Plant Molecular Biology 31: 205-211, 1996. © 1996 Kluwer Academic Publishers. Printed in Belgium.

205

Cloning of Brassica napus CTP:phosphocholine cytidylyltransferase cDNAs by complementation in a yeast cct mutant Ikuo Nishida t'2*, Russell Swinhoe 3, Antoni R. Slabas 3 and Norio Murata 1 ~National Institute for Basic Biology, Okazaki 444, Japan; 2 Current address: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyoku, Tokyo 113, Japan (*author for correspondence); 3Department of Biological Sciences, University of Durham, Durham DH1 3LE, UK Received 3 August 1995; accepted in revised form 19 January 1996

Key words: Brassica napus, CDP-choline, cytidylyltransferase, freezing stress, phosphatidylcholine, yeast mutant complementation

Abstract

CTP:phosphocholine cytidylyltransferase is a rate-limiting enzyme in biosynthesis of phosphatidylcholine in plant cells. We have isolated four cDNAs for the cytidylyltransferase from a root c D N A library of Brassica napus by complementation in a yeast cct mutant. The deduced amino-acid sequences of the B. napus enzymes resembled rat and yeast enzymes in the central domain. Although all cytidylyltransferases ever cloned from B. napus and other organisms were predicted to be structurally hydrophilic, the yeast cct mutant transformed with one of the B. napus c D N A clones restored the cytidylyltransferase activity in the microsomal fraction as well as in the soluble fraction. These results are consistent with a recent view that yeast cells contained a machinery for targeting the yeast cytidylyltransferase to membranes, and may indicate that the B. napus enzyme was compatible with the machinery.

In recent years there has been a rapid growth in our understanding of the molecular mechanism of the biosynthesis of fatty acids [26] and their desaturation [2, 18] in plants. However, the role of complex lipids in plant physiology and cellular metabolism has remained to be studied. The biosynthesis of phosphatidylcholine (PC) in plants is of particular interest to us because it is a major lipid accounting for 32-67 Yo in plant membranes [4, 7, 33]. Desaturation of 18:1 to 18:2 is now firmly established to occur on a PC backbone [21], and freezing tolerance has been correlated

with changes in phospholipid content in a number of plant systems [24]. Clearly a greater understanding of the physiological role of PC in plants would be advanced by the availability of appropriate cDNAs to the genes in question, which would permit direct observations to phenomena when their expression level was manipulated. PC in plant cells can originate mainly from CDP-choline [17] but minutely from phosphatidylethanolamine [27] and phosphatidylserine [ 13]. The CDP-choline pathway for synthesis involves 3 enzymatic steps: (1) choline kinase (EC

The nucleotide and amino-acid sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers D58404 (CCT1), D63166 (CCT2), D63167 (CCT3) and D63168 (CCT4).

206 2.7.1.32; CKI) catalyzing the synthesis of phospho(ryl)choline, (2) CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15; CCT) catalyzing the synthesis of CDP-choline, and (3) CDPcholine:diacylglycerol cholinephosphotransferase (EC 2.7.8.2; CPT) catalyzing the synthesis of PC. CCT is apparently rate limiting in this pathway [12, 19, 30] but to date no plant CCT c D N A has been cloned. Reports on the properties of CCTs purified from plants differ depending on the sources of the plant material [20, 31 ]. Recently, the yeast cki and cpt mutations, which are defective in the first and the last reactions of the CDPcholine pathway, have been complemented by human [9] and soybean c D N A clones [3], respectively. Here we report that plant CCT cDNAs from a root c D N A library ofBrassica napus have successfully complemented the yeast cct mutation and describe a first investigation of the structure of plant CCT in comparison with mammal and fungus CCTs. We also describe subcellular localization of B. napus CCT in yeast cells. PC in yeast cells can be synthesized mainly via phosphatidylserine (PS) and subsidiarily via CDP-choline or CDP-ethanolamine. If the biosynthesis of PS is blocked by mutation in the gene for phosphatidylserine synthase (the CH01 gene), the resultant chol strain exhibits phenotypic choline (or ethanolamine) auxotrophy [8] owing to the presence of CCT (or ECT). The logic used in this study was to construct a chol cct strain, so that a foreign CCT could be isolated by complementation of the cct mutation. However, cell viability was a major problem with the double mu-

tation and therefore we constructed the chol cct strain INY103, which was viable in the presence of galactose owing to the plasmid YCpGPS S [ 6] (see below). The yeast strain, INY 103 (MA Ta, CHOIA::HIS3, CCTA::LEU2, YCpGPSS), was constructed from a diploid yeast strain, Saccharomyces cerevisiae YPH501 (MATa/~, ura3-52/ ura3-52, l y s 2 - 8 o l a m b e r / l y s 2 - 8 0 1 amber, ade2lOl°chre/ade2-1Ol°Chre, trp1-A63/trp1-A63, his3A2OO/his3-A200, leu2-A1/leu2-A1) [25]: YPH501 was transformed with a CHOIA::HIS3 cassette 1 according to Gietz et aL [5] and a haploid His + Cct ÷ strain, INY102B (MATa, CHOIA::HIS3), was isolated according to Hikiji et al. [8]; INY102B was transformed as above [5] with YCpGPSS (TRP1) [6], which contains a yeast CH01 gene under the control of a GAL7 promoter; and the resultant Trp + strain INY102B/ YCpGPSS grown in a YPADGal medium (YPA [23] plus 1~o glucose and 2~o galactose) was transformed [5] with a CCTA:.'LEU2 cassette 2. The resultant Leu + C c t - strain INY103 showed the galactose-dependent Chol + phenotype as well as the C c t - phenotype [29], i.e., it could grow on minimal agar plates (SD) [32] (containing 100 #M myo-inositol with appropriate supplements described in Kiyono et al. [14]) supplemented with either 2~o galactose or 1 mM ethanolamine, but not on those supplemented with or without 0.1 mM choline. All the above gene disruptions were confirmed by D N A / D N A hybridization analysis (data not shown). INY103 was transformed with the pFL61

1 A CHOIA::HIS3 cassette for disruption of CHO1 in yeast was constructed as follows. D N A fragments of CHO1 were amplified from the genomic D N A of yeast by PCR using the following combination of primers, i.e., a 5'-CH01 fragment from 5 ' - A A A G A A T I ' C G A T A C C C T A A C A T C A A T C C C - 3 vs. 5 ' - A A A C C C G G G T T T T T T A A T A T A T A G T T T T A T T T T T G - 3 ' and a 3'-CHOI fragment from 5 ' - A A A T C T A G A A A A C T A C A T T C G A T G T C A T G A - 3 ' vs. 5 ' - A A A G T C G A C C A G C T G G C A A G A A T T G A G T A A - 3 ' . The 5'- and 3'-CH01 fragments were subcloned between EcoRI and SmaI sites and between XbaI and Sall sites of pTZ18R, respectively, and the resultant plasmid was designated pTZ 18R/CHO1A. A BamHI/XhoI fragment of a HIS3 gene from pJJ215 [ 10] was blunt-ligated into the BamHI site o f p T Z 18R/CHO 1A, and the resultant plasmid gave a CHOIA:.'HIS3 cassette by EcoRV/PvulI digestion. 2 A CCTA::LEU2 cassette for disruption of CCT in yeast was constructed as follows. A D N A fragment of CCT resulting from PCR with primers 5 ' - G G C A A A C C C A A C A A C A G G G A A G T C C - 3 ' a n d 5 r - C G T A A A G T T G C T G A G C G T C - 3 ' was subcloned into the HindlII site of pBluescript (SK + ) and the resultant plasmid was designated pBS/CCT. A Sall fragment of a LEU2 gene from pJJ283 [ 10] was blunt-ligated into the MscI site of pBS/CCT, then a 0.9-kb XbaI/HpaI fragment of CCT was deleted. The resultant plasmid gave a CCTA::LEU2 cassette by HindlII digestion.

207 ( U R A 3 ) [16] c D N A library c o n s t r u c t e d f r o m roots o f Brassica napus L. cv. Jet Neuf. T h e cct m u t a t i o n o f I N Y 1 0 3 w a s c o m p l e m e n t e d by 13 p F L 6 1 plasmids containing foreign c D N A s . Preliminary nucleotide sequence analysis suggested that these c D N A clones were g r o u p e d into 4 distinct c D N A s designated C C T 1 , C C T 2 , C C T 3 and C C T 4 . T h e y c o n t a i n e d 6, 5, 1, and 1 clone(s), respectively. Nucleotide sequences o f C C T 1 , C C T 2 , C C T 3 a n d ' C C T 4 were determined with representative clones. Nucleotide sequences o f C C T 1 , C C T 2 , C C T 3 and C C T 4 c o n t a i n e d 1382, 1321, 1099 and 1196 bp with o p e n - r e a d i n g frames ( O R F ) o f 990, 996, 975, 981 bp, respectively. T h e O R F s of C C T 1 and C C T 3 were full-length b e c a u s e these c D N A c o n t a i n e d an in-frame stop c o d o n in the 5 ' u n t r a n s l a t e d sequences. T h e O R F s o f C C T 2 and C C T 4 were also likely to be the full-length as judged f r o m extensive identities ( m o r e t h a n 95 ~ ) o f their a m i n o - a c i d sequences to the other two clones (see below). H o w e v e r , the 5 ' - u n t r a n s l a t e d sequences o f C C T 2 and C C T 4 were still in-frame and therefore we could not exclude the possibility that the O R F s o f these clones might not be full-length. Southern hybridization analysis with a radiolabeled p r o b e derived f r o m a 0.35 k b E c o R V - X h o I fragment o f C C T 3 c D N A shows that the g e n o m e o f B. napus cv. Jet N e u f contained multiple copies o f sequences hybridizable to the p r o b e (Fig. 1), suggesting that isolated C C T c D N A s were originated f r o m the plant. T o confirm that the isolated c D N A clones enc o d e d C C T , the e n z y m e activity was tested in cell-free system [28]. T h e cell free extracts f r o m yeast strains were i n c u b a t e d with p h o s p h o r y l [ m ethyl-14C]choline and C T P in the presence of MgC12 and the radioactive product, which was separated by silica gel thin-layer c h r o m a t o g r a p h y ( T L C ) [28], was identified as described in the legend o f Fig. 2. U n d e r these conditions, we confirmed that C D P - c h o l i n e was synthesized with the cell-free extracts f r o m I N Y 1 0 2 B / Y C p G P S S ( C C T , positive control) but not with that f r o m I N Y 1 0 3 ( C C T A : : L E U 2 , negative control) ( d a t a not shown). Figure 2 (lane 1) shows a typical exa m p l e that C D P - c h o l i n e was synthesized with

Fig. 1. Southern blot of leaf genomic DNA from B. napus Jet

Neuf hybridized at high stringency [ 1] to a probe derived from a 0.35 kbp EcoRV-Xhol fragment of B. napus CCT3 cDNA. Lanes each contained 10 /~g of DNA. Lanes: 1, BamHI; 2, XbaI; 3, EcoRV; 4, HindlII; 5, XhoI; 6, PstI; 7, ClaI. Seeds of Brassica napus L. cv. Jet Neuf were immobilized on

sterile agarose plates with low-melting point agarose and the plates were incubated upside-down at 25 °C under illumination. After 3-4 days roots were excised and RNA was purified [ 1]. cDNA was synthesized with a cDNA synthesis kit (Pharmacia) and purified on a SizeSep 400 span column (Pharmacia) before ligation into the plasmid pFL61 (URA3) [ 16], which can express foreign cDNAs in yeast under the control of a constitutive PGK promoter. A cDNA library represented by 5 x 105 independent clones was constructed, and was amplified in Escherichia coli DH5~ competent cells (Life Technologies, Tokyo). INY103 was transformed with 75/tg of the cDNA library and the resultant Ura + transformants were selected on minimal agar plates supplemented with 0.1 mM choline. From 2.8 x 10 6 Ura + transformants 16 clones were isolated; 13 clones demonstrated the Cct ÷ phenotype [29], whereas the remaining 3 clones showed the Cct- phenotype. The latter clones have not been studied further, however they possibly encode either phosphatidylserine synthetase as pointed out earlier [29] or a trans factor acting on the GAL7 promoter. Plasmids were isolated from yeast [ 1] and rescued in E. coli HB101 competent cells (Takara, Japan). Nucleotide sequences were determined by a dideoxy chain termination method [22] with a BcaBEST dideoxy sequencing kit (Takara, Japan).

the cell-free extracts f r o m I N Y 1 0 3 t r a n s f o r m e d with B. napus C C T 1 c D N A . T h e s e results indicate that the isolated c D N A encodes the enzyme. D e d u c e d a m i n o - a c i d sequences o f C C T 1 ,

208

Fig. 2. Detection of the CCT activity in the cell-free system. The CCT activity in cell-free extracts was measured according to Tsukagoshi et al. [28]. INY103 (CCTA::LEU2) was transformed with B. napus CCT1 cDNA, and the cell homogenate was fractionated by differential centrifugation. Lanes: 1, 17000 x g 10 min supernatant (42 #g protein); 2, 17000140000 x g 60 min pellet (20 #g protein); 3, 140000 × g 60 min supernatant (38 ~g protein); 4, phosphoryl[methyl-14C]choline (6750 dpm). The protein samples were incubated at 30 °C for 30 min in reaction mixtures (20/~1) containing 50 mM TrisHC1 (pH 8.0), 5 mM CTP, 25 mM MgC12, 0.1 mM phosphoryl[methyl-14C]choline (20000 dpm/nmol) (Amersham, England). The reaction was stopped by adding 10/~1 10% trichloroacetic acid, and 5/~1 aliquots with additional 50 #g CDP-choline were subjected to thin-layer chromatography [28] on silica gel 60 F-254 plate (Merck 5715). The location of the radioactive product, which was detected with a phosphoimage analyzer (BAS2000, FujiFilm, Tokyo), was completely superimposed on the location of authentic CDP-choline (Sigma), which was visualized on the TLC plate under ultraviolet illumination.

CCT2, CCT3 and CCT4 contained 329, 331,324 and 326 amino acid residues with calculated molecular masses of 38,258, 38,716, 37,767 and 38,024, respectively. Identity of amino-acid sequences between CCT1 and CCT2 and that between CCT3 and CCT4 were 95.5~'o and 99.4~o, respectively. On the other hand, between CCT1 (or CCT2) and CCT3 (or CCT4) the sequence

identity was less than 82.4~o and substitutions and deletions of amino-acid residues were prominent in both the amino- and carboxyl-terminal domains (Fig. 3). It was a unique feature of B. napus CCTs that they contained clusters of acidic amino-acid residues in the carboxyl terminal domains (Fig. 3). Figure 3 also compares the deduced aminoacid sequences of representative B. napus CCT with those of mammal and fungus CCTs. Both CCT1 and CCT3 sequences resembled the rat and yeast sequences in the central domain, i.e., between 34p and 19°L for CCT1 and between 27p and 183L for CCT3, respectively. Within this domain 46 ~o of amino-acid residues were conserved among the three organisms. On the contrary, the sequences further upstream of this domain were totally diverse among them. The B. napus sequence of CCT1 between 2°5R and 235E and that of CCT3 between 196Rand 227E showed a small identity of 33 ~o with the mammal sequence. The B. napus CCT3 contained a unique sequence, 275QREDTEEQ, and interestingly the fungus CCT contained a similar sequence, 32QREETEEQ; the plant sequence between 249T and 282Q showed an identity of 40~o with the fungus sequence between 5T and 39Q (Fig. 3b). This sequence must be unrelated to the enzymatic activity but may be involved in some regulation of CCT. Hydrophilicity analysis [ 15] predicted that the B. napus CCTs were hydrophilic proteins as is predicted for other CCTs [11, 29]. However, a preliminary experiment suggested that the INY103 transformed with a pFL61 plasmid containing a B. napus CCT1 c D N A restored the enzyme activity in the microsomal fraction as well as in the soluble fraction (Fig. 2, lanes 2 and 3). Tsukagoshi et al. [28] have observed that the yeast CCT expressed in Escherichia coli is totally recovered in the soluble fraction whereas it is exclusively recovered in the microsomal fraction in yeast cells. They concluded that yeast cells are likely to have a machinery for targeting the native CCT to membranes. Our results, being consistent with their finding, may indicate that B. napus CCT was compatible with the machinery in yeast cells.

209 Ca)

Yeast

I:NANPTTGKSSIRAKLSNSSLSNLFKKNKNKRQREETEEQDNEDKDESKNQDENKDTQLTPRKRRRLTKEFEEK

CCT1

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TT ....

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I:N .....

TNVI"G-D-R ......

NG . . . .

DG . . . . . .

R-STAV ....

P....

S--D

Rat

I:NDAQSSAKVNSRKRRKEVPGPNGATEEDGIPSKVQRCAVGL ....

Yeast

74:E .....

ARYTN-ELP ......

o

CCTI CCT3

KEL---RKY

ox

.....

R-PKGF ....

oo

T---ESSP

...........

R---QPAPFSOEIEVDFSKPYVRVTMEE R---FNLP

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T--D

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x

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