Pi 4 5 p2

Liposomes 55% 16:0 PC, 25% 16:0 PS, 18% 16:0 PE, and 5% 18:1 (0.33 mM final) PI phosphates (PI(3,5)P₂ or PI(4,5)P₂; Avanti Polar lipids, Alabaster, AL) were prepared according to Banerjee and Kane, 2017 (link). Liposomes were resuspended in 9:20:1 CH₃OH:CHCl₃:H₂O, lyophilized for 30–40 min at 35°C, rehydrated in ice-cold 25 mM NaCl (pH 7.4) and 50 mM Tris-HCl, freeze-thawed five times, and extruded through a 100 nm filter twenty times. Liposomes and 1 µM PFN1 or mAp-PFN1 were incubated for 30 min at room temperature, and pelleted at 400,000 × g. Collected supernatants and pellets were resuspended in equal volumes (100 µL) of buffer, precipitated in 10%_(v/v) trichloroacetic acid, washed with cold 100% acetone, and dissolved in 50 µL of 50 mM Tris-HCl (pH 6.8), 8 M urea, 5% SDS, and 1 mM EDTA. Blots were probed as described in figure legends. Densitometry was performed in Fiji (Schindelin et al., 2012 (link)).

Lipids: DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), 18:1 biotinyl cap PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)), DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), cholesterol (ovine wool), PI(4,5)P2 (L-α-phosphatidylinositol-4,5-bisphosphate, brain, porcine), TopFluor TMR (1-oleoyl-2-(6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino)hexanoyl)-sn-glycero-3-) PI(4,5)P2 and PS were purchased from Avanti Polar Lipids. Lipophilic tracer DiD, NeutrAvidin, biotinylated bovine serum albumin, were from Thermo Fisher Scientific.

TopFluor conjugated PI, PS, and PI(4,5)P₂ were obtained from Avanti Polar Lipids Inc. (810187P, 810283P, and 810184P, respectively). Lipid-BSA complexing was accomplished using 5 µM (0.34 mg/ml) fatty acid–free BSA (Sigma; 126575) dissolved in PBS with 3 mM EGTA. Fluorescent lipids were sonicated in methanol at a concentration of 1 mM before injecting 5 µl into 1 ml BSA/PBS mixture and vortexed at medium to high speed and incubated at 37°C for 20–30 min. Back-extraction media were made using 17 mg/ml fatty acid–free BSA (i.e., 250 µM, five times the final concentration applied to cells) in serum-free imaging media and filtered.

DOPE (Avanti Polar Lipids; 850725), DOPC (Avanti Polar Lipids; 850375), NBD-PS (Avanti Polar Lipids; 810198), Liss Rhod-PE (Avanti Polar Lipids; 810150), DGS-NTA(Ni) (Avanti Polar Lipids; 790404), PI4,5P2 (Avanti Polar Lipids; 840046), glyceryl trioleate (Sigma-Aldrich; T7140), BODIPY 558/568 C12 (Invitrogen; D3835), Imperial protein stain (Thermo Scientific; 24617), Flag M2 resin (Sigma-Aldrich; A2220), WIPI-4-TurboGFP (Origene; WDR45-tGFP transcript variant 1, RG209654), BSA-Oleate monounsaturated fatty acid complex and BSA control (Cayman Chemical; 29557 and 29556), rabbit anti-ATG2A (Cell Signalling; 15011), rabbit anti-ATG2B (Sigma-Aldrich; HPA019665), mouse anti-WIPI-4 (Santa Cruz Biotechnology; sc398272), rabbit anti-LC3B (D11) (Cell Signalling; 3868), rabbit anti-p62 (Cell Signalling; 95697), rabbit anti-Plin3 (Sigma-Aldrich; HPA006427), rabbit anti-beta-actin (Cell Signalling; 4970), mouse anti-Flag M2 (Millipore; F1804), rabbit anti-GFP (Cell Signalling; 2956), secondary antibodies, AlexaFluor 647 goat anti-mouse (Life Technologies; A21236), sodium dithionite Sigma-Aldrich (157953), DMEM (Gibco; 11965-092), MEM (Gibco; 31985-070), Expi293 medium (Gibco; A14351-01), 1x EBSS (Gibco; 24010-043).

Syntaxin 1 TMD (residues 266–288; sx-1 TMD Rattus norvegicus sequence) and syntaxin 1 TMD mutant (sx-1 TMD with the following mutations: K265A and K266A) were synthesized using Fmoc solid-phase synthesis as described in Ref. 16 (link). The fluorescent dyes Atto647N NHS-ester (Atto-Tec) and Rhodamine red succinimidyl ester (Life Technologies) were coupled to the N termini of the peptides during the Fmoc synthesis.
DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine, and PI(4,5)P₂ (1,2-dioleoyl-sn-glycero-3-phosphatidyl-(1′-myo-inositol-4′,5′- bisphosphate)) were purchased from Avanti Polar Lipids. Atto647N labeled at the SN1 position of PI(4,5)P₂ and Atto590 coupled to ceramide were gifts from Dr. Vladimir Belov (MPI-BPC, Göttingen, Germany).

POPC (#850457), POPE (#850757), POPS (#840034) and PI(4,5)P₂ (#850155) were purchased from Avanti Polar Lipids, Inc and stored in chloroform and/or methanol at −20°C until use. For large unilamellar vesicle (LUV) preparation, lipid mixtures were prepared at the indicated compositions (see SPR section), dried down to lipid films under a continuous stream of N₂, and stored at −20 °C until further use. On each day of experiments, LUVs were brought to room temperature, hydrated in SPR buffer (10 mM Hepes, pH 7.4 containing 150 mM NaCl), vortexed vigorously, and extruded through a 100 nm filter 17 times.