Supporting Information. Lipid-dependent bimodal MCL1 membrane activity

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1 Supporting Information Lipid-dependent bimodal MCL1 membrane activity Olatz Landeta 1ǂ, Juan GarciaValero 1ǂ, Hector Flores-Romero 1, Itsasne Bustillo- Zabalbeitia 1, Ane Landajuela 1, Miguel Garcia-Porras 1, Oihana Terrones 1, Gorka Basañez 1 * ǂ Both authors contributed equally to the work *Corresponding author: gorka_basanez@ehu.es This PDF includes Supplemental Figures S1-S5 Supplemental Methods 1

2 Figure S1. (a) Effect of wild-type (wt) and mutant (mt) BAX, BAK C and cbid proteins in ANTS/DPX release from MOM-like LUV. Protein concentrations were 150nM, and ANTS/DPX extents were taken at 1000s. Data correspond to mean ± S.E.M. (n=3-4). (b) Intrinsic fluorescence spectra of MCL1 N C, MCL1 N Cmt1 and MCL1 N Cmt2. (c) Dose-dependent effect elicited by indicated combinations of BCL2 family proteins and drugs in vesicular ANTS/DPX release (n=2-3). (d) Effect oftw37, AT101, and Obatoclax/GX (GX15) on MCL1 Ν C inhibition of vesicular ANTS/DPX release from MOM-like LUV (n=3). BAX, cbid concentration were 150nM, MCL1 Ν C concentration was 600nM, and drug concentration was 1200nM 2

3 Figure S2. Analysis of MCL1 Ν C conformation in MOM-like membranes of different composition by site-specific NBD labeling and fluorescence spectroscopy.average NBD fluorescence intensity ratios of monocysteine MCL1 Ν C mutants incubated with indicated proteins andluv composed of 55PC/35PE/10PImol/mol (0CL), 35PC/35PE/30PI mol/mol (20PI), 30PC/35PE/10PI/10CL/25CHOLmol/mol (25CHOL). Data correspond to mean ± S..E. M. (n=3-4). 3

4 Fig. S3. (a) Representative NBD fluorescence spectra of indicated monocysteine MCL1 Ν C mutant incubated with 100CL LUV incorporating 20mol% Dox5-PC, 20mol% Dox14-PC, or no lipid quencher. (b) Dose-dependent effect of Dox5- and Dox14-mediated quenching of indicated NBD-labeled monocysteine MCL1 C mutants. Data correspond to mean ± S.E.M. (n=2-3). 4

5 Fig. S4. (a) Analysis of MCL1-derived peptide insertion into MOM-like (10%CL) or 100%CL monolayers. (b) Effect of MCL1α5 and BCLXLα5 peptides in ANTS/DPX release from LUV composed of 100%CL (CL), 80%CL720%LPC (LPC), 80%CL/20%DAG (DAG), or 75%CL/25%CHOL (CHOL). Data are shown as mean ± S.E.M. (n=2-4). (c) Dose- and time-dependent effect of MCL1α5 in the release of cyt c from BAX/BAK DKO MEF mitochondria. (d) Dose-dependent effect of BCLXLα5 peptide in the release of cyt c from BAX/BAK DKO MEF mitochondria. (e) Dosedependent effect of different MCL1-derived peptides in the release of cyt c from BAX/BAK DKO MEF mitochondria. Cyt c release data are representative of at least two independent experiments. 5

6 Fig. S5. (a) Analysis of MCL1α5 effect on plasma membrane integrity of DKO MEF assessed by Propidium Iodide (PI) staining. (b) Effect of MCL1-derived peptides in the release of cyt c from semi-intact BAX/BAK DKO fibroblasts.data shown are representative of two independent experiments. 6

7 Supplemental Methods Materials. Egg phosphatidylcholine (PC), egg phosphatidylethanolamine (PE), rat liver phosphatidylinositol (PI), bovine heart cardiolipin (CL), Cholesterol (CHOL), egg lysophosphatidylcholine (LPC), egg diacylglycerol (DAG), 1-palmitoyl-2-stearoyl-(5- doxyl)-sn-glycero-3-phosphocholine(5-no-pc), and 1-palmitoyl-2-stearoyl-(14-doxyl)- sn-glycero-3phosphocholine (14-NO-PC) were purchased from Avanti Polar Lipids.pyPC was a generous gift of Hermann Muller. KCl, CaCl 2, HEPES, EDTA, TCEP, CsA, dodecyl octaethylene glycol monoether (C 12 E 8 ), alamethicin, FD10, and FD70 were obtained from Sigma (Cambridge, UK). NBD, 1, 3, 6, aminonaphtalene-trisulfonate (ANTS) and p-xilene-bis-dipicolyinicacis (DPX) were purchased from Molecular Probes. TW37, AT101 and Obatoclax were purchased from SelleckChem.com. Anti-cytochrome c 7H8.2C-12 antibody and anti-tom monoclonal antibodies were from BD life Sciences. Synthetic peptides (>85% purity) were purchased from Biomatik. Peptide sequences were as follows: MCL1α1: 170 EEDELYRQSLEIISRYLREQATGA 193 ; MCL1α2: 200 RSGATSRKALETLRRVGDGVQRN 223 ; MCL1α3: 224 HETAFQGMLRKLD 236 ; MCL1α4: 237 IKNEDDVKSLSRVMIHVFSDG 257 ; MCL1α5: 258 VTNWGRIVTLISFGAFVAKHLKTINQ 283 ; MCL1α6: 284 ESCIEPLAESITDVLVLRT 302 ; MCL1α7α8: 303 KRDWLVKQRGWDGFVEFFHVEDLE 326 ; MCL1α9: 327 GGIRNVLLAFAGVAGVGAGLAYLIR 350 ; MCL1α5 K276E,K279E : 258 VTNWGRIVTLISFGAFVAEHLETINQ 283 ; BCLXLα5: 134 GVNWGRIVAFFSFGGALCVESVDKEM 159 Labeling of Cysteine-substituted proteins with IANBD: In a typical labeling reaction, mg of a cysteine-substituted MCL1 C derivative was first diluted to 1ml in KHE+1mM TCEP, incubated for 30min at room temperature, and passed over a desalting pre-packed PD10 column (biorad) equilibrated and eluted with KHE to remove TCEP. The eluted protein was concentrated to approximately 1ml 7

8 using aamiconmicroconcentrator, and IANBD dissolved in DMSO was then added to the protein solution to give a final probe/protein molar ration of at least 10:1. The mixture was incubated at room temperature for 2h or at 4ºC overnight. Next, the reaction was quenched by the addition of 1mM TCEP, and the mixture was passed again over a PD-10 column equilibrated with KHE. The molar concentrations of the protein and conjugated NBD were determined from the absorbance at 280nm and 488nm, using the molar extinction coefficients (ε M ) of 19,300 M -1 cm -1 and 26,500 M - 1 cm -1, respectively. 31 P-NMR Measurements Samples for 31P NMR were prepared by dispersing 15 µmol of dry lipid mixtures in either 0.5 ml of KHE buffer alone or 0.5 ml of KHE buffer containing the protein/peptide of interest. Multilamellar vesicle suspensions were freeze-thawed 3 times in liquidn 2 to disperse the added proteins in the lipid membranes, and the liposomes were spun down in an Eppendorf centrifuge (14,000 rpm, 15 min, 4 C). Pellets were loaded directly into 5-mm Pyrex NMR tubes. Samples were equilibrated for 20 min at each temperature before data acquisition.high power, proton noisedecoupled 31P NMR spectra were recorded on a Bruker AV-500 spectrometer operating at MHz using 5-mm broadband inverse probes with z-gradient equipment free induction decays were averaged using a 2-s recycle delay. Spectra were processed and evaluated using TOPSPIN 1.3 (Bruker) and plotted with 80-kHz line broadening. Monolayer surface pressure measurements: Measurements were carried out with a MicroTrough-S system from Kibron (Helsinki, Finland) at 37 C with constant stirring. The lipid, dissolved in chloroform, was gently spread over the surface and kept at a constant surface area. The desired initial surface pressure, πi, was attained by changing the amount of lipid applied to the airwater interface. After 10 min to allow for solvent evaporation, the protein was injected through a hole connected to the subphase. The final protein concentration in the Langmuir trough was 0.5 µm. The change in surface pressure, π,was recorded as a function of time until a stable signal was obtained. The subphase buffer was 1.0 ml of KHE buffer (ph 7.0). The linear plot of π as a function of πi can be extrapolated to a 8

9 πi of 0 to give the critical pressure, πc, which is a measure of the relative penetration capacity of a protein into the monolayer. Cell culture: Wildtype(WT) and SV40 transformed BAX -/- /BAK -/- DKO mouse embryonic fibroblasts (MEFs) were kindly provided by Dr. I Marzo (University of Zaragoza, Spain). MEFs were grown in Dulbecco's Modified Eagle Medium (Invitrogen, U.S.A); supplemented with 10% fetalbovine serum (Invitrogen), 1% Penicylin-Streptomicyn-Ionomycin solution (Invitrogen) and 1% of non-essential amino acids (Invitrogen). Cells were transfected using JetPrime according to the manufacturer s instructions ( cells per/plate, cultured overnight before transfection). Cell membrane integrity assay: DKO MEF cells were treated with increasing concentrations of MCL1α5 peptide in the presence of propidium iodide (PI, 1µg/mL) and Hoescht (0,5µg/mL, 5 min, 37 o C). Cell permeabilization was followed by fluorescence microscopy upon PI nuclear staining. PI is cell membrane-impermeable and stains the nuclei of cells with damaged membranes with strong red fluorescence. Hydrogen peroxide (H 2 O 2, 3 mm) was added as a positive control for permeabilized cells. 9