Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 Apr 1;307(Pt 1):115–121. doi: 10.1042/bj3070115

Temperature regulation of the Tetrahymena mimbres glycosylphosphatidylinositol-anchored protein lipid composition.

Y G Ko 1, C Y Hung 1, G A Thompson Jr 1
PMCID: PMC1136752  PMID: 7717965

Abstract

By incubating Tetrahymena mimbres cells with [3H]myristic acid, [3H]ethanolamine, [3H]inositol, and [3H]mannose, proteins having apparent molecular masses of 23 and 63 kDa were identified as the cells' principal glycosylphosphatidylinositol (GPI)-anchored proteins. These proteins accounted for as much as 2-5% of the whole cell proteins, with the higher levels being recovered from non-growing cells. The two proteins, gpi 23 and gpi 63, were purified to near homogeneity through Triton X-114/water partitioning followed by preparative SDS/PAGE. The lipid components of the GPI anchors were determined by chemical and enzymic hydrolysis. Both proteins were anchored by ceramides, with the principal long-chain base being C18 sphinganine containing an O-methyl group at the 3 position. O-Methylation was shown not to be an artifact of hydrolysis. When T. mimbres was cultured at 15 degrees C, the ceramide fatty acid component of the GPI anchors was principally palmitic acid (75% in gpi 23 and 76% in gpi 63). GPI anchors from 28 degrees C-grown cells contained mainly stearic acid (79% in gpi 23 and 70% in gpi 63). Temperature change had little effect on the long-chain-base composition. The direction of temperature-induced lipid change in the protein-bound anchors was the same as found in the inositolphosphorylceramide putative precursors of the protein anchors described in the accompanying paper [Hung, Ko and Thompson (1995) Biochem. J. 307, 107-113], but the detailed fatty acid compositions of the precursors and the protein-bound lipids were quite different. The precise metabolic regulation of anchor lipid chain length supports the concept that composition of the lipid anchor is important in the function and/or metabolism of the anchored protein.

Full text

PDF
115

Images in this article

118Figure 3
on p.118
119Figure 4
on p.119

Selected References

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

  1. BATTAGLIA F. C., MESCHIA G., BARRON D. H. A method for the determination of free myo-inositol in biological fluids and tissues. Biochim Biophys Acta. 1960 Nov 4;44:354–355. doi: 10.1016/0006-3002(60)91573-0. [DOI] [PubMed] [Google Scholar]
  2. Brown D. A. Interactions between GPI-anchored proteins and membrane lipids. Trends Cell Biol. 1992 Nov;2(11):338–343. [PubMed] [Google Scholar]
  3. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  4. Doerder F. P., Berkowitz M. S. Purification and partial characterization of the H immobilization antigens of Tetrahymena thermophila. J Protozool. 1986 May;33(2):204–208. doi: 10.1111/j.1550-7408.1986.tb05590.x. [DOI] [PubMed] [Google Scholar]
  5. Fankhauser C., Homans S. W., Thomas-Oates J. E., McConville M. J., Desponds C., Conzelmann A., Ferguson M. A. Structures of glycosylphosphatidylinositol membrane anchors from Saccharomyces cerevisiae. J Biol Chem. 1993 Dec 15;268(35):26365–26374. [PubMed] [Google Scholar]
  6. Haynes P. A., Gooley A. A., Ferguson M. A., Redmond J. W., Williams K. L. Post-translational modifications of the Dictyostelium discoideum glycoprotein PsA. Glycosylphosphatidylinositol membrane anchor and composition of O-linked oligosaccharides. Eur J Biochem. 1993 Sep 15;216(3):729–737. doi: 10.1111/j.1432-1033.1993.tb18192.x. [DOI] [PubMed] [Google Scholar]
  7. Hooper N. M., Bashir A. Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114. Biochem J. 1991 Dec 15;280(Pt 3):745–751. doi: 10.1042/bj2800745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hung C. Y., Ko Y. G., Thompson G. A., Jr Temperature-induced alteration of inositolphosphorylceramides in the putative glycosylated lipid precursors of Tetrahymena mimbres glycosylphosphatidylinositol-anchored proteins. Biochem J. 1995 Apr 1;307(Pt 1):107–113. doi: 10.1042/bj3070107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ko Y. G., Thompson G. A., Jr Immobilization antigens from Tetrahymena thermophila are glycosyl-phosphatidylinositol-linked proteins. J Protozool. 1992 Nov-Dec;39(6):719–723. doi: 10.1111/j.1550-7408.1992.tb04454.x. [DOI] [PubMed] [Google Scholar]
  10. Ko Y. G., Thompson G. A., Jr Purification of glycosylphosphatidylinositol-anchored proteins by modified triton X-114 partitioning and preparative gel electrophoresis. Anal Biochem. 1995 Jan 1;224(1):166–172. doi: 10.1006/abio.1995.1024. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Low M. G. The glycosyl-phosphatidylinositol anchor of membrane proteins. Biochim Biophys Acta. 1989 Dec 6;988(3):427–454. doi: 10.1016/0304-4157(89)90014-2. [DOI] [PubMed] [Google Scholar]
  13. McConville M. J., Ferguson M. A. The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem J. 1993 Sep 1;294(Pt 2):305–324. doi: 10.1042/bj2940305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pak Y., Ryals P. E., Thompson G. A., Jr Phosphatidylinositol glycan formation and utilization by the ciliate Tetrahymena mimbres. J Biol Chem. 1991 Aug 15;266(23):15054–15059. [PubMed] [Google Scholar]
  15. Peitzsch R. M., McLaughlin S. Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins. Biochemistry. 1993 Oct 5;32(39):10436–10443. doi: 10.1021/bi00090a020. [DOI] [PubMed] [Google Scholar]
  16. Ryals P. E., Thompson G. A., Jr Protein acylation in Tetrahymena. Arch Biochem Biophys. 1988 Nov 1;266(2):408–415. doi: 10.1016/0003-9861(88)90272-x. [DOI] [PubMed] [Google Scholar]
  17. Silvius J. R., Zuckermann M. J. Interbilayer transfer of phospholipid-anchored macromolecules via monomer diffusion. Biochemistry. 1993 Mar 30;32(12):3153–3161. doi: 10.1021/bi00063a030. [DOI] [PubMed] [Google Scholar]
  18. Smith D. L., Berkowitz M. S., Potoczak D., Krause M., Raab C., Quinn F., Doerder F. P. Characterization of the T, L, I, S, M and P cell surface (immobilization) antigens of Tetrahymena thermophila: molecular weights, isoforms, and cross-reactivity of antisera. J Protozool. 1992 May-Jun;39(3):420–428. doi: 10.1111/j.1550-7408.1992.tb01475.x. [DOI] [PubMed] [Google Scholar]
  19. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  20. Thompson G. A., Jr Studies of membrane formation in Tetrahymena pyriformis. I. Rates of phospholipid biosynthesis. Biochemistry. 1967 Jul;6(7):2015–2022. doi: 10.1021/bi00859a020. [DOI] [PubMed] [Google Scholar]
  21. Weinhart U., Thomas J. R., Pak Y. B., Thompson G. A., Jr, Ferguson M. A. Structural characterization of a novel glycosyl-phosphatidylinositol from the protozoan Tetrahymena mimbres. Biochem J. 1991 Oct 15;279(Pt 2):605–608. doi: 10.1042/bj2790605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wolfe J., Mpoke S., Tirone S. F. Cilia, ciliary concanavalin A-binding proteins, and mating recognition in Tetrahymena thermophila. Exp Cell Res. 1993 Dec;209(2):342–349. doi: 10.1006/excr.1993.1319. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES