Extended Data Fig. 9: Functional characterization of IST2 constructs by yeast complementation assays.
From: Structural basis for lipid transport at membrane contact sites by the IST2–OSH6 complex
a, Scheme of the IST2/OSH6 complex bridging the ER and the PM. The residue range of the indicated parts of IST2 is shown. b, Normalized mean gray value quantification of spot assay dilution series of IST2 transformed into ist2/psd1 KO cells under inducing conditions (2% Galactose, n = 27). Dashed line indicates 1:100 dilution, which was used for evaluation and comparison of all other constructs. For each dilution series, values were normalized to the maximum and minimum mean gray value. Data was fit with a sigmoidal model using GraphPad prism. c-f, Yeast spot dilution series used for FIJI-based evaluation shown in Fig.6. Shown are c, WT in comparison to the construct IST2921 lacking the C-terminal attachment site to the PM and constructs where the OSH binding site was moved by 83 residues in N or C-terminal direction of the linker, d, mutations in the OSH6 binding region, e, constructs with decreasing liker length, f, fusions of OSH6 into the linker. Images show representative 10-fold dilution series of n = 3×3 (n = 2×3 for Fusion trunc. and Fusion ext.) replicates for plates containing 2% galactose and 1 mM choline for each IST2 construct transformed into ist2/psd1 KO cells. Neg. refers to cells transfected with pYESnSM3 vector only. Plates were imaged with a Vilber Fusion FX detection system and the background was subtracted using FIJI. g, Size exclusion chromatogram of the purified IST2-OSH6 fusion construct (Fusion) in comparison to IST2 WT.
