Molecular and fluorescent sterol approaches to probing lysosomal membrane lipid dynamics.
Although the most exogenous lipids enter the 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-phosphatidic 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 as compared to controls. SCP-2, sphingomyelinase, low density lipoprotein, and high density lipoprotein directly enhanced the initial rates of sterol transfer from isolated lysosomal membranes by 50-, 12-, 4-, and 5-fold, respectively. In contrast, albumin and cholesterol esterase had no effect on lysosomal sterol transfer. Spontaneous sterol was very slow, t(1/2)>4 days, regardless of the source of the lysosomal membrane, while SCP-2 added in vitro induced formation of 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.
Gallegos
Adalberto M
AM
Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 77843-4466, USA.
Atshaves
Barbara P
BP
Storey
Stephen
S
Schoer
Jonathan
J
Kier
Ann B
AB
Schroeder
Friedhelm
F
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D007425:Intracellular Membranes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008247:Lysosomes / Q000737:chemistry / Q000648:ultrastructure*; D051379:Mice; D013261:Sterols / Q000378:metabolism* / Q000493:pharmacokinetics; D014162:Transfection
D005456:Fluorescent Dyes; D008055:Lipids; D013261:Sterols