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

Abstract

AbstractClathrin/dynamin-independent endocytosis of ordered plasma membrane domains (orderedmembranedomainendocytosis, OMDE) can be far more powerful than conventional endocytosis. Nevertheless, it remains controversial how OMDE takes place, in which cell processes it is important, and how it is regulated. Using patch clamp, we have described that OMDE becomes massive in multiple cell types in response to Ca elevations, G protein activation by non-hydrolyzable GTP analogs, and enhanced oxidative metabolism. We describe here that cytoplasmic succinate and pyruvate, but not β-hydroxybutyrate, can induce OMDE of 75% of the plasma membrane in murine marrow macrophages (BMMs) within 2 min. OMDE is decreased by 70% in BMMs lacking the acyltransferase, DHHC5, by treatment with carnitine to shift long- chain acyl groups from cytoplasmic to mitochondrial acyl-CoA’s, by bromopalmitate/albumin complexes to block DHHCs, and by the cyclosporin, NIM811, to block permeability transition pores that may release mitochondrial coenzyme A into the cytoplasm. Using T-REx293 cells, plasma membrane loss amounts to 40% with pyruvate or GTPγS, whereby OMDE is then highly sensitive to actin cytoskeleton disruption. Using fluorescent 3kD dextrans to monitor constitutive endocytosis in unperturbed cells without serum or bicarbonate, multiple results suggest that OMDE is constitutively active in T-REx293 cells, but not in BMMs. In T-REx293 cells, substrates that generate metabolites activated by coenyzme A (CoA), e.g. pyruvate and hydroxybutyrate, inhibit endocytosis as expected for a shift of long-chain acyl-CoA to other CoA metabolites. In the presence of bicarbonate, however, pyruvate strongly enhances OMDE, and OMDE is then blocked by β-hydroxybutyrate, bromopalmitate/albumin complexes, and cyclosporines. After pyruvate responses, T-REx293 cells grow normally without evidence of apoptosis. Low concentrations of fatty acid (FA)-free albumin inhibit basal endocytosis in T-REx293 cells, but not BMMs, as do cyclosporines, carnitine, and RhoA blockade. However, constitutive endocytosis (i.e. in the absence of substrates and bicarbonate) is not inhibited by siRNA knockdown of the acyltransferases, DHHC5 or DHHC2, which support OMDE responses activated in patch clamp experiments. We verify biochemically that small CoA metabolites cause a decrease of long-chain acyl-CoA’s and that several palmitoylated membrane proteins are depalmitoylated during treatments that decrease long-chain acyl-CoA’s. STED microscopy reveals that vesicles formed in constitutive OMDE in T-REX293 cells have diameters of 90 to 130 nm. In summary, palmitoylation-dependent OMDE is likely a major G-protein-dependent endocytic mechanism that can be constitutively active, albeit not in BMMs. It depends on the activities of different DHHC acyltransferases in different cellular circumstances and can be limited by local supplies of fatty acids, CoA, and long-chain acyl-CoA’s.