Abstract
PurposeCaveolae are cholesterol and sphingolipids rich subcellular domains on plasma membrane. Caveolae contain a variety of signaling proteins which provide platforms for signaling transduction. In addition to enriched with cholesterol and sphingolipids, caveolae also contain a variety of fatty acids. It has been well-established that acylation of protein plays a pivotal role in subcellular location including targeting to caveolae. However, the fatty acid compositions of caveolae and the type of acylation of caveolar proteins remain largely unknown. In this study, we investigated the fatty acids in caveolae and caveolin-1 bound fatty acids.MethodsCaveolae were isolated from Chinese hamster ovary (CHO) cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS). The caveolin-1bound fatty acids were immunoprecipitated by anti-caveolin-1 IgG and analyzed with GC/MS.ResultsIn contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids, 0.48 µg palmitic acid, 0.61 µg stearic acid and 0.83 µg oleic acid/caveolae preparation/5×107 cells. Unexpectedly, GC/MS analysis indicated that caveolin-1 was not acylated by myristic acid; instead, it was acylated by palmitic acid and stearic acid.ConclusionCaveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1 was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1 may be important for caveolae formation and for maintaining the function of caveolae.
Highlights
Caveolae are cholesterol and sphingomyelin-rich plasma membrane microdomains presented in most types of mammalian cells and tissues[1,2,3,4,5]_ENREF_13
In contrast to the whole Chinese hamster ovary (CHO) cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids, 0.48 mg palmitic acid, 0.61 mg stearic acid and 0.83 mg oleic acid/caveolae preparation/56107 cells
gas chromatograph-mass spectrometer (GC/MS) analysis indicated that caveolin-1 was not acylated by myristic acid; instead, it was acylated by palmitic acid and stearic acid
Summary
Caveolae are cholesterol and sphingomyelin-rich plasma membrane microdomains presented in most types of mammalian cells and tissues[1,2,3,4,5]_ENREF_13. Caveolae were originally identified as 50–100 nm flask-shaped, non-clathrin-coated invagination of the plasma membrane and found to be involved in endocytosis [6] and potocytosis [7]. Changes in the components of caveolae may have a profound effect on cellular functions. Caveolin-1, a 22-kDa protein, is the principal structural component of caveolae and a determinant for caveolae formation [13,14]. A deficiency of caveolin-1 in mice eliminated caveolae, which subsequently impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone [15,16]
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