Constitutive Plasma Membrane Turnover in T-REx293 cells via Ordered Membrane Domain Endocytosis under Mitochondrial Control

Clathrin/dynamin-independent endocytosis of ordered plasma membrane domains (ordered membrane domain endocytosis, OMDE) can become massive in response to cytoplasmic Ca elevations, G protein activation by non-hydrolyzable GTP analogs, and enhanced oxidative metabolism. In patch-clamped murine bone marrow macrophages (BMMs), cytoplasmic succinate and pyruvate, but not {beta}-hydroxybutyrate, induce OMDE of 75% of the plasma membrane within 2 min. The responses require palmitoylation of membrane proteins, being decreased by 70% in BMMs lacking the acyltransferase, DHHC5, by treatment with carnitine to shift long-chain acyl groups from cytoplasmic to mitochondrial acyl-CoAs, by bromopalmitate/albumin complexes to block DHHCs, and by the mitochondria-specific cyclosporin, NIM811, to block permeability transition pores that may release mitochondrial coenzyme A into the cytoplasm. Using T-REx293 cells, OMDE amounts to 40% with succinate, pyruvate, or GTP{gamma}S, and it is inhibited by actin cytoskeleton disruption. Pyruvate-induced OMDE is blocked by the hydrophobic antioxidant, edaravone, which prevents permeability transition pore openings. Using fluorescent 3kD dextrans to monitor endocytosis, OMDE appears to be constitutively active in T-REx293 cells but not in BMMs. After 1 h without substrates or bicarbonate, pyruvate and hydroxybutyrate inhibit constitutive OMDE, as expected for a shift of CoA from long-chain acyl-CoAs to other CoA metabolites. In the presence of bicarbonate, pyruvate strongly enhances OMDE, which is then blocked by {beta}-hydroxybutyrate, bromopalmitate/albumin complexes, cyclosporines, or edaravone. After pyruvate responses, T-REx293 cells grow normally with no evidence for apoptosis. Fatty acid-free albumin (15 M) inhibits basal OMDE in T-REx293 cells, as do cyclosporines, carnitine, and RhoA blockade. Surprisingly, OMDE in the absence of substrates and bicarbonate is not inhibited by siRNA knockdown of the acyltransferases, DHHC5 or DHHC2, which are required for activated OMDE in patch clamp experiments. We verify biochemically that small CoA metabolites decrease long-chain acyl-CoAs. We verify also that palmitoylations of many PM-associated proteins decrease and increase when OMDE is inhibited and stimulated, respectively, by different metabolites. STED microscopy reveals that vesicles formed during constitutive OMDE in T-REX293 cells have 90 to 130 nm diameters. In summary, OMDE is likely a major G-protein-dependent endocytic mechanism that can be constitutively active in some cell types, albeit not BMMs. OMDE depends on different DHHC acyltransferases in different circumstances and can be limited by local supplies of fatty acids, CoA, and long-chain acyl-CoAs.