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. 2002 Jun 15;364(Pt 3):795–805. doi: 10.1042/BJ20020071

Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene.

Adrian P Brown 1, Simon Carnaby 1, Clare Brough 1, Melissa Brazier 1, Antoni R Slabas 1
PMCID: PMC1222629  PMID: 12049644

Abstract

Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens. The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels. The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22 daf (days after flowering). Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards. LAT1 is present at a level of 27 pg/microg of membrane protein in leaf tissue and <or=12.5 pg/microg of membrane protein in developing embryos. The amount of LAT2 reaches a peak at 305 pg/microg of membrane protein 25 daf and is not expressed 20 daf or before. This is the first study to quantify these membrane-bound proteins in a plant tissue. The maximal level of LAT2 protein coincides with the maximal level of erucic acid synthesis in the seeds. Both full-length proteins were expressed in the Escherichia coli LPAAT mutant JC201, and membranes from these strains were used to investigate the substrate selectivity of these two enzymes, demonstrating that they are different. Finally, we report that LAT2 and a maize LPAAT enzyme (MAT1) can functionally replace the E. coli plsC gene after its deletion in the chromosome, whereas LAT1 and a coconut LPAAT (Coco1) cannot. This is probably due to differences in substrate utilization.

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Selected References

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  1. Brown A. P., Brough C. L., Kroon J. T., Slabas A. R. Identification of a cDNA that encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii. Plant Mol Biol. 1995 Oct;29(2):267–278. doi: 10.1007/BF00043651. [DOI] [PubMed] [Google Scholar]
  2. Brown A. P., Coleman J., Tommey A. M., Watson M. D., Slabas A. R. Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases. Plant Mol Biol. 1994 Oct;26(1):211–223. doi: 10.1007/BF00039533. [DOI] [PubMed] [Google Scholar]
  3. Chersi A., Di Modugno F., Rosano L. Aims and limitations in the use of antipeptide antibodies in molecular biology. Biol Chem. 1997 Jul;378(7):635–640. [PubMed] [Google Scholar]
  4. Coleman J. Characterization of Escherichia coli cells deficient in 1-acyl-sn-glycerol-3- phosphate acyltransferase activity. J Biol Chem. 1990 Oct 5;265(28):17215–17221. [PubMed] [Google Scholar]
  5. Coleman J. Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3-phosphate acyltransferase (plsC). Mol Gen Genet. 1992 Mar;232(2):295–303. doi: 10.1007/BF00280009. [DOI] [PubMed] [Google Scholar]
  6. Eccleston V. S., Harwood J. L. Solubilisation, partial purification and properties of acyl-CoA: glycerol-3-phosphate acyltransferase from avocado (Persea americana) fruit mesocarp. Biochim Biophys Acta. 1995 Jun 27;1257(1):1–10. doi: 10.1016/0005-2760(95)00054-g. [DOI] [PubMed] [Google Scholar]
  7. Hanke C., Wolter F. P., Coleman J., Peterek G., Frentzen M. A plant acyltransferase involved in triacylglycerol biosynthesis complements an Escherichia coli sn-1-acylglycerol-3-phosphate acyltransferase mutant. Eur J Biochem. 1995 Sep 15;232(3):806–810. [PubMed] [Google Scholar]
  8. Heacock P. N., Dowhan W. Construction of a lethal mutation in the synthesis of the major acidic phospholipids of Escherichia coli. J Biol Chem. 1987 Sep 25;262(27):13044–13049. [PubMed] [Google Scholar]
  9. Kater M. M., Koningstein G. M., Nijkamp H. J., Stuitje A. R. The use of a hybrid genetic system to study the functional relationship between prokaryotic and plant multi-enzyme fatty acid synthetase complexes. Plant Mol Biol. 1994 Aug;25(5):771–790. doi: 10.1007/BF00028873. [DOI] [PubMed] [Google Scholar]
  10. Knutzon D. S., Lardizabal K. D., Nelsen J. S., Bleibaum J. L., Davies H. M., Metz J. G. Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates. Plant Physiol. 1995 Nov;109(3):999–1006. doi: 10.1104/pp.109.3.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kume K., Shimizu T. cDNA cloning and expression of murine 1-acyl-sn-glycerol-3-phosphate acyltransferase. Biochem Biophys Res Commun. 1997 Aug 28;237(3):663–666. doi: 10.1006/bbrc.1997.7214. [DOI] [PubMed] [Google Scholar]
  12. Lassner M. W., Levering C. K., Davies H. M., Knutzon D. S. Lysophosphatidic acid acyltransferase from meadowfoam mediates insertion of erucic acid at the sn-2 position of triacylglycerol in transgenic rapeseed oil. Plant Physiol. 1995 Dec;109(4):1389–1394. doi: 10.1104/pp.109.4.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lewin T. M., Wang P., Coleman R. A. Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction. Biochemistry. 1999 May 4;38(18):5764–5771. doi: 10.1021/bi982805d. [DOI] [PubMed] [Google Scholar]
  14. Manaf A. M., Harwood J. L. Purification and characterisation of acyl-CoA: glycerol 3-phosphate acyltransferase from oil palm (Elaeis guineensis) tissues. Planta. 2000 Jan;210(2):318–328. doi: 10.1007/PL00008140. [DOI] [PubMed] [Google Scholar]
  15. Murata N., Tasaka Y. Glycerol-3-phosphate acyltransferase in plants. Biochim Biophys Acta. 1997 Sep 4;1348(1-2):10–16. doi: 10.1016/s0005-2760(97)00115-x. [DOI] [PubMed] [Google Scholar]
  16. Nieboer M., Vis A. J., Witholt B. Overproduction of a foreign membrane protein in Escherichia coli stimulates and depends on phospholipid synthesis. Eur J Biochem. 1996 Oct 15;241(2):691–696. doi: 10.1111/j.1432-1033.1996.00691.x. [DOI] [PubMed] [Google Scholar]
  17. Ohlrogge J., Browse J. Lipid biosynthesis. Plant Cell. 1995 Jul;7(7):957–970. doi: 10.1105/tpc.7.7.957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Peterson G. L. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal Biochem. 1979 Dec;100(2):201–220. doi: 10.1016/0003-2697(79)90222-7. [DOI] [PubMed] [Google Scholar]
  19. Scheideler M. A., Bell R. M. Efficiency of reconstitution of the membrane-associated sn-glycerol 3-phosphate acyltransferase of Escherichia coli. J Biol Chem. 1986 Aug 25;261(24):10990–10995. [PubMed] [Google Scholar]
  20. Sun C., Cao Y. Z., Huang A. H. Acyl coenzyme a preference of the glycerol phosphate pathway in the microsomes from the maturing seeds of palm, maize, and rapeseed. Plant Physiol. 1988 Sep;88(1):56–60. doi: 10.1104/pp.88.1.56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Swartley J. S., Balthazar J. T., Coleman J., Shafer W. M., Stephens D. S. Membrane glycerophospholipid biosynthesis in Neisseria meningitidis and Neisseria gonorrhoeae: identification, characterization, and mutagenesis of a lysophosphatidic acid acyltransferase. Mol Microbiol. 1995 Nov;18(3):401–412. doi: 10.1111/j.1365-2958.1995.mmi_18030401.x. [DOI] [PubMed] [Google Scholar]
  22. Turnbull A. P., Rafferty J. B., Sedelnikova S. E., Slabas A. R., Schierer T. P., Kroon J. T., Simon J. W., Fawcett T., Nishida I., Murata N. Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase. Structure. 2001 May 9;9(5):347–353. doi: 10.1016/s0969-2126(01)00595-0. [DOI] [PubMed] [Google Scholar]
  23. Verwoert I. I., Brown A., Slabas A. R., Stuitje A. R. A Zea mays GTP-binding protein of the ARF family complements an Escherichia coli mutant with a temperature-sensitive malonyl-coenzyme A:acyl carrier protein transacylase. Plant Mol Biol. 1995 Feb;27(3):629–633. doi: 10.1007/BF00019329. [DOI] [PubMed] [Google Scholar]
  24. Weigert R., Silletta M. G., Spanò S., Turacchio G., Cericola C., Colanzi A., Senatore S., Mancini R., Polishchuk E. V., Salmona M. CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid. Nature. 1999 Nov 25;402(6760):429–433. doi: 10.1038/46587. [DOI] [PubMed] [Google Scholar]
  25. West J., Tompkins C. K., Balantac N., Nudelman E., Meengs B., White T., Bursten S., Coleman J., Kumar A., Singer J. W. Cloning and expression of two human lysophosphatidic acid acyltransferase cDNAs that enhance cytokine-induced signaling responses in cells. DNA Cell Biol. 1997 Jun;16(6):691–701. doi: 10.1089/dna.1997.16.691. [DOI] [PubMed] [Google Scholar]

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