THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1989 hy The American Society for Biochemistry and Molecular Biology, Inc

Val. 264. No 4, Issue of February 5. pp. 2053-2059,1969 Printed in U.S.A.

Cloning and Characterization of the Yeast CKI Gene Encoding Choline Kinase and Its Expression in Escherichia coZi*

(Received for publication, April 7, 1988)

Kohei Hosaka, Tsutomu Kodaki, and Satoshi Yamashita From the Department of Biochemistry, Gunma University School of Medicine, Maebashi 371, Japan

Using a mutant defective in choline kinase (Hosaka, K., and Yamashita, S. (1980) J. Bacteriol. 143, 176- 181; Hosaka, K., and Yamashita, S. (1987) Eur. J. Biochem. 162, 7-13), the structural gene (CKI) for choline kinase of the yeast, Saccharomyces cerevisiae, was isolated by means of genetic complementation. Within its sequence there was an open reading frame capable of encod.ing 582 amino acids with a calculated molecular weight of 66,316. The primary translation product contained a segment closely related to the phosphotransferase consensus sequence (Brenner, S. (1987) Nature 329, 21). A yeast transformant carry- ing CKI in multiple copies exhibited very high choline kinase activity as well as ethanolamine kinase activity. In-frame insertion of the CKI coding frame into lacZ’ on the pUCl9 vector led to efficient expression. of choline kinase in Escherichia coli cells in the presence of a lac inducer, isopropylthiogalactoside, proving that CKI is the structural gene for choline kinase. Concom- itantly, ethanolamine kinase activity was also ex- pressed. When the CKI locus in the wild-type yeast genome was inactivated by its replacement with the in vitro disrupted cki gene, the yeast cells lost virtually all of the choline kinase activity and most of the etha- nolamine kinase activity. Thus, it is concluded that choline kinase is mono-cistronic and that the ethanol- amine kinase activity is a second activity of choline kinase in the yeast.

Phosphatidylcholine is the most abundant phospholipid in eucaryotes, and it plays important roles as to the structure and function of cellular membranes. The synthesis of phos- phatidylcholine occurs through the CDP-choline pathway and the phosphatidylethanolamine methylation pathway. Whereas the methylation pathway operates in limited types of cells, such as hepatocytes, adipocytes, pituitary cells, and yeast (1-4), the CDP-choline pathway is widely distributed in eucaryotic cells. This pathway begins with the phosphoryla- tion of choline to phosphorylcholine, followed by conversion to CDP-choline and then to phosphatidylcholine through the transfer of the phosphorylcholine moiety of CDP-choline to diacylglycerol (5-7).

Choline kinase, which catalyzes the initial phosphorylation step of the CDP-choline pathway, was first characterized in yeast extracts, by Wittenberg and Kornberg in 1953 (5), but it was only recently that extensive enzyme purification was

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted

504454. to the GenBankTM/EMBL Data Bank with accession number(s)

achieved from African green monkey lung and rat kidney (8, 9). Because the initial enzyme preparation from yeast was found to catalyze the phosphorylation of ethanolamine as well (5), many studies have been performed to determine whether a single enzyme or separate enzymes are responsible for the phosphorylation of choline and ethanolamine (10-16). Al- though the results of several studies suggested the separate entities of choline kinase and ethanolamine kinase, highly purified enzyme preparations obtained from mammalian sources exhibited both of these related activities, supporting the idea that a single enzyme is responsible for these two activities (8, 17). Ishidate et al. (9) reported that the rat kidney enzyme showed a minimum molecular weight of 42,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since the molecular size of the native enzyme was estimated to be 75,000-80,000, they concluded that the enzyme is a dimeric protein. But detailed analysis was not carried out to determine whether or not the native enzyme is composed of two identical subunits.

Unlike the mammalian enzyme, yeast choline kinase has not been extensively purified yet. Brostrom and Browning (18) obtained a 300-fold purified enzyme and estimated its molecular weight to be 67,000 by Sephadex G-200 chromatog- raphy. Their kinetic experiments revealed that yeast choline kinase follows the random Bi Bi mechanism. But further information concerning the molecular structure and proper- ties of the enzyme is still lacking. What is the primary structure of yeast choline kinase? Is the enzyme mono-cis- tronic or poly-cistronic? Does the yeast enzyme phosphorylate both choline and ethanolamine? A promising approach to answer these questions is to clone and characterize the struc- tural gene encoding the enzyme.

Previously, we isolated several mutant strains of the yeast, Saccharomyces cerevisiae, with reduced choline kinase activity (19, 20). The availability of these mutants prompted us to perform an initial cloning study on the structural gene (CKZ) for choline kinase. The present paper describes 1) cloning of the CKZ gene by means of genetic complementation using a choline kinase mutant, 2) determination of the complete nucleotide sequence of the gene and the deduced amino acid sequence of its product, 3) expression of the gene in Esche- richia coli cells and determination of the substrate specificity of the enzyme, and 4) construction of a yeast null mutant by targeted disruption of the CKZ locus in the genome to deter- mine the effect of the loss of choline kinase on phospholipid synthesis. The present results show that yeast choline kinase is composed of 582 amino acids with a calculated molecular weight of 66,316. I t is also shown that the enzyme is mono- cistronic and catalyzes the phosphorylation of both choline and ethanolamine.

EXPERIMENTAL PROCEDURES

Materi~k-[[a-~*P]dCTP (400 Ci/mmol) and a nick translation kit were purchased from Amersham Corp. [methyl-14C]Choline chloride

2053

2054 Yeast Choline Kinase Gene

(52.0 Ci/mol) and [1,2-“C]ethanolamine (4.3 Ci/mol) were products of Du Pont-New England Nuclear. Restriction endonucleases, T4 DNA ligase, and bacterial alkaline phosphatase were obtained from Takara Shuzo (Kyoto) and Nippon Gene (Toyama). Calf intestine alkaline phosphatase was from Boehringer Mannheim. Zymolyase 60,000 was obtained from Kirin Brewery (Takasaki).

Yeast Strains and Culture-The genotypes and sources of the S. cereuisiae strains used in this study are listed in Table I. The com- positions of the minimum media containing various concentrations of myo-inositol, M0.2i (0.2 gg/ml), M2i (2 pglml), and M20i (20 rg/ ml), and complex medium WaD were as described previously (21). When used, L-leucine, L-histidine, and choline chloride were added to the medium at concentrations of 20, 20, and 2 pg/ml, respectively. Yeast cells were grown aerobically at 30 “C.

Bacterial Strains and Culture-E. coli K12 HBlOl (22) was used for amplification of all plasmids except for M13 recombinant phages, which were propagated in E. coli JM103. JM103 was also used for expression of the yeast CKI gene. Bacterial cells were cultured in Luria broth (22) or 2 X YT medium (23) at 37 “C. Ampicillin was used at a concentration of 50 pg/ml. The absorbance of cultures was measured at 550 nm in 18-mm-diameter test tubes with a Hitachi 101 spectrophotometer.

Transformation of Yeast and E. coli-Unless otherwise stated, yeast cells were transformed by the method of Beggs (24) with the modifi- cation that spheroplasts were prepared by treating cells with 0.1 mg/ ml Zymolyase 60,000 for 30 min at 30 “C. E. coli was transformed as described by Bolivar and Backman (22).

DNA Preparations-Yeast chromosomal DNA was prepared as described by Cryer et al. (25). Yeast plasmids were prepared as described previously (26). The E. coli plasmids were prepared as described by Marko et al. (27), followed by Sepharose 4B column chromatography. Rapid preparation of E. coli plasmids was carried out as described by Birnboim and Doly (28).

DNA Sequencing-The DNA sequence was determined by the

TABLE I List of the yeast strains used

~

Genotype Source (Ref.) Strain

X2180-1A

X2180-1B

AH22

D302-1A 172 172B1 172B2 186

186B1

186B2

D173-5A

D333-7A D334-3A

689 786 786B1 788 788B2 D366-3A D336-1D

D372-10C

D302-3C

2014

a SUC2 mal me1 gal2

a SUC2 mal me1 gal2

a leu2-3 leu2-112 his4-519

a leu2-3 leu2-I12 his4-519 a ise a ise a ise a cki ise a cki a cki

CUP1

CUP1

canl

CY cki

a cki ise a cki ise leu2-3 leu2-112

his4-519 a CSEl a CSEl cki a cki a CSEl cki a cki a cki ise a ise leu2-3 leu2-112 his4-

519 a cki ise leu2-3 leu2-112

his4-519

canl

canl cki::LEU2

a leu2-3 leu2-112 his4-519

a leu2-3 leu2-112 his4-519

YGSC“

YGSC

N. Gungeb

AH22 X X2180-1B This laboratory (21) X2180-1A X 172 X2180-1A X 172 This laboratory (19) X2180-1A X 186 X2180-1A X D173-

5A

5A X2180-1A X D173-

172B1 X 186B1 D333-7A X D302-1A

This laboratory (20) This laboratory (20)

This laboratory (20)

172B1 X 788B2

X2180-1A X 786

X2180-1A X 788

172B1 X D302-1A

D366-3A X D336-ID

AH22 X X2180-1B

Present study -

~

Yeast Genetic Stock Center, University of California, Berkeley. Kumamoto Institute for Technology. Strain “X” X strain “Y” indicates a segregant from a cross of

strain “X” with strain “Y”.

dideoxy chain-termination method (29) after subcloning into the M13 vectors, mplO and mpll (23), using the DNA sequencing kit (Takara Shuzo).

Enzyme Assays-Yeast cells were cultured and then harvested under the indicated conditions. The soluble fraction of the cells was prepared as described previously (21) and used for assaying the choline kinase and ethanolamine kinase activities. The choline kinase activity was determined as described previously (21) with some mod- ifications. The reaction was carried out for 20 min at 30 “C in 50 mM glycine-NaOH buffer, pH 9.6, 10 mM MgSO,, 10 mM ATP, 2 mM [methyZ-’4C]choline (220 cpm/nmol), and 50-100 pg of protein, in a total volume of 250 pl. The phosph~ryl-[rnethyl-’~C]choline produced was isolated by the method of Burt and Brody (30). The ethanolamine kinase activity was assayed in a 25-pl reaction mixture containing 50 mM glycine-NaOH buffer, pH 9.6,lO mM MgSO4, 10 mM ATP, 5 mM [1,2-14C]ethanolamine (6500 cpm/nmol), and 5-20 pg of protein. The incubation was carried out for 20 min at 30 “C, and then a 10-pl aliquot of the incubation mixture was spotted onto a Silica Gel 60 plate (Merck) and developed with 2% NH40H, 95% ethanol (l:l, v/ v). The produced phosphorylethanolamine was located by autoradi- ography and then quantitated by scraping off and counting the gel in a toluene/Triton X-100 scintillant (31).

Incorporation of Labeled Choline and Ethanolamine into Lipids- After incubation with labeled precursors under the indicated condi- tions, cells were disrupted by sonication in the presence of glass beads, and then lipids were extracted with a mixture of chloroform and methanol as described previously (32). Individual phospholipids were separated on a Silica Gel 60 plate (Merck) with chloroform/ methanol/acetic acid/88% formic acid/water (35:15:62:0.25, by vol- ume), and the gel containing the indicated phospholipids was scraped off and then counted in a toluene/Triton X-100 scintillant.

Construction of a CKI-expressing Plusmid-For the expression of the yeast CKI gene in E. coli, in-frame insertion of CKI into hcZ’ of pUC19 was performed as follows. A 2.7-kb’ PstIIHindIII fragment was excised from plasmid pCKlD and then digested with MoaI. The resulting 1.8-kb MuaI fragment was end-repaired and then ligated into the HincII site of pUC19. The orientation of the insert was checked by mapping the unique BglII site in the insert as an internal reference point. pUCK5 contained the insert in the proper orienta- tion.

Gene Disruption-The 2.7-kb PstIIHindIII fragment of pCKlD was cloned into pUC19. The resulting plasmid, pUK1, was cleaved with EcoRV, and the EcoRV fragments were removed by electropho- resis. The gap thus formed was filled in with the 2.2-kb yeast LEU2 gene (33) to yield pKD1. pKDl was linearized by digestion with PstI and HindIII, and the resulting 4.0-kb fragment was purified by electrophoresis and then used for transformation of yeast D302-3C by the lithium acetate method of Ito et al. (34) to yield strain 2014 containing disrupted cki in its CKI locus.

RESULTS

Characterization of Choline Kinase Mutants-For cloning of the gene encoding choline kinase, it is desirable to use a structural gene mutant as the cloning host. Previously, we isolated several choline kinase mutant strains using two dif- ferent procedures (19, 20). In one method, strain 172 (ise) with a mutational alteration in the control of the phosphati- dylethanolamine-methylating enzymes was used as the parent (19). The methyltransferases in the wild-type yeast were repressed on the addition of both myo-inositol and choline (20 pg/ml each), whereas the enzymes in the ise mutant were significantly repressed on the addition of myo-inositol alone at a concentration of 20 pg/ml (M20i medium) (35). Conse- quently, in a medium containing myo-inositol, the ise mutant required choline for growth. A choline kinase mutant, strain 186, was obtained from a mutagenized culture of k e by se- lecting a strain not growing in a myo-inositol-containing medium even if choline was supplemented. The recessive character of this mutation was ascertained by backcrossing two derivatives of strain 186, D333-7A, with 172B2 (ke).

In the other method, strain 689 (CSEl; “choline-sensitive”)

The abbreviations used are: kb, kilobase pairs; bp, base pairs; IPTG, isopropylthiogalactoside.

Yeast Choline Kinase Gene 2055

was utilized as the parent (20). The growth of this parent was strongly inhibited by choline. This peculiar phenotype was due to mutationally decreased phosphatidylinositol synthesis in the mutant. When choline was added, more diacylglycerol was utilized for phosphatidylcholine synthesis, and therefore, less CDP-diacylglycerol was available for phosphatidylinositol synthesis. This decreased the already lowered phosphatidyli- nositol synthesis in CSEl mutants and led to the cessation of their growth. We could obtain choline kinase mutants, 786 and 788, by selecting choline-resistant revertants from CSEl (20). Their choline kinase defects were shown to be recessive.

We first subjected these three choline kinase mutants to the complementation test. A derivative of strain 788 (D372- 10C) was crossed with derivatives of strains 786 (786B1) and 186 (186B2), respectively. The choline kinase activities of the resulting diploids were invariably less than 2% of that of a wild-type diploid (AH22 x X2180-1A). Thus, all these muta- tions, obtained by different procedures, fall into a single complementation group. This was further supported by posi- tive complementation of the mutants with a cloned gene (see below). The mutation is referred to as cki.

To determine whether or not cki is a structural gene mu- tation, we isolated and analyzed a revertant. Strain D333- 7AR22 obtained from cki ise strain D333-7A grew on choline- supplemented M20i medium. Its choline kinase activity amounted to 18% of the wild-type level. An alteration in choline kinase was strongly suggested by a change in apparent K, for ATP. Whereas the value for the wild type was 3.9 mM, that for the revertant was 8.3 mM. These results support the view that cki is an alteration in the structural gene for choline kinase.

Isolation of the CKI Gene That Encodes Choline Kinase-A strain harboring only the cki mutation was not a convenient host for the cloning of the choline kinase gene because it showed no apparent growth phenotype. We used a cki ise double mutant, D372-10C (a cki ise leu2-3 leu2-112 his4-519), as the cloning host. D372-10C was transformed with a yeast gene library (36) constructed on YEpl3 (37) carrying the yeast LEU2 gene, and the colonies that grew on M20i medium containing choline and L-histidine, but not on M20i medium supplemented with L-histidine alone, were selected. This screening gave CKI transformants but excluded ISE trans- formants. Plasmid DNA was isolated from the transformants and then subjected to restriction enzyme analysis. Two kinds of plasmids, pCKl and pCK3, were obtained, which contained 7.2- and 9.0-kb inserts, respectively (Fig. 1A). Comparison of their restriction maps indicated that pCKl is part of pCK3. As shown in Table 11, the choline kinase activities of the transformants harboring pCKl and pCK3 were 16 and 13 times as high as that of the wild type, respectively. The overproduction of the enzyme is thought to reflect a gene dosage effect because both pCKl and pCK3 are 2-pm DNA- based plasmids known to be maintained in multiple copies in yeast cells (38).

Subcloning-The pCK1 insert was cleaved with BglII, HindIII, PstI, and Sal] , separately or in combination, and the fragments obtained were subcloned into the YEpM4 yeast-E. coli shuttle vector, which was composed of the 2-pm replica- tion origin, the LEU2 selectable marker, and part of the pUC18 sequence containing muticloning sites (39). Each sub- clone was tested as to its ability to complement D372-10C. pCKlA and pCKlD positively complemented the cki muta- tion, but pCKlB, pCKlC, and pCKlG did not (Fig. 1B). As shown in Table 111, like the original clones, subclones pCKlA and pCKlD overproduced choline kinase. In contrast, its activity was not detected in the pCKlG transformant. Thus,

A

pCK I

x HHP @q P BgSB H P v Pv H H

pCK3 uf ’ I I If I( I f I

- Ikb 0

pCKl

P BgSB HPv P v H H X H n I I U +

pCKlG - Ikb -

FIG. 1. Structure of the CKI gene: A , restriction maps of the pCKl and pCK3 inserts; B, subclones derived from pCK1. The thin lines, wavy lines, and boxes represent the YEpl3 vector, YEpM4 vector, and inserts, respectively. + and - indicate the ability and inability of the respective plasmids to complement the cki mu- tation, respectively. Restriction enzyme sites: B, BamHI; Bg, BglII; H, HindIII; P, PstI; Pu, PuuII; S, SalI; X , XbaI.

TABLE I1 Choline kinase actiuities of wild-type, mutant, and transformant cells

The wild type and transformants (D372-10C [pCKl] and D372- 1OC [pCK3]) were cultured in M0.2i medium supplemented with L- histidine. The mutant, D372-10C, was cultured in M0.2i medium supplemented with L-leucine and L-histidine. The cells were har- vested at the late logarithmic phase and then their choline kinase activities were assayed as described under “Experimental Proce- dures.”

~

Strain Activity ~~

nmol/min/mg protein X2180-1A 19.4 D372-10C 0.7 D372-10C [pCKl] D372-10C IpCK31

311 260

TABLE I11 Choline kinase activities of the transformants

carrying pCK1 subclones The wild type (X2180-1A) and transformants (D372-10C harboring

the indicated subclones) were cultured in M0.2i medium supple- mented with L-histidine. The mutant (D372-10C) was cultured in M0.2i medium supplemented with L-histidine and L-leucine. The cells were harvested at the late logarithmic phase and then their choline kinase activities were determined as described under “Exper- imental Procedures.”

Strain Activity

nmol/min/mg protein X2180-1A 32.6 D372-1OC 0.3 D372-10C [pCKl] 670 D372-10C [pCKlA] 454 D372-10C [pCKlD] 722 D372-10C [pCKlG] 0.9

the CKI gene was located within the 2.7-kb PstI/HindIII segment of pCK1D. pCKlD also effectively complemented another choline kinase mutant, D334-3A, derived from strain 186 (for the construction, see Table I) and overproduced choline kinase, supporting the above conclusion that choline

2056 Yeast Choline Kinase Gene

kinase mutants, so far obtained, fall into a single complemen- tation group.

Table IV shows the close relationship between choline kinase and ethanolamine kinase. pCKlD, which overproduced choline kinase, also enhanced the ethanolamine kinase activ- ity of the transformant. Furthermore, pCKlG, which failed to restore the choline kinase activity, was also ineffective in elevating the ethanolamine kinase activity. These results strongly suggest that the CKI gene product mediates the phosphorylation of both choline and ethanolamine.

Nucleotide Sequence of the CKI Gene and the Deduced Amino Acid Sequence of Choline Kinase-The nucleotide se- quence of the 2,698-bp PstI/HindIJI fragment of pCKlD was determined by the dideoxy-chain termination method (29). Fig. 2 shows the sequencing strategy and the detailed restric- tion map of the CKZ gene. The complete nucleotide sequence of the CKI gene and the deduced amino acid sequence of its primary translate are shown in Fig. 3. The CKZ gene contained an open reading frame capable of encoding 582 amino acids with a calculated molecular weight of 66,316. There is a TATA box-like sequence (40) in the 5’-flanking region as well as the putative transcription termination signal, AATAAA (41), in the 3”flanking region. The sequence surrounding the initiator codon is A X X E G , which was proposed to facilitate trans- lational initiation (42). There are two putative phosphoryla- tion sites, Arg-Arg-His-Ser and Arg-Arg-Ala-Ser (43), near the amino terminus (amino acid positions 27 and 82) .

ATP is one of the substrates for the choline kinase reaction. But the Gly-X-Gly-X-X-Gly ....... Ala-X-Lys sequence, which can often be seen in ATP-binding proteins like protein kinases and phosphotransferases (44,45), could not be found. Instead, we noted the presence of a sequence closely related

TABLE IV Choline kinase and ethanolamine kinase actiuities in wild-type,

mutant, and transformant cells The wild type (X2180-1A) and the transformant (D372-10C

[pCKlD] and D372-10C [pCKlG]) were cultured in M0.2i medium supplemented with L-histidine. The mutant (D372-10C) was cultured in M0.2i medium supplemented with L-histidine and t-leucine. The cells were harvested at the logarithmic phase and then their enzyme activities were determined as described under “Experimental Proce- dures.”

Strain Choline kinase Ethanolamine kinase

nmoljmin jmg protein X2180-1A 6.3 1.6 D372-10C <o. 1 0.4 D372-10C [pCKlD] 148.3 41.7 D372-10C [pCKIG] <o. 1 0.2

PE M b EVBg E SBEV Sa EV E M A SaMH > r I ’I1 I I I

I IU

0 500 1000 1500 2000 2500 Base pair

FIG. 2. DNA sequencing strategy and restriction endonu- clease sites in the CKI gene. The arrows in the upper part of the figure indicate the direction and length of the DNA fragments se- quenced. The arrowhead denotes the 3’ end of each fragment. The open arrow indicates the CKZ coding region. The direction of tran- scription is from left to right. Restriction enzyme sites; A, AccII; B, BarnHI; Bg, BglII; E, EcoRI; EV, EcoRV; H , HindIII; Hp, HpaI; M, MuaI; P, Pstl; S, SalI; Sa, S a d

CTGCAGATATGAATTCCATAGGCCAGTCTTCTTCTAAGCGATTGGTAACCTCCTTCACTT

GGTGCTCTACCGTTTTTCTTGTCGGCCCAGCCGGTTAAATClCGAAGAGACAGAATAGAA T A G A A C A C C A C C A A C A G A T C G T T C T C T T G T T C T T T G T T C T T T A T G G ~ T A T T C A C A T

GCTCTGTGGCTGTAAGTAAGGATAAGCCAAATGGAAAAGTTATAGAATTAGGACAACAAT A T A C A G T T T T T T T T T G T A C T C T T A T A G A A T A C A C A C A C A T A G A T A C G C A C G T A A A A T T A G A G C A A A A G A T G G T A C A A G A A T C A C G T G ~ ~ - ~ L ; G A G T G T A A G A A G T T A C T ~ G G T C G G T T A C C

AAGCAAGGTCCAGATCGAGTTCTCAAAGAAGACATTCGTTAACACGCCAACGTTCCTCGC

M V Q E S R P G S V R S Y S V G Y

Q A R S R S S S Q U L T R Q R S S AAAGACTGATTAGAACCATCAGTATCGAGTCTGATGTGTCTAATATTACTt iACGATGACG Q R L I R T I S I E S D V S N I T D D D ATTTGAGAGCTGTCAATGAGGGAGTAGCGGGTGTGCAACTGGACGTCTCTGAAACCGCAA D L R A V N E G V A G V O L D V S E T A

N K G F U A T D V I D S L G S T S ATAAGGGACCAAGAAGAGCATCAGCAACTGATGTCACAGATAGTTTGGGTTCGACTTCGT

CGGAATATATTGAGATTCCCTTTGTTAAGGAAACATTGGATGCAAGTTTACCTTCGGATT S E Y I E I P F V K E T L D A S L P S D

Y L K Q D I L N L I Q S L K I S K W Y N

ACAAGAAAATCCAACCGGTAGCACAAGATATGAACTTAGTCAAGATCTCTGGTGCGATGA N K K I Q P V A Q O M N L V K I S G A M CAAACGCAATTTTCAAAGTTGAATACCCTAAGTTACCATCGTTGCTATTGAGAATATACG

GACCGAATATTGATAATATCATTGACAGGGAATATGAATTGCAGATTTTGGCTAGGCTTT G P N I D N J I D R E Y E L O I L A R L CATTGAAAAATATAGGTCCTTCCCTTTACGGCTGTTTTGTAAACGGTAGATTTGAGCAGT S L K N I G P S L Y G C F V N G R F E Q

TTCTGGAGAATTCTAAGACTTTAACAAAAGACGACATTAGAAACTGGAAGAACTCTCAAA F L E N S K T L T K D D l R N W K N S Q GGATTGCAAGGAGAATGAAGGAGTTACATGTAGGTGTTCCTCTCTTGAGTTCAGAAAGGA R I A R R M K E L H V G V P L L S S E R AGAACGGGTCGGCTTGTTGGCAAAAGATTAACCAGTGGTTGCGCACGATTGAGAAAGTCG K N G S A C W Q K I N Q W L R T L E K V

ACCAArGGGTGGGGGATCCTAAAAACATTGAAAACTCTTTATTATGTGAGAATTGGTCCA D Q W V G D P K N I E N S L L C E N W S

K F M D I V D R Y H K W L I S Q E Q G I AGTTTATGGATATTGTCGATAGATATCACAAGTGGCTTATTTCTCAAGAACAGGGTATAG

AGCAAGTCAACAAAAATCTTATATTCTGCCATAATGATGCCCAATACGGCAATTTACTTT E Q V N K N L I F C H N O A Q Y G N L L TCACTGCTCCTGTGATGAACACACCGAGCCTATACACTGCACCTTCGTCTACATCATTGA F T A P V M N T P S L Y T A P S S T S L CTTCCCAATCAAGTTCCTTATTTCCTTCGAGCTCCAATGTCATTGTAGATGATATAATCA T S Q S S S L F P S S S N V I V O D I I ACCCGCCAAAGCAGGAGCAAAGCCAAGATTCCAAATTGGTCGTCArTGATTTTGAATATG N P P K Q E O S Q D S K L V V I D F E Y CAGGTGCCAATCCCGCCGCATATGATTTAGCGAATCATCTTTCCGAGTGGATGIATGATT A G A N P A A Y D L A N H L S E W M Y D

Y N N A K A P H Q C H A O R Y P D K E Q ACAACAATGCTAAGGCCCCACATCAGTGCCACGCTGATAGATATCCCGATAAAGAACAGG

TTTTGAATTTCTTATACTCTTATGTTTCGCATCTAAGGGGTGGTGCTAAGGAACCCATAG V L N F L Y S Y V S H L R G G A K E F I

D E E V Q R L Y K S I I Q W R P T V Q L

F W S L W A I L Q S G K L E K K E A S T CCATCACTAGAGAAGAAATTGGACCCAATGGAAAAAAATATATCATCAAGACTGAACCCG A I T R E E I G P N G K K Y I I K T E P AATCCCCTGAAGAAGACTTTGTTGAAAATGACGACGAGCCT~AAGCTGGCGTCAGCATTG E S P E E D F V E N D D E P E A G V S I

ACACGTTCGATTATATGGCTTATGGTCGTGACAAGATTGCGGTCTTTTGGGGCGACCTCA D T F D Y M A Y G R D K I A V F W G D L TTGGCTTAGGCATAATCACCGAAGAAGAATGCAAAAATTTCAGCTCTTTCAAGTTCCTCG I G L G I I T E E E C K N F S S F K F L A T A C T A G T T A T T T G T ~ A T A C G T A T A C G A A T T C C T T C A A C A A A G G C C A A G G A ~ G C A D T S Y L

ATGTGAAGCAGGACATATTAAATCTCATTCAGAGTTTGAAGATATCCAAATGGTATAACA

~ N A I F K V E Y P K L P S L I . L R I Y

ATGAAGAGGTTCAAAGACTCTATAAGTCAATCATTCAATGGAGACCCACTGTACAACTAT

TTTGGTCGCTCTGGGCCATCCTACAAAGTGGTAAATTAGAGAAAAAAGAAGCCTCCACTG

120 60

1 80 240 300 360

420

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1020

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1140

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2040

2100

AATAACAATAACACCATTATTTTAATTTTTTTTCTATTACTGTCGCTAACACCTGTATGG 2160 TTGCAACC.n_c~GAGAATCCTTCTGAlGCATACTTTATGCGTTTATGCGTTTTGCGCCCC 2220 T T G G A A A A A A A T T G A T T C T C A T C G T A A A T G C A T A C T A C A T G C G T T T ~ T G G G A A A A G C C T C 2280 CATATCCAAAGGTCGCGTTTCTTTTAGAAAAACTAATACGTAAACCTGCATTAAGGTAAG 2340 ATTATATCAGAAAATGTGTTGCAAGAAATGCATTATGCAATTTTTTGATTATGACAATCT 2400 CTCGAAAGAAATTTCATATGATGAGACTTGAATAATGCAGCGGCGCTTGCTAAAAGAACT 2460 TGTAIATAAGAGCTGCCATTCTCGATCAATATACTGTAGTAAGTCCTTTCCTCTCTTTCT 2520

AACAAAATGTCTGAAATAACCTTAGGTAAATATTTACTTTGAAAGATTGAGCCAAGTCAA 2640 TATTACACTTATTTCACATAATCAATCTCAAAGAGAACAACACAATACAATAACAAGAAG 2580

CTGTAACACCGTCTTCGGTTTGCCAGGTGACTTTAACTTGTCTCTTTTGGATAAGCTT 2698

FIG. 3. Nucleotide sequence of the CKZ gene and the de- duced amino acid sequence of choline kinase. The longest open reading frame is translated into an amino acid sequence. The putative TATA box in the 5“flanking region and the potential transcription termination signal in the 3”flanking region are underlined. The sequences with dashed underlining are the MuaI sites which were used for the expression of CKI in E. coli cells. The amino acid sequences with wauy underlining are potential phosphorylation sites.

Yeast Choline Kinase Gene 2057

FIG. 4. Protein sequence homology. The sequences were aligned for maximum matching and gaps were introduced as indicated by dashes. The numbers in parentheses indicate the positions of the first amino acids in the sequences shown, from the amino termini. Identical amino acids are boxed. Dashed boxes indicate hydrophobic amino acids. CKI, yeast choline kinase; Aphl, B. circulans aminogly- coside phosphotransferase (47); Aph2, Tn903 aminoglycoside phos- photransferase (48); cdc2+, Schizosaccharomyces pombe cdc2+ gene product (49); CDC28, S. cereuisiae CDC28 gene product (50); SNFl, S. cereuisiae SNFI gene product (51); Phk-y, the y subunit of phos- phorylase kinase from rabbit skeletal muscle (52); src, pp60""" of the Prague C strain of Rous sarcoma virus (53).

A H8

CKI(2.7kb)

M U

I L i g a t h

m ..... *.. . . . . . . .

I 'r'

FIG. 5. Construction of the CKI-expressing plasmid, pUCK5, ( A ) and the amino-terminal sequence of its transla- tion product (B) . The plasmid construction is described under "Experimental Procedures." The dotted box is the CKI coding region and the striped box the CKI flanking region. The open box is the E. coli lac gene. p and o denote its promoter and operator, respectively. Amino acid sequences are shown in the one-letter notation. The dotted underlining denotes the Shine-Dalgarno sequence (54). The wavy and straight underlining indicate the HincII and MuaI sites used for the construction, respectively. Restriction sites: Hi, HincII; M, MuaI.

to the consensus sequence recently reported by Brenner (46) for protein kinases and a group of phosphotransferases in- volved in the conferral of antibiotic resistance. Fig. 4 compares the partial sequence of the CKI product with those of phos- photransferases such as Bacillus circulans aminoglycoside phosphotransferase (47) and Tn903 aminoglycoside phospho- transferase (kanamycin resistance determinant) (48), and protein kinases such as the cdc2+ product (49), CDC28 product (50), SNFl product (51), the y subunit of phosphorylase kinase (52), and pp60"" (53). The observed sequence homology suggests that the CKI encodes a phosphotransferase.

Expression of the Yeast CKI Gene in E. coli-In order to confirm that CKI is the structural gene for choline kinase more directly, we attempted the expression of the CKI gene in E. coli cells completely lacking choline kinase activity by means of the strategy shown in Fig. 5. The 1840-bp MvaI

SF -

-cDp-cho

"P-cho

0- .I - - I 0 li -Cho

1 2 3 4 5 6 7

FIG. 6. Expression of the yeast CKI gene in E. coli cells. E. coli strain JM103 cells harboring pUC19 (vector) or pUCK5 (CKI- expressing plasmid) were cultured in 100 ml of LB medium containing ampicillin to an absorbance of 0.6-0.7 at 550 nm and then IPTG was added to a final concentration of 0.75 mM, where indicated. After further culturing for 2 h, the cells were harvested, washed with cold saline, and then disrupted by sonication for a total of 1.5 min at 0.5- min intervals in 2 ml of 50 mM Tris-HC1 buffer, pH 7.5, containing 1 mM EDTA and 20% (v/v) glycerol on ice. The homogenate was centrifuged at 10,000 X g for 20 min. The resulting supernatant was used for the choline kinase assay. The assay mixture (10 p l ) contained the same ingredients as given under "Experimental Procedures" except that the concentration of [methyl-l4C]choline (52 Ci/mol) was 0.6 mM. After 20 min at 30 "C, a 9-pl aliquot of the reaction mixture was spotted directly onto a Silica Gel-60 plate, and then the plate was developed with 95% ethanol, 2% ammonium hydroxide (l:l, v/ v). Autoradiography was carried out for 1 day at room temperature. Lane 1, no enzyme; lane 2, 10 pg of the extract from pUC19-carrying JM103 cells cultured in the presence of IPTG; lane 3, 10 pg of the extract from pUCK5-carrying JM103 cells cultured in the absence of IPTG; lanes 4-7, 9.3 pg of the extract from JM103 cells harboring pUCK5 cultured in the presence of IPTG. The assay conditions were as follows: lanes 1-4 and 7, complete assay mixture; lane 5, minus A T P lane 6, minus MgS04 plus 1 mM EDTA. In the experiment shown in lane 7, the reaction mixture was boiled for 1 min at the end of the incubation and then treated with 10 units of calf intestine alkaline phosphatase for 10 min at 37 "C. SF, solvent front; 0, origin; CDP-cho, CDP-choline; P-cho, phosphorylcholine; Cho, choline.

fragment containing the entire CKI coding region except for the first seven amino acids was isolated from pCKlD, treated with Klenow fragment to repair its ragged ends, and then inserted at the HincII site of pUC1S to yield pUCK5. The chimeric gene thus constructed encoded a CKI protein which contained the first 13-amino acid residues of E. coli 0-galac- tosidase fused to the carboxyl-terminal 575-amino acid resi- dues of CKI.

The transformant carrying pUCK5 was grown in the pres- ence and absence of a gratuitous lac inducer, IPTG, and the choline kinase activity was assayed in the 10,000 x g super- natant. The sensitivity of the assay was increased by using [methyl-'4C]choline with high specific radioactivity and by separating the reaction product by thin layer chromatography. As shown in Fig. 6 (lane 2) , no choline kinase activity was found in the extract obtained from the pUC19-carrying E. coli cells. In contrast, the extract obtained from the pUCK5- carrying cells cultured in the presence of IPTG mediated the ATP- and MP-dependent formation of phosphorylcholine (lanes 4-6). The identity of the reaction product was con- firmed by alkaline phosphatase treatment, which converted the product completely to choline (lane 7). The efficient expression of CKI required the presence of IPTG during the culture. The activity expressed in its absence amounted to only one-fifth that expressed in its presence (lanes 3 and 4) . When fully expressed, the choline kinase activity reached a level of 21 nmol/min/mg protein, which was comparable to that of the wild-type yeast. Thus, it was shown that the yeast CKI gene directed the synthesis of choline kinase in E. coli cells. These results provide conclusive evidence that CKI is the structural gene for choline kinase. Furthermore, since the

2058 Yeast Choline Kinase Gene

A EV EV

H

B 1 2 - start

40kb - 27kb -

- 231 - 9.4 - 6.6 - 44 - 2.3 - 2.0

- 0.6 FIG. 7. Disruption of the genomic CKI locus. A, in vitro dis-

ruption of the CKZ gene. Construction of the pKDl plasmid contain- ing the disrupted cki gene and disruption of the CKZ locus in the genome of strain D302-3C are described under “Experimental Pro- cedures.” The dotted box is the CKZ coding region and the striped box its flanking region. The open box is the yeast LEU2 gene. B, Southern blot analysis. Genomic DNAs from parental strain D302-3C and cki- disrupted strain 2014 were digested with PstI and HindIII, electro- phoresed on a 0.7% agar gel, transferred to a nitrocellulose sheet, and then hybridized with the 32P-labeled 2698-bp PstIIHindIII fragment of pCK1D. Lane 1, DNA from parental strain D302-3C; lane 2, DNA from cki-disrupted strain 2014. Restriction sites: EV, EcoRV, H, HindIII; P, PstI. HindIII fragments were used as size markers.

TABLE V Choline kinase and ethanolamine kinase activities of the wild-type

and the cki::LEU2 disruptant The parent (D302-3C) and the cki::LEU2 disruptant (2014) were

cultured in WaD medium and then harvested at the late logarithmic phase. The enzyme activities were assayed as described under “Ex- uerimental Procedures.”

Strain Choline kinase ~ ~~

Ethanolamine kinase

nmollminlmg protein D302-3C 13.0 1.8 2014 co.1 0.3

CKI-coding frame expressed a fully active enzyme in E. coli cells, yeast choline kinase was concluded to be mono-cistronic.

E. coli harboring pUCK5 exhibited not only choline kinase activity but also significant ethanolamine kinase activity. When fully expressed, the ethanolamine kinase activity of E. coli cells was determined to be 4.8 nmol/min/mg protein, being comparable to that of the wild-type yeast cells. The activity was absolutely dependent on ATP and M$+. Control cells containing pUC19 instead of pUCK5 did not exhibit ethanolamine kinase activity at all. Thus, yeast choline kinase mediated the phosphorylation of both choline and ethanola- mine.

Disruption of the Genomic CKI Locus-The knowledge of the complete CKI sequence prompted us to disrupt the CKI locus in the yeast genome and to construct a null choline kinase mutant. The disruption strategy is diagrammatically shown in Fig. 7. We used two EcoRV sites found at positions 701 and 1600 within the CKI coding region. The 2.7-kb PstI/

TABLE VI Incorporation of [methyl-“Clcholine and [l,2-’4C]ethanolamine into

lipids in the cki::LEU2 mutant Cells of strains 2014 (cki::LEU2) and D302-3C (parent) were grown

in M2i medium supplemented with L-leucine and L-histidine at 30 “C to cell densities of 111 and 114 pg protein/ml, respectively. To 1 ml of each culture was added 0.05 pCi [methyl-’4C]choline (10 Ci/mol) or 1 pCi [1,2-“C]ethanolamine (4 Ci/mol) and then the mixtures were incubated at 30 “C for 30 min with shaking. Lipids were ex- tracted and then separated as described under “Experimental Proce- dures.” The gel containing the indicated phospholipids were scraped off and then counted. Abbreviations: PC, phosphatidylcholine; PE, phosphatidylethanolamine; PMME, phosphatidylmonomethyletha- nolamine; PDME, phosphatidyldimethylethanolamine.

[“C]Choline Strain incorporation

into PC

[“C]Ethanolamine incorporation

into PE, PMME, PDME, and PC

cpmlmg protein D302-3C 30,852 14,938 201 4 2.016 3.887

HindIII fragment of pCKlD, which contained the entire CKI gene, was first cloned into pUC19. The plasmid thus obtained (pUK1) was treated with EcoRV to remove a 899-bp sequence from the CKI coding region. The 2.2-kb yeast LEU2 marker (33) was inserted into this gap to yield pKD1. Thus, the CKI coding region was partially replaced by LEU2. pKDl was digested with PstIIHindIII, and then the 4.0-kb linear DNA containing disrupted cki was isolated electrophoretically and introduced into yeast strain D302-3C (a leu2-3 leu2-112 his4- 519 canl) to disrupt the CKI locus in its genome. After transformation, stable Leu+ transformants were selected and purified on a minimum plate supplemented with histidine. To confirm that gene disruption had occurred at the CKI locus, the transformant’s chromosomal DNA was isolated and then analyzed by the Southern blot technique as follows. DNA was digested with PstI and HindIII, separated by agarose gel electrophoresis, transferred to a nitrocellulose membrane, and then hybridized with the 32P-labeled 2.7-kb PstIIHindIII frag- ment from pCBlD as a probe. Fig. 7B (lane 1) shows that the probe hybridized to the expected 2.7-kb fragment in parental strain D302-3C. In the case of transformant 2014, however, the same probe hybridized to a 4.0-kb fragment (lane 2), indicating that this strain contained the cki::LEU2 gene. As shown in Table V, virtually no choline kinase activity was detected in the disruptant, indicating that CKI is the only gene capable of encoding choline kinase in the yeast. Likewise, the ethanolamine kinase activity greatly decreased on the disruption of the CKI locus. We noted that slight activity still remained. The present results are consistent with the above conclusion that the CKI product catalyzes both the choline kinase and ethanolamine kinase activities.

Flux of the CDP-Choline and CDP-Ethanolamine Pathways in the cki Disruptant-We determined the fluxes of the CDP- choline pathway and the CDP-ethanolamine pathway in a cki::LEU2 mutant by measuring the incorporation of labeled choline and ethanolamine into lipids. Table VI shows that the rate of [14C]choline incorporation into lipids decreased greatly on disruption of the CKI locus (less than 7% of the wild-type level). The rate of [“Clethanolamine incorporation into lipids was also markedly reduced in the disruptant. However, low, but measurable, activity as to incorporation of ethanolamine into lipids remained after disruption (26% of the wild-type level). Thus, the enzyme levels were correlated with the overall rates of the CDP-choline pathway and the CDP-ethanolamine pathway in the disruptant.

Yeast Choline Kinase Gene 2059

DISCUSSION

This is the first report of the molecular cloning and char- acterization of the gene encoding choline kinase (CKZ). The evidence indicating that CKI is the structural gene can be summarized as follows. First, CKZ, under the control of the E. coli lac promoter, directed the synthesis of choline kinase in E. coli cells originally devoid of this enzyme. Second, the deduced CKI product contains a segment resembling the phosphotransferase consensus sequence (46). Third, the cal- culated molecular weight of the CKI product (66,316) was well consistent with the value determined for the partially purified yeast choline kinase by Sephadex G-200 column chromatog- raphy (67,000) (18). Thus, yeast choline kinase is very likely a monomeric protein. The present data exclude the possibility that the CKZproduct is an activator protein which stimulates the expression or activity of choline kinase.

It is important to note that the extract obtained from the E. coli cells expressing yeast choline kinase efficiently cata- lyzed the phosphorylation of ethanolamine as well. These results provide firm evidence for the identity of choline kinase and ethanolamine kinase in the yeast. This conclusion was supported by two additional lines of evidence: 1) the intro- duction of the CKI gene into yeast cells in multiple copies caused the overproduction of both choline kinase and etha- nolamine kinase, and 2) the disruption of the CKI locus in the wild-type yeast led to concomitant losses of the choline kinase and ethanolamine kinase activities. Thus, the CDP- choline pathway and the CDP-ethanolamine pathway share the same enzyme, in their initial steps, in the yeast.

Although it is evident that the phosphorylation of ethanol- amine is mostly attributable to the activity of choline kinase in yeast cells, it should be pointed out that slight ethanolamine kinase activity still remained after complete disruption of the choline kinase gene. This can be accounted for by postulating that yeast cells contain a very low level of an enzyme which phosphorylates ethanolamine, but not choline. This is remi- niscent of the finding of Weinhold and Rethy (11) that the activity of rat liver ethanolamine kinase was separated into two peaks on DEAE-cellulose column chromatography; the major fraction phosphorylated both choline and ethanola- mine, whereas the minor fraction only ethanolamine.

Acknowledgment-We would like to thank Dr. J. Nikawa for the YE-pM4 vector and his valuable suggestions as to the expression of the choline kinase gene in E. coli cells.

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