Gene, 108 (1991) 253-258 0

1991 Elsevier

GENE

Science

Publishers

253

B.V. All rights reserved. 0378-l 119/91/$03.50

06176

Synthesis of human initiation factor-2a in Saccluzromyces (Recombinant

DNA;

yeast; translation

factor;

eIF-2cc; heterologous

cerevisiae gene expression)

Simon R. Greena*, Alison Spalding”, Tony Ashford b, Christopher G. Proud b and Mick F. Tuite” ” Biological Laboratory, Universityof Kent, Canterbury, Kent, CT2 7NJ (U.K.): and b Department OfBiochemistry, Universityof BristolMedical School, University Walk, Bristol, BS8 1 TD (U.K.) Tel. (44-272)303030,

ext. 4959

Received by J.K.C. Knowles: 6 March 1991 Revised/Accepted: 16 August/27 August 1991 Received at publishers: 19 September 1991

SUMMARY

A human elF-2a cDNA (encoding a-subunit of the eukaryotic initiation factor-2) was expressed under the control of the galactose-regulated GALI, promoter, in Saccharomyces cerevisiae, in order to study the possible interactions of human eIF-2a with the yeast protein synthesis apparatus. Isoelectric focusing coupled with Western-blot analysis demonstrated that the human eIF-2a subunit synthesized in yeast under a variety of growth conditions was detected as two bands which co-migrated with the phosphorylated and unphosphorylated forms of rabbit eIF-2a, suggesting covalent modification in vivo. Cell fractionation studies further demonstrated that the synthesised human eIF-2a protein, though present in the cytoplasm, was largely associated with the yeast ribosomes, but could be removed from these by washing with 0.3 M KCl. This possible association of the synthesised human subunit into a three-subunit (a, /I and y) eIF-2 complex was further examined by partial purification of the yeast eIF-2 complex and estimation of the molecular mass of this complex. Immunoreactive eIF-2a was found in fractions with eIF-2 activity and the estimated molecular mass (130 kDa) corresponded to that predicted for the eIF-2 trimer. These analyses suggest that human eIF-2a subunit synthesised in yeast can become involved with the yeast protein synthetic apparatus, though whether this is a functional incorporation requires further genetic studies.

INTRODUCTION

The a-subunit of the eukaryotic translational initiation factor-2 (eIF-2) plays a key role in the regulation of mam-

Correspondencefo: Dr. M.F. Tuite, Biological Kent, Canterbury,

Laboratory,

University

of

Kent, CT2 7NJ (England)

Tel. (44-227)764000,

ext. 3699; Fax (44-227)763912.

* Current address: Cold Spring Harbor Spring Harbor, NY 11724 (U.S.A.)

Laboratory,

P.O. Box 100, Cold

Tel. (l-516)367-8333. Abbreviations:

aa, amino acid(s); bp, base pair(s); ds, double strand(ed);

ds1, ds RNA-activated inhibitor (an elF-2a kinase); eIF-Za, a-subunit of eukaryotic initiation factor-2; eIF-2a, gene (cDNA) encoding eIF-2a; kb, kilobase initiator

or 1000 bp; nt, nucleotide(s); tRNA;

plasmid-carrier

YGal, state.

galactose-based

S., Saccharomyces; minimal

medium;

tRNA,

[ 1, denotes

malian protein synthesis (Pain, 1986; Hershey, 1989). Phosphorylation of a Sersl residue (Colthurst et al., 1987) in eIF-2a results in the inhibition of the activity of a guanine nucleotide exchange factor (GEF) to catalyse the regeneration of eIF-2 : GTP from eIF-2 : GDP (Proud, 1986, a review). Recent analysis of the primary aa sequence of eIF-2a from a range of mammalian species, namely human, rat (Ernst et al., 1987), and bovine (S.R.G., J. Fullekrug, K. Sauer and M.F.T., submitted), reveals a high degree (> 99%) of conservation of the primary aa sequence between these three species. However, the derived amino acid sequence of eIF-2a from the lower eukaryote S. cerevisiae (yeast) shows significant deviation from the mammalian eIF-2a sequence; there is only 58% aa identity between S. cerevisiae and human eIF-2a (Cigan et al., 1989). Significantly, the sequence of yeast eIF-2a is highly conserved around the mammalian phosphorylation site

254 (Ser”) with complete identity over a 19 aa sequence encompassing this site (Cigan et al., 1989) suggesting that an analogous phosphorylation system might exist in yeast. That the role of the eIF-2cc subunit in regulating translation in S. cerevisiue may not be analogous to its role in mammalian cells comes from a report that the S. cerevisiae

A

E

SC I

4

0

H I

7'

v

0

E

+hRI linker

E

SC

eIF-2cr subunit is phosphorylated throughout the yeast growth cycle without any obvious impairment of translation initiation (Romero and Dahlberg, 1986). This observation has however been challenged by the recent work of Cigan et al. (1989) using the cloned S. cerevisiue eZF-2a gene. The phosphorylation state of yeast eIF-2a is still therefore a matter for study as is the existence and nature of the corresponding eIF-2a kinase. It is known that protein synthesis in S. cerevisiae cell-free lysates is not inhibited by the presence of low levels of dsRNA (Prescott et al., 1988); the reticulocyte cell-free system is inhibited by dsRNA due to the activation of a specific kinase (ds1) that phosphorylates eIF-2cc (Hunter et al., 1975). In this paper we demonstrate that a mammalian eIF-2cr can be expressed in S. cerevisiue, that it is covalently modified and that it is incorporated into the yeast translational machinery.

Fig. 1. Engineering yeast

under

a cDNA

control

1.4-kb EcoRI-HincII

encoding

by inserting

site located

in

cDNA

eIF-2a

(A) kindly pro-

of California,

Davis) was en-

fragment

encoding

(University

an EcoRI linker [GGAATTCC]

125 bp downstream

codon. The resulting

eIF-2cr for expression GALZJO

vided by Dr. J.W.B. Hershey gineered

human

of the galactose-inducible

promoter.

at the unique DraI

from the eIF-2a translation

l.l-kb EcoRI-EcoRI

fragment(B)

A

termination

was then gel-puri-

tied and inserted into the unique EcoRI site of pBM272 (C) kindly provided by Dr. M. Romanos (Wellcome Biotechnology Ltd.) which is RESULTS

AND DISCUSSION

(a) Expression of a human eZF-2a cDNA in yeast A cDNA encoding the human eIF-2or subunit was engineered for expression in the yeast S. cerevisiae by linking it to the highly regulated GALl,IO promoter in pBM272 (Fig. 1). Transcription of the human eZF-2ct gene was initially detected by Northern blotting of total RNA samples prepared from transformed cultures. The 1.5-kb human eZF-2a transcript was first detected in cells transformed with pUKC552 5 h after induction by growth on galactose (data not shown). No cross-hybridisation with the endogenous yeast eZF-2a mRNA was detected at the high stringency used presumably because the homology at the nt level between the mammalian and yeast mRNAs is low (Cigan et al., 1989). Analysis of total protein extracts from cells transformed with pUKC552, using Western blotting with a monoclonal antibody raised rabbit eIF-2cc, detected a 36-kDa protein (the size predicted for human eIF-2cc from the cDNA sequence; Ernst et al., 1987) in cells that had been grown on galactose for at least 8 h (Fig. 2). No protein of similar molecular weight was detected in the control cells transformed with pBM272 alone or with pUKC551 (pBM272 containing the engineered eZF-2a cDNA in the wrong orientation for expression). These results confirm that the yeast eIF-2cr subunit does not crossreact with an anti-mammalian eIF-2cr antibody presumably due to the low homology at the aa level (58 “/, ; Cigan et al., 1989).

approx.

10 bp downstream

GAL10

gene (Johnson

designated the

pUKC552.

strain

trpC9830 scribed

MC1066 h&R)

using

by Maniatis

EcoRI; H,HincII;

from the transcriptional

and Davis,

All E. coli manipulations (IacIPOZYA

start

1984). The resulting galU galK

recombinant

DNA

et al. (1982).

Symbols:

point of the plasmid

were carried

was

out using

strA pyr74::Tn5

methods

essentially

B, BarnHI;

leuB6 as de-

D, DraI;

E,

P, PstI; S, SalI; SC, ScaI. In (A), open box is the eWZa

coding sequence. In (C), open boxes are yeast sequences as indicated open arrows show the GALI GAL10 transcriptional promoters.

and

(b) Covalent modification of human eIF3a synthesised in yeast One-dimensional isoelectric focusing coupled with Western-blot analysis (Cox et al., 1988) was used to investigate the properties of the expressed human eIF-2a protein in yeast. As shown in Fig. 3, two forms of the synthesised human protein were found under all growth conditions investigated. On the gel system used these two forms focused at a p1 equivalent to that observed for a mammalian eIF-2cl polypeptide phosphorylated in vitro by the kinase ds1 which phosphorylates Ser” in mammalian eIF-2cr (Fig. 3, lane 1). Thus the human eIF-2a protein is subject to a post-translational modification in vivo, possibly phosphorylation by an endogenous yeast protein kinase such as GCN2 (Ramirez et al., 1991) although as yet other types of covalent modification cannot be completely ruled out. The eIF-2a does not contain any potential N-linked glycosylation sites (Asn-X-Ser/Thr) nor has it been reported to be subject to any other form of post-translational modification.

255

Oh, --8h,18h, Ia b da b cia b clhn

polypeptide

I

somes

was associated isolated

from

with yeast ribosomes. galactose-grown

Ribo-

pUKC552-

transformed cells and washed with progressively higher KC1 concentrations (0.15 M, 0.3 M and 0.8 M) to liberate protein factors associated with the ribosome, but which are not an integral part of the ribosome, e.g., eIF-2, which is known to be mostly released from the mammalian ribosome

kDa -116 * 84 - 58 - 48 -

were

by 0.3 M KC1 and completely removed by 0.5 M KC1 (Merrick, 1979). Western blotting of the ribosomes and the various salt washes (Fig. 4a and b) clearly demonstrates that the human eIF-2a protein was associated with yeast ribosomes and was washed off the ribosomes most effec-

36 26

tively by 0.3 M KCl. Blotting the same protein samples with a polyclonal antibody against the soluble nonribosomal associated polypeptide phosphoglycerate kinase, gave a dif-

Fig. 2. Galactose-regulated formed

with plasmid

expression

pUKC552.

of human

Yeast

cultures

eIF-2~4 in yeast transwere grown

in a 2%

glucose-based

minimal medium (YGlu; 2% glucose 0.67% yeast nitrogen

base without

aa) for 18 h at 3O”C, the cells harvested

centrifugation

and resuspended

medium (YGal; 2% galactose0.67%

yeast nitrogen

yeast

urd-52

strain

transformed pBM272

used (DBY745 by the method

(sample

MATa

(sample

was extracted

from these transformants Western

blotting

human

was

Pane1 A is the Coomassie

(sample

in YGal and frac-

SDS gel and analyzed blue-stained

in panel B indicates

by

eIF-2x mono-

gel; pane1 B is the

(sizes in kDa) in lane M are indicated

The large arrow

c).

by Mellor et al. (1983)

(Towbin et al., 1979) using an anti-rabbit

blot. The A4, markers

the small arrows.

leu2-3-112)

b) and pUKC552

after 0, 8 and 18 h growth

on a 12.5% polyacrylamide-0.1%

Western

base without aa). The

a&l-100

as described

tionated

clonal antibody.

by

minimal

of Beggs (1978) with the following plasmids:

a), pUKC551

Total soluble protein

and washed

in 50 ml 2% galactose-based

the position

by of

eIF-2cL.

(c) Subcellular location of human eIF-2a in yeast During the initiation of protein synthesis in eukaryotes the eIF-2 trimer (eIF-2a/?y), after forming a ternary complex with Met-tRNA, and GTP, binds to the 40s ribosomal subunit to form a 43 S pre-initiation complex. This complex is then joined by the 60s subunit to form the 80s initiation complex (reviewed in Pain, 1986). To determine whether the human eIF-2a polypeptide was incorporated into an eIF-2 trimer we first ascertained whether the human eIF-2a

ferent profile, although trace amounts could be detected in the ribosome salt washes (Fig. 4~). To confirm that the observed ribosome association of the human eIF-2a subunit was due to its incorporation into a functional eIF-2 complex, rather than nonspecific binding to the ribosome, the yeast eIF-2a complex was partially purified by successive chromatographic steps on phosphocellulose, DE-52, and Mono Q ion exchange columns essentially as described by Ahmad et al. (1985). Active eIF-2 complex was assayed in the eluates by the formation of the ternary complex (eIF-2.GTP.[ 35S]Met tRNA) formation assay (Proud and Pain, 1982) and the human eIF-2a protein was detected by Western blotting of the same protein samples using a monoclonal antibody raised against rabbit eIF-2a (see section a above). As shown in Fig. 5, the human protein was clearly detectable in fractions that contained eIF-2 activity suggesting that the human eIF-2a subunit does associate with an active eIF-2 complex. To study this further the molecular weight of the complex containing human eIF-2a was estimated by gel filtration on Sephacryl S300. Fractions containing the human eIF-2a subunit were analyzed by dot-blotting using the anti-mammalian eIF-2a monoclonal antibody, and the results indicated that the majority of the human subunit was found in fractions corresponding to the predicted M, of three subunit eIF-2, namely 130 kDa (Fig. 6). No human eIF-2a was detected as monomers (predicted A4, 36 kDa) either due to complete incorporation of the subunit into the high A4, complex and/or due to degradation of the unincorporated heterologous subunit by endogenous proteases. Interestingly, a low level of human eIF-2a was also detected in a very high A4,.complex (fractions 77-8 1, > 400 kDa) which may correspond to the formation of a large complex with various GCN4 regulating proteins as predicted by the recent results of Cigan et al. (1991) for endogenous S. cerevisiae eIF-2. While these data do not unequivocally demonstrate a functional role for the synthesised human eIF-2a in yeast they do strongly suggest that it becomes incorporated into

256 Fig. 4.

pUKC 552

I

p UKC 551 II

d2345671234567'.

Fig. 3. 1234

Fig. 3. One-dimensional extracts

were prepared

mid-exponential

isoelectric

focusing

from 50 ml cultures

phase (l-2

antibody

Samples

to mammalian

Fig. 4. Subcellular The primary previously

a Western arrows

of the human

were prepared

phase (l-2

in yeast

strain

transformant

concentrations

with pUKC552.

expressed

x 10’ cells/ml) in YGal, then subjected

( + P) and dephosphorylated

initially by ultracentrifugation

1986). The ribosomes

of strain

eIF-2a

partially

by Western

phosphorylated

blotting

DBY745

transformed

x 10’ cells/ml) grown on YGal as described

to first produce

blot of the gel developed

with an anti-yeast

were analyzed

blot of the gel developed

phosphoglycerate

x

in vitro

with a monoclonal are indicated.

with either plasmid in the legend to Fig. 2.

gfor 2 h at 4°C) and then sequentially

by 12.5% polyacrylamide-0.1%

with the anti-mammalian

kinase polyclonal

to

an S30 lysate (lane 2) then an SIOO lysate (lane 3) as

were pelleted from the SlOO lysate (40000

of the 0.8 M KC1 salt wash. All proteins

blue. (Panel B), a Western

protein

to a 42°C heat

(-P) forms of the rabbit eIF-2a subunit

in yeast. Total soluble lysates phase cultures (l-2

Total soluble

in the legend to Fig. 2; lane 2, grown

of KCl; 0.15 M KC1 (lane 4), 0.30 M KC1 (lane 5) and finally 0.8 M KC1 (lane 6); the salt wash fractions

after removal

with Coomassie

phase (l-2

transformed

as described

by Cox et al. (1988) and eIF-2a was identified

of the phosphorylated

eIF-2cc subunit

DBY745

cultures

x lo8 cells/ml) in YGal. Lane 1 is a sample of rabbit

from 50 ml mid-exponential

(Tuite and Plesset,

the ribosomes

the gel stained

expressed pUKC552

and run as described

lysate (lane l), was then fractionated described

with increasing 7 contains

were prepared

eIF-2cc. The positions

location

pUKC55 1 or pUKC552

eIF-2a

of the following

x 10’ cells/ml) in YGal; lane 3, grown to mid-exponential

shock for 30 min; lane 4, grown to stationary with the dsI kinase.

of human

antibody.

eIF-2u monoclonal

The M, markers

washed

are shown. Lane

SDS gels. (Panel A), antibody.

(lane M) are indicated

(Panel C), by small

with the M, (in kDa) indicated.

a complex with many of the physical properties of three subunit eIF-2. A direct demonstration of a functional role for the human eIF-2a subunit in yeast would come from demonstrating that its synthesis could complement a deletion of the single host eZF-2cr gene SUZ2 (Cigan et al., 1989). (d) Conclusions (I) Human eIF-2a subunit can be synthesised in the yeast S. cerevisiae from an eZF-2a cDNA expressed using the galactose-regulated GALl,lO promoter. (2) S. cerevisiae has an endogenous post-translational

modifying activity that will modify the human eIF-2% subunit under a variety of growth conditions. The analysis of the modified forms of eIF-2a by one-dimensional isoelectric focusing suggests that this modification is phosphorylation. (3) The human eIF-2a synthesised in yeast becomes associated with the ribosomes of S. cerevisiae through assembly into a three subunit eIF-2 complex. Whether this complex is functional in protein synthesis in vivo remains to be determined, for example, by genetic complementation experiments.

257

234567

1

kDa -116 - a4 -

58

-48

- 36

Fig. 5. Partial purification from an exponentially involved

sequential

promote

ternary

-

26

-

36 26 Fig. 5.

El&ton

of the eIF-2 complex from yeast strain DBY745 transformed

growing

culture

fractionation

complex

of strain DBY74S[pUKCS52]

of an S 100 lysate on phosphocellulose,

formation

in the presence

from the latter stages of the purification, with 0.35 M KCI; lanes 3-7,

which showed

fractions

from mono-Q

maximal showing

eIF-2 activity.

amide-O.1 y0 SDS gel, the lower panel is the same gel blotted with an anti-rabbit are shown

columns.

by Ahmad

The eIF-2 activity was assayed

unbound

of fractions

eIF-2a monoclonal

antibody.

by its ability to

of the yeast lysate

to DE-52; lane 2, fraction

The upper panel is the Coomassie

eluted from DE-52

blue-stained

The positions

purified

et al. (1985) and

12.5% polyacryl-

of the M, markers

(in kDa)

in both panels.

Fig. 6. Determination cells of a pUKCSS2 ribosomes

of the molecular transformant

and then the ribosomes

and proteins

concentration.

apparatus

and the nitrocellulose

alcohol dehydrogenase

weight of human eIF-2a synthesised

essentially removed

eluted in I ml fractions.

of protein

In addition

as described

20 pg of protein

blotted

in S. cereviriae. An S30 lysate was prepared

The resulting

of a rabbit

reticulocyte

beginning

using an anti-rabbit and 36 kDa (eIF-2a

lysate (20 pg) is indicated

fraction

eIF-2a

number

monoclonal

monomer)

70 and ending fraction from 70-164 antibody.

was bound

The column

(44 kDa) and soybean

are indicated

170, the A,,,

was calibrated

trypsin inhibitor

by the arrows

was determined

to nitrocellulose

bound to the S300 column as a measure

using a BioRad

with /I-galactosidase

‘Dot-Blot’ (486 kDa),

(20 kDa) and the predicted

labelled T and M, respectively.

A control

elution sample

by the open box

initiation

ACKNOWLEDGEMENTS

subunit.

This work was supported by SERC grant no. GR/D/55313. S.R.G. held an SERC research studentship. We are grateful to members of the Proud laboratory for assistance with the eIF-2 purification and analysis, and to Dr. John W.B. Hershey (University of California, Davis) for providing the human eZF-2a cDNA clone.

factor

sequence

identity

a

with the human

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D.R., Campbell,

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