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
Data files
Aug 28, 2025 version files 302.72 MB
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Data_Ist2.zip
302.71 MB
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README.md
9 KB
Abstract
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.
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
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Author Information
A. Principal Investigator Contact Information
Name: Raimund Dutzler
Institution: University of Zurich
Address: Winterthurerstrasse 190, Zurich 8057, Switzerland
Email: dutzler@bioc.uzh.chB. Alternate Contact Information
Name: Melanie Arndt
Institution: University of Zurich
Address: Winterthurerstrasse 190, Zurich 8057, Switzerland
Email: m.arndt@bioc.uzh.ch -
Date of data collection March 2021 - October 2025
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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
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.