Abstract
Apolipoprotein (apo)E is well established as a secreted protein that plays an important role in systemic lipoprotein metabolism and vascular wall homeostasis. Recently, endogenous expression of apoE in adipocytes has been shown to play an important role in adipocyte lipoprotein metabolism and gene expression consistent with a nonsecreted cellular itinerary for apoE. We designed studies to evaluate if adipocyte apoE was retained as a constituent protein in adipocytes and to identify a cellular retention compartment. Using confocal microscopy, coimmunoprecipitation, and sucrose density cellular fractionation, we establish that endogenous apoE shares a cellular itinerary with the constituent protein caveolin-1. Altering adipocyte caveolar number by modulating cellular cholesterol flux or altering caveolin expression regulates the distribution of cellular apoE between cytoplasmic and plasma membrane compartments. A mechanism for colocalization of apoE with caveolin was established by demonstrating a noncovalent interaction between an aromatic amino acid-enriched apoE N-terminal domain with the caveolin scaffolding domain. Absent apoE expression in adipocytes alters caveolar lipid composition. These observations provide evidence for an interaction between two proteins involved in cellular lipid metabolism in a cell specialized for lipid storage and flux, and rationalize a biological basis for the impact of adipocyte apoE expression on adipocyte lipoprotein metabolism.
Highlights
ApolipoproteinE is well established as a secreted protein that plays an important role in systemic lipoprotein metabolism and vascular wall homeostasis
ApoE is a phospholipid binding protein that is well characterized as a secreted protein from hepatocytes and macrophages and that has an important role in systemic lipoprotein metabolism and vessel wall homeostasis
In order to evaluate this question, caveolar fractions from adipocytes harvested from WT and apoE knockout (EKO) mouse adipose tissue were isolated on discontinuous sucrose gradients and equal amounts of caveolar protein were extracted for lipid analysis on HPLTC
Summary
Apolipoprotein (apo)E is well established as a secreted protein that plays an important role in systemic lipoprotein metabolism and vascular wall homeostasis. Incubation of intact 3T3-L1 cells with CH increases the amount of apoE and cav-1 detected in plasma membrane sheets and leads to substantial colocalization of apoE and cav-1.
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