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, After 634 four hours, the suspension was visualized by confocal microscopy. Experiment was also 635 performed with inverse color combination with similar results. Bar = 5 µm. 636 (D-F) Analysis of heterotypic tethering 637 (D) 50 nM N-PH-?CC-FFAT or PH-?CC-FFAT labeled with Alexa568 (red) was mixed with 638 50 nM VAP-A-His labeled with Alexa488 (green). Golgi-like GUVs (2% PI(4)P, Atto390-639 DOPE) and ER-like GUVs (2% DGS-NTA-Ni, no color) were added and the sample was 640 very gently mixed. After 30 min, Alexa568 (red) were incubated with Golgi-like GUVs (2% PI(4)P, Atto390-DOPE)
, Photobleaching was performed on a circular area 644 (diameter 2 µm) in the middle of the GUV interface. Means ± SD of one representative 645 experiment is shown, Bar = 5 µm
, Same as in (E) but FRAP was conducted on VAP-A-His labelled with Alexa-488
, Predictor of Natural Disordered Regions (PONDR®) Molecular Kinetics
, , 1991.
,
, RRID:AB_2676401)) and mouse monoclonal V9 Vimentin antibody (Sigma-Aldrich Cat# V6389, 661 RRID:AB_609914 ) were from Sigma-Aldrich. Secondary Alexa Fluor-conjugated antibody 662 (Thermo Fisher Scientific Cat# A32723, RRID:AB_2633275) were from Invitrogen and 663 secondary HRP-conjugated antibody were from Jackson ImmunoResearch (Jackson 664 ImmunoResearch Labs Cat#, Rabbit polyclonal antibody against OSBP (Atlas Antibodies Cat# HPA039227, vol.660
dansyl-PE (1,2-dioleoyl-sn-glycero-3-666 phosphoethanolamine-N-(5-dimethylamino-1-naphthalenesulfonyl)), rhodamine-PE [1,2-667 dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)], and DGS-668 NTA(Ni), vol.1 ,
, Methyl-?-cyclodextrin (MCD) and G418 were from 671, Cholesterol, dehydroergosterol (DHE)
,
, Texas Red-DHPE, Oregon Green-DHPE and Atto390-DHPE were from Invitrogen
, We used the following sequences of human ORPs (Uniprot access number): OSBP1 (P22059), p.676
, ORP9 678 (Q96SU4). Notably, our ORP4 sequence (gift from N. Ridgway) started at M39 (as referred to 679 UNIPROT Q969R2:ORP4-OSBP2 sequence), therefore, in all this study M1 corresponds to M39 680 of the UNIPROT reference sequence. Order/disorder composition of different ORPs was 681 determined with "Predictor of Natural Disordered Regions (PONDR®)" web server 682, OSBP2/ORP4 (Q969R2), ORP5 (Q9H0X9), ORP8L (Q9BZF1), ORP10 (Q9BXB5), ORP11 677 (Q9BXB4), ORP6 (Q9BZF3), ORP7 (Q9BZF2), ORP1 (Q9BXW6), ORP3 (Q9H4L5)
, For phylogenetic analysis, protein sequences of higher eukaryotes most similar to human OSBP 688 were obtained from the UniProt database. The phylogenetic tree was created using the
The sequences of each OSBP domain 690 were then aligned and compared to that of the corresponding human domain using Clustal 691, 2008. ,
A percent identity matrix was calculated for each domain, p.692, 2011. ,
sequences shorter than 20 amino acids were not included in the identity analysis. The (residues 1-408) and human ORP4 sequences ,
, PH-FFAT (residues 144-474)] were cloned into the BamHI site of 702 pmCherry-N1 vector using the GeneArt TM Seamless Cloning and Assembly Kit, PH-FFAT (residues 1-474)
, The siRNA-resistant OSBP constructs (full-length and ?N) were prepared by PCR using the 704 corresponding pmCherry-N1 plasmids as template with primers
,
, For protein expression, cells were transfected with Lipofectamine 2000 reagent (Invitrogen) or 707 by electroporation with Nucleofector Solution (Lonza) using the Amaxa Nucleofector device 708 (Lonza), pp.18-24
, Construction, expression and purification of OSBP and ?N-OSBP
For??N-OSBP, the pFastBac TM HTA vector (Invitrogen) was 714 modified by successive mutations to allow the insertion of a PCR amplified sequence upstream 715 and in frame with the 6His tag, Full-length (1-807) human OSBP and ?N-OSBP (88-807) were purified from baculovirus-712 infected Sf9 cells, 2013. ,
BamHI site into 2 stop codons (F oligo sequence ,
OSBP ?N (88-807) + thrombin site] DNA 719 sequence was PCR amplified using the pENTR/D-(OSBP-FL-thrombin site) as matrix and cloned 720 into the BamHI-digested pFastBac HTA modified vector using the GeneArt TM Seamless Cloning 721 and Assembly Kit (Invitrogen), ) insertion of a new BamHI site upstream of the His tag (F oligo sequence: 718 ,
Recombinant bacmides were selected as described in Bac to Bac R Expression System user 723 manual (Invitrogen) and used to produce recombinant Baculovirus ,
EDTA-free protease inhibitors and (Thermo Scientific), submitted to 3 washes with lysis buffer supplemented with 800, 550, and 730 300 mM NaCl, respectively, and then eluted with 250 mM imidazole-containing buffer. OSBP 731 fractions were pooled, concentrated on Amicon Ultra (cut-off 30 kDa), and submitted to 732 thrombin cleavage, Cell pellets were resuspended in lysis 726 buffer (20 mM Tris pH 7.5, 300 mM NaCl, 20 mM imidazole, 2013. ,
, All 734 steps were performed at 4°C. The purified protein fractions were pooled, concentrated, 735 supplemented with 10 % glycerol, flash-frozen in liquid nitrogen and stored at -80°C, HK16/70 column (GE Healthcare) using an AKTÄ chromatography system (GE Healthcare)
, Construction, expression and purification of OSBP and ORP4 fragments
,
, The corresponding constructs were prepared using pET.His6.StrepII.TEV.LIC (2HR-T, Addgene 740 plasmid # 29718) and pET.His10.TEV.LIC (2B-T-10
-408) and OSBP PH-FFAT (76-408) fragments were first inserted into 744 pET.His6.StrepII.TEV.LIC vector, subcloned into pET16b, and then expressed as N-terminal 6His 745 tag ,
, =PH-FFAT] were PCR 747 amplified using the pmCherry-ORP4 FL as matrix. The host plasmid pET16b, ORP4 DNA sequences, vol.4, pp.128-475
76-408) by a His10.TEV.LIC fragment. In addition, the SspI site of 750 pET16b was mutated (AATATT into AATAGC). Last, the ORP4 N-PH-FFAT or PH-FFAT PCR 751 fragments were inserted into the SspI digested pET16b ,
, After protein expression, bacteria 753 were lysed with a French Press (SLM AMINCO) and incubated for 30 min on ice with DNAse, Seamless Cloning and Assembly Enzyme Mix (Invitrogen), p.754
, MgCl2 (5mM) before ultracentrifugation (125 000 g)
, Protein fractions were pooled and submitted to TEV 756 protease cleavage overnight at 4°C. Digested proteins were purified on a SourceQ HR 10/10 757 column (GE Healthcare) with a 0-1M NaCl, p.2
, Purified proteins were pooled, concentrated, supplemented with 10% glycerol, flash-760 frozen in liquid nitrogen and stored at -80°C
T1 (GE Healthcare) 762 plasmids expressing the OSBP (1-408) or (76-408) sequence. A NaeI restriction was introduced 763 by site directed mutagenesis to remove the coiled-coil (207-329) region by digestion / ligation 764 taking advantage of another NaeI site ,
The preparation of NBD-PHFAPP1 and VAP-A have been described previously, p.772 ,
, The TEV protease plasmid, p.774, 2001.
, Minerva Biolabs) and were incubated at 37°C in a 779 5% CO2 humidified atmosphere. For hTERT-RPE1 cells (ATCC Cat# CRL-4000, 780 RRID:CVCL_4388); hereafter RPE1 cells), DMEM was replaced by DMEM/F12 (Gibco). RPE1 781 cells stably expressing EGFP-PHOSBP were selected using G418 (Sigma). Surviving colonies were 782 isolated using cloning cylinders, HeLa cells were cultured in DMEM medium with glutaMAX (Gibco) supplemented with 10% 778 fetal calf serum, 1% antibiotics, p.783
, For microscopy, cells were seeded at suitable 785 density to reach 50-90% confluence on the day of imaging. SF9 cells were cultured at 27°C in SF-786 900 II media supplemented with 1,5% FCS in absence of antibiotic. For protein expression SF9 787 cells were infected at 10 6 cells/ml and an MOI of 0.1 in 0.5 l CELLSPIN Spinner. After 72h, cells 788 were collected by centrifugation at 300xg for 15 mn, BD Biosciences). RPE1 cells stably expressing EGFP-PHOSBP, EGFP-P4MSidM were cultured in 784 medium supplemented with G418 (500 µg/ml)
, For endogenous OSBP silencing and simultaneous expression of siRNA resistant OSBP, RPE-1 792 cells stably expressing GFP-PH OSBP were electroporated with 90 pmol siRNA and 1 ?g siRNA-793 resistant OSBP plasmid using RNAiMAX (Invitrogen) and plated on 6-well plate or on µ-Dish 35mm 794 (Ibidi)
, NTA, p.875
8300 spectrofluorimeter using a cylindrical quartz cuvette (600 µl) equilibrated at 876 37°C and equipped with a magnetic bar for continuous stirring. The cuvette initially contained 877 NBD-PHFAPP1 (300 nM) and VAP-A-His (3 µM) in HKM buffer. NBD emission was measured at 878 510 nm (excitation 460 nm). Golgi liposomes (300 µM lipid supplemented with 4% PI(4)P and 879 2% rhodamine-PE) ,
, For sedimentation assays comparing the binding properties of N-PH-?CC-FFAT and PH-?CC
, 120 mM 888 potassium acetate, and 1 mM MgCl2 (HKM buffer) at room temperature for 30 min in a total 889 volume of 50 µL, FFAT, we used sucrose-loaded Golgi-like liposomes containing (mol%) egg PC (61), liver PE 885 (17), brain PS (5), cholesterol (10), vol.14, p.890
, The pellets resuspended in 50µl HKM buffer before analysis on 13% SDS-PAGE by Sypro
, Lipid 896 film was rehydrated at 1 mM in 50 mM Hepes pH 7, 120 mM K-acetate and liposomes were 897 formed by 2 minutes vortex. Liposomes were diluted at 30 ?M with 600 nM N-PH-FFAT or PH-898 FFAT. After 5 minutes incubation, a 5 µL drop of the solution was deposited on a glow 899 discharged lacey carbon electron microscopy grid, CHCl3 composed of Egg PC/brain PS/brain PI, p.901
Image acquisition was 903 performed under low dose conditions of 10 e -/Å 2 at a magnification of 50,000 or 29, Samples were imaged using a Tecnai G2, vol.500 ,
, , p.907
Phylogeny.fr: robust phylogenetic 908 analysis for the non-specialist, Nucleic Acids Res, vol.36, pp.465-474, 2008. ,
URL : https://hal.archives-ouvertes.fr/lirmm-00324099
, , p.911
Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid 912 bilayers, The Journal of Cell Biology, vol.195, pp.965-978, 2011. ,
, , 2001.
, Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with 915 wild-type catalytic proficiency, Protein Eng, vol.14, pp.993-1000
Predicting Coiled Coils from Protein Sequences, Science, vol.917, pp.1162-1164, 1991. ,
, , p.919, 2013.
, Step Cycle Driven by PI(4)P Hydrolysis Directs Sterol/PI(4)P Exchange by the ER-Golgi Tether 920 OSBP, Cell, vol.155, pp.830-843
Sterol transfer, 922 PI4P consumption, and control of membrane lipid order by endogenous OSBP, The EMBO 923 Journal, vol.36, pp.3156-3174, 2017. ,
, , p.925
Fast, scalable generation of high-926 quality protein multiple sequence alignments using Clustal Omega, Molecular Systems Biology, vol.7, p.539, 2011. ,
Expression and purification of soluble His(6)-tagged 929 TEV protease, Methods Mol Biol, vol.498, pp.297-307, 2009. ,
, After 30 min, an excess 1000 of PH-FFAT (Alexa568, red) and N-PH-FFAT (Alexa568, red) was added, Golgi-like GUVs (2% PI(4)P, labeled with Atto390-DOPE) were preincubated with 50nM 999 of N-PH-FFAT (Alexa488, green) or PH-FFAT (Alexa488, green)
, Confocal microscopy was performed after additional 30 min of incubation. Bar = 5 µm