Oxidized high-density lipoprotein (oxHDL) reduces the ability of cells to mediate reverse cholesterol transport and also shows atherogenic properties. Palmitoylation of cluster of differentiation 36 (CD36), an important receptor mediating lipoprotein uptake, is required for fatty acid endocytosis. However, the relationship between oxHDL and CD36 has not been described in mechanistic detail. Here, we demonstrate using acyl-biotin exchange analysis that oxHDL activates CD36 by increasing CD36 palmitoylation, which promotes efficient uptake in macrophages. This modification increased CD36 incorporation into plasma lipid rafts and activated downstream signaling mediators, such as Lyn, Fyn, and c-Jun N-terminal kinase, which elicited enhanced oxHDL uptake and foam cell formation. Furthermore, blocking CD36 palmitoylation with the pharmacological inhibitor 2-bromopalmitate decreased cell surface translocation and lowered oxHDL uptake in oxHDL-treated macrophages. We verified these results by transfecting oxHDL-induced macrophages with vectors expressing wildtype or mutant CD36 (mCD36) in which the cytoplasmic palmitoylated cysteine residues were replaced. We show that cells containing mCD36 exhibited less palmitoylated CD36, disrupted plasma membrane trafficking, and reduced protein stability. Moreover, in ApoE−/−CD36−/− mice, lipid accumulation at the aortic root in mice receiving the mCD36 vector was decreased, suggesting that CD36 palmitoylation is responsible for lipid uptake in vivo. Finally, our data indicated that palmitoylation of CD36 was dependent on DHHC6 (Asp-His-His-Cys) acyltransferase and its cofactor selenoprotein K, which increased the CD36/caveolin-1 interaction and membrane targeting in cells exposed to oxHDL. Altogether, our study uncovers a causal link between oxHDL and CD36 palmitoylation and provides insight into foam cell formation and atherogenesis.

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