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Journal of Lipid Research, Vol. 43, 36-44, January 2002
Copyright © 2002 by Lipid Research, Inc.

ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL

Jeffrey W. Chisholma, Ellen R. Burlesona, Gregory S. Shelnessa, and John S. Parksa
a Section on Comparative Medicine, Department of Pathology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1040

Correspondence to: John S. Parks, To whom correspondence should be addressed., jparks{at}wfubmc.edu (E-mail)

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 [35S]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 ~7.5 nm. These and additional data suggested that ~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, J. W., E. R. Burleson, G. S. Shelness, and J. S. Parks. ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL. J. Lipid Res. 2002. 43: 36–44.

Supplementary key words: apolipoprotein A-I, carbonate extraction, HDL metabolism, HDL secretion, HDL biosynthesis, human, lipid-free apoA-I, lipid-poor apoA-I, ultracentrifugation


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