Artemia salina embryos - PNAS

Proc. Nat. Acad. Sci. USA Vol. 72, No. 10, pp. 3947-3951, October 1975

Biochemistry

Polypeptide chain initiation in eukaryotes: Initiation factor MP in Artemia salina embryos* (cysts/brine shrimp eggs/eukaryotic initiation factors/protein synthesis/development)

WITOLD FILIPOWICZt, JOSE M. SIERRA, AND SEVERO OCHOA Roche Institute of Molecular Biology, Nutley, New Jersey 07110

Contributed by Severo Ochoa, July 31, 1975.

ABSTRACT The activity of IF-MP, a polypeptide chain initiation factor that forms a ternary complex with eukaryotic initiator Met-tRNA and GTP and promotes binding of the initiator to 40S ribosomes, is very low in undeveloped Artemia salina embryos but increases over 20-fold following resumption of development upon hydration of the cysts. The factor is present in both the ribosomal salt wash and highspeed supernatant. Its specific activity is 50 times higer in the wash but its total activity is only about twice as high in the wash as in the supernatant. As is true of IF-MP from other eukaryotic sources, the A. salina factor is specific for eukaryotic Met-tRNAj and sensitive to SH-reagents, and its activity is GTP dependent.

The prokaryotic protein synthesis initiation factor IF-2 has two eukaryotic counterparts: IF-Ml and IF-MP in the Anderson nomenclature (1, 2). IF-Ml, discovered in 1970 in salt washes of rabbit reticulocyte ribosomes (3, 4), is widely distributed (5-10). It catalyzes the AUG-dependent binding of pro- or eukaryotic fMet-tRNAi and less effectively Met-tRNAj (11), as well as the poly(U)-dependent binding of prokaryotic acPhe-tRNA and less effectively Phe-tRNA (12), to eukaryotic cytoplasmic 40S ribosomes in the absence of GTP or other nucleotides. Upon addition of 60S ribosomes and puromycin to the rather unstable 40S complex, aminoacyl-puromycin is formed in good yield. IF-MP, discovered in 1971 in ribosomal salt washes of mouse fibroblasts (13), is present in rabbit reticulocytes (14-16) and other cells (17, 18). Its distribution is roughly the same as that of IF-Mi. IF-MP promotes the binding of Met-tRNAj to 40S ribosomes via formation of a Met-tRNA1. factor-GTP ternary complex in the presence or absence of template (13-20). IF-MP, unlike IF-Ml, (a) requires GTP for activity, (b) is specific for eukaryotic Met-tRNAj and less Abbreviations: There is no standard nomenclature for initiation faceukaryotic cytoplasmic origin. Factors from mammalian cells are designated as IF-Mi, IF-M2 (M2A and M2B), IF-M3, and IF-MP in papers from Anderson's laboratory (1, 2). The more general abbreviation EIF (for eukaryotic initiation factor) has been suggested (1). Thus, IF-Mi and EIF-i are different abbreviations for the same factor (see ref. 11). The terms "embryos" and "cysts" refer to encapsulated Artemia salina embryos commonly known as brine shrimp eggs. Met-tRNAj is used to designate the initiator species of eukaryotic cytoplasmic Met-tRNA in general. The Artemia initiator species is not formylatable by E. coli transformylase (see ref. 7). The remaining abbreviations are as follows: Mg(AcO)2, magnesium acetate; Hepes, N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid; GMPPCP, 5'-guanylyl-methylenediphosphonators of

te.

This is paper no. V in the series, "Polypeptide Chain Initiation-in Eukaryotes." Preceding paper in this series: Zasloff, M. & Ochoa, S. (1973) "Purification and properties of supernatant initiation factor from Artemia salina embryos," J. Mol. Biol. 73, 65-76. t Permanent address: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-532 Warsaw, Poland. *

3947

reactive with the formylated species, (c) forms a stable 40S complex from 40S or 80S ribosomes, and (d) requires template and additional factors for formation of the 80S initiation complex and Met-puromycin synthesis. It appears that only IF-MP may be functional in the eukaryotic cytoplasm because mRNA translation in Artemia systems that contain IF-Ml but are free of other initiation factors (21) requires IF-MP and is not inhibited by antibody against A. salina IF-Ml (paper in preparation). This is consistent with the reversal by IF-MP of the inhibition of globin synthesis observed in reticulocyte lysates in the absence of hemin (22, 23). Since the occurrence of IF-MP in A. salina had not previously been reported, we looked for this factor in ribosomal washes and supernatants of undeveloped and developing embryos. IF-MP activity is very low in undeveloped embryos but increases markedly with development.

MATERIALS AND METHODS Preparation of IF-MP. IF-MP was partially purified from a 0.5 M KC1 ribosomal wash of developing A. salina embryos. Development and preparation of the unwashed ribosomal fraction were as described (21) except that the ribosomes were pelleted by centrifugation for 4 hr at 125,000 X g in a Spinco 60 Ti rotor. The ribosomal pellet from 300 g of cysts is gently suspended in 100 ml of low salt buffer A [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonlc acid (Hepes), pH 7.6, 70 mM KCI, 9 mM Mg(AcO)2, 0.1 mM EDTA, 1 mM dithiothreitol, 5% glycerol], stirred for 2.5 hr at 00, and centrifuged for 20 min at 55,000 X g to remove glycogen. The clear portion of the supernatant is collected and centrifuged at the same speed for 10 hr. The low salt ribosomal pellet is then suspended in 60 ml of buffer B [20 mM Hepes, pH 7.6, 0.1 mM EDTA, 0.5 M KC1, 5 mM Mg(AcO)2, 2 mM dithiothreitol, 5% glycerol], stirred for 4 hr at 00, and finally centrifuged for 5 hr at 125,000 X g. The high salt wash is dialyzed overnight against buffer C (20 mM Hepes, pH 7.6, 80 mM KCI, 0.1 mM EDTA, 1 mM dithiothreitol, 5% glycerol) and applied to a DEAE-cellulose column (Whatmann DE-52, 1.5 X 7 cm) equilibrated with the same buffer. The column is washed with buffer C containing 80 mM KC1, and the IF-MP activity is eluted with buffer C containing 180 mM KC1. This preparation also contained other initiation factors. For assay of IF-MP levels in cytosol and ribosomal washes of undeveloped and developed embryos (Table 1), the high speed supernatant (S-125) and the 0.5 M KC1 ribosomal wash fractions were applied to a DEAE-cellulose column in buffer C containing 100 mM KC1, and IF-MP was eluted with buffer C containing 350 mM KC1. Protein was determined by the method of Lowry et al. (24) using bovine serum albumin as standard. Other Preparations. A. salina 40S and 60S ribosomal sub-

3948

Biochemistry: Filipowicz et al.

Proc. Nat. Acad. Sci. USA 72 (1975)

Table 1. IF-MP levels in cytosol and ribosomal wash fractions from undeveloped and developing embryos

[35S] Met-tRNAi retained on Difference

Total units*

IF-MP specific activityt

0.003 0.060 0.02 0.50

5 101 7.4 204

0.017 0.360 0.7 18.5

Millipore filters (pmol)

Stage of development

Factor fraction derived from

-GTP

+GTP

Undeveloped Developing Undeveloped Developing

Cytosol (0.18 mg) Cytosol (0.17 mg) Wash (0.028 mg) Wash (0.026 mg)

0.043 0.043 0.06 0.10

0.046 0.103 0.08 0.60

Standard assay was with A. salina Met-tRNA1 (4.15 pmol) and factor fractions as indicated. * Per 100 g of dry cysts. t Units/mg of protein.

units were prepared essentially as described (7). A. salina (undeveloped embryos) and rat liver aminoacyl-tRNA synthetases were prepared by DEAE-cellulose chromatography (7) of postribosomal supernatant fractions. E. coli aminoacyl-tRNA synthetases (25) were kindly supplied by Dr. J. Ofengand of this Institute. Crude A. salina tRNA was isolated from postribosomal supernatant of undeveloped embryos. It was separated from contaminating high-molecular-weight RNA by Sephadex G-100 chromatography. Partially purified tRNA1Met was prepared by BD-cellulose chromatography of crude tRNA (26). A. salina [s5S]Met-tRNAi (12,000 to 30,000 cpm/pmol) was prepared by acylation of crude tRNA with A. salina aminoacyl-tRNA synthetases. A. salina [14C]Met-tRNAi (450 cpm/pmol) was prepared by acylation of partially purified tRNAiMet with E. coli aminoacyl-tRNA

synthetases. Rabbit reticulocyte [35S]Met-tRNAi (12,000 cpm/pmol), f[35S]Met-tRNAi (12,000 cpm/pmol), and [3H]Met-tRNAi (1060 cpm/pmol) were prepared by acylation of crude rabbit reticulocyte tRNA (kindly supplied by Dr. N. K. Gupta, University of Nebraska) with E. coli aminoacyl-tRNA synthetases. Leucovorin was used as formyl donor in acylation mixtures when f[a5S]Met-tRNA1 was prepared. Rabbit liver [l4C]Met-tRNAmMet (450 cpm/pmol) was prepared by acylation of purified Met-tRNAmMet (kindly supplied by Drs. W. C. Merrick and W. F. Anderson, NIH, Bethesda, Md.) with rat liver aminoacyl-tRNA synthetases. E. coli [14C]Met-tRNAf (450 cpm/pmol) and ['4C]PhetRNA (850 cpm/pmol) were prepared by acylation of crude E. coli tRNA (Schwarz/Mann) with E. coli aminoacyl-tRNA synthetases. Highly purified E. coli f[s5S]Met-tRNAf (31,900 cpm/pmol) was similarly prepared from E. coli tRNAfMet (about 95% pure) kindly supplied by Dr. G. D. Novelli, Oak Ridge National Laboratory. Homogeneous rabbit reticulocyte IF-MP (2) was the kind gift of Drs. W. C. Merrick and W. F. Anderson. Ternary Complex Assay. Unless otherwise noted, samples contain, in a final volume of 0.05 ml, Hepes buffer pH 7.3, 20 mM; KC1, 100 mM; Mg(AcO)2, 3 mM; dithiothreitol, 1 mM; partially purified IF-MP, about 35 ,ug of protein; A. salina [s5S]Met-tRNA1, 3.3 pmol; and GTP, 0.14 mM when present. The reaction components are added at 00 in the order listed above. After incubation for 5 min at 300 the reaction is stopped by dilution with 4 ml of cold wash buffer [20 mM Tris-HCl pH 7.5, 100 mM KC1, 3 mM Mg(AcO)2], and the samples are filtered through nitrocellulose membranes (Millipore Hawpo 2500, 0.45 ,m pore size). The filters are washed twice each with 4 ml of the same buffer and

dried, and the retained radioactivity is measured in Omni-

fluor in a Beckman LS-100 Scintillation counter. One unit is

taken as the amount of factor causing a GTP-dependent binding of 1 pmol of Met-tRNA1 under the conditions of the assay. Sucrose Density Gradient Centrifugation Analysis. For 40S initiation complex assay, samples containing, in a final volume of 0.1 ml, Hepes buffer pH 7.3, 20 mM; KC1, 100 mM; Mg(AcO)2, 3 mM; dithiothreitol, 1 mM; IF-MP, 84 Mg of protein; A. salina [35S]Met-tRNAi, 10 pmol; and GTP or 5'-guanylyl-methylenediphosphonate (GMPPCP), 0.14 mM when present, are first incubated for 5 min at 30'. Then,

0

E C/)

cc

w -. -J LL -j

-j

-J 0

z

0

[Mg 2+] (mM)

IF-MP

(p&g)

z w

cc z 4)

cn

-A

2-

MINUTES [35S] Met-tRNA; (pmol) FIG. 1. Ternary complex formation as a function of the concentration of (A) Mg2+, (B) IF-MP, and (C) Met-tRNAi, and (D) as a function of time. Curves 1 (-), with GTP; curves 2 (0), without GTP. Panel A, curve 3, (A), with GTP but without IF-MP. The wash buffer for each of the panel A assays had the same Mg2+ concentration as the corresponding assay sample. Conditions were of the standard assay except for the variable component and for the fact that panel C samples contained only 21 ,ug of IF-MP. The batch of IF-MP used for panels A and D was different from that used for B and C.



3949

Table 2. Effect of N-ethylmaleimide on ternary complex formation

[3-SJ Met-tRNAi retained on Millipore filters (pmol) Inhibition

0

x

E c0 E

In

z

C%4

c) tn in

2-

System

-GTP

Complete Complete + NEM (1 mM) Complete + NEM (5 mM) Complete + NEM (20 mM)

0.05

0.90

0.85

0

0.05

0.58

0.53

38

0.05

0.05

0

100

0.04

0.04

0

100

Complete + dithiothreitol (40 mM) + NEM (20 mM)

0.04

0.83

0.78

+GTP Difference

(%)

8

Standard assay samples were preincubated either without or with N-ethylmaleimide (NEM), or dithiothreitol and N-ethylmaleimide, at the stated concentrations for 15 min at 250, prior to adding A. salina [35S]Met-tRNA1 (3.3 pmol) and GTP (0.14 mM). Unreacted N-ethylmaleimide was then inactivated with excess dithiothreitol (40 mM), and the assay was conducted as described in Materials and Methods. The last sample contained dithiothreitol throughout the assay.

diluted and filtered, and its radioactivity is measured as described for ternary complex assay.

20 4 FRACTION NUMBER FIG. 2. Formation of ternary and 40S complexes

as assayed by density gradient centrifugation. Assay conditions were as described in Materials and Methods. Sedimentation was from left to right. Upper panels, with GTP; lower panels, with GMPPCP. A and C, no subunits; B and D, complete system. A255 nm (-); 35S radioactivity (--- ---); 35S radioactivity in assays (panel B) without GTP or IF-MP (-o-o-).

sucrose

after addition of A. salina 40S subunits (0.85 A2 0 unit), they further incubated for 7 min at 30'. After incubation the samples are chilled in ice, layered on 4.5 ml of a linear 1525% sucrose gradient in Hepes buffer pH 7.3, 20 mM; KCI, 80 mM; Mg(AcO)2, 5 mM; dithiothreitol, 0.5 mM, and centrifuged for 2 hr at 55,000 rpm and 2° in a Spinco SW 56 rotor. The gradients are monitored at 254 nm in an ISCO fractionator, and 0.2-ml fractions collected. Each fraction is are

RESULTS IF-MP in undeveloped and developing embryos Preparations from developing, but not from undeveloped, A. salina embryos can translate exogenous mRNA, e.g., brome mosaic virus, globin mRNA (21). Since undeveloped embryo preparations promote chain elongation, they must be deficient in a factor(s) required for initiating translation of mRNA. As seen in Table 1, IF-MP activity as measured by ternary complex formation with A. salina [35S]Met-tRNAj, is very low in undeveloped embryos but increases markedly during development. The factor is present in both the highspeed supernatant and the ribosomal wash fraction to the extent of about 30% and 70% of the total, respectively, but its specific activity is about 50-fold higher in the ribosomal wash.

Table 3. Initiator specificity aa-tRNA retained on Millipore filters (pmol) aa-tRNA

[35S]Met-tRNAi

[14C] Met-tRNAi

[3H]Met-tRNAi

[35S] Met-tRNAi

f[35S]Met-tRNAi

[14C] Met-tRNAm [14C]Met-tRNAf f[35S]Met-tRNAf [14C]Phe-tRNA

[35S]Met-tRNAi

Source of tRNA

-GTP

+GTP

Difference

A. salina A. salina Rabbit reticulocytes Rabbit reticulocytes Rabbit reticulocytes Rabbit liver E. coli E. coli E. coli A. salina

0.17 0.12 0.12 0.12 0.05 0.10 0.15 0.21 0.10 0.01

1.26 1.10 1.50 1.00 0.52 0.11 0.16 0.18 0.11 0.92

1.09 0.98 1.38 0.88 0.47 0.01 0.01 0 0.01 0.91

Assay conditions as described in Materials and Methods with 6-10 pmol of aminoacyl-tRNA/sample. All assays except the last were with partially purified A. salina IF-MP (about 50 1g of protein per sample). Last assay was with highly purified rabbit reticulocyte IF-MP (2.5 ,gg of protein per sample).

3950


Properties of factor As found for IF-MP from other sources (13-20, 27), the retention of eukaryotic Met-tRNAr by nitrocellulose filters in the presence of the A. salina factor is markedly GTP-dependent (Fig. 1). GDP (not shown) was inactive. The effect of GTP depends on the Mg2+ concentration. Under our conditions maximal stimulation by GTP occurred at 3 mM Mg2+ (Fig. IA), a concentration that is optimal for natural mRNA translation in cell-free A. salina systems (21). Fig. 1B (curve 1) shows that formation of the ternary complex is proportional to the concentration of IF-MP within a wide concentration range. Under our conditions the concentration of A. salina Met-tRNAj giving maximal binding was about 0.1 tM (Fig. iC), and formation of the ternary complex (Fig. ID, curve 1) was essentially complete in 10 min. Like IF-MP from other sources (17, 18), the A. salina factor is sensitive to N-ethylmaleimide (Table 2). In further agreement with the properties of other preparations of this factor (2, 13-15, 18), A. salina IF-MP forms a ternary complex with eukaryotic Met-tRNA1, but not Met-tRNAm, prokaryotic Met (or fMet)-tRNAf, or Phe-tRNA (Table 3). As shown by others (2, 14, 15), rabbit reticulocyte IF-MP is also unreactive towards eukaryotic aminoacyl-tRNAs other than Met-tRNAi. Since A. salina IF-MP forms ternary complexes with Met-tRNA&, whether from A. salina or rabbit reticulocytes (Table 3), it would appear that this factor is not species specific but only discriminates between prokaryotic and eukaryotic initiator tRNA. As further seen in Table 3, rabbit reticulocyte fMet-tRNAj is about one-half as active as the nonformylated species in ternary complex formation. Under our conditions (Table 3) binding of A. salina MettRNAj by homogeneous rabbit reticulocyte IF-MP (2) is fully dependent on the presence of GTP. Formation of 40S initiation complex Ternary complex formation and subsequent transfer of the bound [35S]Met-tRNAi to 40S ribosomes, as analyzed by sucrose density gradient centrifugation, are illustrated in Fig. 2. In the absence of 40S subunits (Fig. 2A) there is only one peak of radioactivity near the top of the gradient. This peak undoubtedly corresponds to the [35S]Met-tRNAj-IF-MP-GTP ternary complex, for it decreases approximately to the extent that a new radioactive peak appears in the 40S region of the gradient upon addition of 40S subunits (Fig. 2B). No radioactive peaks are seen (Fig. 2B, open circles) in the absence of either IF-MP or GTP. Fig. 2 (C and D) further shows that the GTP analog, GMPPCP, can efficiently substitute for GTP in formation of both the ternary and the 40S initiation complex. However, the 40S complex formed with GMPPCP cannot be converted to an 80S complex (19, 20). Note that a 40S complex is formed in the absence of added template (16, 19, 20) despite the fact that ribosomal subunits prepared from undeveloped A. salina embryos are devoid of messenger (21).

DISCUSSION Our results with A. salina IF-MP are in full accord with those reported with factor from other sources (13-20). Interestingly, the level of this factor in undeveloped A. salina embryos is very low and undergoes a pronounced increase during development. In a subsequent communication (paper in preparation) we shall show that other initiation factors are also present in smaller amounts before development. IF-MP is found not only in the ribosomal wash, but also in the high-


speed supernatant fraction. This would be expected if, like its prokaryotic counterpart IF-2 (28), IF-MP recycles between ribosomes and cytosol. Isolation of IF-MP from wheat germ cytosol has been reported (18). We found that nucleoside triphosphates other than GTP promoted retention of [a5S]Met-tRNAi by Millipore filters with the partially purified A. salina but not with homogeneous rabbit reticulocyte IF-MP (see ref. 18). The effect of nucleoside triphosphates is due to the presence of nucleosidediphosphate kinase (EC 2.7.4.6). This enzyme catalyzes the transfer of the terminal phosphate of nucleoside triphosphates to diphosphates (e.g., NTP + GDP NDP + GTP) and is present along with GDP in the A. salina but not the homogeneous reticulocyte preparation (paper in preparation). A similar observation with partially purified rabbit reticulocyte IF-MP was recently reported (29). We thank Christa Melcharick for her excellent technical assistance. J.M.S. is a postdoctoral trainee of the Institute of Molecular

Biology, Madrid, Spain.

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14.

triphosphate," Proc. Nat. Acad.

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