Both Sphingolipids and Cholesterol Participate in the Detergent Insolubility of Alkaline Phosphatase, a Glycosylphosphatidylinositol-anchored Protein, in Mammalian Membranes (*)
- From the (1)Department of Biochemistry and Cell Biology, National Institute of Health, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162, Japan and
- the (2)Department of Embryology, Carnegie Institution, Baltimore, Maryland 21210
- § To whom correspondence and reprint requests should be addressed. Present address: Dept. of Biochemistry and Cell Biology, National Institute of Health, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162, Japan. Fax: 81-3-5285-1157.
Abstract
SPB-1, a Chinese hamster ovary cell variant defective in serine palmitoyltransferase activity for sphingolipid synthesis, provides a useful system for studying the effects of sphingolipids and/or cholesterol deprivation on cellular functions and membrane properties. To investigate whether there was an interaction among sphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins in biological membranes, we introduced human placental alkaline phosphatase (PLAP) in SPB-1 and in wild type cells by stable transfection and examined the effects of sphingolipid and/or cholesterol deprivation on the solubility of PLAP in Triton X-100. Although the PLAP solubility of the membranes isolated from the control cells in Triton X-100 was only 10%, deprivation of sphingolipid and cholesterol further enhanced the solubility, which reached 50% when both sphingolipids and cholesterol were deprived. The enhanced solubility was suppressed to the control level by metabolic complementation with exogenous sphingosine and cholesterol. The sphingolipid and cholesterol content of the isolated membranes changed independently, eliminating the possibility that sphingolipid deprivation induced a reduction in cellular cholesterol and enhanced PLAP solubility and vice versa. It was also unlikely that the enhanced solubility was due to structural changes in PLAP molecules since, regardless of sphingolipid and cholesterol deprivations, almost all PLAP had the GPI-anchor moiety and there were no differences in the apparent molecular weight of the protein in supernatant and precipitate fractions of the detergent-treated membranes. In addition, the expression level of caveolin in the isolated membranes was not significantly affected by sphingolipids and/or cholesterol depletion. These results indicated that both sphingolipids and cholesterol were involved in the PLAP insolubility and suggested that these lipids coordinately played a role in formation of Triton X-100resistant complexes.
Footnotes
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↵* This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan and also by a long-term fellowship from International Human Frontier Science Program (to K. H.) and United States Public Health Service Grant R37 GM22942 (to R. E. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- GPI
-
glycosylphosphatidylinositol
- BSA
-
bovine serum albumin
- CHO
-
Chinese hamster ovary
- G
-
sialyllactosylceramide
- PBS
-
Ca
/Mg
-free phosphate-buffered saline
- PI-PLC
-
phosphatidylinositol-specific phospholipase C
- PLAP
-
human placental alkaline phosphatase
- CHAPS
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.
-
↵2 D. Brown, personal communication.
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↵3 K. Hanada, unpublished observations.
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- Received October 19, 1994.
- Revision received December 23, 1994.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
