Extended Data Fig. 5: Ammonia binds to SCAP stimulating SCAP/SREBP activation.
a, Representative confocal images of U87 cells in response to glutamine (4 mM), glucose (5 mM) or NH4Cl (4 mM) stimulation for 12 hr with/without CB-839 (100 nM) under serum-free culture conditions. Scale bars, 10 μm. b, In vitro SCAP ER-budding assay. H1299 cells were stimulated with/without glutamine (4 mM) or NH4Cl (4 mM) for 4 hr under serum-free medium (5 mM glucose). Microsomes were purified and incubated at 37 °C for 15 min or on ice (as time 0) with cytosol extracts from rat liver in the presence of ATP and GTP (left panel). Alternatively, microsomes purified from H1299 cells cultured with glucose (5 mM) alone (2 hr) were incubated with NH4Cl (1 mM) or NaCl (1 mM) at 37 °C or on ice together with liver extracts as above (right panel). The mixtures were centrifuged to separate budded vesicles from the ER membrane fractions, which were then analyzed by Western blot by using indicated antibodies. c, Co-solvent NH3 computational mapping of SCAP. d, Alignment of the SCAP protein fragment. e, A schematic model for the sequential binding of NH4+ to SCAP obtained from the co-solvent ammonia mapping and NH4+-bound SCAP simulations. f, Western blot analysis of HEK293T cells transfected with GFP, wild-type or different GFP-SCAP mutants together with full-length Flag-SREBP-1c for 24 hr and then stimulated with glutamine (4 mM) for 12 hr under serum-free conditions (5 mM glucose). g. Co-solvent ammonia mapping for SCAP bound with 25-HC. Right panel shows the biochemical analysis of GFP-SCAP-bound ammonia in HEK293T cells stimulated with NH4Cl (4 mM) for 2 hr with/without pretreatment with 25-HC (10 µg/ml, 1 hr) using an ammonia assay kit. Top panel shows by western blot that equal amounts of proteins were purified. The results are presented as mean ± SEM (n = 3). Significance was determined by unpaired and two-tailed Student’s t-test. h, i, Western blot analysis of H1299 cells cultured with NH4Cl (4 mM) (h) or glutamine (4 mM) (i) for 12 hr in serum-free medium (5 mM glucose) together with a cholesterol/25-hydroxycholesterol mixture (sterols).
