Fig. 1.

Scap, the cholesterol sensor that controls SREBP activation, shares identical sterol binding specificity with ALO, a bacterial toxin that forms membrane pores. (A) Overview of the SREBP pathway. (Left) When cellular cholesterol levels are low, Scap escorts SREBPs from ER to Golgi, where two proteases (S1P and S2P) sequentially cleave the SREBPs, releasing their bHLH transcription factor domains that then travel to the nucleus and activate genes involved in lipid production. (Right) When cholesterol levels rise above a threshold concentration, the sterol binds to Scap and changes Scap’s conformation to promote binding to Insigs, which retain Scap in the ER. As a result, transport of SREBPs to the Golgi is halted, thus reducing transcriptional activation of lipogenic genes. (B) Structure-based models of Scap and ALO. (Left) Scap is a polytopic membrane protein that can be divided into three domains—i) a luminal domain (purple) composed of an intertwined complex of Loop1 (L1) with Loop7 (L7) that contains a cholesterol-binding site in L1; ii) a transmembrane domain (red) that binds Insigs when cholesterol is bound to L1; and iii) a cytosolic domain (gray) that binds SREBPs regardless of whether L1 is bound to cholesterol or not. (Right) ALO is a soluble protein that harbors a cholesterol-binding site in its Domain 4 (D4, gold). The remainder of the ALO protein (blue) facilitates its oligomerization once cholesterol is bound to D4. (C) Competitive binding of unlabeled sterols to His₆-Scap(L1)-FLAG. Each reaction, in a final volume of 200 µL of buffer A with 0.004% NP-40 and 0.002% FC-13, contained 0.2 µg of His₆-Scap(L1)-FLAG, 1 µg of BSA, 150 nM [³H]cholesterol (110,000 dpm/pmol), and varying concentrations of the indicated unlabeled sterol. After incubation for 4 h at 4 °C, bound [³H]cholesterol was measured as described in SI Appendix, Methods. The 100% control value, determined in the absence of competitor, was 544 fmol/tube. (D) Sterol specificity for inhibition of hemolysis by His₆-ALO. Each reaction, in a final volume of 50 µL of buffer A, contained 1 nM of His₆-ALO and varying amounts of the indicated sterols solubilized in DMSO [final concentration of DMSO in each reaction tube was 4% (v/v)]. After incubation for 1 h at room temperature, 450 µL of rabbit erythrocytes (isolated and resuspended in buffer C as described in SI Appendix, Methods) was added to each reaction mixture. After incubation for 10 min at room temperature, the extent of hemolysis was quantified as described in SI Appendix, Methods by measuring the release of hemoglobin (absorbance at 540 nm). The amount of hemoglobin released after treatment with 1% (w/v) Triton X-100 detergent was set to 100%, and all values were normalized to this set-point. (C and D) Each data point represents the average of three assays and error bars represent the SE. When not visible, error bars are smaller than the size of the symbols.