Extended Data Fig. 3: Structural features of CLSTN3β.

a, Electron microscopy of immortalized brown adipocytes (day 4 of differentiation) stably expressing (left) no APEX2 or (right) an N-terminal APEX2-CLSTN3β fusion. APEX2-CLSTN3β is poorly expressed and thus its localization is not visualized. b, Biochemical extraction of CLSTN3β-Flag (integral membrane protein) and S6 (peripheral membrane protein) from membranes of HEK293T cells treated with OA (250 µM, overnight). P = pellet, S = supernatant. c, Protease protection assay on membranes from HEK293T cells transfected with CLSTN3β-Flag (C-terminal tag) and treated with OA (250 µM, overnight). Calreticulin = inside ER, S6 = outside ER. d, Helical wheel plots of representative α-helices from CLSTN3β, generated using HeliQuest. e, Summary of CLSTN3β secondary structure predictions. Putative hydrophobic hairpins and β-strand domain are underlined. Positively charged and hydrophobic residues in the β-strand domain are colored green and yellow, respectively. f, RoseTTAFold model of full-length CLSTN3β. (Left) Model positioned to highlight hairpin domain (red) and TM domain (green). (Right) Model positioned to highlight hairpin 1 (cyan), hairpin 2 (magenta), hairpin 3 (yellow), and TM domain (green). g, Schematic of CLSTN3β hairpin mutants. h, Confocal microscopy of OA-treated (1 mM, overnight) HeLa cells transfected with ∆H1, ∆H2, ∆H3, ∆H12, ∆H13, or ∆H23 CLSTN3β-Flag constructs and stained with BODIPY 488 or LipidTOX 647. Scale bar = 5 µm.