DOI: 10.5061/dryad.8w9ghx40q
title: 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

description: 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. 
description: <strong>Lipid Scrambling Assay</strong><br>
 <br> 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.<br> <br> <strong>Yeast
 Growth Assays</strong> IST2 constructs were
 transformed into a <em>S. cerevisiae</em>
 <em>ist2/psd1</em> 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 OD<sub>600</sub> 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.
description: # 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. Through liposome-based lipid
 scrambling assays, we demonstrate that IST2, a member of the TMEM16
 family, acts as a constitutively active scramblase, which is lacking
 Ca2+-regulation otherwise common within the TMEM16 family. In these
 assays, we reconstitute the transmembrane domain of IST2 (IST2 1-716) or
 the controls nhTMEM16 (positive, Ca2+-dependent) and TMEM16A (negative)
 into liposomes spiked with fluorescently labeled lipids and record the
 decrease in fluorescence after addition of a membrane-impermeable reducing
 agent bleaching the lipids in the outer leaflet. Fluorescence decays to
 below 50% of the initial value indicate scrambling. The extended
 C-terminus binds to the plasma membrane and the PS/PI4P exchanger OSH6 and
 acts as a linker between the ER and the plasma membrane. Through cellular
 growth assays (yeast spot assays) in a growth-deficient S.cerevisiae
 ist2/psd1 KO strain, we show that OSH6 remains associated with IST2 during
 lipid shuttling between membranes. We use complementation of growth as a
 readout for lipid transport. S.cerevisisae ist2/psd1 KO strains were
 transformed with plasmids encoding for various IST2 tail mutants and
 overexpression of the IST2 constructs was induced by adding 2% Galactose
 to solid media. After 3-5 days, plates were imaged. For better
 visualization of the data, quantification of the 1:100 dilution was
 performed as follows: images were imported into FIJI, the background was
 removed. The mean gray value of the spot corresponding to the 1:100
 dilution was quantified using a circular mask of equal size for all
 conditions. The data was exported to excel and normalized to the average
 mean gray value of the WT (IST2fl) control condition for each biological
 replicate. Biological replicates correspond to experiments performed on
 different days with different transformants. Each biological replicate was
 performed as three technical replicates to account for pipetting errors. #
 General Information 1. Author Information A. Principal Investigator
 Contact Information Name: Raimund Dutzler Institution: University of
 Zurich Address: Winterthurerstrasse 190, Zurich 8057, Switzerland Email:
 [dutzler@bioc.uzh.ch](mailto:dutzler@bioc.uzh.ch) B. Alternate Contact
 Information Name: Melanie Arndt Institution: University of Zurich Address:
 Winterthurerstrasse 190, Zurich 8057, Switzerland Email:
 [m.arndt@bioc.uzh.ch](mailto:m.arndt@bioc.uzh.ch) 2. Date of data
 collection March 2021 - October 2025 3. Geographic location of data
 collection : Winterthurerstrasse 190, Zurich 8057, Switzerland ##
 Description of the data and file structure Data_Ist2.zip: Folder
 containing data listed below Dataset1_Scrambling_Assay: *
 Data_Scrambling_Assay_Unprocessed.csv (contains raw fluorescence traces,
 each column corresponds to one trace. Headers of each column indicate the
 reconstituted proteins, addition/absence of 1mM Ca2+ and the replicate
 number. Numbers (1,2,3) correspond to "biological replicates"
 from independent reconstitutions, letters (a,b,c) to the technical
 replicate of each reconstitution. * Data_Scrambling_Assay_Processed.csv
 (Data normalized to fluorescence intensity at the datapoint before
 reducing agent addition after 1min) Dataset2_Yeast_Growth_Assay:
 -Raw_Image_Data (Folder) \- Bindingsite_mutants (Folder, contains images
 used for quantification of IST2fl, empty vector, and two different
 mutations in the Osh6 binding site as .tiff files. The abbreviations for
 the transformants plated in a specific row (scrd for scrambled binding
 site and R750A for point mutant) are writting at the edge of the plate) \-
 Fusions\ (Folder, contains images used for quantification of vector,
 IST2fl, IST2_OSH6_fusion (FUS) and a fusion construct with shortened
 linker (Fus 1/2) and extended linker (Fus GS) as .tiff files. The
 abbreviations for the transformants plated in a specific row are written
 at the edge of the plate. Note that rows labeled with 1/2 were not used in
 the analysis. \- Linker_truncations (Folder, contains images used for
 quantification of vector, IST2fl, and truncations of the Ist2 IDR by 75%
 (3/4), 50% (1/2), 33% (1/3) and 25% (1/4) as .tiff files. The
 abbreviations for the transformants plated in a specific row are written
 at the edge of the plate.) \- Move_bindingsite_C-term_truncation (Folder,
 contains images used for quantification of vector, IST2fl, re-placing of
 the OSH6 binding site within the IST2 IDR to the N-Terminus (MoveN) or
 C-Terminus (MoveC) and c-terminal truncations of IST2 (IST2_716, pMA10 and
 IST2_921, pMA50) as .tiff files. The abbreviations for the transformants
 plated in a specific row are written at the edge of the plate, any other
 cells on the plates except the ones mentioned are not included in the
 analysis.) Every subfolder contains the images from the inducing
 conditions at 2% Galactose ("Gal") and a control image of a
 plate containing 1mM Choline-Bitartrate ("Cho"), which rescues
 the growth defect of the ist2/psd1 KO strain. Numbers (1,2,3) correspond
 to biological replicates from independent transformants, letters (a,b,c)
 to the technical replicates. -Quantification_Unprocessed.xlsx (Contains
 Mean Grey Values measured in FIJI for all replicates of all constructs)
 Explanation:\ The data contains a header indicating the biological (1-3)
 and technical (a-c) replicate number. n/a means no data was recorded.\
 Data is labeled as follows: | Label | Explanation | |
 :---------------------------- |
 :------------------------------------------------------------------ | |
 vec ctrl truncation | negative control for constructs 0.25, 0.33, 0.75 | |
 Ist2fl ctrl truncation | positive control for constructs 0.25, 0.33, 0.75
 | | 0.25 | IDR truncation to 25% | | 0.33 | IDR truncation to 33% | | 0.5
 | IDR truncation to 50% | | 0.75 | IDR truncation to 75% | | vec ctrl
 mutants | negative control for constructs scrd and R750A | | Ist2fl ctrl
 mutants | positive control for constructs scrd and R750A | | scrd | IDR
 with scrambled OSH6 binding site | | R750A | IDR with R750A mutation | |
 vec ctrl move C-term trunc | negative control for constructs Ist2 716,
 Ist2 921, MoveN and MoveC | | Ist2fl ctrl move C-term trunc | positive
 control for constructs Ist2 716, Ist2 921, MoveN and MoveC | | Ist2 716 |
 C-terminal truncation of IDR after residue 716 | | Ist2 921 | C-terminal
 truncation of IDR after residue 921 | | MoveN | OSH6 binding site moved
 towards N-terminus of IDR | | MoveC | OSH6 binding site moved towards
 C-terminus of IDR | | vec ctrl fusions | Negative control for constructs
 Fus, Fus trunc, Fus ext | | Ist2fl ctrl fusions | Positive control for
 constructs Fus, Fus trunc, Fus ext | | Fus | OSH6 sequence fused into IDR
 | | Fus trunc | OSH6 sequence fused into IDR with IDR truncated by 50% | |
 Fus ext | OSH6 sequence fused into IDR with flanking GS linker sequence |
 | 
 
 | 
 
 | -Quantification_Processed.xlsx\ (Contains the mean gray value
 quantifications obtained from FIJI, as in
 *Quantification_Unprocessed.xlsx*, with additional normalization analysis
 applied for the data used in the publication figure.) ## Sharing/Access
 information N/A ## Code/Software Raw data from the lipid scrambling assay
 was curated and normalized in Microsoft Excel. For visualization, data was
 imported to GraphPad Prism ver 10.4.1. Raw image data from the yeast
 growth assays was imported, processed and quantified in FIJI as described
 above. Data was curated and normalized in Excel. For visualization, data
 was imported to GraphPad Prism ver 10.4.1