Expression and intracellular processing of the 58 kDa sterol carrier protein-2/3-oxoacyl-CoA thiolase in transfected mouse L-cell fibroblasts.
Although the sterol carrier protein 2 (SCP-2) gene encodes for two proteins, almost nothing is known of the function and potential processing of the larger transcript corresponding to the 58 kDa sterol carrier protein-2/3-oxoacyl-CoA thiolase (SCP-x), in intact cells. L-cell fibroblasts transfected with cDNA encoding for the 58 kDa SCP-x protein had a 4.5-fold increase in SCP-x mRNA transcript levels. Western blot analysis showed SCP-x protein expression reached 0.011% of total protein, representing a 4.1-fold increase over basal levels. Surprisingly, the 13.2 kDa SCP-2 protein also increased 2-fold in the transfected cells. This was consistent with part of the 58 kDa SCP-x being proteolytically processed to 13.2 kDa SCP-2 as there was no evidence of an mRNA transcript corresponding to a 13.2/15.2 kDa gene product in the transfected L-cell clones. Confocal immunofluorescence microscopy of transfected L-cells showed that SCP-x/SCP-2 co-localized in highest concentration with catalase in peroxisomes, but significant amounts appeared extra-peroxisomal. Overexpression of SCP-x significantly altered cholesterol uptake and metabolism. Uptake of exogenous [3H]cholesterol and total cholesterol mass were increased 1.9- and 1.4-fold, respectively, in SCP-x expressors. Although cholesterol ester mass was unaltered, incorporation of exogenous [3H]cholesterol and [3H]oleic acid into cholesteryl esters increased 2.3- and 2.5-fold, respectively. These results from intact cells suggest the 13.2 kDa SCP-2 can arise from the larger SCP-2 gene product and indicate a role for the 58 kDa SCP-x protein in cholesterol uptake and intracellular cycling.
Atshaves
B P
BP
Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 77843-4466, USA.
Petrescu
A D
AD
Starodub
O
O
Roths
J B
JB
Kier
A B
AB
Schroeder
F
F
D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D002788:Cholesterol Esters / Q000378:metabolism; D004951:Esterification; D005347:Fibroblasts / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D007700:Kinetics; D051379:Mice; D008830:Microbodies / Q000378:metabolism; D008970:Molecular Weight; D019301:Oleic Acid / Q000378:metabolism; D010940:Plant Proteins; D014162:Transfection; D014316:Tritium
D002352:Carrier Proteins; D002788:Cholesterol Esters; D010940:Plant Proteins; C072087:sterol carrier proteins; D014316:Tritium; D019301:Oleic Acid; D002784:Cholesterol; D000102:Acetyl-CoA C-Acyltransferase