Abstract
Aminoalcoholphosphotransferases (AAPTases) utilize diacylglycerols and cytidine diphosphate (CDP)-aminoalcohols as substrates in the synthesis of the abundant membrane lipids phosphatidylcholine and phosphatidylethanolamine. A soybean cDNA encoding an AAPTase that demonstrates high levels of CDP-choline:sn-1,2-diacylglycerol cholinephosphotransferase activity was isolated by complementation of a yeast strain deficient in this function and was designated AAPT1. The deduced amino acid sequence of the soybean cDNA showed nearly equal similarity to each of the two characterized AAPTase sequences from yeast, cholinephosphotransferase and ethanolaminephosphotransferase (CDP-ethanolamine:sn-1,2-diacylglycerol ethanolaminephosphotransferase). Moreover, assays of soybean AAPT1-encoded enzyme activity in yeast microsomal membranes revealed that the addition of CDP-ethanolamine to the reaction inhibited incorporation of 14C-CDP-choline into phosphatidylcholine in a manner very similar to that observed using unlabeled CDP-choline. Although DNA gel blot analysis suggested that AAPT1-like sequences are represented in soybean as a small multigene family, the same AAPT1 isoform isolated from a young leaf cDNA library was also recovered from a developing seed cDNA library. Expression assays in yeast using soybean AAPT1 cDNAs that differed only in length suggested that sequences in the 5'leader of the transcript were responsible for the negative regulation of gene activity in this heterologous system. The inhibition of translation mediated by a short open reading frame located 124 bp upstream of the AAPT1 reading frame is one model proposed for the observed down-regulation of gene activity.
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- Becker D. M., Fikes J. D., Guarente L. A cDNA encoding a human CCAAT-binding protein cloned by functional complementation in yeast. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1968–1972. doi: 10.1073/pnas.88.5.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bell R. M., Coleman R. A. Enzymes of glycerolipid synthesis in eukaryotes. Annu Rev Biochem. 1980;49:459–487. doi: 10.1146/annurev.bi.49.070180.002331. [DOI] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Dykes C. W., Kay J., Harwood J. L. Incorporation of choline and ethanolamine into phospholipids in germinating soya bean. Biochem J. 1976 Sep 15;158(3):575–581. doi: 10.1042/bj1580575. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Han S., Navarro J., Greve R. A., Adams T. H. Translational repression of brlA expression prevents premature development in Aspergillus. EMBO J. 1993 Jun;12(6):2449–2457. doi: 10.1002/j.1460-2075.1993.tb05899.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hannapel D. J., Ohlrogge J. B. Regulation of Acyl Carrier Protein Messenger RNA Levels during Seed and Leaf Development. Plant Physiol. 1988 Apr;86(4):1174–1178. doi: 10.1104/pp.86.4.1174. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hjelmstad R. H., Bell R. M. Mutants of Saccharomyces cerevisiae defective in sn-1,2-diacylglycerol cholinephosphotransferase. Isolation, characterization, and cloning of the CPT1 gene. J Biol Chem. 1987 Mar 15;262(8):3909–3917. [PubMed] [Google Scholar]
- Hjelmstad R. H., Bell R. M. The sn-1,2-diacylglycerol cholinephosphotransferase of Saccharomyces cerevisiae. Nucleotide sequence, transcriptional mapping, and gene product analysis of the CPT1 gene. J Biol Chem. 1990 Jan 25;265(3):1755–1764. [PubMed] [Google Scholar]
- Hjelmstad R. H., Bell R. M. The sn-1,2-diacylglycerol ethanolaminephosphotransferase activity of Saccharomyces cerevisiae. Isolation of mutants and cloning of the EPT1 gene. J Biol Chem. 1988 Dec 25;263(36):19748–19757. [PubMed] [Google Scholar]
- Hjelmstad R. H., Bell R. M. sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases in Saccharomyces cerevisiae. Nucleotide sequence of the EPT1 gene and comparison of the CPT1 and EPT1 gene products. J Biol Chem. 1991 Mar 15;266(8):5094–5103. [PubMed] [Google Scholar]
- Khalili K., Brady J., Khoury G. Translational regulation of SV40 early mRNA defines a new viral protein. Cell. 1987 Feb 27;48(4):639–645. doi: 10.1016/0092-8674(87)90242-x. [DOI] [PubMed] [Google Scholar]
- Kozak M. An analysis of vertebrate mRNA sequences: intimations of translational control. J Cell Biol. 1991 Nov;115(4):887–903. doi: 10.1083/jcb.115.4.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
- Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. doi: 10.1083/jcb.108.2.229. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
- Macher B. A., Mudd J. B. Biosynthesis of phosphatidylethanolamine by enzyme preparations from plant tissues. Plant Physiol. 1974 Feb;53(2):171–175. doi: 10.1104/pp.53.2.171. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Murray M. G., Thompson W. F. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980 Oct 10;8(19):4321–4325. doi: 10.1093/nar/8.19.4321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ohlrogge J. B., Browse J., Somerville C. R. The genetics of plant lipids. Biochim Biophys Acta. 1991 Feb 26;1082(1):1–26. doi: 10.1016/0005-2760(91)90294-r. [DOI] [PubMed] [Google Scholar]
- Okuley J., Lightner J., Feldmann K., Yadav N., Lark E., Browse J. Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell. 1994 Jan;6(1):147–158. doi: 10.1105/tpc.6.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prud'homme M. P., Moore T. S. Phosphatidylcholine synthesis in castor bean endosperm : free bases as intermediates. Plant Physiol. 1992 Nov;100(3):1527–1535. doi: 10.1104/pp.100.3.1527. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Slack C. R., Roughan P. G., Browse J. Evidence for an oleoyl phosphatidylcholine desaturase in microsomal preparations from cotyledons of safflower (Carthamus tinctorius) seed. Biochem J. 1979 Jun 1;179(3):649–656. doi: 10.1042/bj1790649. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Somerville C., Browse J. Plant lipids: metabolism, mutants, and membranes. Science. 1991 Apr 5;252(5002):80–87. doi: 10.1126/science.252.5002.80. [DOI] [PubMed] [Google Scholar]
- Sparace S. A., Wagner L. K., Moore T. S. Phosphatidylethanolamine synthesis in castor bean endosperm. Plant Physiol. 1981 May;67(5):922–925. doi: 10.1104/pp.67.5.922. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stymne S., Stobart A. K., Glad G. The role of the acyl-CoA pool in the synthesis of polyunsaturated 18-carbon fatty acids and triacylglycerol production in the microsomes of developing safflower seeds. Biochim Biophys Acta. 1983 Jul 12;752(2):198–208. doi: 10.1016/0005-2760(83)90113-3. [DOI] [PubMed] [Google Scholar]
- Stymne S., Stobart A. K. The biosynthesis of triacylglycerols in microsomal preparations of developing cotyledons of sunflower (Helianthus annuus L.). Biochem J. 1984 Jun 1;220(2):481–488. doi: 10.1042/bj2200481. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tillman T. S., Bell R. M. Mutants of Saccharomyces cerevisiae defective in sn-glycerol-3-phosphate acyltransferase. Simultaneous loss of dihydroxyacetone phosphate acyltransferase indicates a common gene. J Biol Chem. 1986 Jul 15;261(20):9144–9149. [PubMed] [Google Scholar]
- Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
- Werner M., Feller A., Messenguy F., Piérard A. The leader peptide of yeast gene CPA1 is essential for the translational repression of its expression. Cell. 1987 Jun 19;49(6):805–813. doi: 10.1016/0092-8674(87)90618-0. [DOI] [PubMed] [Google Scholar]