Originally published 24 July 2015; corrected 5 October 2015

www.sciencemag.org/content/349/6246/428/suppl/DC1

Supplementary Material for

PI4P/phosphatidylserine countertransport at ORP5- and ORP8-

mediated ER–plasma membrane contacts

Jeeyun Chung, Federico Torta, Kaori Masai, Louise Lucast, Heather Czapla, Lukas B.

Tanner, Pradeep Narayanaswamy, Markus R. Wenk, Fubito Nakatsu,*

Pietro De Camilli*

*Corresponding author. E-mail: pietro.decamilli@yale.edu (P.D.); nakatsu@med.niigata-u.ac.jp (F.N.)

Published 24 July 2015, Science 349, 428 (2015)

DOI: 10.1126/science.aab1370

This PDF file includes:

Materials and Methods

Figs. S1 to S16

Table S1

Legends for Movies S1 to S3

Other Supplementary Material for this manuscript includes the following:

(available at www.sciencemag.org/content/349/6246/428/suppl/DC1)

Movies S1 to S3

Correction: The number of phosphatidylserine molecules in the original fig. S16 did not

reflect their higher concentration in the plasma membrane relative to the ER membrane.

The figure has been corrected here. In addition, legends for the supplementary movies

have been added.

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Materials and Methods

Special reagents and antibodies

The A1 compound (25) was a kind gift from Tamas Balla (National Institutes of Health). Rapamycin was purchased from Calbiochem. Oxo-M and Atropine were purchased from Sigma-Aldrich. All electron microscopy reagents were purchased from Electron Microscopy Sciences (Hatfield, PA). All lipids were purchased from Avanti Polar Lipids.

Primary antibodies used in this study were: monoclonal anti-GFP (BD Biosciences), monoclonal anti-FLAG (Sigma-Aldrich, clone M2), polyclonal anti-ORP5 (Abcam, ab59016), polyclonal anti-ORP8 (Abcam, ab99069), polyclonal anti PIPKIγ (our lab(34)), Sac1 (our lab (35)), and monoclonal anti-α-tubulin (Sigma Aldrich, clone B-5-1-2).

Plasmid construction

The following reagents were kind gifts: mRFP-Sec61β from Tom Rapoport (Harvard University), M1 muscarinic acetylcholine receptor (M1R) from Bertil Hille (University of Washington), GFP-PHPLCδ from Antonella De Matteis (Telethon Institute of Genetics and Medicine), CFP-PM-FRB from Tamas Balla (National Institutes of Health), mCherry-ORP8 from Hongyuan Yang (University of New South Wales), and GFP-evt2-2XPH from Hiroyuki Arai and Tomohiko Taguchi (University of Tokyo). LactC2–GFP (#22852), mRFP-FKBP-PJ (#37999), and iRFP-P4M (#51470) were obtained from Addgene. GFP-PI4KIIIα, HA-EFR3B, and FLAG-TTC7B were previously generated in our lab (20). Protein-coding cDNA clones corresponding to human ORP5 (clone ID 5769002) was from Thermo Fisher Scientific. pEGFP-N1 and pEGFP-C1 vectors were purchased from Clontech Laboratories. p3XFLAG-CMV-10 vector was purchased from Sigma Aldrich.

2XPHORP5-GFP was generated by overlap extension PCR. Two separate cDNAs encoding the PH domain of ORP5 (PHORP5) were amplified by PCR from ORP5 cDNA using the following primer pairs: 5'- GCACA CTCGAG GCC ACC ATG GGG TCA GAC AAG GAA TGT GTG TCC-3' and 5'- GCACA GGTACC GGT GCC CAG TCT CAG TAG GCT AG-3'; 5'-GCACA GGTACC GGG TCA GAC AAG GAA TGT GTG TCC-3' and 5'-GCACA AAGCTT GGT GCC CAG TCT CAG TAG GCT AG-3'. These two PCR amplicons were then used as a template for an overlap PCR reaction and amplified with the primer pair 5'- GCACA CTCGAG GCC ACC ATG GGG TCA GAC AAG GAA TGT GTG TCC -3' and 5'- GCACA AAGCTT GGT GCC CAG TCT CAG TAG GCT AG -3'. The overlap PCR amplicons, encoding the tandem PH domain were sub-cloned into pEGFP-N1 vector using XhoI and HindIII sites.

ΔORDORP5-GFP was generated by overlap extension PCR. ORP5 (1-334) fragment and ORP5 (751-879) were amplified by PCR from ORP5 cDNA using the following primer pairs: ORP5 (1-334), 5'- GCACA CTCGAG CT ATG AAG GAG GAG GCC TTC CTC CGG-3' and 5'- GCCAGACCTCTCGTGCCT CCCAGGGGTCTCTGACTG-3'; ORP5 (751-879), 5'-

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CAGTCAGAGACCCCTGGG AGGCACGAGAGGTCTGGC-3' and 5'- GCACA AAGCTT CTA TTT GAG GAT GTG GTT AAT GAA C-3'. The PCR amplicons were then used as a template for an overlap PCR reaction and amplified with the primer pair 5'- GCACA CTCGAG CT ATG AAG GAG GAG GCC TTC CTC CGG -3' and 5'- GCACA AAGCTT CTA TTT GAG GAT GTG GTT AAT GAA C -3'. The overlap PCR amplicons were sub-cloned into pEGFP-C1 vector using XhoI and HindIII sites.

The cloning strategies of the other plasmids were summarized in Table S1.

Generation of PI4KIIIα conditional KO MEFs PI4KIIIα conditional KO MEFs were generated as previously described (20). Gene recombination in these cells was induced by treatment with 0.5 µM (Z)-4-hydroxytamoxifen (4-OHT), or with vehicle (ethanol) as a control, for 24–36 h, followed by replacement of the media and subsequent growth for a total period of 7–10 days.

Cell culture, Transfection, and RNA Interference

HeLa cells and PI4KIIIα conditional KO MEFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, and 1% penicillin/streptomycin supplement at 37 °C and 5% CO2. Expi293F cells (Life Technologies) were cultured in Expi293TM expression medium (Life Technologies) on the orbital shaker platform with 125rpm, at 37°C and 8% CO2.

For transfection of DNA plasmids, MEFs were electroporated using the Amaxa Nucleofector method (Lonza). HeLa cells were transfected using either Lipofectamine 2000 (Life Technologies, for immunoprecipitation experiment) or FuGENE HD (Promega, for live cell imaging) as transfection reagents. Expi293F cells were transfected with plasmids using ExpiFectamineTM 293 transfection kit (Life Technologies).

Specific knockdown of Sac1 in HeLa cells was performed by transfection of small interfering RNA (siRNA) duplexes with the following sequences with Lipofectamine RNAiMAX (Life Technologies): control (NC1 negative control duplex from Integrated DNA Technologies), Sac1 (5'-rArArUrCrCrArUrGrCrArArUrUrGrCrUrUrCrGrGrArArCrArCrGrC-3' and 5'-rGrUrGrUrUr CrCrGrArArGrCrArArUrUrGrCrArUrGrGrATT-3' (36), ORP5 (5’-rGrGrArUrUrArCrArCrCrCrUrU rArCrCrArUrGrCrCrCrUAC-3’ and 5’-rGrUrArGrGrGrCrArUrGrGrUrArArGrGrGrUrGrUrA rArUrCrCrUrC), and ORP8 (5’-rGrArGrUrGrGrUrCrUrUrGrCrArArArUrUrArUrUrUrGrAAC-3’ and 5’-rGrUrUrCrArArArUrArArUrUrUrGrCrArArGrArCrCrArCrUrCrUrU-3’) from Integrated DNA Technologies.

Immunoprecipitation, and Immunoblotting

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For protein level analysis of PI4KIIIα KO MEFs (Fig 2B), cells were lysed in 1% SDS lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% SDS, and cOmplete Protease Inhibitor Cocktail tablet, EDTA-Free (Roche)). After sonication, cell lysates were mixed with Laemmli sample buffer and heated for 15 min at 98 °C prior to SDS-PAGE. For immunoprecipitation in Fig 1G, cells were lysed in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP40, and cOmplete Protease Inhibitor Cocktail tablet, EDTA-Free (Roche)). After incubation on ice for 30 min, the cell lysates were centrifuged at 16,000 g for 10 min at 4 °C, and the supernatants were collected. The supernatants were incubated with Chromotek GFP-trap agarose beads (Allele Biotech) and the solubilized bead-bound material was processed for SDS-PAGE and standard immunoblotting procedure. All Western blots were developed by chemiluminescence using the SuperSignal West Pico or Dura reagents (Thermo Fisher Scientific).

Live cell imaging

Cells were plated on 35 mm glass-bottom dishes (MatTek Corp). Imaging was carried out at 37 ºC approximately 18 hours after transfection. Before imaging, cells were transferred to imaging buffer (136 mM NaCl, 25 mM KCl, 2 mM CaCl2, 1.3 mM MgCl2, 10 mM HEPES, pH 7.4) that had been prewarmed to 37 ºC.

Spinning-disc confocal microscopy was performed using an UltraVIEW VoX spinning disc confocal microscope (Perkin Elmer) equipped with Perfect Focus, temperature-controlled stage, 14-bit EMCCD camera (Hamamatsu C9100-50) and controlled by Volocity software (Improvision). All images were acquired through a 60× objective (1.4 NA, CFI Plan Apo VC). Green fluorescence was excited with a 488 nm/50 mW diode laser (Coherent) and collected by a BP 527/55 filter. Red fluorescence was excited with a 561 nm/50 mW diode laser (Cobolt) and collected by a BP 615/70 filter. Multicolor images were acquired sequentially.

TIRF microscopy was performed at 37 °C using an objective-type inverted microscope (Ti-E;Nikon) fitted with a 60×, NA 1.49 TIRF microscopy lens (Nikon) and controlled by iQ software (Andor Technology). Laser lines (488, 561 nm, and 640nm) from diode-pumped solid state lasers (Coherent and Cobolt) were coupled to the TIRF microscopy condenser through an optical fiber. The calculated evanescent field depth was ∼100 nm. Cells were typically imaged without binning and with 0.2–0.3-s exposures and detected with a back-illuminated electron-multiplying charge-coupled device camera (512 × 512 pixels; 16 bit; iXon+ DU-897-BV; Andor Technology).

ORP8-ORD purification

3XFLAG-ORDORP8 was expressed in suspension cultures of Expi293F cells (Life Technologies) for 3 days. Cells were lysed by freeze–thawing in buffer containing 20mM Tris pH 8.0, 150mM NaCl, Halt protease and phosphatase inhibitor cocktail (Thermo Scientific). 3XFLAG-ORDORP8 was purified on anti-FLAG M2 affinity gel (Sigma-Aldrich) and eluted with 3 × FLAG peptides (Sigma-Aldrich) before further purification using size-exclusion chromatography.

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GST-ORDORP8-WT and GST-ORDORP8-H478/479A were expressed in BL21-CondonPlus (DE3)-RIPL cells (Agilent Technologies) grown in LB medium by induction (at D600=0.6) with 0.1mM isopropyl β-D-1-thigalactopyranoside at 18°C for overnight. Collected cells were suspended in the lysis buffer (50mM Tris pH8.0, 500mM NaCl, 0.5mM DTT, protease inhibitor cocktail (Complete, EDTA-free; Roche)) and lysed by sonication. The expressed proteins in the soluble fractions were captured on Glutathione Sepharose 4 Fast Flow beads (GE Healthcare) and cleaved by PreScission Protease (GE Healthcare) at 4°C for 18h.

In vitro lipid-exchange assay

Phosphatidylcholine (PC) and additional lipids were mixed in the indicated proportions and dried into thin films in glass-vials using nitrogen gas and then in the vacuum oven for at least 2h. The ‘heavy’ liposomes (400 nm) composed of PC/PI4P (90:10 mol/mol) were formed by rehydrating the lipid films in the rehydration buffer (50 mM Hepes, pH 7.2, 120 mM potassium-acetate, 1 mM DTT, and 0.75M sucrose) and the ‘light’ liposomes (100nm) composed of PC/PS (90:10 mol/mol) were formed by rehydrating the lipid films in the rehydration buffer without sucrose to the total lipid concentration of 2 mM at 37°C for 30 min. The suspension was then frozen and thawed five times (using liquid nitrogen and a water bath) and extruded sequentially through polycarbonate filters of 400-, 200-, and 100-nm (pore size) using a mini-extruder (Avestin). Additionally, ‘Heavy’ liposomes were pelleted at 16,100g for 15 min and washed twice with the rehydration buffer to clean out the excessive sucrose.

Recombinant ORDORP8 (5 µM) was incubated with the heavy and light liposomes (each corresponding to 2.0 mM total lipids) in 200 µl assay buffer (50 mM Hepes, pH 7.2, 120 mM potassium-acetate, and 1 mM DTT) at 25°C under agitation for 15 min. Heavy liposomes were then separated from light liposomes by centrifugation at 16,000g for 15 min. Levels of phosphoinositides in the pellet and in the supernatant (light liposomes) were then analyzed by separating glycerol-inositol phosphates with HPLC after phospholipid deacylation as described previously (37). Briefly, each fraction was subsequently incubated with 50% MeOH/ 1 M HCl/ 2 mM AlCl3 and CHCl3 to cause a phase separation. The lipid phase were washed with ice-cold 100% MeOH : 2 mM oxalic acid (1 : 0.9; vol/vol), and then dried under N2 gas. Resuspended lipids were deacylated by incubation with MeOH : 40% methylamine : n-butanol : water (47 : 36 : 9 : 8; vol/vol) at 50 °C for 45 min, dried and then resuspended in n-butanol : petroleum ether : ethyl formate (20 : 40 : 1; vol/vol), followed by the extraction with distilled water. Aqueous fractions were dried, resuspended in distilled water and then subjected to anion-exchange HPLC on an Ionpac AS11-HC column (Dionex, Sunnyvale, CA). Negatively charged glycerol head groups were eluted with a 1.5–86 mM KOH gradient and detected by suppressed conductivity in a Dionex Ion Chromatography system equipped with an ASRS-ultra II self-regenerating suppressor. Individual peaks were identified based on standards included in the run and peak areas were calculated using the Chromeleon software (Dionex). Data were shown as the mean of PS and PI4P levels (peak areas) from three independent experiments.

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In vitro lipid transport assay

PC, PI4P, PS and NBD-PE were mixed in the indicated proportions and dried into thin films in glass-vials using nitrogen gas and then in the vacuum oven for at least 2h. The ‘heavy’ liposomes (400 nm diameter) were formed by rehydrating the lipid films in the rehydration buffer (50 mM Hepes, pH 7.2, 120 mM potassium-acetate, 1 mM DTT, and 0.75M sucrose) and the ‘light’ liposomes (100nm diameter) were formed by rehydrating the lipid films in the rehydration buffer without sucrose to the total lipid concentration of 2 mM at 37°C for 30 min. The suspension was then frozen and thawed five times (using liquid nitrogen and a water bath) and extruded sequentially through polycarbonate filters of 400-, 200-, and 100-nm (pore size) using a mini-extruder (Avestin). Additionally, ‘Heavy’ liposomes were pelleted at 16,100g for 15 min and washed twice with the rehydration buffer to clean out the excessive sucrose.

For the PI4P transport assay, recombinant ORDORP8 (5 µM) was incubated with the ‘heavy’ PC/PI4P (90:10 mol/mol) liposome and ‘light’ PC/ NBD-PE (99:1 mol/mol) or PC/PS/ NBD-PE (89:10:1 mol/mol/mol) (each corresponding to 2.0 mM total lipids) in 200 µl assay buffer (50 mM Hepes, pH 7.2, 120 mM potassium-acetate, and 1 mM DTT) at 25°C under agitation for 15 min. For the PS transport assay, recombinant ORDORP8 (5 µM) was incubated with the ‘heavy’ PC/PS (90:10 mol/mol) liposome and ‘light’ PC/ NBD-PE (99:1 mol/mol) or PC/PI4P/ NBD-PE (89:10:1 mol/mol/mol) (each corresponding to 2.0 mM total lipids) in 200 µl assay buffer (50 mM Hepes, pH 7.2, 120 mM potassium-acetate, and 1 mM DTT) at 25°C under agitation for 15 min. Light liposomes were then separated from heavy liposomes by centrifugation at 16,000g for 15 min. Levels of phosphoinositides in the supernatant (light liposomes) were then analyzed by separating glycerol-inositol phosphates with HPLC after phospholipid deacylation as described above. NBD-PE intensities of the supernatants were measured by a fluorometer (Molecular Device) and these values were used to normalize total lipid contents of supernatants.

Mass spectrometry analysis of proteins and protein-associated lipids

For intact protein analyses, after purification the samples were desalted with a Vivaspin 10,000 MWCO device (GE Healthcare) and the initial buffer was exchanged with 20 mM ammonium acetate, pH 6.8. The sample was then directly infused at a flow of 1 µl/min and a concentration of 0.2 mg/ml into a quadrupole time-of-flight (QTOF) 6550 Agilent (Santa Clara, USA) mass spectrometer equipped with an Agilent 1260 series system. Native mass spectrometry analyses were performed in positive ion mode with the spray voltage set to 1500 V, temperature 150°C, drying gas flow 14 l/min and fragmentor 175 V. The samples were injected in 20 mM ammonium acetate, pH 6.8, and spectra acquired at a scan rate of 1 spectra/sec. Data were collected over a mass range of 1000-10000 m/z and deconvoluted using Bioconfirm software (Agilent) and the Maximum Entropy deconvolution algorithm. Denaturing mass spectrometry, to identify the possible ligands and to confirm the theoretical mass of the purified proteins, was performed injecting the proteins in positive and negative ion mode using conditions that could

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break the protein-ligand complex (acidic solutions containing 5% acetic acid in 20mM ammonium acetate or 25% acetonitrile or 30 eV collision energy) and denature the protein. Data were acquired between 100 and 1000 m/z (both positive and negative mode) to identify the ligands and between 1000 and 10000 m/z (positive mode) to monitor the protein conformational states. The structural identity of ligands was confirmed by collision-induced dissociation (CID) in positive and negative ion mode after selection of precursor ions in MSMS mode and fragmentation with collision energy values of 10, 20 and 40 eV (data not shown).

For lipid identification and relative quantification, a lipid extract obtained after sonicating (10 minutes) the protein (2 µg) in 100 µl of methanol or butanol/methanol 1:1 was collected after centrifugation of the sample and protein precipitation. For phosphoinositides (PIP) analysis, the sample was resuspended in solvent A (see below) and analysed by LC-MSMS in negative ion mode with the same mass spectrometer connected with Agilent 1260 series ChipLC system and using a C8 chip (Zorbax 300SB, Agilent). Solvents used for C8 reversed phase HPLC: 40% acetonitrile in water containing 5mM ammonium acetate (solvent A), 90% isopropanol/10% acetonitrile containing 5mM ammonium acetate (solvent B). LC gradient used: 10% to 20% B from 0 to 2 mins, 20 to 98% B from 2 to 7 mins, 98% B from 7 to 10 mins, 98 to 10% B from 10 to 10.1 min, 10% B to 15 min. The 6550 QTOF mass spectrometer was operated in negative ion mode; electrospray voltage was set to 1580 V (Vcap), temperature 200°C, drying gas 14 L/min, fragmentor voltage 175 V. The instrument was operated in auto MS/MS mode with MS acquisition rate of 4 spectra/sec and MS/MS acquisition rate of 2 spectra/sec. Data were collected over a mass range of 200-1300 m/z. For the other lipids the conditions were the same but the instrument was operated in positive ion mode. Relative quantification of the phosphoinositides released from ORP5/8 was confirmed by LC-MSMS (MRM mode) using a 6490 QQQ (Agilent) mass spectrometer, equipped with a 1260 series ChipLC system and a C8 chip (data not shown). The same chromatographic conditions previously developed for QTOF analysis were used. The QQQ mass spectrometer was operated in negative ion mode; electrospray voltage was set to 1580 V (Vcap), temperature 185°C, drying gas 12 L/min. The MRM transitions monitored included the doubly charged precursors and the fragments corresponding to the inositol headgroup (m/z 241) and the specific fatty acids. Relative quantification of the PS present in the extract was confirmed by LC-MSMS (MRM mode) using a 6460 QQQ (Agilent) mass spectrometer, equipped with a 1290 UHPLC series system and a C18 Agilent Zorbax RRHD Eclipse plus, 2.1 x 50mm, 1.8 µm (data not shown). Solvents used for C18 reversed phase UHPLC: 40% acetonitrile in water containing 10mM ammonium formate (solvent A), 90% isopropanol/10% acetonitrile containing 10mM ammonium formate (solvent B). LC gradient used: 20% to 60% B from 0 to 2 mins, 60 to 100% B from 2 to 7 mins, 100% B from 7 to 9 mins, 100 to 20%B from 9 to 9.01, 20% B from 9.01 to 10.8 min. The 6460 QQQ mass spectrometer was operated in positive ion mode; capillary voltage was set to 3500 V (Vcap), gas temperature 300°C, gas flow 5 l/min, nebulizer 45 psi, sheath gas temperature 250 0C, sheath gas flow 11 l/min. The MRM transitions monitored for PS included the fragments generated by neutral loss of phosphoserine (m/z 185). Lipid standards: 1,2-dioctanoyl-sn-

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glycero-3-phospho-(1'-myo-inositol) (PI 8:0/8:0), 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC 17:0/17:0), 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (PE 17:0/17:0), 1,2-diheptadecanoyl-sn-glycero-3-phospho-L-serine (PS 17:0/17:0), 1,2-diheptadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (PG 17:0/17:0), N-hexanoyl-D-erythro-sphingosylphosphorylcholine [SM (d18:1/6:0)], N-octanoyl-D-erythro-sphingosine [Ceramide (d18:1/8:0)] and D-glucosyl-ß-1,1' N-octanoyl-D-erythro-sphingosine [Glucosyl Ceramide (d18:1/8:0)] were purchased from Avanti polar lipids (Alabaster, AL, USA), 1,2-dioctanoyl-sn-glycero-3-phospho-(1'-myo-inositol) was purchased from Echelon Biosciences (USA).

Quantitative analysis of phospholipids of PI4KIIIα KO MEFs by HPLC/MS

Individual classes of phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid (PA) and sphingomyelin (SM) were analyzed by multiple reaction monitoring (MRM) as described previously (38, 39). Briefly, cell lipid extracts were dissolved in chloroform:methanol (C:M) 1:1 and separated using an Agilent 1200 HPLC system before introduction into a 3200 Q-Trap mass spectrometer (Applied Biosystems). Only HPLC grade solvents were used and lipid standards were obtained from Avanti Polar Lipids Inc. (Alabama, USA). Signal intensities for each lipid species were normalized to corresponding internal standards (dimyristoyl-glycero-phosphoserine (DMPS), 1,2-dimyristoyl-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-glycero-3-phosphocholine (DMPC), 1,2-dioctanoyl-glycero-3-phosphoinositol (C8PI), 1,2-ditetradecanoyl-sn-glycero-3-phosphate (DMPA) & lauroyl sphingomyelin (L-SM)) and represented as molar fractions of the total amount of measured phospholipids.

Electron microscopy

Control HeLa cells and HeLa cells expressing GFP-ORP5 (~90% confluent) were grown in 60 mm dishes and fixed in 2% glutaraldehyde-0.1M sodium cacodylate buffer pH 7.4. Cells were then scraped from the dishes, post-fixed with 1% OsO4 in 1.5% K4Fe(CN)6-0.1M sodium cacodylate buffer, followed by en bloc staining with 2% uranyl acetate in 50mM sodium maleate buffer pH 5.2, dehydration and embedding in Embed 812. Randomly selected cells were imaged at low magnification and cell perimeter was measured using iTEM by ©Olympus (average perimeter measured per cells: 84.5 µm). Number and length of ER-PM appositions were then counted in higher magnification images of these cells.

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Fig. S1. Schematic representation of the major classes of human ORP proteins. PH, pleckstrin homology domain; FFAT, two phenylalanines in an acidic tract motif; ORD, OSBP-related domain; ANK, ankyrin repeats; SUO, sequence unrelated to ORD; TM, trans-membrane segment. [modified from Olkkonen et al, 2013 (12)].

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Fig. S2. Sequence alignments of ORP and yeast Osh proteins demonstrating conservation of amino acids that crystallographic studies of ORDs have shown to be critical for PI4P (9) and PS binding (11), respectively. (A) Domain structures of ORP5 and ORP8 and of the two yeast Osh proteins, Osh6 and Osh7, whose ORDs are the most closely related to the ORDs of ORP5 and ORP8 (11, 12) (top). The structure of ORD of Osh6p in complex with PS (blue) is also shown

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(PDB code 4B2Z) (11). The N-terminal portion of the ORD, which represents the lipid “nesting” module (green), is the region most conserved between the ORDs of ORP5 and ORP8. Vertical black lines within the ORD of ORP5 (top) indicate location of conserved motifs important for PI4P or PS recognition as indicated in B and C. (B and C) a.a. sequences involved in the recognition of the head group of PI4P in yeast Kes1 are aligned with corresponding sequences in human ORP5 and ORP8 using the MegAlign program (DNASTAR, ClustalW method). Conservation is indicated by color scale. Identical residues are shown in black. Residues that directly contact PI4P are indicated with asterisks. (D and E) a.a. sequences involved in the recognition of the head group of PS in yeast Osh6 and Osh7 are aligned with corresponding sequences in human ORP5 and ORP8 using the MegAlign program (DNASTAR, ClustalW method). Conservation is indicated by color scale. Identical residues are shown in black. Residues that directly contact PS are indicated with asterisks.  

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Fig. S3. Comparison of the localizations of GFP-ORP8L and GFP-ORP5 with the localization of the ER marker mRFP-Sec61β. Live-cell imaging of HeLa cell co-transfected with mRFP-Sec61β and GFP-ORP5 or GFP-ORP8L. GFP-ORP8L colocalizes throughout the ER with mRFP-Sec61β. GFP-ORP5, also an ER protein, is concentrated next to the plasma membrane. Scale bars, 20 µm.

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Fig. S4. Accumulation of ORP5 at the plasma membrane requires its PH domain. (A) Schematic view of GFP-ORP5 constructs used for B. (B) Confocal live-cell imaging of HeLa cell transfected with GFP-ΔORDORP5 and GFP-ΔPHORP5. Scale bars, 20 µm.  

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Fig. S5. The N-terminal extension of ORP8L confers an overall negative charge to the a.a. sequence of ORP8 upstream of its PH domain. Isoelectric points (pI) of the N-terminal sequences of ORP5, ORP8L and ORP8S. The ORP8L specific sequence (a.a. 1-42) confers negative charge to this region. Theoretical pI for each protein was calculated using ExPASy (web.expasy.org/compute_pi/).  

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Fig. S6. LC-MS/MS-based quantification of PS species in control and PI4KIIIα KO MEFs (mean ± s.e.m.; n= 3).

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Fig. S7. Presence of the counter-lipid in the acceptor liposomes facilitates lipid transport from the donor liposomes. (A) PI4P transport assay. Sucrose-loaded ‘heavy’ PC/PI4P liposomes (90:10 mol/mol, 2mM lipids, 400 nm diameter) and ‘light’ PC/PS/NBD-PE liposomes (89:10:1 mol/mol/mol, 2mM lipids, 100 nm diameter) were incubated with no protein (-) or with 5µM wild-type ORPORP8 for 15min at 25°C. After centrifugation (16,000g for 15 min), supernatant fractions were collected and the relative amount of PI4P recovered in them (light liposomes) was assessed by HPLC-based lipid analysis (mean ± s.e.m.; *P<0.05, t-test, n=3). PI4P in supernatants was normalized by NBD-PE signal. (B) PS transport assay. Sucrose-loaded ‘heavy’ PC/PS liposomes (90:10 mol/mol, 2mM lipids, 400 nm diameter) and ‘light’ PC/PI4P/NBD-PE liposomes (89:10:1 mol/mol/mol, 2mM lipids, 100 nm diameter) were incubated with no protein (-) or with 5µM wild-type ORPORP8 for 15min at 25°C. After centrifugation (16,000g for 15 min), supernatant fractions were collected and the relative amount of PS recovered in them was assessed by HPLC-based lipid analysis (mean ± s.e.m.; ****P<0.0001, t-test, n=3). PS in supernatants was normalized by NBD-PE signal.

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Fig. S8. Impact of the transfection of mCherry-tagged WT ORP5 and of ORP5 with ORD mutations expected to prevent PI4P or PS binding on the localization of co-transfected monomeric (top) or dimeric (bottom) GFP-tagged PH domain of ORP5. Overexpression in HeLa cells of WT ORP5, but not of mutant ORP5, induced a major dissociation of the monomeric PH domain from the PM (with its resulting accumulation in the nucleus), consistent with a decrease of PM PI4P. The dimeric PH domain, however, remained associated with the PM, indicating residual presence of PI4P, which could be detected by the increased avidity of this construct for a PI4P containing membrane. Confocal live-cell imaging. Scale bars, 10 µm.

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Fig. S9. Increased levels of PS at the PM upon ORP5 overexpression. Ratio of GFP fluorescence visible in the TIRF (basal PM associated fluorescence) versus epifluorescence fields (entire cytosolic fluorescence) of cells transfected with GFP-LactC2 (PS probe) and either mCh-ORP5WT or various mCh-ORP5mutants (mean ± s.e.m.; P<0.0001, t-test, n=26 to 36 cells).

   

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Fig. S10. Mutations in ORP5 and ORP8L expected to prevent PS or PI4P binding to their ORD lock them at ER-PM contact sites. (A) Confocal live-cell imaging of HeLa cells transfected with either GFP-ORP5 constructs (top panel) or GFP-ORP8L constructs (bottom panel). Not only ORP5 and even more so the two ORP5 mutants, but also the two ORP8L mutants accumulate at the cell periphery, indicating increased PI4P levels in the PM upon expression of these mutants constructs. Scale bars, 10 µm. (B) Ratio of GFP fluorescence visible in the TIRF (basal PM associated fluorescence) versus epifluorescence fields (entire cytosolic fluorescence) of cells transfected as in (A) (mean ± s.e.m.; P<0.0001, t-test, n=16 to 29 cells)    

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Fig. S11. The accumulation of the PS reporter (GFP-evt2-2XPH) at the PM is reduced upon the double KD (DKD) of ORP5 and ORP8. (A) Confocal live imaging of control or double KD HeLa cells expressing with GFP-evt2-2XPH and (B) ratio of the GFP fluorescence visible in the TIRF (basal PM associated fluorescence) versus epifluorescence fields (entire cytosolic fluorescence). The PM accumulation of GFP-evt2-2XPH is reduced in the double KD cells. Scale bars, 10 µm. (mean ± s.e.m.; ****P<0.0001, t-test, n= 20 to 28 cells)    

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Fig. S12. The accumulation of the PS marker (GFP-evt2-2XPH) at the PM is reduced in PI4KIIIα KO MEFs. (A) Confocal live imaging of control and PI4KIIIα KO MEFs transfected with GFP-evt2-2XPH revealed that the accumulation of GFP-evt2-2XPH was reduced in PI4KIIIα KO MEFs. mCh-PI4KIIIα expression rescued this phenotype. Scale bars, 20 µm. (B) Ratio of GFP fluorescence visible in the TIRF (basal PM associated fluorescence) versus epifluorescence fields (entire cytosolic fluorescence) of cells transfected as in (A) (mean ± s.e.m.; ****P<0.0001, t-test, n= 12 to 16 cells)  

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Fig. S13. Cartoon depicting WT ORP5 and ORP8 and the constructs used for the rapamycin-dependent heterodimerization assay. Rapamycin triggers dimerization of mRFP-FKBP-ΔPH-ORP5 with PM-FRB-CFP and thus induces formation of ER-PM tethers. mRFP-FKBP-ΔPH-ORP5-ΔTM is also recruited to the PM, but cannot induce formation of ER-PM tethers.  

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Fig. S14. Acute PM recruitment of FKBP12-ORP5 fusion proteins with mutations in the ORD that affect PI4P or PS binding had no effect on the localization of the PS reporter GFP-evt2-2XPH. Quantification of GFP-evt2-2XPH fluorescence in the TIRF field upon rapamycin-induced recruitment of mRFP-FKBP-ΔPH-ORP5WT, mRFP-FKBP-ΔPH-ORP5H478/479A, or mRFP-FKBP-ΔPH-ORP5L389D, as indicated. (mean ± s.e.m., n=11 to 16 cells).  

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Fig. S15. Different kinetics of PM recruitment in response to rapamycin of FKBP12 fusion proteins with or without an ER transmembrane anchor. The construct lacking the C-terminal transmembrane anchor, mRFP-FKBP-ORP5WTΔTM (a cytosolic protein) was recruited to the PM in response to rapamycin much faster than the ER anchored construct, mRFP-FKBP-ORP5WT. The graphs show quantification of the mRFP fluorescence in the TIRF field upon rapamycin-induced recruitment (mean ± s.e.m., n=4).  

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Fig. S16. Schematic representation of the proposed model of PI4P/PS countertransport mediated by ORP5 and ORP8 at the ER-PM contact sites followed by PI4P dephosphorylation in the ER membrane by Sac1.

 

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Table S1. Cloning strategies used in this paper Plasmid Template for

PCR Primer set Vector

backbone enzyme sites

GFP-ORP5 ORP5 cDNA 5’-GCACA CTCGAG CT ATG AAG GAG GAG GCC TTC CTC CGG-3’ / 5’-GCACA AAGCTT CTA TTT GAG GAT GTG GTT AAT GAA C-3’

pEGFP-C1 XhoI/ HindIII

GFP-ORP5-ΔN-PH

ORP5 cDNA 5’-GCACA CTCGAG CT TGC AAG CCG GGC CGA GAC GGG GAG-3’ / 5’-GCACA AAGCTT CTA TTT GAG GAT GTG GTT AAT GAA C-3’

pEGFP-C1 XhoI/ HindIII

ORP5-ΔTM-GFP ORP5 cDNA 5’-GCACA CTCGAG GCC ACC ATG AAG GAG GAG GCC TTC CTC CGG-3’ / 5’-GCACA AAGCTT CGG TGC CTG TGC TGC CCG TGC CG-3’

pEGFP-N1

XhoI/ HindIII

ORP5-N-PH-GFP ORP5 cDNA 5’-GCACA CTCGAG GCC ACC ATG AAG GAG GAG GCC TTC CTC CGG-3’ / 5’-GCACA AAGCTT GGT GCC CAG TCT CAG TAG GCT AG-3’

pEGFP-N1

XhoI/ HindIII

ORP5-PH-GFP ORP5 cDNA 5’-GCACA CTCGAG GCC ACC ATG GGG TCA GAC AAG GAA TGT GTG TCC-3’ / 5’-GCACA AAGCTT GGT GCC CAG TCT CAG TAG GCT AG-3’

pEGFP-N1 XhoI/ HindIII

ORP5(1-252)-GFP

ORP5 cDNA 5’-GCACA CTCGAG GCC ACC ATG AAG GAG GAG GCC TTC CTC CGG-3’ / 5’-GCACA AAGCTT GGT GCC CAG TCT CAG TAG GCT AG-3’

pEGFP-N1 XhoI/ HindIII

GFP-H478/479A GFP-ORP5 5’-GCA GAG CAG GTG TCC GCC GCC CCG CCC GTG TCT GCC-3’ / 5’-GGC AGA CAC GGG CGG GGC GGC GGA CAC CTG CTC TGC-3’

Site-directed mutagenesis

GFP-L389D GFP-ORP5 5’-CTG TCC CGC GTG GTG GAT CCC ACG TTC GTA CTG-3’ / 5’-CAG TAC GAA CGT GGG ATC CAC CAC GCG GGA CAG-3’

Site-directed mutagenesis

mRFP-FKBP-ΔPH-ORP5WT

GFP-ORP5 5’-GCACA AAGCTT CG TGC AAG CCG GGC CGA GAC GGG GAG C-3’ / 5’-GCACA TCTAGA CTA TTT GAG GAT GTG GTT AAT GAA C-3’

mRFP-FKBP-PJ

HindIII/ XbaI

mRFP-FKBP-ΔPH-ORP5HH

mRFP-FKBP-ΔPH-ORP5WT

5’-GCA GAG CAG GTG TCC GCC GCC CCG CCC GTG TCT GCC-3’ / 5’-GGC AGA CAC GGG CGG GGC GGC GGA CAC CTG CTC TGC-3’

Site-directed mutagenesis

mRFP-FKBP- mRFP-FKBP- 5’-CTG TCC CGC GTG GTG GAT CCC ACG TTC GTA CTG-3’ / 5’-CAG

Site-

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ΔPH-ORP5LD ΔPH-ORP5WT TAC GAA CGT GGG ATC CAC CAC GCG GGA CAG-3’

directed mutagenesis

mRFP-FKBP-ΔPH-ORP5ΔTM

GFP-ORP5 5’-GCACA AAGCTT CG TGC AAG CCG GGC CGA GAC GGG GAG C-3’ / 5’-GCACA TCTAGA CTA CGG TGC CTG TGC TGC CCG TGC CG-3’

mRFP-FKBP-PJ

HindIII/ XbaI

mRFP-FKBP-ΔPH-ORP5-HHΔTM

mRFP-FKBP-ΔPH-ORP5ΔTM

5’-GCA GAG CAG GTG TCC GCC GCC CCG CCC GTG TCT GCC-3’ / 5’-GGC AGA CAC GGG CGG GGC GGC GGA CAC CTG CTC TGC-3’

Site-directed mutagenesis

mRFP-FKBP-ΔPH-ORP5-LDΔTM

mRFP-FKBP-ΔPH-ORP5ΔTM

5’-CTG TCC CGC GTG GTG GAT CCC ACG TTC GTA CTG-3’ / 5’-CAG TAC GAA CGT GGG ATC CAC CAC GCG GGA CAG-3’

Site-directed mutagenesis

GFP-ORP8L mCh-ORP8 Subcloned from mCh-ORP8 pEGFP-C1 BglII/ SalI

GFP-ORP8S GFP-ORP8L 5’-GCACA AGATCT ATG AGT CAG CGC CAA GGA AAA GAA GC-3’ / 5’-GCACA GTCGAC CTA CTT GAA CAT GAA GTT TAT TAT GAC TTG -3’

pEGFP-C1 BglII/ SalI

GFP-ORP8-ΔPH mCh-ORP8 5’-GCACA AGATCT CAT GAC CTG AGC GTT TCA TCA GAT AG-3’ / 5’- GCACA GTCGAC TG CTT GAA CAT GAA GTT TAT TAT GAC-3’

pEGFP-C1 BglII/ SalI

ORP8-ΔTM-GFP mCh-ORP8 5’-GCACA AGATCT GCC ACC ATG GAG GGA GGT TTG GCA GAT GG-3’ / 5’-GCACA GTCGAC TG CGG TGT GCT TGA AAC TAA ATG ATT TCG-3’

pEGFP-N1

BglII/ SalI

ORP8-N-PH-GFP mCh-ORP8 5’- GCACA AGATCT GCC ACC ATG GAG GGA GGT TTG GCA GAT GG -3’ / 5’- GCACA GTCGAC TG TTC CTT TCC TTC TCT GAT CAT TGT AC -3’

pEGFP-N1

BglII/ SalI

ORP8-PH-GFP mCh-ORP8 5’-GCACA AGATCT GCC ACC ATG TCA ACT TCA AGC AAA CTC ACA AAA AAA G-3’ / 5’-GCACA GTCGAC TG TTC CTT TCC TTC TCT GAT CAT TGT AC-3’

pEGFP-N1 BglII/ SalI

ORP8(1-280)-GFP

mCh-ORP8 5’-GCACA AGATCT GCC ACC ATG GAG GGA GGT TTG GCA GAT GG-3’ / 5’-GCACA GTCGAC TG TTC CTT TCC TTC TCT GAT CAT TGT AC-3’

pEGFP-N1 BglII/ SalI

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GFP-H514/515A GFP-ORP8 5’-GCT GAA CAG GTG TCC GCT GCT CCA CCA ATA TCT GCC-3’ / 5’-GGC AGA TAT TGG TGG AGC AGC GGA CAC CTG TTC AGC-3’

Site-directed mutagenesis

GFP-L425D GFP-ORP8 5’-CTA TCC AAG GTG GTT GAC CCT ACA TTT ATT TTG-3’ / 5’- CAA AAT AAA TGT AGG GTC AAC CAC CTT GGA TAG-3’

Site-directed mutagenesis

3XFLAG-ORP8 GFP-ORP8 5’-GCACA GCGGCCGC G ATG GAG GGA GGT TTG GCA GAT GG-3’ / 5’-GCACA TCTAGA CTACTTGAACATGAAGTTTATTATGACTTG-3’

p3XFLAG-CMV-10

NotI/ XbaI

3XFLAG-ORDORP8

GFP-ORP8 5’-GCACA GCGGCCGC CCT GTT GAG CCT TTA AAG GAG ACT AC-3’ / 5’-GCACA TCTAGA CTA TCC AGA GGA GTC TTG AAT GTC AGG -3’

p3XFLAG-CMV-10

NotI/ XbaI

GST-ORDORP8 GFP-ORP8 5’-GCACA G GTCGAC CCT GTT GAG CCT TTA AAG GAG ACT AC-3’ / 5’-GCACA GCGGCCGC CTA TCC AGA GGA GTC TTG AAT GTC AGG-3’

pGEX6P-1 SalI/ NotI

GST-ORDORP8-HH GST-ORDORP8 5’-GCT GAA CAG GTG TCC GCT GCT CCA CCA ATA TCT GCC-3’ / 5’-GGC AGA TAT TGG TGG AGC AGC GGA CAC CTG TTC AGC-3’

Site-directed mutagenesis

Movie legends

Movie S1. Dissociation of GFP-ORP5 from ER-PM contacts upon A1 treatment. Confocal live

imaging of HeLa cells expressing GFP-ORP5 (green) and mRFP-PHPLCδ (red). 10-s intervals

image acquisition. 100nM A1 was added at 30 sec. Scale bar, 10μm.

Movie S2. Dissociation of GFP-ORP8S from ER-PM contacts upon A1 treatment. Confocal live

imaging of HeLa cells expressing GFP-ORP8S (green) and mRFP-PHPLCδ (red). 10-s intervals

image acquisition. 100nM A1 was added at 30 sec. Scale bar, 10μm.

Movie S3. Dissociation of iRFP-P4M, but not mRFP-PHPLCδ, from ER-PM contacts upon A1

treatment. Confocal live imaging of HeLa cells expressing iRFP-P4M (cyan) and mRFP-

PHPLCδ (red). 10-s intervals image acquisition. 100nM A1 was added at 30 sec. Scale bar,

10μm.