Although the functional significance of caveolae/lipid rafts in cellular signaling and cholesterol transfer is increasingly recognized, almost nothing is known regarding the lipids, cholesterol dynamics, and factors regulating these properties in caveolae/lipid rafts as opposed to nonlipid raft domains of the plasma membrane. The present findings demonstrate the utility of con-A affinity chromatography for simultaneous isolation of caveolae/lipid raft and nonlipid raft domains from plasma membranes of L-cell fibroblasts. These domains differed markedly in both protein and lipid constituents. Although caveolae/lipid rafts were enriched in total lipid, cholesterol, and phospholipid as well as other markers for these domains, the cholesterol/phospholipid ratio of caveolae/lipid rafts did not differ from that of nonlipid rafts. Nevertheless, spontaneous sterol transfer was 7-12-fold faster from caveolae/lipid raft than nonlipid raft domains of the plasma membrane. This was largely due to the near absence of exchangeable sterol in the nonlipid rafts. SCP-2 dramatically and selectively enhanced sterol transfer from caveolae/lipid rafts, but not from nonlipid rafts. Finally, overexpression of SCP-2 significantly altered the sterol dynamics of caveolae/lipid rafts to facilitate retention of cholesterol within the cell. These results established for the first time that (i) caveolae/lipid rafts, rather than the nonlipid raft domains, contain significant levels of rapidly transferable sterol, consistent with their role in spontaneous sterol transfer from and through the plasma membrane, and (ii) SCP-2 selectively regulates how caveolae/lipid rafts, but not nonlipid raft domains, mediate cholesterol trafficking through the plasma membrane.

Although it is hypothesized that long-chain fatty acyl CoAs (LCFA-CoAs) and long-chain fatty acids (LCFAs) regulate transcription in the nucleus, little is known regarding factors that determine the distribution of these ligands to nuclei of living cells. Immunofluorescence colocalization showed that liver fatty acid-binding protein (L-FABP; binds LCFA-CoA as well as LCFA) significantly colocalized with PPARalpha in nuclei of transfected L-cell fibroblasts. Colocalization with a DNA binding dye (SYTO59) revealed that, within the nucleus of control L-cells, the nonhydrolyzable fluorescent LCFA-CoA (BODIPY-C16-S-S-CoA) was distributed primarily in a punctate pattern throughout the nucleoplasm, while nonmetabolizable fluorescent LCFAs (BODIPY-C16 and BODIPY-C12) were localized primarily near the nuclear envelope membranes. L-FABP overexpression selectively increased the targeting of BODIPY-C16-S-S-CoA by 1.9- and 2.7-fold into the nuclear membrane and nucleoplasm, respectively. L-FABP also increased the targeting of fluorescent LCFAs (especially long-chain-length BODIPY-C16) by 1.7-fold to the nuclear membrane and 7.4-fold into the nucleoplasm. A cis-parinaric acid displacement assay showed that L-FABP bound BODIPY-C12 and BODIPY-C16 with K(i)s of 10.1 +/- 2.5 and 20.7 +/- 1.5 nM, respectively, in the same range as naturally occurring LCFAs. Finally, solid-phase extraction and HPLC analysis revealed that, depending on the fatty acid content of the culture medium, L-FABP expression also increased the cellular LCFA-CoA pool size and altered the LCFA-CoA acyl chain composition. Thus, L-FABP may function as a carrier for selectively enhancing the distribution of LCFA-CoA, as well as LCFA, to nuclei for potential interaction with nuclear receptors.

Very long chain fatty acids (VLCFA) and branched-chain fatty acids (BCFA) are potent inducers of the peroxisome proliferator-activated receptor PPARα, a nuclear receptor that enhances transcription of peroxisomal enzymes mediating β-oxidation of these potentially toxic fatty acids. However, it is not known whether the respective free fatty acids or their activated metabolites, i.e. CoA thioesters: (i) are the endogenous high-affinity PPARα ligands, (ii) alter PPARα conformation, and (iii) alter recruitment of coregulatory proteins to PPARα. As shown by quenching of PPARα intrinsic amino acid fluorescence, PPARα exhibited high affinity (3-29nM K d s) for the CoA thioesters of the common (C20-C24) VLCFA. In contrast, with the exception of arachidonic acid (K d =20nM), PPARα only weakly bound the VLCFA. PPARα also exhibited higher affinity for the CoA thioesters of BCFA (phytanoyl-CoA, pristanoyl-CoA; K d s near 11nM) than for the respective free branched-chain fatty acids. As shown by circular dichroism, the high affinity VLCFA-CoA and BCFA-CoA strongly altered PPARα conformation. Likewise, the high affinity VLCFA-CoA and BCFA-CoA altered cofactor recruitment to PPARα as shown by co-immunoprecipitation from liver homogenates. In contrast, nearly all the respective free fatty acids elicited only weak conformational changes in PPARα and did not alter co-factor recruitment to PPARα. In summary, the CoA thioesters of verylong-chain and branched-chain fatty acids are much more potent PPARα ligands than the free acids, resulting in altered PPARα conformation and cofactor recruitment. Since these are hallmarks of ligands-activated nuclear receptors, this suggests that the CoA thioesters are the active forms of these PPARα ligands.

Although studies in vitro and in yeast suggest that acyl-CoA binding protein ACBP may modulate long-chain fatty acyl-CoA (LCFA-CoA) distribution, its physiological function in mammals is unresolved. To address this issue, the effect of ACBP on liver LCFA-CoA pool size, acyl chain composition, distribution, and transacylation into more complex lipids was examined in transgenic mice expressing a higher level of ACBP. While ACBP transgenic mice did not exhibit altered body or liver weight, liver LCFA-CoA pool size increased by 69%, preferentially in saturated and polyunsaturated, but not monounsaturated, LCFA-CoAs. Intracellular LCFA-CoA distribution was also altered such that the ratio of LCFA-CoA content in (membranes, organelles)/cytosol increased 2.7-fold, especially in microsomes but not mitochondria. The increased distribution of specific LCFA-CoAs to the membrane/organelle and microsomal fractions followed the same order as the relative LCFA-CoA binding affinity exhibited by murine recombinant ACBP: saturated > monounsaturated > polyunsaturated C14-C22 LCFA-CoAs. Consistent with the altered microsomal LCFA-CoA level and distribution, enzymatic activity of liver microsomal glycerol-3-phosphate acyltransferase (GPAT) increased 4-fold, liver mass of phospholipid and triacylglyceride increased nearly 2-fold, and relative content of monounsaturated C18:1 fatty acid increased 44% in liver phospholipids. These effects were not due to the ACBP transgene altering the protein levels of liver microsomal acyltransferase enzymes such as GPAT, lysophosphatidic acid acyltransferase (LAT), or acyl-CoA cholesterol acyltransferase 2 (ACAT-2). Thus, these data show for the first time in a physiological context that ACBP expression may play a role in LCFA-CoA metabolism.

It is demonstrated that single-molecule tracking of a fluorescently labeled protein undergoing transient binding to model membranes presents a useful method of obtaining fluid properties. The labeled ACBP protein was tracked during its binding to free-standing giant unilamellar vesicles (GUVs) and supported bilayers prepared from the GUVs in the same environment. The analysis of images that are blurred as a result of fast probe diffusion was discussed. An examination of the lateral diffusion trajectories revealed a homogeneous diffusion on the top segments of the GUVs with D = 6.9 +/- 0.3 microm(2)/s. The supported bilayer experiments revealed two diffusion processes, one with Df = 3.1 +/- 0.4 microm(2)/s and the other with Ds = 0.078 +/- 0.001 microm(2)/s. The 2-fold difference in the lipid bilayer mobility for the free-standing and fast components in the supported bilayers is attributed to the known effect of frictional coupling with the solid support. The slow mobile fraction in the bilayer is suggested to be associated with the migration of pore-like structures, originating from the interaction of the membrane with the glass support.