Gene, 120 (1992) 43-49 0 1992 Elsevier Science

GENE

Publishers

B.V. All rights reserved.

43

037%1119/92/$05.00

06662

The cell division cycle gene CDC60 synthetase in Saccharomyces cerevisiae (Gene cloning;

sequence

Stefan Hohmann Laboratorium Received

analysis;

sequence

comparison;

encodes

aminoacyl-tRNA

synthetase;

cytosolic

leucyl-tRNA

cell cycle regulation;

yeast)

and Johan M. Thevelein

moor Moleculaire Celbiologie. Instituut voor Plantkunde,

by J. Marmur:

2 April 1992; Revised/Accepted:

Katholieke

Universiteit te Leuven. B-3001 Leuven-Heverlee.

1 May 1992; Received

at publishers:

Flanders. Belgium

11 June 1992

SUMMARY

The cdc60 mutation (for cell c&vision cycle) of the yeast, Saccharomyces cerevisiue, confers arrest at the START point of the cell cycle upon shift to the restrictive temperature [Bedard et al., Curr. Genet. 4 (1981) 205-2141. We have cloned the CDC60 gene by complementation of the temperature-sensitive phenotype. Sequence analysis revealed a single open reading frame of 3270 bp and the deduced amino acid sequence showed 50.5% sequence identity to the cytosolic leucyl-tRNA synthetase (LeuRS) from Neurosporu crussu, implying that CDC60 encodes the corresponding yeast protein. Thus, CDC60 does not appear to be involved directly in the regulation of the cell cycle. Rather, the cdc60 mutation leads to cell-cycle arrest at the nutrient control point START due to a deficiency of charged leucyl-tRNA. The CDC60 gene product also shows homology to LeuRSs from other organisms and to aminoacyl-RS for isoleucine, valine and methionine.

CDC2

INTRODUCTION

The transition from the Gl- to the S-phase is a major control point in the cell cycle of eukaryotic cells (for review, see Forsburg and Nurse, 1991). This point has been termed START (Hartwell, 1974). During the last years the genetic analysis of the regulation of the cell cycle in the yeasts S. cerevisiue and Schizosuccharomyces pombe has greatly enhanced our understanding of the molecular events. The key component appears to be a protein kinase encoded by

Correspondence to: Dr. S. Hohmann, biologie, Kardinaal

Instituut

voor Plantkunde,

Mercierlaan

Laboratorium Katholieke

voor Moleculaire Universiteit

92, B-3001 Leuven-Heverlee,

Cel-

te Leuven,

Belgium.

Tel. (32-16) 220931, ext. 1516; Fax (32-16) 221855. Abbreviations: aa, amino acid(s); aaRS, CDC, cell division cycle; kb, kilobase nucleotide(s); Saccharomyces;

ORF, Sz.,

open

reading

aminoacyl-RS; bp, base pair(s); or 1000 bp; N., Neurospora; nt,

frame;

Schizosaccharom~~es;

RS, tRNA START,

synthetase;

S..

see INTRODUC-

TION; ts, temperature sensitive; YCp, yeast centromeric yeast episomal plasmid; YIP, yeast integrating plasmid.

plasmid;

YEp,

in Sz. pombe and CDC28 in S. cerevisiue that is activated by different cyclins. These cyclins, termed according to their periodic appearance during the cell cycle, are specific for the Gl- to S-phase transition or for the other major control point, the transition from G2 to mitosis (Forsburg and Nurse, 1991). In S. cerevisiue two environmental signals of central importance for this organism are integrated at the START point. In haploid cells the presence of the mating pheromone of the opposite mating type results in arrest of the cell cycle and the cells become competent for mating (BiickingThrom et al., 1973; Marsh et al., 1991). Depletion of nutrients results in arrest at START in both haploids and diploids and cells enter a quiescence phase termed GO (Hartwell, 1974; Johnston et al., 1977; Pringle and Hartwell, 198 1). In attempts to identify additional components of the mechanisms controlling START several additional temperature-sensitive CDC mutants were identified using different approaches (Bedard et al., 1981; Reed et al., 1988; Hadwiger et al., 1989; Prendergast et al., 1990). One of these is the cdc60 mutant that appeared to arrest at

44 START in a fashion analogous to cells starved for nutrients (Bedard et al., 1981). Such mutants are called class-II START mutants and they are unable to mate when shifted to the restrictive temperature indicating a stop of cell growth in addition to the arrest in cell proliferation and thus a possible defect in biosynthetic capacity. In contrast, class-I START mutants are able to mate at the restrictive temperature (Reed, 1980; Forsburg and Nurse, 1991). Thus, class-I START mutants are candidates for being defective in START control itself, while class-II START mutants may be either affected directly in the control mechanisms regulating the cell cycle like in sensing the availability of nutrients or they could be defective in processing the required nutrients and in macromolecular biosynthesis (Prendergast et al., 1990; Forsburg and Nurse, 1991). The work presented here implies that CDC60 is of the latter type. This gene encodes the cytosolic LeuRS from yeast.

RESULTS

AND DISCUSSION

(a) Cloning of CDC60 The gene CDC60 was cloned by complementation of the ts phenotype of the cdc60 mutant. The cdc60 mutant strain 8003 (MATa leu2 uru3 trpl his3 nde8 cdc60) was transformed (Ito et al., 1983) with a yeast genomic library (Carlson and Botstein, 1982) cloned into the multi-copy vector YEp24 (Botstein et al., 1979) and nine transformants potentially carrying the CDC60 gene were identified by their ability to grow at 37’ C. These transformants turned out to carry either of two plasmids. Restriction analysis showed that the two plasmids had inserts of 10.5 kb (pSGl-3-40) and 10.4 kb (pSGl-l-11), respectively and these inserts overlapped by 9.6 kb (Fig. 1A). To narrow the region which contains CDC60 and to check whether the cloned gene suppresses the growth defect at 37 “C even when located on a single-copy plasmid, several different fragments (Fig. 1A) were subcloned into the centromere based vector YCplac33 (Gietz and Sugino, 1988) and again transformed into the cdc60 mutant. The suppressing gene was identified on a 5.7-kb EcoRI-PstI fragment which contains 4.7 kb of the insert and 1 kb of the vector YEp24. Suppression was equally efficient with the single and the multi-copy vector, indicating that the cloned gene was not a multi-copy suppressor. However, it has happened occasionally that genes suppress the growth defect of a mutant defective in a different gene even when placed on a single-copy vector (e.g., Van Aelst et al., 1991). Therefore, we proved that we had cloned the CDC60 gene by integrating a URA3 marker into the yeast genome at the location of the cloned gene. For this approach, a 1.2-kb EcoRI-HindI fragment of the complementing fragment (Fig. 1A) was subcloned into the integrative vector YIplac2 11 (Gietz and Sugino, 1988). This

construct was linearized with BglII to trigger integration into the yeast genome at the location of the cloned gene (Orr-Weaver et al., 1981). The cdc60 mutant strain was transformed with this plasmid and Ura+ -prototrophic transformants were checked for the proper integration event by Southern blot analysis (not shown). One of the transformants was then crossed with a CDC60 wild-type strain defective in URA3 and tetrad analysis was performed. In all 38 complete tetrads analysed the URA3 marker segregated with the ts phenotype. Thus, the cloned DNA fragment contains CDC60 or it is at least very closely linked to CDC60. The 5.7-kb EcoRI-PstI fragment was used as a probe in Northern blot analysis and it hybridized to a mRNA of 3.5 kb (Fig. 2). (b) Sequence analysis The sequence of the complementing fragment (for sequencing strategy see Fig. 1B) revealed a single 3270-bp ORF coding for a 1090-aa protein with A4, of 124 135 (Fig. 3). The size of the ORF is in good correlation with the length of the mRNA (Fig. 2). Yeast mRNAs are normally about 200 nt longer than the corresponding ORF (Hereford and Rosbash, 1977; Cigan and Donahue, 1987). The 5’noncoding region contains two TATAAA sequences at nt positions -30 and -8 1 that could serve as TATA-elements for transcription initiation (Struhl, 1989). About 60 bp further upstream is a stretch of 16 A-residues flanked by further A-rich sequences. Such sequence elements have been shown to serve as promoter elements for constitutive expression (Struhl, 1989). The sequence showed 50.5u, identity on both aa and nt level to the N. crassa gene encoding the cytosolic form of LeuRS (Chow and Rajbhandary, 1989). The comparison of the yeast and the Neurosporu aa sequences is shown in Fig. 4. The ValRS from yeast and Neurosporu (for references to sequence data see legend to Fig. 5) are 52’4 identical, suggesting that the degree of sequence identity for these type of proteins between the two fungi is about 50”/, Therefore, we conclude that CDC60 most likely encodes the cytosolic LeuRS from yeast protein. The codon usage is only moderately biased. The codon bias index (Bennetzen and Hall, 1982) which gives an estimation of the biased use of 22 codons that are preferred in highly expressed yeast genes and which has a maximum value of 1.00, is 0.31 for CDC60. The codon usage resembles the one for weakly expressed yeast genes (Sharp et al., 1986). This is similar to other aaRS in yeast: the codon bias indices for the IleRS, ValRS and MetRS are 0.38,0.46 and 0.29, respectively. These data also fit with the observation that the signal for the CDC60 mRNA is weaker than the one for the actin gene (Fig. 2).

45

A s

B

S

I i ! i i i! i W/B EWSeXoSp

i EcN

B

It

E

if i! Xo S

i! ;

BScX9Ep

i ! i ?M XOEQWWHE~J

E

I .I I’I’

I

SCEVXO

NEEt

ea

i

@a

. I

,I.

i !

NEEt

. 1-i. I

I’ll’l’l’

H

Pv

I’

1 . I

*I I’

EV

N

H

&y

I I

,I

H

Fv

.I I’

XoBgEV~HBC1

H

if

I’

EV

H

I

E’/

i sat% H

I

I’

H

I’

@

I

wm!B

_ +

+ f

B

eo I

H

I I ’

Ii

E

St

N

I

I

Ev

K

K

H

I I’ I

.I. I’I

H

I

K&H

salr,

---“=?!A -+

lkb

X --=s

ACTIN

t-

1.4

ttt+-Fig. 1

Fig. 2.

Fig. 1. Restriction pSG 1-l-1

maps and subcloning

1 which complement

and sequencing

the c&60 mutation

striction

maps and plus or minus symbols

termined

by seyucncc

analysis

is marked

indicate

strategies.

The fragments complementation

with an arrow pointing

(A) Restriction subcloned

maps of the inserts

into YCplac33

of the ts phentotype

into the direction

(Gietz and Sugino, of the c&60 mutant.

of the ORF. The EroRI-Hind111

YIplac211 is shown at the bottom. B, BarnHI; Bg, &/II; E, EcoRI; EV, EcoRV; H, HindIII; K, @I; St, SruI; Xb, X&I; Xo, X/WI. (B) Sequencing strategy. Appropiate restriction fragments were subcloned 1982) and sequenced

according

according

sequence data were used as primers.

to available

only and are therefore

to Sanger et al. (1977). In addition

not represented

to the universal

For restriction

of the plasmids

Ml3 sequencing

Numerals

The location fragment

(upper

map) and

as lines below the rc-

of the CDC60 gene as dcused to integrate

the plasmid

N, NcoI; Pv, PvuII; S, SalI; SC, &cl; Sp, SphI; into M13mp18 or M13mp19 (Vieira and Messing,

primer additional

oli~odcoxyribonucle(~tidcs

enzymes see panel A legend. The &vrI sites were determined

designed

by sequence analysis

in panel A.

Fig. 2. Northern blot analysis. Yeast mRNA (10 ng) was electrophoresed in an I?, agarose gel, transferred radiolabelled 5.7-kb EcoRI-PstI fragment carrying the CDC60 gene (Sambrook et al., 1989). The radiolabelled was used as a control.

pSGl-3-40

1988) are shown

refer to kb.

to a nylon membrane

and hybridized

yeast actin gent (Gallwita

to the

and Sures, 1980)

46

1 193

His

89 481

Phe TTT

105 529

CYS Thr

Gly Met

TGT ACA

CYS

GCT ATG

TGT

CAC Gl” GAA 01” GA?.

AAli

Ala GCC

+.=qGlY

Gl” GAA

Gl” CAG

Gl” GAA

=Ys

CCT GGT

His CAT “al GTA

Fig. 3. The nt sequence isks mark

Gl” GAA

IA”

Phe

Al.3 GCA

LYS AAA

m

of CLtC60, and the deduced

the stop codon.

GenBank/EMBL

Tie ATT

databases

The sequence under accession

cm

aa sequence.

has been deposited No. X62878.

Asterwith the

47 ORQANISM

mzym.3 pas. MOTIFI.

MOTIF2

KS.

7-aa VBRPCAD~ 753 S. cerevidae Idelm 728 SKLk5KsMN I?.crassa LeuRs 756 BKUSKSTQN 7-aa vKkm3mAAR781 B. c0.u 7-aa VBRYOADTVR I.euP.s 618 sIa6sKsRNN 643 EKMSKSKYN -/-aa s. cerevisiee lnt. Lams 645 TmH(IPDATR 670 696 IKK9KsIMN 7-aa N.clas8a mt LeuRs mQYGA!xTR 721

: :.::..:..

. :..::.:::.:::.:..::.:

..:

.::::.:.

EIEnFGQEFERYKEDEWEGMP”“’ ~KTKEOLTKFNAKKGK~~~~Q~Q~L~ 140

150

:::: .:: ::::..:::

170

160

. . . . ..‘.

:..:

180

:..::::::::

190

250

:.:

::::...:...

:.:

300

310

:::I.::::::

.::::

NRLLELNKIKFGKRYTlYSII(DGQPCHDHDRSEGEGVLPL 260 270 290 280

IM4.S 11&S MeetR.3 IdetRE MCms "al&s vams "alrls

601 601 332 299 524 702 487 553

Rw69KsLRN R.msKsIl3N AKMSKSRGT RmllsKTLQN OKpSKsR(N RKMSKSLCIN RKHSKSLaN QKhlSKsKGN

Fig. 5. The KMSKS-motifandflanking

:::::::::::::.::::

SLGIP”SEIHKFADPQYWLH~~~~~=~~~L~~G~~~-Q~~~~*~~*-~Q~ 200 210 220 230 240

. .:::::.::::::

S.cereviaiae Z.coli .E.cdi T. themphilus S.cerevLsiae S.cerevL9i.w N.creeaa E.coli

position .::.

in the aa sequence.

7-aa 7-88 7-aa 7.83

LNKyQAIlm MNiaaAD)Im LNHFDADSLR LBRIORDALR

sequences

MOTIF

ofseveral

1 and MOTIF

626 626 358 324

cit. 1 2 3 4 5 6 7 8 9 10 11 12 13

aaRS. POS.,

2 are two conserved

sequences separated by 7 aa. mt., mitochondrial; Cit., source of data: (1) this work; (2) Chow and Rajbhandary (1989); (3) HBrtlein and Madern

.

(1987); (4) Tzagoloff

et al. (1988); (5) Chow et al. (1989); (6) Englisch

et al. (1987); (7) Webster et al. (1984); (8) Dardel et al. (1984); (9) Nureki

. . . . . . :. .:::::::::::.::::.:...::.:.:... --KGKLPEGANVYLC*ATLR~~~~GQ”~~~G~~L~*G”~-~~~~~~~~ 320 330 350 340

::..:::: 360

et al. (1991); (10) Walter

:::.

Kubelik,

370

A.R., Turcq,

sults in the GenBank

et al. (1983); (11) Jordana

B. and Lambowitz, database,

accession

et al. (1987);

(12)

A.M. (1991): unpublished

re-

No. M64703;

(13) Hartlein

et al. (1987).

Comparison with other aaRS We have compared the predicted CDC60 product with other aaRS, in particular with the enzymes LeuRS, IleRS, ValRS and MetRS which have been suggested to comprise a related subfamily within the aaRS (Englisch et al., 1987; Jordana et al., 1987; Heck and Hatfield, 1988). The CDC60 product shares with other class I aaRS the HIGH-region, a short sequence located in the N-terminal part that appears to be involved in adenylate binding (Schimmel, 1987). This sequence is located in CDC60 at aa position 66-77, the Ile of the consensus sequence HIGH is replaced by an Ala as in the Neurospora counterpart. Another short conserved peptide is the KMSKS-motif found in several aaRS (Englisch et al., 1987; Heck and Hatfield, 1988). This sequence contains a Lys that could be cross-linked to the tRNA in the case of E. coli MetRS and thus this sequence appears to be involved in tRNA-binding (Hountondij et al., 1985; 1986). This sequence starts at aa 728 in the CDC60 product. The sequences immediately flanking this motif are well conserved in the LeuRS from other sources as well as in IleRS, MetRS and ValRS. The first three types of enzymes also share a well conserved sequence motif 7 aa downstream from the extended KMSKS-motif that contains the almost perfectly conserved tripeptide GAD and a perfectly conserved Arg that have not been pointed out previously (Fig. 5). This sequence motif is present in the MetRS from E. coli and Thermus thermophilus but not from yeast and is lacking in all three ValRS. (For an aligment of the entire sequences for the LeuRS from yeast mitochondria, Neurospora mitochondria and from E. coli see Chow et al., 1989.) The conserved KMSKS-motif of CDC60 is surrounded by additional sequences with weak homology. The CDC60 product shows 19% identity over 230 aa to the E. coli LeuRS and between 16 and 19% identity over more (c)

Fig. 4. Alignment of the deduced aa sequences of the N. crassa cytosolic LeuRS and of CDC60. SC, S. cerevisiue; NC, Neurospora CMSSCI.Identical aa are indicated

by colons

and exchanges

or hydrophobicity by dots. Dashes indicate merals are aligned with corresponding aa.

conserving deletions.

aa side-chain

size

Last digits of nu-

48

than 300 aa at the C terminus to the mitochondrial enzyme from Neurosporu. The similarity to the yeast mitochondrial enzyme appears to be even less. In general, the nuclear encoded mitochondrial aaRS from fungi resemble more the bacterial enzymes than the cytosolic counterpart of the same organism (Schimmel, 1987; Chow et al., 1989). One known exception is ThrRS where both forms of the yeast enzyme are about equally similar to the counterpart from E. coli (Schimmel, 1987). Interestingly, there are some yeast aaRS where both the cytosolic as well as the mitochondrial form are encoded by one and the same gene, like HisRS (Natsoulis et al., 1986) and ValRS (Jordana at al., 1987). The CDC60 product is 16% identical to the yeast IleRS over 327 aa, 20% identical over 131 aa to the corresponding enzyme from E. coli and 2 1 y0 identical over 195 aa to the MetRS from E. coli. These homologies are very weak but may be regarded significant since they run over long stretches of sequences in similar positions relative to the highly conserved sequences discussed above. The homologies in the sequences surrounding the KMSKS-motif to the other aaRS listed in Fig. 5 were even less.

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(d) Conclusions CDC60 encodes the cytosolic LeuRS in yeast. Thus, the CDC60 gene product does not appear to be directly involved in cell-cycle regulation. The cdc60 mutant is arrested at START at the restrictive temperature because protein synthesis is blocked due to a lack of charged leu-tRNA. Also the genes encoding IleRS (Hartwell and McLaughlin, 1968) and MetRS (Unger and Hartwell, 1976) as well as the gene encoding the translation initiation factor 4E, CDC33 (Brenner et al., 1988), have been identified as CDC mutants that are arrested at START in a fashion analogous to nutrient-starved cells. A block of protein synthesis apparently causes a signal similar to nutrient deprivation and this leads in certain mutants to a similar cell cycle arrest (Wheals, 1987). Thus, protein synthesis or protein synthesis initiation may serve as a sensing device for the availability of a nitrogen source in cell-cycle regulation (Thevelein, 1992).

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