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. 1991 Jul;173(14):4325–4332. doi: 10.1128/jb.173.14.4325-4332.1991

Cloning and characterization of LCB1, a Saccharomyces gene required for biosynthesis of the long-chain base component of sphingolipids.

R Buede 1, C Rinker-Schaffer 1, W J Pinto 1, R L Lester 1, R C Dickson 1
PMCID: PMC208092  PMID: 2066332

Abstract

The existence of auxotrophic mutants of Saccharomyces cerevisiae having an absolute requirement for the long-chain base (lcb) component of sphingolipids suggests that sphingolipids are crucial for viability and growth. One mutant, termed the lcb1-1 mutant, lacks the activity of serine palmitoyltransferase, the first enzyme in the pathway for long-chain base synthesis. Here, we present evidence that LCB1 has been molecularly cloned. The size of the LCB1 transcript, the direction of transcription, and transcription initiation sites were determined. In addition, the coding region and its 5' and 3' flanking regions were sequenced. Analysis of the DNA sequence revealed a single open reading frame of 1,674 nucleotides, encoding a predicted peptide of 558 amino acids. The hydropathy profile of the predicted peptide suggests a hydrophobic, globular, membrane-associated protein with two potential transmembrane helices. Comparison of the predicted amino acid sequence to known protein sequences revealed homology to 5-aminolevulinic acid synthase and to 2-amino-3-ketobutyrate coenzyme A ligase. These homologies, the similarity of the chemical reactions catalyzed by the three enzymes, and the finding that LCB1 restores serine palmitoyltransferase activity to an lcb1-defective strain indicate that serine palmitoyltransferase or a subunit of the enzyme is the most likely product of LCB1. Homology of the LCB1 predicted protein to the Escherichia coli biotin synthetase was also observed, but the biological significance of this observation is not clear. A role for sphingolipids in sporulation is implicated by our finding that diploids homozygous for lcb1 failed to sporulate.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aronson B. D., Ravnikar P. D., Somerville R. L. Nucleotide sequence of the 2-amino-3-ketobutyrate coenzyme A ligase (kbl) gene of E. coli. Nucleic Acids Res. 1988 Apr 25;16(8):3586–3586. doi: 10.1093/nar/16.8.3586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bawden M. J., Borthwick I. A., Healy H. M., Morris C. P., May B. K., Elliott W. H. Sequence of human 5-aminolevulinate synthase cDNA. Nucleic Acids Res. 1987 Oct 26;15(20):8563–8563. doi: 10.1093/nar/15.20.8563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borthwick I. A., Srivastava G., Day A. R., Pirola B. A., Snoswell M. A., May B. K., Elliott W. H. Complete nucleotide sequence of hepatic 5-aminolaevulinate synthase precursor. Eur J Biochem. 1985 Aug 1;150(3):481–484. doi: 10.1111/j.1432-1033.1985.tb09047.x. [DOI] [PubMed] [Google Scholar]
  4. Brennan P. J., Lösel D. M. Physiology of fungal lipids: selected topics. Adv Microb Physiol. 1978;17:47–179. doi: 10.1016/s0065-2911(08)60057-0. [DOI] [PubMed] [Google Scholar]
  5. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  6. Chang Y. D., Dickson R. C. Primary structure of the lactose permease gene from the yeast Kluyveromyces lactis. Presence of an unusual transcript structure. J Biol Chem. 1988 Nov 15;263(32):16696–16703. [PubMed] [Google Scholar]
  7. Dale R. A. Catabolism of threonine in mammals by coupling of L-threonine 3-dehydrogenase with 2-amino-3-oxobutyrate-CoA ligase. Biochim Biophys Acta. 1978 Dec 18;544(3):496–503. doi: 10.1016/0304-4165(78)90324-0. [DOI] [PubMed] [Google Scholar]
  8. Dickson R. C., Wells G. B., Schmidt A., Lester R. L. Isolation of mutant Saccharomyces cerevisiae strains that survive without sphingolipids. Mol Cell Biol. 1990 May;10(5):2176–2181. doi: 10.1128/mcb.10.5.2176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eisenberg D., Schwarz E., Komaromy M., Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984 Oct 15;179(1):125–142. doi: 10.1016/0022-2836(84)90309-7. [DOI] [PubMed] [Google Scholar]
  10. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  11. Hakomori S. Aberrant glycosylation in tumors and tumor-associated carbohydrate antigens. Adv Cancer Res. 1989;52:257–331. doi: 10.1016/s0065-230x(08)60215-8. [DOI] [PubMed] [Google Scholar]
  12. Hannun Y. A., Bell R. M. Functions of sphingolipids and sphingolipid breakdown products in cellular regulation. Science. 1989 Jan 27;243(4890):500–507. doi: 10.1126/science.2643164. [DOI] [PubMed] [Google Scholar]
  13. Komatsubara S., Murata K., Kisumi M., Chibata I. Threonine degradation by Serratia marcescens. J Bacteriol. 1978 Aug;135(2):318–323. doi: 10.1128/jb.135.2.318-323.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Lev M., Milford A. F. The 3-ketodihydrosphingosine synthetase of Bacteroides melaninogenicus: induction by vitamin K. Arch Biochem Biophys. 1973 Aug;157(2):500–508. doi: 10.1016/0003-9861(73)90668-1. [DOI] [PubMed] [Google Scholar]
  16. Maguire D. J., Day A. R., Borthwick I. A., Srivastava G., Wigley P. L., May B. K., Elliott W. H. Nucleotide sequence of the chicken 5-aminolevulinate synthase gene. Nucleic Acids Res. 1986 Feb 11;14(3):1379–1391. doi: 10.1093/nar/14.3.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marshall R. D. Glycoproteins. Annu Rev Biochem. 1972;41:673–702. doi: 10.1146/annurev.bi.41.070172.003325. [DOI] [PubMed] [Google Scholar]
  18. McClung C. R., Somerville J. E., Guerinot M. L., Chelm B. K. Structure of the Bradyrhizobium japonicum gene hemA encoding 5-aminolevulinic acid synthase. Gene. 1987;54(1):133–139. doi: 10.1016/0378-1119(87)90355-6. [DOI] [PubMed] [Google Scholar]
  19. Merrill A. H., Jr, Nixon D. W., Williams R. D. Activities of serine palmitoyltransferase (3-ketosphinganine synthase) in microsomes from different rat tissues. J Lipid Res. 1985 May;26(5):617–622. [PubMed] [Google Scholar]
  20. Mukherjee J. J., Dekker E. E. Purification, properties, and N-terminal amino acid sequence of homogeneous Escherichia coli 2-amino-3-ketobutyrate CoA ligase, a pyridoxal phosphate-dependent enzyme. J Biol Chem. 1987 Oct 25;262(30):14441–14447. [PubMed] [Google Scholar]
  21. Otsuka A. J., Buoncristiani M. R., Howard P. K., Flamm J., Johnson C., Yamamoto R., Uchida K., Cook C., Ruppert J., Matsuzaki J. The Escherichia coli biotin biosynthetic enzyme sequences predicted from the nucleotide sequence of the bio operon. J Biol Chem. 1988 Dec 25;263(36):19577–19585. [PubMed] [Google Scholar]
  22. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Petko L., Lindquist S. Hsp26 is not required for growth at high temperatures, nor for thermotolerance, spore development, or germination. Cell. 1986 Jun 20;45(6):885–894. doi: 10.1016/0092-8674(86)90563-5. [DOI] [PubMed] [Google Scholar]
  24. Riddle R. D., Yamamoto M., Engel J. D. Expression of delta-aminolevulinate synthase in avian cells: separate genes encode erythroid-specific and nonspecific isozymes. Proc Natl Acad Sci U S A. 1989 Feb;86(3):792–796. doi: 10.1073/pnas.86.3.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rose M. D. Isolation of genes by complementation in yeast. Methods Enzymol. 1987;152:481–504. doi: 10.1016/0076-6879(87)52056-0. [DOI] [PubMed] [Google Scholar]
  26. Schoenhaut D. S., Curtis P. J. Nucleotide sequence of mouse 5-aminolevulinic acid synthase cDNA and expression of its gene in hepatic and erythroid tissues. Gene. 1986;48(1):55–63. doi: 10.1016/0378-1119(86)90351-3. [DOI] [PubMed] [Google Scholar]
  27. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Smith S. W., Lester R. L. Inositol phosphorylceramide, a novel substance and the chief member of a major group of yeast sphingolipids containing a single inositol phosphate. J Biol Chem. 1974 Jun 10;249(11):3395–3405. [PubMed] [Google Scholar]
  29. Snell E. E., Dimari S. J., Brady R. N. Biosynthesis of sphingosine and dihydrosphingosine by cell-free systems from Hansenula ciferri. Chem Phys Lipids. 1970 Oct;5(1):116–138. doi: 10.1016/0009-3084(70)90013-7. [DOI] [PubMed] [Google Scholar]
  30. Srivastava G., Borthwick I. A., Maguire D. J., Elferink C. J., Bawden M. J., Mercer J. F., May B. K. Regulation of 5-aminolevulinate synthase mRNA in different rat tissues. J Biol Chem. 1988 Apr 15;263(11):5202–5209. [PubMed] [Google Scholar]
  31. Steiner S., Smith S., Waechter C. J., Lester R. L. Isolation and partial characterization of a major inositol-containing lipid in baker's yeast, mannosyl-diinositol, diphosphoryl-ceramide. Proc Natl Acad Sci U S A. 1969 Nov;64(3):1042–1048. doi: 10.1073/pnas.64.3.1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stoffel W. Studies on the biosynthesis and degradation of sphingosine bases. Chem Phys Lipids. 1970 Oct;5(1):139–158. doi: 10.1016/0009-3084(70)90014-9. [DOI] [PubMed] [Google Scholar]
  33. Urban-Grimal D., Volland C., Garnier T., Dehoux P., Labbe-Bois R. The nucleotide sequence of the HEM1 gene and evidence for a precursor form of the mitochondrial 5-aminolevulinate synthase in Saccharomyces cerevisiae. Eur J Biochem. 1986 May 2;156(3):511–519. doi: 10.1111/j.1432-1033.1986.tb09610.x. [DOI] [PubMed] [Google Scholar]
  34. Wells G. B., Lester R. L. The isolation and characterization of a mutant strain of Saccharomyces cerevisiae that requires a long chain base for growth and for synthesis of phosphosphingolipids. J Biol Chem. 1983 Sep 10;258(17):10200–10203. [PubMed] [Google Scholar]
  35. Williams R. D., Wang E., Merrill A. H., Jr Enzymology of long-chain base synthesis by liver: characterization of serine palmitoyltransferase in rat liver microsomes. Arch Biochem Biophys. 1984 Jan;228(1):282–291. doi: 10.1016/0003-9861(84)90069-9. [DOI] [PubMed] [Google Scholar]
  36. Wray L. V., Jr, Witte M. M., Dickson R. C., Riley M. I. Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Mar;7(3):1111–1121. doi: 10.1128/mcb.7.3.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yamamoto M., Kure S., Engel J. D., Hiraga K. Structure, turnover, and heme-mediated suppression of the level of mRNA encoding rat liver delta-aminolevulinate synthase. J Biol Chem. 1988 Nov 5;263(31):15973–15979. [PubMed] [Google Scholar]

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