Fig. 1: Structural and functional properties of the IST2–OSH6 complex.

a, SEC profile of a sample containing IST2 and OSH6 at a 1.5× molar excess (yellow). Inset, the peak fraction (*) contains the complex of both proteins as shown by SDS–PAGE. The red trace represents free OSH6. b, Scheme composed of the cryo-EM density of IST2 and OSH6 as observed in their complex with the flexible linker that is not defined in the structure sketched as line. The C-terminal parts interacting with the PM are shown in green and the membrane boundaries are indicated. c, Cryo-EM density of the N-terminal membrane-inserted part of the IST2 dimer at 4.9 Å. d, Cryo-EM density of OSH6 at 10.5 Å with a placed ribbon representation of its structure (PDB 4B2Z) shown for comparison. e–h, Lipid-scrambling properties of the N-terminal construct IST2⁷¹⁶ assayed in liposomes by the irreversible bleaching of fluorescent lipids facing the outside. e, Absence of lipid-scrambling activity in murine TMEM16A-containing proteoliposomes. Data show averages of three measurements obtained from one reconstitution. f, Ca²⁺-dependent scrambling in proteoliposomes containing nhTMEM16. In e,f, liposomes not containing any protein (neg.) are shown as a control. g, Ca²⁺-independent scrambling in proteoliposomes containing IST2⁷¹⁶. Traces of empty liposomes and nhTMEM16 (displayed in b) are displayed as dotted lines for comparison. In f,g, data show the averages of nine measurements obtained from three independent reconstitutions. In e–g, traces depict the fluorescence decrease in tail-labeled NBD–PE lipids after the addition of dithionite (t = 0) in the absence and presence of 1 mM Ca²⁺. The dashed gray line indicates the 0.5 threshold. h, Comparison of fluorescence 150 s after the addition of dithionite in proteoliposomes containing nhTMEM16 or IST2⁷¹⁶. Bars show the average of nine experiments (displayed as dots). The fluorescence difference in the presence of Ca²⁺ is significant in the case of nhTMEM16 (P < 0.001) but not IST2⁷¹⁶ (P > 0.9999) as calculated by a two-way ANOVA using Šidák’s multiple comparisons test. For the statistical analysis, values from the same reconstitution were matched to account for differences in the reconstitution efficiency. In e–h, errors are the s.e.m.

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