ABSTRACT
Intracellular sterol transport occurs largely by non-vesicular mechanisms in which sterol transport proteins extract sterol from one membrane and transfer it to another across the cytoplasm. Here we describe a suite of complementary assays to measure intracellular sterol transport in the model eukaryote Saccharomyces cerevisiae, as well as to quantify protein-mediated sterol transport between populations of vesicles in vitro. The in vivo assays can be adapted to study sterol transport in other cell types.
Subject(s)
Saccharomyces cerevisiae/metabolism , Sterols/metabolism , Acetyl-CoA C-Acetyltransferase/metabolism , Biological Transport , Carrier Proteins/metabolism , Cell Membrane/metabolism , Endoplasmic Reticulum/metabolism , Intracellular Space/metabolism , Microscopy, Fluorescence , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Sphingolipid and cholesterol metabolism are closely associated at the structural, biochemical, and functional levels. Although HDL-associated sphingosine-1-phosphate (S1P) contributes to several HDL functions, and S1P signaling regulates glucose and lipid metabolism, no study has addressed the involvement of S1P in cholesterol efflux. Here, we show that sphingosine kinase (Sphk) activity was induced by the LXR agonist 22(R)-hydroxycholesterol and required for the stimulation of ABCA1-mediated cholesterol efflux to apolipoprotein A-I. In support, pharmacological Sphk inhibition and Sphk2 but not Sphk1 deficiency abrogated efflux. The involved mechanism included stimulation of both transcriptional and functional ABCA1 regulatory pathways and depended for the latter on the S1P receptor 3 (S1P3). Accordingly, S1P3-deficient macrophages were resistant to 22(R)-hydroxycholesterol-stimulated cholesterol efflux. The inability of excess exogenous S1P to further increase efflux was consistent with tonic S1P3 signaling by a pool of constitutively generated Sphk-derived S1P dynamically regulating cholesterol efflux. In summary, we have established S1P as a previously unrecognized intermediate in LXR-stimulated ABCA1-mediated cholesterol efflux and identified S1P/S1P3 signaling as a positive-feedback regulator of cholesterol efflux. This constitutes a novel regulatory mechanism of cholesterol efflux by sphingolipids.
