Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1986 Mar;165(3):901–910. doi: 10.1128/jb.165.3.901-910.1986

Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae.

K Kuchler, G Daum, F Paltauf
PMCID: PMC214514  PMID: 3005242

Abstract

Using highly enriched membrane preparations from lactate-grown Saccharomyces cerevisiae cells, the subcellular and submitochondrial location of eight enzymes involved in the biosynthesis of phospholipids was determined. Phosphatidylserine decarboxylase and phosphatidylglycerolphosphate synthase were localized exclusively in the inner mitochondrial membrane, while phosphatidylethanolamine methyltransferase activity was confined to microsomal fractions. The other five enzymes tested in this study were common both to the outer mitochondrial membrane and to microsomes. The transmembrane orientation of the mitochondrial enzymes was investigated by protease digestion of intact mitochondria and of outside-out sealed vesicles of the outer mitochondrial membrane. Glycerolphosphate acyltransferase, phosphatidylinositol synthase, and phosphatidylserine synthase were exposed at the cytosolic surface of the outer mitochondrial membrane. Cholinephosphotransferase was apparently located at the inner aspect or within the outer mitochondrial membrane. Phosphatidate cytidylyltransferase was localized in the endoplasmic reticulum, on the cytoplasmic side of the outer mitochondrial membrane, and in the inner mitochondrial membrane. Inner membrane activity of this enzyme constituted 80% of total mitochondrial activity; inactivation by trypsin digestion was observed only after preincubation of membranes with detergent (0.1% Triton X-100). Total activity of those enzymes that are common to mitochondria and the endoplasmic reticulum was about equally distributed between the two organelles. Data concerning susceptibility to various inhibitors, heat sensitivity, and the pH optima indicate that there is a close similarity of the mitochondrial and microsomal enzymes that catalyze the same reaction.

Full text

PDF
901

Images in this article

904Image
on p.904

Selected References

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

  1. Ackrell B. A., Kearney E. B., Singer T. P. Mammalian succinate dehydrogenase. Methods Enzymol. 1978;53:466–483. doi: 10.1016/s0076-6879(78)53050-4. [DOI] [PubMed] [Google Scholar]
  2. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  3. Barańska J. Phosphatidylserine biosynthesis in mitochondria from Ehrlich ascites tumor cells. Biochim Biophys Acta. 1980 Aug 11;619(2):258–266. doi: 10.1016/0005-2760(80)90074-0. [DOI] [PubMed] [Google Scholar]
  4. Belendiuk G., Mangnall D., Tung B., Westley J., Getz G. S. CTP-phosphatidic acid cytidyltransferase from Saccharomyces cerevisiae. Partial purification, characterization, and kinetic behavior. J Biol Chem. 1978 Jul 10;253(13):4555–4565. [PubMed] [Google Scholar]
  5. Carman G. M., Matas J. Solubilization of microsomal-associated phosphatidylserine synthase and phosphatidylinositol synthase from Saccharomyces cerevisiae. Can J Microbiol. 1981 Nov;27(11):1140–1149. doi: 10.1139/m81-179. [DOI] [PubMed] [Google Scholar]
  6. Carroll M. A., Morris P. E., Grosjean C. D., Anzalone T., Haldar D. Further distinguishing properties of mitochondrial and microsomal glycerophosphate acyltransferase and the transmembrane location of the mitochondrial enzyme. Arch Biochem Biophys. 1982 Mar;214(1):17–25. doi: 10.1016/0003-9861(82)90003-0. [DOI] [PubMed] [Google Scholar]
  7. Christiansen K. Triacylglycerol synthesis in lipid particles from baker's yeast (Saccharomyces cerevisiae). Biochim Biophys Acta. 1978 Jul 25;530(1):78–90. doi: 10.1016/0005-2760(78)90128-5. [DOI] [PubMed] [Google Scholar]
  8. Christiansen K. Utilization of endogenous diacylglycerol for the synthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine by lipid particles from baker's yeast (Saccharomyces cerevisiae). Biochim Biophys Acta. 1979 Sep 28;574(3):448–460. doi: 10.1016/0005-2760(79)90241-8. [DOI] [PubMed] [Google Scholar]
  9. Cobon G. S., Crowfoot P. D., Linnane A. W. Biogenesis of mitchondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions. Biochem J. 1974 Nov;144(2):265–275. doi: 10.1042/bj1440265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Daum G., Böhni P. C., Schatz G. Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem. 1982 Nov 10;257(21):13028–13033. [PubMed] [Google Scholar]
  11. Daum G. Lipids of mitochondria. Biochim Biophys Acta. 1985 Jun 12;822(1):1–42. doi: 10.1016/0304-4157(85)90002-4. [DOI] [PubMed] [Google Scholar]
  12. Dennis E. A., Kennedy E. P. Intracellular sites of lipid synthesis and the biogenesis of mitochondria. J Lipid Res. 1972 Mar;13(2):263–267. [PubMed] [Google Scholar]
  13. Haid A., Suissa M. Immunochemical identification of membrane proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methods Enzymol. 1983;96:192–205. doi: 10.1016/s0076-6879(83)96017-2. [DOI] [PubMed] [Google Scholar]
  14. Hosaka K., Yamashita S. Choline transport in Saccharomyces cerevisiae. J Bacteriol. 1980 Jul;143(1):176–181. doi: 10.1128/jb.143.1.176-181.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hostetler K. Y., Zenner B. D., Morris H. P. Altered subcellular and submitochondrial localization of CTP:phosphatidate cytidylyltransferase in the Morris 7777 hepatoma. J Lipid Res. 1978 Jul;19(5):553–560. [PubMed] [Google Scholar]
  16. Hostetler K. Y., Zenner B. D., Morris H. P. Phosphatidylserine biosynthesis in mitochondria from the Morris 7777 hepatoma. J Lipid Res. 1979 Jul;20(5):607–613. [PubMed] [Google Scholar]
  17. Hostetler K. Y., van den Bosch H. Subcellular and submitochondrial localization of the biosynthesis of cardiolipin and related phospholipids in rat liver. Biochim Biophys Acta. 1972 Mar 23;260(3):380–386. doi: 10.1016/0005-2760(72)90052-5. [DOI] [PubMed] [Google Scholar]
  18. Jelsema C. L., Morré D. J. Distribution of phospholipid biosynthetic enzymes among cell components of rat liver. J Biol Chem. 1978 Nov 10;253(21):7960–7971. [PubMed] [Google Scholar]
  19. Johnston J. M., Paltauf F. Lipid metabolism in inositol-deficient yeast, Saccharomyces carlsbergensis. II. Incorporation of labeled precursors into lipids by whole cells and activities of some enzymes involved in lipid formation. Biochim Biophys Acta. 1970 Dec 15;218(3):431–440. [PubMed] [Google Scholar]
  20. Kanfer J. N. The base exchange enzymes and phospholipase D of mammalian tissue. Can J Biochem. 1980 Dec;58(12):1370–1380. doi: 10.1139/o80-186. [DOI] [PubMed] [Google Scholar]
  21. Kanoh H., Ohno K. Solubilization and purification of rat liver microsomal 1,2-diacylglycerol: CDP-choline cholinephosphotransferase and 1,2-diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase. Eur J Biochem. 1976 Jun 15;66(1):201–210. doi: 10.1111/j.1432-1033.1976.tb10440.x. [DOI] [PubMed] [Google Scholar]
  22. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  23. LaBelle E. F., Jr, Hajra A. K. Biosynthesis of acyl dihydroxyacetone phosphate in subcellular fractions of rat liver. J Biol Chem. 1972 Sep 25;247(18):5835–5841. [PubMed] [Google Scholar]
  24. Labeyrie F., Baudras A., Lederer F. Flavocytochrome b 2 or L-lactate cytochrome c reductase from yeast. Methods Enzymol. 1978;53:238–256. doi: 10.1016/s0076-6879(78)53030-9. [DOI] [PubMed] [Google Scholar]
  25. Lehle L., Tanner W. Formation of lipid-bound oligosaccharides in yeast. Biochim Biophys Acta. 1975 Aug 13;399(2):364–374. doi: 10.1016/0304-4165(75)90265-2. [DOI] [PubMed] [Google Scholar]
  26. Lord J. M., Kagawa T., Moore T. S., Beevers H. Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm. J Cell Biol. 1973 Jun;57(3):659–667. doi: 10.1083/jcb.57.3.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moore T. S., Lord J. M., Kagawa T., Beevers H. Enzymes of phospholipid metabolism in the endoplasmic reticulum of castor bean endosperm. Plant Physiol. 1973 Jul;52(1):50–53. doi: 10.1104/pp.52.1.50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. OGUR M., ROSEN G. The nucleic acids of plant tissues; the extraction and estimation of desoxypentose nucleic acid and pentose nucleic acid. Arch Biochem. 1950 Feb;25(2):262–276. [PubMed] [Google Scholar]
  29. Percy A. K., Moore J. F., Carson M. A., Waechter C. J. Characterization of brain phosphatidylserine decarboxylase: localization in the mitochondrial inner membrane. Arch Biochem Biophys. 1983 Jun;223(2):484–494. doi: 10.1016/0003-9861(83)90613-6. [DOI] [PubMed] [Google Scholar]
  30. RACKER E. Spectrophotometric measurements of the enzymatic formation of fumaric and cis-aconitic acids. Biochim Biophys Acta. 1950 Jan;4(1-3):211–214. doi: 10.1016/0006-3002(50)90026-6. [DOI] [PubMed] [Google Scholar]
  31. Riezman H., Hay R., Gasser S., Daum G., Schneider G., Witte C., Schatz G. The outer membrane of yeast mitochondria: isolation of outside-out sealed vesicles. EMBO J. 1983;2(7):1105–1111. doi: 10.1002/j.1460-2075.1983.tb01553.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Robinson M. L., Carman G. M. Solubilization of microsomal-associated phosphatidylinositol synthase from germinating soybeans. Plant Physiol. 1982 Jan;69(1):146–149. doi: 10.1104/pp.69.1.146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. SCHATZ G., KLIMA J. TRIPHOSPHOPYRIDINE NUCLEOTIDE: CYTOCHROME C REDUCTASE OF SACCHAROMYCES CEREVISIAE: A "MICROSOMAL" ENZYME. Biochim Biophys Acta. 1964 Mar 9;81:448–461. doi: 10.1016/0926-6569(64)90130-0. [DOI] [PubMed] [Google Scholar]
  34. Schlossman D. M., Bell R. M. Glycerolipid biosynthesis in Saccharomyces cerevisiae: sn-glycerol-3-phosphate and dihydroxyacetone phosphate acyltransferase activities. J Bacteriol. 1978 Mar;133(3):1368–1376. doi: 10.1128/jb.133.3.1368-1376.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schnaitman C., Greenawalt J. W. Enzymatic properties of the inner and outer membranes of rat liver mitochondria. J Cell Biol. 1968 Jul;38(1):158–175. doi: 10.1083/jcb.38.1.158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sparace S. A., Moore T. S. Phospholipid metabolism in plant mitochondria: submitochondrial sites of synthesis. Plant Physiol. 1979 May;63(5):963–972. doi: 10.1104/pp.63.5.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Steiner M. R., Lester R. L. In vitro studies of phospholipid biosynthesis in Saccharomyces cerevisiae. Biochim Biophys Acta. 1972 Feb 21;260(2):222–243. doi: 10.1016/0005-2760(72)90035-5. [DOI] [PubMed] [Google Scholar]
  38. Yamada K., Okuyama H., Endo Y., Ikezawa H. Acyltransferase systems involved in phospholipid metabolism in Saccharomyces cerevisiae. Arch Biochem Biophys. 1977 Sep;183(1):281–289. doi: 10.1016/0003-9861(77)90441-6. [DOI] [PubMed] [Google Scholar]
  39. van Golde L. M., Raben J., Batenburg J. J., Fleischer B., Zambrano F., Fleischer S. Biosynthesis of lipids in Golgi complex and other subcellular fractions from rat liver. Biochim Biophys Acta. 1974 Aug 22;360(2):179–192. doi: 10.1016/0005-2760(74)90168-4. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES