Cholesterol, but not its oxidized derivatives, is essential for the transport activity of ASC1 (SLC7A10)

The neutral amino acid transporter ASC1 (SLC7A10) plays a key role in the regulation of amino acid homeostasis in neurons and adipose tissue, thereby contributing to neurotransmission, metabolic balance and redox homeostasis. Although the 3D structure of ASC1 has recently been solved, regulatory properties remain largely unexplored. Here, we investigated the impact of sterols on ASC1 stability, trafficking, and transport activity using an integrated in silico, in vitro, and ex vivo approach. Functional reconstitution of purified ASC1 into proteoliposomes revealed that cholesterol increased the Vmax by approximately ten-fold without significantly affecting Km, indicating that the sterol facilitates conformational transitions associated with the transport cycle. The orientation and incorporation efficiency of ASC1 in proteoliposomes were unchanged by cholesterol, reinforcing the hypothesis of a direct effect on the transporter. In intact cells, cholesterol depletion reduced ASC1 thermal stability and transport activity; moreover, the thermal stability of the ancillary protein CD98, required for proper membrane targeting, was decreased confirming the physiological relevance of cholesterol also in the native context. Replacement of cholesterol with enzymatic or non-enzymatic oxysterols impaired ASC1 activity suggesting that redox stress may target ASC1 functionality via cholesterol sites. In agreement, molecular docking analyses identified sterol-binding pockets. The data on both cholesterol and oxysterols suggests that ASC1 regulation may rely on a structurally specific sterol-protein interaction rather than general alterations of membrane biophysics, with potential relevance for conditions characterized by altered membrane cholesterol composition in cholesterol-rich tissues.