Molecular and fluorescent sterol approaches to probing lysosomal membrane lipid dynamics

Although the Most exogenous lipids enter (lie cell via the LDL-receptor pathway, the mechanism(s) whereby lipids leave the lysosome for transport to intracellular sites are not clearly resolved. As shown herein, expression of sterol carrier protein-2 (SCP-2) in transfected L-cells altered lysosomal membrane lipid distribution, dynamics. and response to lipid transfer proteins, SCP-2 expression decreased the mass of cholesterol and lyso-bis-phospliatidic acid [LBPA], as well as the ratios of cholesterol phospholipid and polyunsaturated/monounsaturated fatty acids esterified to lysosomal membrane phospholipids. Concomitantly, a fluorescent sterol transfer assay showed that SCP-2 expression decreased the initial rates of spontaneous and SCP-2-mediated sterol transfer 5.5- and 3.8-fold. respectively, from lysosomal membranes isolated from SCP-2 expressing cells Lis compared to controls. SCP-2, sphingomyelinase, low high density lipoprotein directly enhanced the initial rates of sterol transfer from isolated density lipoprotein. and high density lipoprotein directly enhanced the initial rates of sterol transfer from isolated lysosomal membranes by 50-, 12-, 4-, Mid 5-fold. respectively. In contrast, albumin and cholesterol esterase had no effect on lysosomal sterol transfer. Spontaneous sterol was very slow. t(3 2) > 4 days, regardless of the source of the lysosomal membrane. while SCP-2 added in vitro induced formation or rapid and slowly transferable sterol pools in lysosomal membranes of control cells. In contrast, SCP-2 did not induce formation of a rapidly transferable sterol domain in lysosomal membranes isolated from SCP-2 expressing cells, These data suggest that SCP-2 expression selectively shifted the distribution of lipids (cholesterol, LBPA, esterified polyunsaturated fatty acids) away from lysosomal membranes. Furthermore. the cholesterol depleted lysosomal membrane isolated from SCP-2 expressing cells was resistant to additional direct action of SCP-2 to further enhance sterol transfer and induce rapidly transferable sterol pools in the lysosomal membrane. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.