ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL.
The goal of this study was to determine whether apolipoprotein A-I (apoA-I) is lipidated before secretion by HepG2 cells. ApoA-I was extracted from microsomes after radiolabeling with [(35)S]Met/Cys. After ultracentrifugal flotation, d < 1.25 g/ml and d > 1.25 g/ml fractions were immunoprecipitated and analyzed by SDS-PAGE. Under steady state radiolabeling conditions, 20% of extracted microsomal apoA-I floated at d < 1.25 g/ml. Pulse-chase experiments demonstrated that the percentage of microsomal apoA-I associated with lipid peaked between 2 and 8 min postsynthesis. Density gradient ultracentrifugation, and nondenaturing gradient gel electrophoresis of HepG2 cell medium, indicated that 50% of secretory apoA-I existed as small HDL particles with a diameter of approximately 7.5 nm. These and additional data suggested that approximately 20% of newly secreted apoA-I is lipidated intracellularly and another 30% is secreted in lipid-free or lipid-poor form but acquires sufficient lipid to become small HDL within 1 h of secretion, with little further maturation over the time course of the incubation (2 h).These results indicate that a process exists for the presecretory intracellular assembly of apoA-I with lipid in HepG2 cells and that apoA-I is secreted in both lipid-poor and lipidated forms.
Chisholm
Jeffrey W
JW
Section on Comparative Medicine, Department of Pathology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1040, USA. jeffreyc@cvt.com
Burleson
Ellen R
ER
Shelness
Gregory S
GS
Parks
John S
JS
D016632:Apolipoprotein A-I / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular; D002499:Centrifugation, Density Gradient / Q000379:methods; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D050356:Lipid Metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism*; D010316:Particle Size; D014407:Tumor Cells, Cultured
D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL