Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3DGV, specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1–3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cavDGV to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

In this study, we compared the transport of newly synthesized cholesterol with that of influenza virus hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. The arrival of cholesterol on the cell surface was monitored by cyclodextrin removal, and HA transport was monitored by surface trypsinization and endoglycosidase H digestion. We found that disassembly of the Golgi complex by brefeldin A treatment resulted in partial inhibition of cholesterol transport while completely blocking HA transport. Further, microtubule depolymerization by nocodazole inhibited cholesterol and HA transport to a similar extent. When the partitioning of cholesterol into lipid rafts was analyzed, we found that newly synthesized cholesterol began to associate with low-density detergent-resistant membranes rapidly after synthesis, before it was detectable on the cell surface, and its raft association increased further upon chasing. When cholesterol transport was blocked by using 15°C incubation, the association of newly synthesized cholesterol with low-density detergent-insoluble membranes was decreased and cholesterol accumulated in a fraction with intermediate density. Our results provide evidence for the partial contribution of the Golgi complex to the transport of newly synthesized cholesterol to the cell surface and suggest that detergent-resistant membranes are involved in the process.

Cholesterol-sphingolipid rich plasma membrane domains, known as rafts, have emerged as important regulators of signal transduction. The adipocyte insulin receptor (IR) is localized to and signals via caveolae that are formed by polymerization of caveolins. Caveolin binds to IR and stimulates signalling. We report that, in liver-derived cells lacking caveolae, autophosphorylation of the endogenous IR is dependent on raft lipids, being compromised by acute cyclodextrin-mediated cholesterol depletion or by antibody clustering of glycosphingolipids. Moreover, we provide evidence that IR becomes recruited to detergent-resistant domains upon ligand binding and that clustering of GM2 ganglioside inhibits IR signalling apparently by excluding the ligand-bound IR from these domains. Our results indicate that, in cells derived from liver, an important insulin target tissue, caveolae are not required for insulin signalling. Rather, the dynamic recruitment of the ligand-bound IR into rafts may serve to regulate interactions in the initiation of the IR signalling cascade.

Chicken avidin and bacterial streptavidin are proteins familiar from their use in various (strept)avidin-biotin technological applications. Avidin binds the vitamin biotin with the highest affinity known for non-covalent interactions found in nature. The gene encoding avidin (AVD) has homologues in chicken, named avidin-related genes (AVRs). In the present study we used the AVR genes to produce recombinant AVR proteins (AVRs 1, 2, 3, 4/5, 6 and 7) in insect cell cultures and characterized their biotin-binding affinity and biochemical properties. Amino acid sequence analysis and molecular modelling were also used to predict and explain the properties of the AVRs. We found that the AVR proteins are very similar to avidin, both structurally and functionally. Despite the numerous amino acid substitutions in the subunit interface regions, the AVRs form extremely stable tetramers similar to those of avidin. Differences were found in some physico-chemical properties of the AVRs as compared with avidin, including lowered pI, increased glycosylation and, most notably, reversible biotin binding for two AVRs (AVR1 and AVR2). Molecular modelling showed how the replacement Lys(111)-->isoleucine in AVR2 alters the shape of the biotin-binding pocket and thus results in reversible binding. Both modelling and biochemical analyses showed that disulphide bonds can form and link monomers in AVR4/5, a property not found in avidin. These, together with the other properties of the AVRs described in the present paper, may offer advantages over avidin and streptavidin, making the AVRs applicable for improved avidin-biotin technological applications.