Abstract

To elucidate the relationship between the structure and function of CTP:phosphoethanolamine cytidylyltransferases (ECf, EC 2.7.7.14) in plants, we cloned and characterized the cDNA of an ECT from Hordeum vulgare. This HvECT1 cDNA is 1783 bp long, and includes an open reading frame (ORF) of 1263 b that encodes a protein of 421 amino acids. The predicted protein sequence of HvECT1 is $73\%$ identical to and $84\%$ similar with Arabidopsis thaliana AtECT1; it also shares 57, 55, and $37\%$ similarities with human, rat, and yeast ECTs, respectively. Its 252-b 5'-noncoding leader contains a putative upstream ORF of 36 nucleotides, possibly encoding a putative peptide enriched in Ala and Pro residues. Alignment of the N-terminal and C-terminal halves of HvECT1 revealed a large internal repetitive sequence. Both halves contain the HXGH motif known to be involved in the catalytic activity of cytidylyltransferases. The RTXGVSTT sequence and Asp residues are also conserved. Our hydropathy profile showed that HvECT1 contains a signal sequence that is absent in yeast or animal ECTs. Results from reverse transcriptase-PCR indicated that HvECT1 is expressed highly in the leaves, stems, and roots of one-week-old plants; its expression is not regulated by low temperatures. After transforming a yeast mutant ect1 and labeling those transformants with radioactive ethanolamine, we identified HvECT1 cDNA through in vivo analyses of the enzymatic reaction products.

참고문헌 (55)

1. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides SignalP 3.0. J Mol Biol 340: 783-795 
2. Chow M, Der CJ, Buss JE (1992) Structure and biological effects of lipid modifications on proteins. Curr Opin Cell Biol 4: 629-636 
3. Harwood JL, Jones AL, Perry HJ, Rutter AJ, Smith KL, Williams M (1994) Changes in plant lipids during temperature adaptation. In AR Cossins, ed, Temperature Adaptation of Biological Membranes. Portland Press, London, pp 107-118 
4. Hauser H, Pascher I, Pearson RH, Sundell S (1981) Preferred conformation and molecular packing of phosphatidylethanolamine and phosphatidylcholine. Biochim Biophys Acta 650: 21-51 
5. Hosaka K, Yamashita S (1980) Choline transport in Saccharomyces cerevisiae. J Bacteriol 143: 176-181 
6. Kennedy EP, Weiss SB (1956) The function of cytidine coenzymes in the biosynthesis of phospholipids. J Biol Chem 222: 193-214 
7. Kinney AJ (1993) Phospholipid head groups. In TS Moore, ed, Lipid Metabolism in Plants. CRC, Boca Raton, pp 259-284 
8. Kinney AJ, Clarkson DT, Loughman BC (1987) Phospholipid metabolism and plasma membrane morphology of warm and cool rye roots. Plant Physiol Biochem 25: 769-774 
9. Lynch DV, Steponkus PL (1987) Plasma membrane lipid alterations associated with cold acclimation of winter rye seedlings (Secale cereale L. cv. Puma). Plant Physiol 83: 761-767 
10. Park YS, Gee P, Sanker S, Schurter EJ, Zuiderweg ER, Kent C (1997) Identification of functional conserved residues of CTP:glycerol-3-phosphate cytidylyltransferase: Role of histidines in the conserved HXGH in catalysis. J Biol Chem 272: 15161-15166 
11. Qi Q, Huang YF, Cutler AJ, Abrams SR, Taylor DC (2003) Molecular and biochemical characterization of an aminoalcoholphosphotransferase (AAPT1) from Brassica napus: Effects of low temperature and abscisic acid treatments on AAPT expression in Arabidopsis plants and effects of over-expression of BnAAPT1 in transgenic Arabidopsis. Planta 217: 547-558 
12. Sparace SA, Wagner LK, Moore TS (1981) Phosphatidylethanolamine synthesis in castor bean endosperm. Plant Physiol 67: 922-925 
13. Menon AK, Stevens VL (1992) Phosphatidylethanolamine is the donor of the ethanolamine residue linking a glycosylphosphatidylinositol anchor to protein. J Biol Chem 267: 15277-15280 
14. Hunt AG (1994) Messenger RNA 3' end formation in plants. Annu Rev Plant Physiol Plant Mol Biol 45: 47-60 
15. Bladergroen BA, Houweling M, Geelen MJH, van Golde LMG (1999) Cloning and expression of CTP:phosphoethanolamine cytidylyltransferase cDNA from rat liver. Biochem J 343: 107-114 
16. Chapman KD, Moore TS (1993) Catalytic properties of a newly discovered acyltransferase that synthesizes Nacylphosphatidylethanolamine in cottonseed (Gossypium hirsutum L.) microsomes. Plant Physiol 102: 761-769 
17. Choi S-B, Lee K-W, Cho SH (1997) Cloning of CTP:phosphocholine eytidylyltransferase cDNA from Arabidopsis thaliana. Mol Cells 7: 58-63 
18. Justin AM, Demandre C, Tremolieres A, Mazliak P (1985) No discrimination by choline- and ethanolaminephosphotransferase from potato tuber microsomes in molecular species of endogenous diacylglycerols. Biochim Biophys Acta 836: 1-7 
19. Belyavsky AT, Vinogradova T, Rajewsky K (1989) PCR-based cDNA libraries at the level of a few cells. Nucl Acids Res 17: 2919-2932 
20. Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85: 8998-9002 
21. Macher BA, Mudd JB (1974) Biosynthesis of phosphatidylethanolamine by enzyme preparations from plant tissues. Plant Physiol 53: 171-175 
22. Choi YH, Lee JK, Lee C-H, Cho SH (2000) cDNA cloning and expression of an aminoalcoholphosphotransferase isoform in Chinese cabbage. Plant Cell Physiol 41: 1080-1084 
23. Harwood JL (1998) Environmental effects on plant lipid biochemistry. In JL Harwood, ed, Plant Lipid Biosynthesis. Cambridge University Press, Cambridge, pp 305-347 
24. Kyte J, Doolittle RF (1982) A simple method displaying the hydropathic character of a protein. J Mol Biol 157: 105-132 
25. Mizoi J, Nakamura M, Nishida I (2003) A study of the physiological function of phosphatidylethanolamine in Arabidopsis. In N Murata, M Yamada, I Nishida, H Okuyama, J Sekiya, H Wada, eds, Advanced Research on Plant Lipids. Kluwer Academic Publishers, Dordrecht, pp 377-380 
26. Yang W, Mason CB, Pollock SV, Lavezzi T, Moroney JV, Moore TS (2004) Membrane lipid biosynthesis in Chlamydomonas reinhardtii. Expression and characterization of CTP:phosphoethanolamine cytidylyltransferase. Biochem J 382: 51-57 
27. Sundler R (1975) Ethanolaminephosphate cytidylyltransferase: Purification and characterization of the enzyme from rat liver. J Biol Chem 250: 8585-8590 
28. Innis MA, Holland MJ, McCabe PC, Cole GE, Wittman VP, Tal R, Watt KWK, Gelfand DH, Holland JP, Meade JH (1985) Expression, glycosylation, and secretion of an Aspergillus glucoamylase by Saccharomyces cerevisiae. Science 228: 21-26 
29. Lee JK, Choi VH, Bae VG, Lee WS, Cho SH (2001) Cloning and expression of a cDNA encoding aminoalcoholphosphotransferase from Pimpinella brachycarpa. J Plant Biol 44: 27-32 
30. Rho M-S, Kawamata Y, Nakamura H, Ohta A, Takagi M (1996) Isolation and characterization of ECT1 gene encoding CTP:phosphoethanolamine cytidylyltransferase of Saccharomyces cerevisiae. J Biochem 120: 1040-1047 
31. van Hellemond JJ, Slot JW, Geelen MJH, van Golde LMG, Vermeulen PS (1994) Ultrastructural localization of CTP:phosphoethanolamine cytidylyltransferase in rat liver. J Biol Chem 269: 15415-15418 
32. Bladergroen BA, van Golde LMG (1997) CTP:phosphoethanolamine cytidylyltransferase. Biochim Biophys Acta 1348: 91-99 
33. Gurr MI, Harwood JL, Frayn KN (2002) Lipids in cellular structures, In Lipid Biochemistry. Blackwell Science, Oxford, pp 215-266 
34. Chapman KD (2000) Emerging physiological roles for Nacylphosphatidylethanolamine metabolism in plants: Signal transduction and membrane protection. Chem Phys Lipids 108: 221-230 
35. Dewey RE, Wilson RF, Novitzky WP, Goode JH (1994) The MPT1 gene of soybean complements a cholinephos-photransferase-deficient mutant of a yeast. Plant Cell 6: 1495-1507 
36. Chou PY, Fasman GD (1978) Empirical predictions of protein conformation. Annu Rev Biochem 47: 251-276 
37. Mukherjee JJ, Chung T, Ways DK, Kiss Z (1996) Protein kinase C is a major mediator of the stimulatory effect of phorbol ester on phospholipase D-mediated hydrolysis of phosphatidylethanolamine. J Biol Chem 271: 28912-28917 
38. Nishida I, Swinhoe R, Siabas AR, Murata N (1996) Cloning of Brassica napus CTP:phosphocholine cytidylyltransferase cDNAs by complementation in a yeast cct mutant. Plant Mol Biol 31: 205-211 
39. Shin S, Moore TS (1990) Membrane bilayer distribution of phosphatidylethanolamine synthesized by the ethanolaminephosphotransferase and ethanolamine exchange reactions. Plant Physiol 93: 154-159 
40. Craig L, Johnson JE, Cornell RB (1994) Identification of the membrane-binding domain of rat liver CTP:phosphocholine eytidylyltransferase using chymotrypsin proteolysis. J Biol Chem 269: 3311-3317 
41. Kozak M (1987b) Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol 7: 3438-3445 
42. Phillips MC, Finer EG, Hauser H (1972) Differences between conformations of lecithin and phosphatidyle- thanolamine polar groups and their effects on interactions of phospholipid bilayer membranes. Biochim Biophys Acta 290: 397-402 
43. Veitch DP, Gilham D, Cornell RB (1998) The role of histidine residues in the HXGH site of CTP:phosphocholine cytidylyltransferase in CTP binding and catalysis. Eur J Biochem 255: 227-234 
44. Inatsugi R, Nakamura M, Nishida I (2002) Phosphatidylcholine biosynthesis at low temperature: Differential expression of CTP:phosphorylcholine cytidylyltransferase isogenes in Arabidopsis thaliana. Plant Cell Physiol 43: 1342-1350 
45. Kalmar GB, Kay RJ, LaChance A, Aebersold R, Cornell RB (1990) Cloning and expression of rat liver CTP:phosphocholine cytidylyltransferase: An amphipathic protein that controls phosphatidylcholine synthesis. Proc Natl Acad Sci USA 87: 6029-6033 
46. Kozak M (1987a) An analysis of 5' -noncoding sequences from 699 vertebrate messenger RNAs. Nucl Acids Res 15: 8125-8148 
47. Lord JM (1975) Evidence that phosphatidylcholine and phosphatidylethanolamine are synthesized by a single enzyme present in the endoplasmic reticulum of castor bean endosperm. Biochem J 151: 451-453 
48. Morris DR, Geballe AP (2000) Upstream open reading frames as regulators of mRNA translation. Mol Cell Biol 20: 8635-8642 
49. Hill J, Donald KA, Griffiths DE (1991) DMSO-enhanced whole cell yeast transformation. Nucl Acids Res 19: 5791 
50. Wang X, Moore TS (1991) Phosphatidylethanolamine synthesis by castor bean endosperm: Intracellular distribution and characteristics of CTP:ethanolaminephosphate cytidylyltransferase. J Biol Chem 266: 19981-19987 
51. Houslay MD, Stanley KK (1982) Dynamics of Biological Membranes. John Wiley and Sons, New York, pp 39-138 
52. Joshi CP (1987a) An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucl Acids Res 15: 6643-6653 
53. Joshi CP (1987b) Putative polyadenylation signals in nuclear genes of higher plants: A complication and analysis. Nucl Acids Res 15: 9627-9640 
54. Kim KS, Park JH, Cho SH (2004) Cloning and expression of a eDNA, AAPT3, encoding aminoalcoholphosphotransferase isoform from Chinese cabbage. Kor J Biol Sci 8: 105-109 
55. Nakashima A, Hosaka K, Nikawa J (1997) Cloning of a human cDNA for CTP-phosphoethanolamine cytidylyltransferase by complementation in vivo of a yeast mutant. J Biol Chem 272: 9567-9572 

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