40S ribosomal subunit-Met-tRNAf complexes also are preparation of purified eIF-2 (12), and the preparation of [3S]- transformed into 80S -mRNA-Met-tRNAf complexes, Met-tRNAf (100,000 cpm/pmol) (12, 13). termed 80S initiation complexes. In vanadate-treated samples, Inhibition of Protein Synthesis by Vanadate. Rabbit retic- however, these 80S initiation complexes are defective and unable ulocyte lysate-based protein-synthesis reaction mixtures (25 .ul) to proceed beyond this step. containing 10 puM hemin were incubated at 30°C with various concentrations of vanadate. At intervals, aliquots (5 ,ud) were The requirement of vanadium in trace amounts as an essential removed and protein synthesis was assayed (12). The vanadate nutrient has been recognized for some time (1, 2). The vana- solutions used in this study were prepared fresh each day in dium compounds in moderately high levels can be highly toxic deionized distilled water. (1, 2). Although the biological role of vanadium at the molecular Assay of Poly(uridylic Acid)-Dependent Polyphenylalanine level is not known, recent interest in vanadate has arisen as a Synthesis in Lysates. Rabbit-reticulocyte-lysate reaction mix- result of the findings of Cantley et aL (3) that vanadate is a po- tures (25 u1) containing 10 puM hemin were incubated at 30°C tent inhibitor of membrane Na+,K+-ATPase. Vanadate also in- with (25 ,ug) or without poly(uridylic acid) in the presence of hibits a variety of other -e.g., ATPase, dy- 8 mM Mg2+. Under these conditions, the endogenous natural nein ATPase, Ca2+-ATPase (sarcoplasmic reticulum), Mg2+- mRNA-dependent protein synthesis is completely suppressed, ATPase, and adenylate kinase (4, 5). Almost all of these en- and maximal poly(uridylic acid)-dependent polyphenylalanine zymes are phosphohydrolases, and frequently a phosphoen- synthesis is observed. At intervals, aliquots were removed and zyme intermediate is involved in the mechanism of action of protein synthesis was assayed (12). these enzymes. Current evidence suggests that vanadate com- Assay of Aminoacylation of tRNA. The aminoacylation assay petes with phosphate for the - (4). was carried out under conditions of protein synthesis (12). Ed- The protein is dependent on a series of reac- eine (5 ,uM) was added to the reaction mixture to block initia- tions that require ATP and GTP hydrolysis-e.g., aminoacyl- tion of protein synthesis (14). At intervals, aliquots (5 ,ul) were ation, GTP- and ATP-dependent initiation of polypeptide, and removed and transferred to 1 ml of 10% cold trichloroacetic the GTP-dependent elongation and termination of polypeptide acid containing 0.5 mM methionine or leucine (see Fig. 4 leg- (6-8). The eukaryotic protein synthesis also is regulated by ATP- end). The precipitate was collected on Millipore filter and was dependent protein kinases that are activated in the presence of washed extensively with cold 5% trichloroacetic acid (15). The double-stranded RNA or by heme deficiency (9-11). These filters were dried and radioactivity was assayed. considerations and the apparent selective inhibition of the The assay of eukaryotic initiation factor eIF-2-dependent by vanadate prompted the examination of the effect ternary complex (eIF-2'GTP'Met-tRNA) formation and the as- of vanadate on protein synthesis in eukaryotes. The results pre- say of the formation of 40S ribosomal subunit-Met-tRNAf com- sented in this report show that vanadate preferentially inhibits plexes in lysates have been described (12, 13, 16). protein chain initiation. Analysis of the Distribution of Polyribosomes. The polyri- bosome distribution in protein-synthesis reaction mixtures was MATERIALS AND METHODS analyzed in sucrose density gradients (10-45%) in buffer A (20 The materials utilized in these studies were obtained from the mM Tris HCl, pH 7.6/80 mM KCl/2 mM magnesium acetate). following sources: ammonium metavanadate (analytical reagent Aliquots of reaction mixture (25 ,ul) were removed and trans- ferred to 100 1,u of ice-cold buffer A. The sample was layered The publication costs of this article were defrayed in part by page charge over a sucrose density gradient and then was centrifuged at 38,000 payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: eIF, eukaryotic initiation factor. 3148 Downloaded by guest on October 2, 2021 Biochemistry: Ranu Proc. Natd Acad. Sci. USA 80 (1983) 3149 rpm in a Spinco SW-50. 1 rotor for 2 hr at 4TC. The absorbance The radioactive peptides were stripped of tRNA by exposure profile was monitored in a ISCO density gradient monitor at to 1% trimethylamine. The samples were applied to Whatman 254 nm. The fractions from the gradients were analyzed for the 3 MM filter paper strips along with internal standards [methi- radioactivity associated with the nascent polypeptide chains ac- onine (10 gg) and methionylvaline (20 pg)] and were subjected cording to Darnbrough et aL (13). to ascending chromatography at room temperature in butanol/ Assay of Formation of 80S Initiation Complexes. The for- acetic acid/H20, 45:5:12.5 (vol/vol) (15). The positions of the mation of 80S ribosome-mRNA-Met-tRNAf complexes, termed markers (methionine and methionylvaline) were located with 80S initiation complexes, was determined by shift assay in ly- ninhydrin. The paper was cut into 1.5-cm pieces and assayed sate protein-synthesis reaction mixture (33 y1) containing 20 for radioactivity. ,M hemin. Incubation with or without 20-30 AuM vanadate was at 30TC for 5 min. Sparsomycin (40 ,M) was then added, RESULTS AND DISCUSSION and incubation was continued for another 3.5 min, at which time The effect of vanadate on eukaryotic protein synthesis was ex- [35S]Met-tRNAf (100,000 cpm) and 2 Ag of globin mRNA were amined in rabbit reticulocyte lysates because in this system added. After 2 min of incubation, the sample was diluted with the in vitro rates of protein chain initiation and elongation ap- 130 td of ice-cold buffer B (10 mM Hepes, pH 7.6/80 mM KCV proach the in vivo rates (12). Moreover, the requirement of heme 2 mM magnesium acetate). The sample was layered over a 5.2- for the maintenance of protein synthesis, first observed in in- ml 10-35% sucrose density gradient in buffer B. The samples tact cells, is preserved in lysates (12). were centrifuged at 45,000 rpm in a Spinco SW-50. 1 rotor for Vanadate strongly inhibited protein synthesis (Fig. 1A). The 2 hr at 2°C. The absorbance profile of the gradients was mon- inhibition of synthesis with 10-40 ,uM vanadate showed a con- itored at 254 nm. The fractions were then analyzed for radio- centration dependence. Beyond these concentrations, the in- activity as described by Darnbrough et aL (13). hibition reached a plateau. The kinetics of inhibition in the Assay of the Formation of Initiation Dipeptide (Methionyl- presence of 10 ,uM vanadate showed that protein synthesis dur- valine) of Globin. The micrococcal nuclease-treated lysate (16) ing the first 4-6 min was maintained at the control rate, fol- protein-synthesis reaction mixtures (40 Al) containing 24 ,uCi lowed by a progressive decline in the rate of synthesis. In the of [3S]methionine and 0.7 Ag of purified globin mRNA were presence of 20 ,uM vanadate, synthesis at the control rate was incubated at 30°C in the presence of sparsomycin (0.2 ,uM) or maintained only for the first 1-2 min, and then there was an vanadate (30 ,AM). A control without added mRNA also was in- abrupt decline in the rate of synthesis. However, after this sharp cluded [the micrococcal nuclease-treated lysate system itself decline, a synthesis at5-10% of the control rate was preserved. shows a high rate of formation of 80S initiation complexes be- These biphasic kinetics of inhibition of protein synthesis cause of the presence of mRNA fragments; these fragments suggest that vanadate inhibits protein chain initiation. The ex- compete with the added globin mRNA in the formation of 80S perimental results in Fig. 2 support this view. The vanadate- initiation complexes (unpublished data; ref. 17)]. The samples induced inhibition of protein synthesis was associated with the were incubated for 10 min. The reaction was stopped by the disaggregation of polyribosomes and a concomitant increase in addition of 180 ,l of ice-cold buffer A containing sparsomycin 80S ribosomes. There was a marked decline in the radioactivity (0.2 AM) or vanadate (30 ,M). The samples were layered on associated with the nascent polypeptides in the polyribosome top of respective sucrose density gradients (10-45% in buffer region. The polyribosome profile and the radioactivity associ- A) containing sparsomycin or vanadate and centrifuged at 49,000 ated with the polyribosome fraction during the first minute of rpm in a Spinco SW-50. 1 rotor for 2 hr at 2°C. Fractions from incubation in the control and vanadate-treated sample were mono- and polyribosome regions of the gradient were pooled. similar (results not shown). After 3 min of incubation, there was The ribosome-bound radioactivity (to tRNA) was extracted with a pronounced disaggregation of polyribosomes into 80S ribo- phenol at pH 5.2 in the presence of carrier tRNA (1 mg/ml). somes and a marked decline in the radioactivity associated with 100 A B

* / ~~~~~2

i=50 /0=04 _S 1 l 3~~~~~~~~~~~~~U

100 200 20 Vanadate, 1M Time, min FIG. 1. Inhibition ofprotein synthesis by vanadate. Protein-synthesis reaction mixtures (25 sAl) containing 10 MM hemin were incubatedat30C for various times with or without the indicated vanadate concentrations, and 5-,l aliquots were removed for assay. (A) Protein synthesis after 40 min of incubation with vanadate at various concentrations. (B) Protein synthesis at indicated incubation times without vanadate (-) or with 10 pM (-) or 20 pM (A) vanadate. Downloaded by guest on October 2, 2021 3150 Biochemistry: Ranu Proc. Natl. Acad. Sci. USA 80 (1983)

U - 1~~~~-

e0.25 A

Fraction

FIG. 2. Effect ofvanadate on polyribosomes. Protein-synthesis reaction mixtures (110 ,l) containing10 ,M hemin and [35S]methioniine (14 ,uCi) were incubated with 20 ,uM vanadate (D, E, or F) or without vanadate (A, B. and C) at 30TC. After 3 (A and D), 6 (B and E), and 12 (C and F) min of incubation, 25-,ul aliquots were removed for the analysis of polyribosomes. (Inset) For protein synthesis assay, 5-,ul aliquots were taken out at 3-, 6-, 12-, 20-, and 40-min intervals. *, Without vanadate; A, with 20 ,uM vanadate.

the polyribosomes in vanadate-treated sample. This finding is trol) of synthesis that is maintained under these conditions (Fig. consistent with the observed kinetics of inhibition shown in Fig. 1B and Fig. 2F Inset). 1B and Fig. 2F Inset. The low level of radioactivity that re- The conclusion drawn from the data in Fig. 1 and Fig. 2 is mained associated in the polyribosomal region in vanadate-treated supported further by the experiments (Fig. 3) in which poly- samples (Fig. 2 E and F) after the onset of inhibition of protein (uridylic acid)-dependent polyphenylalanine synthesis was as- synthesis is a reflection of the reduced rate (5-10% of the con- sayed in lysates in the presence of 8 mM Mg2+. Under these conditions, initiation of protein synthesis dependent on natural mRNA is bypassed. Hence, a measure of the effect of vanadate on polypeptide chain elongation is afforded. The similar ki- co netics of polyphenylalanine synthesis in control and samples 9 treated with 20 ,uM vanadate (Fig. 3B) suggest that vanadate x At -4 has no measurable effect on polypeptide chain elongation. 9 cS P vanadate concentrations higher than 50 ,tM, however, some ,-~E inhibition of polypeptide chain elongation began to emerge (Fig. 3A). The results in Fig. 3B also suggest that vanadate does not QO 3 inhibit the aminoacylation of tRNA of phenylalanine. Similarly, C.) the results in Figs. 1 and 2, in which ["4C]leucine and [3S]me- thionine, respectively, were used for protein-synthesis assay, are indicative of the fact that, here too, the effect on the ami- 50 150 20 noacylation of tRNA of leucine and methionine may not be a Vanadate, AM Time,min factor in the vanadate-dependent inhibition of protein synthe- sis. This conclusion is born out by the experimental results in FIG. 3. Effect of vanadate on poly(uridylic acid)-dependent poly- Fig. 4 showing the similar rate of formation of the Leu-tRNA phenylalanine synthesis. Protein synthesis reaction mixtures (25 ,ul) and the Met-tRNA in control and vanadate-treated samples. were incubated at 3000 with or without vanadate for various time in- The results on and polypeptide chain tervals, and aliquots were removed-for assay. (A) Polyphenylalanine negative aminoacylation synthesis after 60 min of incubation with vanadate at various concen- elongation, the biphasic kinetics of inhibition, and the disag- trations. (B) Polyphenylalanine synthesis at various incubation times gregation of polyribosomes with a concomitant increase in 80S without vanadate (e) or with 20 pM vanadate (A). ribosomes provide compelling evidence that vanadate under Downloaded by guest on October 2, 2021 Biochemistry: Ranu Proc. Natl. Acad. Sci. USA 80 (1983) 3151 AuM vanadate-induced inhibition was not prevented by the ad-

CY3 dition of excess Mg2+ (25-800 AtM) or MgATP (50-400 ,AM; 0 results not shown). Vanadate also inhibited protein synthesis '--4 when addition was made after 10 min of preincubation of pro- x tein-synthesis reaction mixtures at 30TC (results not shown). C) The assembly of an 80S initiation complex in eukaryotes in- volves a series of discrete steps. (i) The formation of a ternary z complex (eIF-2 GTP Met-tRNAf) with Met-tRNAf and GTP is U: 4.. promoted by the initiation factor eIF-2 and several ancillary IFs (6, 9, 18-20). (ii) The ternary complex binds to 40S ribosomal subunit and forms complexes of 40S ribosomal subunit-Met- tRNAf (6, 18). (iii) Insertion of mRNA into the 40S ribosomal subunit-Met-tRNAf preinitiation complexes is promoted by 5 10 5 10 several IFs and the cap binding protein (6). This step also re- quires ATP (6). (iv) Finally, the joining of the 60S ribosomal Time, min subunits to the 40S initiation complex is catalyzed by eIF-5 (6). FIG. 4. Effect of vanadate on the formation ofLeu-tRNA and Met- The effect of vanadate on the formation of the eIF-2-promoted tRNA. Lysate protein synthesis reaction mixture (30 ud) containing ternary complexes was determined: vanadate concentrations that [35S]methionine (2 ,Ci) or ['4C]leucine were incubated at30°C with (A) inhibited protein synthesis had no detectable effect on ternary or without (0) 20 ,uM vanadate. At intervals, aliquots were assayed for complex formation (results not shown). The formation of steady- aminoacylation. state levels of the 40S ribosomal subunit-Met-tRNAf com- plexes also was measured in situ in lysates in the presence and these conditions inhibits protein chain initiation. It should be absence of vanadate. This assay is also a measure of the for- pointed out that the effect of excess magnesium and ATP on mation of ternary complexes under natural conditions, the con- vanadate-induced inhibition of protein synthesis was also ex- ditions under which vanadate strongly inhibits protein synthe- amined (in addition to 2 mM Mg2' and 1 mM ATP already pres- sis (Fig. 1). Here again, vanadate had no measurable effect on ent in the lysate protein-synthesis reaction mixture). The 20 the formation of 40S ribosomal subunit-Met-tRNAf complexes

Control 40S A B 40S II ~~~~8 808 9 80j 0

0.25 °1 0 ii Q 99 4 0 1 0 0 x Va Id ° Vanadate

I' 40 Z~~~C I'~~~~~~~-

Fraction FIG. 5. Formation of 80S initiation complexes. (A and B) Controls incubated without (A) and with (B) 9S globin mRNA. (C and D) Vanadate- treated samples incubated without (C) and with (D) 9S globin mRNA. Downloaded by guest on October 2, 2021 3152 Biochemistry: Ranu Proc. Natl. Acad. Sci. USA 80 (1983) (results not shown; see data in Fig. 5). This finding also is sup- Table 1. Effect of vanadate on the formation of initiation ported by the fact that addition of exogenous eIF-2 did not re- dipeptide (methionylvaline) of globin lieve the vanadate-induced inhibition of protein synthesis (re- [3"S]Met, [3"S]Met-Val, sults not shown) and, therefore, suggests that the inhibition of Exp. Additions cpm cpm lysate protein synthesis by vanadate does not involve the ac- tivation of the heme-regulated protein kinase and the phos- 1 Control 7,517 2,840 phorylation of eIF-2 (9, 12). Globin mRNA + sparsomycin 7,529 2,970 These negative results focus the binding of mRNA and the Globin mRNA + vanadate 9,250 3,408 subsequent joining of 60S ribosomal subunits as the potential 2 Control 6,484 2,280 targets for further investigation into the molecular basis of the Globin mRNA + sparsomycin 7,577 2,723 vanadate-induced inhibition of protein synthesis. The forma- Globin mRNA + vanadate 9,223 3,181 tion of 80S initiation complexes from 40S ribosomal subunit- Met-tRNAfcomplexes in the presence of mRNA represents not only a measure of the formation of 40S ribosomal subunit-Met- the time of initiation and not subsequently. tRNAf-mRNA complexes but also their competence in thejoin- The ability to select an isolated event in reactions involved ing of 60S ribosomal subunits. The results are presented in Fig. in the initiation of polypeptide chain is of considerable value 5. In this experiment, lysates were first incubated with or with- in the elucidation of the mechanism of assembly of an initiation out vanadate for 5 min. They then were incubated for 3.5 min complex. Vanadate may permit the identification of those fac- with sparsomycin before the addition of [wS]Met-tRNAf. Un- tors involved in the formation of functional 80S initiation com- der these conditions there was almost exclusive labeling of 40S plexes involving mRNA and ribosomes. subunits with ['S]Met-tRNAf (Fig. 5 A and C) in agreement This investigation was supported by National Science Foundation Grant with the previous finding (13). The radioactivity associated with PCM 80 21969, Biomedical Research Support Grant 2 SO 7RR 05458- 40S ribosomal subunit in vanadate-treated sample was 2- to 2.5- 20, and the U.S. Department of Agriculture Animal Health and Dis- fold higher, reflecting the fact that under this condition there ease Research Program. were more subunits available. Upon the addition of 9S globin 1. Schwartz, K. (1974) in Trace Elements in Animals, eds. mRNA (Fig. 5 B and D) the formation of 80S initiation com- Hoekstra, W. G., Suttie, J. W., Ganther, H. E. & Mertz, W. plexes was observed. In the vanadate-treated sample there was (University Park Press, Baltimore), Vol. 2, pp. 355-380. a marked increase in the amount of radioactivity associated with 2. Underwood, E. J. (1977) Trace Elements in Human and Animal 80S ribosomes There was also a notable in Nutrition (Academic, London), 4th Ed., pp. 416-424. (Fig. 4D). decrease 3. Cantley, L. C., Jr., Josephson, L., Warner, R., Yanagisama, M., the number of ribosomal subunits and a concomitant increase Lechene, C. & Guidotti, G. (1977)J. Biol Chem. 252, 7421-7423. in 80S ribosomes, as judged by the absorbance profile. This 4. Macara, I. G. (1980) Trends Biochem. Sci. (Pers. Ed.) 5, 92-94. marked increase in the formation of 80S initiation complexes in 5. Simons, T. J. B. (1979) Nature (London) 281, 337-338. vanadate-treated samples supports the view that because of the 6. Jagus, R., Anderson, W. F. & Safer, B. (1981) Prog. Nucleic Acid vanadate-induced inhibition of initiation, more vacant 80S ri- Res. Mot Biol 25, 128-185. and ribosomal subunits are available 7. Miller, D. & Weissback, H. (1977) in Molecular Mechanisms of bosomes for participation , eds. Weissbach, H. & Pestka, S. (Academic, in these reactions of initiation. New York), pp. 324-369. These same results were observed when globin mRNA was 8. Brot, N. (1977) in Molecular Mechanisms of Protein Biosynthesis, replaced by tobacco mosaic virus RNA (data not shown). In the eds. Weissbach, H. & Pestka, S. (Academic, New York), pp. 375- absence of sparsomycin, the radioactivity of [35S]Met-tRNAf in 407. 80S initiation complexes in the control sample moved into the 9. Ochoa, S. & deHaro, C. (1979) Annu. Rev. Biochem. 48, 549-580. not see 10. Ranu, R. S. (1980) FEBS Lett. 112, 211-215. polyribosome region (data shown; Fig. 2), suggesting 11. Ranu, R. S. (1981) Biochem. Biophys. Res. Commun. 97, 1124-1132. that these 80S initiation complexes proceed with the mRNA- 12. Ranu, R. S. & London, I. M. (1979) Methods Enzymol 60, 459- directed assembly of polypeptide. The assay of the formation 484. of initiation dipeptide (methionylvaline) in vanadate-treated 13. Darnbrough, C., Legon, S., Hunt, T. & Jackson, R. J. (1973) J. samples showed that even though there was a noticeable in- Mot Biol 76, 379-403. crease in radioactive Met-tRNAf associated with 80S initiation 14. Obrig, T., Irvin, J., Culp, W. & Hardesty, B. (1971) Eur. J. Bio- complexes compared with a small fraction of this chem. 21, 31-41. controls, only 15. Ranu, R. S. & Kaji, A. (1972)J. Bacteriol. 112, 188-194. radioactivity was transferred into initiation dipeptide (Table 1). 16. Pelham, H. R. B. & Jackson, R. J. (1976) Eur. J. Biochem. 67, 247- In the vanadate-treated samples, therefore, these 80S initiation 256. complexes appear to be defective in one of the subsequent steps 17. Kay, J. E. & Benzie, C. R. (1982) Biochim. Biophys. Acta 698, 218- of translation. The kinetics of protein synthesis (Fig. 1B) show 221. that elongation of globin chains (on ribosomes already engaged 18. Ranu, R. S. & Wool, I. G. (1976)J. Biol Chem. 251, 1926-1935. 19. Ranu, R. S. & London, I. M. (1979) Proc. NatL Acad. Sci. USA 76, in the assembly of polypeptide) can normally proceed in the 1079-1083. presence of vanadate. This observation and the finding that in- 20. Das, A., Ralston, R. O., Grace, M., Roy, R., Ghosh-Dastidar, P., active 80S initiation complexes are formed in vanadate-treated Das, H. K., Yaghmai, B., Palmieri, S. & Gupta, N. K. (1979) Proc. samples suggest that vanadate interacts with ribosomes only at NatL Acad. Sci. USA 76, 5076-5079. Downloaded by guest on October 2, 2021