Abstract 66: SR-BI Mediates Cholesteryl Ester (CE) Sorting in Tetraspanin Microdomains with CD81 Facilitating CE Nano-endocytosis and CE Transport to Lipid Droplets

Abstract

SR-BI functions as a neutral lipid transporter facilitating cholesteryl ester (CE) sorting, internalization and accumulation in LDs. The mechanism of CE transport remains obscure. We hypothesized that HDL lipid transport requires CD81 as a co-receptor and tetraspanin enriched domains (TED) for CE sorting, similar to hepatitis C uptake. Utilizing BODIPY-CE labeled recombinant HDL (rHDL/BP-CE) based on either human apoA-I or an apoA-I mimetic peptide, L-37pA, and confocal and TIRF microscopy, we found that after initially binding to SR-BI at the cellular filopodia and lamellipodia, HDL CE was laterally transported to the bases of filopodia, accumulating in 1-2 μm plasma membrane microdomains. While CE was rapidly internalized, neither apoA-I, L37pA nor SR-BI was, suggesting that these microdomains are an important part of the CE sorting mechanism. Internalized CE was transported in 50-100 nm CE-containing intracellular nano-vesicles. In a process of multiple collisions potentially involving transient nano-vesicle/LD fusion, BODIPY-CE was transferred to LDs. To identify CE-sorting microdomains, SR-BI expressing HeLa cells were transfected with GFP-CD81 or GFP-CD82. After a 1 h incubation with rHDL/BP-CE, CE was co-localized with both tetraspanin proteins demonstrating that these are TED (the observation was confirmed by anti-CD81 and -CD-82 antibody immunostaining). Following a 2 h chase, only GFP-CD81, not CD82, was found internalized and co-localized with CE transporting nano-vesicles. CE transport was not affected by inhibitors of de novo neutral lipid synthesis such as the ACSL-3 inhibitor, triacsin c, ACAT inhibitor, Sandoz 58-035 or ATGL inhibitor, A926500, indicating that CE was directly transported through a novel nano-endocytosis mechanism involving CD81 and not requiring CE hydrolysis and de novo synthesis. We conclude that TED function as CE sorting sites where CD81 helps to form nano-vesicles delivering CE to LDs. Further understanding of their function will help develop novel anti-atherogenic compounds accelerating reverse cholesterol transport.