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. 1989 Apr 1;108(4):1353–1361. doi: 10.1083/jcb.108.4.1353

Subcellular localization of sterol carrier protein-2 in rat hepatocytes: its primary localization to peroxisomes

PMCID: PMC2115497  PMID: 2925789

Abstract

Sterol carrier protein-2 (SCP-2) is a nonenzymatic protein of 13.5 kD which has been shown in in vitro experiments to be required for several stages in cholesterol utilization and biosynthesis. The subcellular localization of SCP-2 has not been definitively established. Using affinity-purified rabbit polyclonal antibodies against electrophoretically pure SCP-2 from rat liver, we demonstrate by immunoelectron microscopic labeling of ultrathin frozen sections of rat liver that the largest concentration of SCP-2 is inside peroxisomes. In addition the immunolabeling indicates that there are significant concentrations of SCP-2 inside mitochondria, and associated with the endoplasmic reticulum and the cytosol, but not inside the Golgi apparatus, lysosomes, or the nucleus. These results were confirmed by immunoblotting experiments with proteins from purified subcellular fractions of the rat liver cells carried out with the anti-SCP-2 antibodies. The large concentration of SCP-2 inside peroxisomes strongly supports the proposal that peroxisomes are critical sites of cholesterol utilization and biosynthesis. The presence of SCP-2 inside peroxisomes and mitochondria raises questions about the mechanisms involved in the differential targeting of SCP-2 to these organelles.

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Selected References

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  1. Beaufay H., Amar-Costesec A., Feytmans E., Thinès-Sempoux D., Wibo M., Robbi M., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods. J Cell Biol. 1974 Apr;61(1):188–200. doi: 10.1083/jcb.61.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chanderbhan R., Noland B. J., Scallen T. J., Vahouny G. V. Sterol carrier protein2. Delivery of cholesterol from adrenal lipid droplets to mitochondria for pregnenolone synthesis. J Biol Chem. 1982 Aug 10;257(15):8928–8934. [PubMed] [Google Scholar]
  3. Chanderbhan R., Tanaka T., Strauss J. F., Irwin D., Noland B. J., Scallen T. J., Vahouny G. V. Evidence for sterol carrier protein2-like activity in hepatic, adrenal and ovarian cytosol. Biochem Biophys Res Commun. 1983 Dec 28;117(3):702–709. doi: 10.1016/0006-291x(83)91654-6. [DOI] [PubMed] [Google Scholar]
  4. DE DUVE C., PRESSMAN B. C., GIANETTO R., WATTIAUX R., APPELMANS F. Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J. 1955 Aug;60(4):604–617. doi: 10.1042/bj0600604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fujiki Y., Fowler S., Shio H., Hubbard A. L., Lazarow P. B. Polypeptide and phospholipid composition of the membrane of rat liver peroxisomes: comparison with endoplasmic reticulum and mitochondrial membranes. J Cell Biol. 1982 Apr;93(1):103–110. doi: 10.1083/jcb.93.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gavey K. L., Noland B. J., Scallen T. J. The participation of sterol carrier protein2 in the conversion of cholesterol to cholesterol ester by rat liver microsomes. J Biol Chem. 1981 Mar 25;256(6):2993–2999. [PubMed] [Google Scholar]
  7. Gould S. G., Keller G. A., Subramani S. Identification of a peroxisomal targeting signal at the carboxy terminus of firefly luciferase. J Cell Biol. 1987 Dec;105(6 Pt 2):2923–2931. doi: 10.1083/jcb.105.6.2923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hagey L. R., Krisans S. K. Degradation of cholesterol to propionic acid by rat liver peroxisomes. Biochem Biophys Res Commun. 1982 Aug;107(3):834–841. doi: 10.1016/0006-291x(82)90598-8. [DOI] [PubMed] [Google Scholar]
  9. Hajra A. K., Bishop J. E. Glycerolipid biosynthesis in peroxisomes via the acyl dihydroxyacetone phosphate pathway. Ann N Y Acad Sci. 1982;386:170–182. doi: 10.1111/j.1749-6632.1982.tb21415.x. [DOI] [PubMed] [Google Scholar]
  10. Hay R., Böhni P., Gasser S. How mitochondria import proteins. Biochim Biophys Acta. 1984 Jan 27;779(1):65–87. doi: 10.1016/0304-4157(84)90004-2. [DOI] [PubMed] [Google Scholar]
  11. Kase B. F., Björkhem I., Hågå P., Pedersen J. I. Defective peroxisomal cleavage of the C27-steroid side chain in the cerebro-hepato-renal syndrome of Zellweger. J Clin Invest. 1985 Feb;75(2):427–435. doi: 10.1172/JCI111717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kase F., Björkhem I., Pedersen J. I. Formation of cholic acid from 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestanoic acid by rat liver peroxisomes. J Lipid Res. 1983 Dec;24(12):1560–1567. [PubMed] [Google Scholar]
  13. Keller G. A., Barton M. C., Shapiro D. J., Singer S. J. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase is present in peroxisomes in normal rat liver cells. Proc Natl Acad Sci U S A. 1985 Feb;82(3):770–774. doi: 10.1073/pnas.82.3.770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Keller G. A., Gould S., Deluca M., Subramani S. Firefly luciferase is targeted to peroxisomes in mammalian cells. Proc Natl Acad Sci U S A. 1987 May;84(10):3264–3268. doi: 10.1073/pnas.84.10.3264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Keller G. A., Pazirandeh M., Krisans S. 3-Hydroxy-3-methylglutaryl coenzyme A reductase localization in rat liver peroxisomes and microsomes of control and cholestyramine-treated animals: quantitative biochemical and immunoelectron microscopical analyses. J Cell Biol. 1986 Sep;103(3):875–886. doi: 10.1083/jcb.103.3.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Keller G. A., Tokuyasu K. T., Dutton A. H., Singer S. J. An improved procedure for immunoelectron microscopy: ultrathin plastic embedding of immunolabeled ultrathin frozen sections. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5744–5747. doi: 10.1073/pnas.81.18.5744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Krisans S. K., Thompson S. L., Pena L. A., Kok E., Javitt N. B. Bile acid synthesis in rat liver peroxisomes: metabolism of 26-hydroxycholesterol to 3 beta-hydroxy-5-cholenoic acid. J Lipid Res. 1985 Nov;26(11):1324–1332. [PubMed] [Google Scholar]
  18. 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]
  19. Lazarow P. B., de Duve C. The synthesis and turnover of rat liver peroxisomes. V. Intracellular pathway of catalase synthesis. J Cell Biol. 1973 Nov;59(2 Pt 1):507–524. doi: 10.1083/jcb.59.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Leighton F., Poole B., Beaufay H., Baudhuin P., Coffey J. W., Fowler S., De Duve C. The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with triton WR-1339. Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions. J Cell Biol. 1968 May;37(2):482–513. doi: 10.1083/jcb.37.2.482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lidström-Olsson B., Wikvall K. The role of sterol carrier protein2 and other hepatic lipid-binding proteins in bile-acid biosynthesis. Biochem J. 1986 Sep 15;238(3):879–884. doi: 10.1042/bj2380879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Morris H. R., Larsen B. S., Billheimer J. T. A mass spectrometric study of the structure of sterol carrier protein SCP2 from rat liver. Biochem Biophys Res Commun. 1988 Jul 15;154(1):476–482. doi: 10.1016/0006-291x(88)90711-5. [DOI] [PubMed] [Google Scholar]
  23. Morton R. E., Zilversmit D. B. Purification and characterization of lipid transfer protein(s) from human lipoprotein-deficient plasma. J Lipid Res. 1982 Sep;23(7):1058–1067. [PubMed] [Google Scholar]
  24. Noland B. J., Arebalo R. E., Hansbury E., Scallen T. J. Purification and properties of sterol carrier protein2. J Biol Chem. 1980 May 10;255(9):4282–4289. [PubMed] [Google Scholar]
  25. Olmsted J. B. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem. 1981 Dec 10;256(23):11955–11957. [PubMed] [Google Scholar]
  26. Pastuszyn A., Noland B. J., Bazan J. F., Fletterick R. J., Scallen T. J. Primary sequence and structural analysis of sterol carrier protein 2 from rat liver: homology with immunoglobulins. J Biol Chem. 1987 Sep 25;262(27):13219–13227. [PubMed] [Google Scholar]
  27. Poorthuis B. J., Wirtz K. W. Increased cholesterol esterification in rat liver microsomes in purified non-specific phospholipid transfer protein. Biochim Biophys Acta. 1982 Jan 15;710(1):99–105. doi: 10.1016/0005-2760(82)90195-3. [DOI] [PubMed] [Google Scholar]
  28. Scallen T. J., Noland B. J., Gavey K. L., Bass N. M., Ockner R. K., Chanderbhan R., Vahouny G. V. Sterol carrier protein 2 and fatty acid-binding protein. Separate and distinct physiological functions. J Biol Chem. 1985 Apr 25;260(8):4733–4739. [PubMed] [Google Scholar]
  29. Thompson S. L., Burrows R., Laub R. J., Krisans S. K. Cholesterol synthesis in rat liver peroxisomes. Conversion of mevalonic acid to cholesterol. J Biol Chem. 1987 Dec 25;262(36):17420–17425. [PubMed] [Google Scholar]
  30. Tokuyasu K. T. Immunochemistry on ultrathin frozen sections. Histochem J. 1980 Jul;12(4):381–403. doi: 10.1007/BF01011956. [DOI] [PubMed] [Google Scholar]
  31. Trzaskos J. M., Gaylor J. L. Cytosolic modulators of activities of microsomal enzymes of cholesterol biosynthesis. Purification and characterization of a non-specific lipid-transfer protein. Biochim Biophys Acta. 1983 Mar 22;751(1):52–65. doi: 10.1016/0005-2760(83)90256-4. [DOI] [PubMed] [Google Scholar]
  32. Vahouny G. V., Chanderbhan R., Noland B. J., Irwin D., Dennis P., Lambeth J. D., Scallen T. J. Sterol carrier protein2. Identification of adrenal sterol carrier protein2 and site of action for mitochondrial cholesterol utilization. J Biol Chem. 1983 Oct 10;258(19):11731–11737. [PubMed] [Google Scholar]
  33. Vahouny G. V., Dennis P., Chanderbhan R., Fiskum G., Noland B. J., Scallen T. J. Sterol carrier protein2 (SCP2)-mediated transfer of cholesterol to mitochondrial inner membranes. Biochem Biophys Res Commun. 1984 Jul 31;122(2):509–515. doi: 10.1016/s0006-291x(84)80062-5. [DOI] [PubMed] [Google Scholar]
  34. Van der Krift T. P., Leunissen J., Teerlink T., Van Heusden G. P., Verkleij A. J., Wirtz K. W. Ultrastructural localization of a peroxisomal protein in rat liver using the specific antibody against the non-specific lipid transfer protein (sterol carrier protein 2). Biochim Biophys Acta. 1985 Jan 25;812(2):387–392. doi: 10.1016/0005-2736(85)90313-x. [DOI] [PubMed] [Google Scholar]
  35. Westerman J., Wirtz K. W. The primary structure of the nonspecific lipid transfer protein (sterol carrier protein 2) from bovine liver. Biochem Biophys Res Commun. 1985 Feb 28;127(1):333–338. doi: 10.1016/s0006-291x(85)80163-7. [DOI] [PubMed] [Google Scholar]
  36. van Amerongen A., Helms J. B., van der Krift T. P., Schutgens R. B., Wirtz K. W. Purification of nonspecific lipid transfer protein (sterol carrier protein 2) from human liver and its deficiency in livers from patients with cerebro-hepato-renal (Zellweger) syndrome. Biochim Biophys Acta. 1987 Jun 2;919(2):149–155. doi: 10.1016/0005-2760(87)90201-3. [DOI] [PubMed] [Google Scholar]
  37. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

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