Lipid scrambling assay and yeast growth assay data demonstrating the ability of IST2 to (i) scramble lipids in vitro and (ii) facilitate lipid transport by Osh6 in S.cerevisiae

In the associated article, we present a comprehensive investigation elucidating structural and mechanistic properties of the tethering protein IST2 and its interaction with the soluble lipid transport protein OSH6. The ER-embedded transmembrane domain of IST2 is homologous to the TMEM16 family and acts as constitutively active lipid scramblase. The extended C-terminus binds to the plasma membrane and the PS/PI4P exchanger OSH6. Through cellular growth assays, biochemical and structural studies, we have elucidated the interaction between both proteins and provide evidence that OSH6 remains associated with IST2 during lipid shuttling between membranes. The dataset features raw data from lipid scrambling and cellular yeast complementation assays. 

Methods
Lipid Scrambling Assay

For the assay, liposomes were diluted to a final concentration of 20 µM in Assay Buffer (80 mM HEPES pH 7.5, 300 mM KCl, 2 mM EGTA) in a quartz glass cuvette. After recording the fluorescence of NBD for 60 s, 30 mM dithionite was added to bleach the fluorescence of NBD in the outer leaflet of the liposomes. The fluorescence was recorded for an additional 6 minutes. Each trace was normalized to the NBD fluorescence level right before the addition of dithionite. Data was recorded using FluorEssence 3.0 (HORIBA Scientific). Statistical analysis was performed using ANOVA in GraphPad Prism. Because the final plateau differs depending on the reconstitution efficiency, for a statistical analysis samples reconstituted from the same batch of destabilized liposomes on the same day were matched in the analysis.

Yeast Growth Assays

IST2 constructs were transformed into a S. cerevisiae ist2/psd1 KO strain (AWY327, provided by Dr. Chris Loewen), using the LiAc/SS carrier DNA/PEG method and plated onto SC-Ura selection medium containing 1mM choline bitartrate. Plates were kept at 4 ºC for 4 weeks maximum. Single colonies were picked and grown in liquid SC-Ura medium containing 1 mM choline to mid-log phase. Cells were harvested by centrifugation and pellet was washed 3 times with sterile water to remove all traces of choline. Cells were subsequently diluted to an OD600 of 0.35 and a 10x dilution series was spotted in three replicates on solid media containing 2% dextrose and 2% galactose or 1 mM choline. Plates were incubated at 30 ºC for 3-4 days. The experiment was repeated 3 times with different transformants for each construct, except otherwise stated. For evaluation, plates were imaged in a Vilber Fusion FX using bright field illumination. For all tranformants grown on galactose-containing The background was subtracted in FIJI and the mean grey value of each spot was quantified using a circular mask of equal size for all replicates and all constructs. The quantification of the 1:100 dilution was chosen for comparison between constructs as it was found in the linear range of the dilution series. For each replicate, all constructs were normalized to IST2 WT. Statistical analysis was performed in GraphPad Prism using ANOVA.