Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo

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1 Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo Supplementary Information Shehab A. Ismail 1, Yong-Xiang Chen 2, Alexandra Rusinova 1, Anchal Chandra 1, 3, Martin Bierbaum 3, Lothar Gremer 4, Gemma Triola 2, Herbert Waldmann 2,5, Philippe I.H.Bastiaens 3 and Alfred Wittinghofer 1 1 Structural Biology Group, 2 Department of Chemical Biology, 3 Department of Systemic Cell Biology, Max Planck Institut für Molekulare Physiologie, Otto-Hahn Str. 11, Dortmund, Germany, 5 Fachbereich Chemische Biologie, Fakultät Chemie Technische Universität Dortmund Otto-Hahn-Strasse 6, Dortmund, Germany ( ) These authors contributed equally to this work ( ) Corresponding author 4 Present address, Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany

2 Supplementary Methods Dual-color fluorescence crosscorrelation spectroscopy (FCCS) Microscope setup: Dual-color fluorescence crosscorrelation spectroscopy (FCCS) was performed on a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss Jena, Jena, Germany) equipped with a ConfoCor 3 unit and a C-Apochromat 40x/1.2 NA water-corrected objective. The sample was simultaneously excited with the 488 nm line of an Argon laser and a 594 nm HeNe Laser through a HFT 405/488/594 beam splitter. The emitted light passed through a pinhole set to a diameter of 72 µm, and was split onto two channels by a NFT 610 beam splitter. Cy5 and GFP fluorescence were detected by avalanche photodiode detectors (APDs) through a LP 655 long pass filter, and a BP IR band pass filter, respectively. The raw intensity data were auto- and crosscorrelated using the microscope manufacturer s software. Curve fitting: Autocorrelation curves were fit with the following model equation G(τ ) = 1+ F exp( τ / τ ) T T F T N 1+ τ / τ D ( ) + 1 α 1+ ( τ /τ D ) α / S 2 where N is the average number of fluorescent particles in the confocal volume, τ D is the characteristic diffusion time of the particles, S gives the ratio between height and width of the confocoal volume, F T is the fraction of fluorophores in a dark state, and τ T is the dark state relaxation time. S was determined from calibration measurements of Alexa Fluor 488 dye and

3 Cy5 dye in solution, yielding S= 5.4 and S= 6.7 for the EGFP and the Cy5 channel, respectively. Sample preparation: Protein samples for FCCS were placed on 8-well LabTek II chambered No. 1 coverglass (Nalge Nunc International, Rochester, NY, USA), which were pretreated by incubation with 1% BSA in PBS. 100 nm GFP-PDE and 170 nm Cy5-labelled Rheb (total concentration, labeling ratio 0.58) were incubated with the indicated concentrations of GppNHp (blue line) or GDP (green line) loaded Arl2 in M-buffer (20 mm Tris ph 7.5, 50 mm NaCl, 5 mm MgCl2, 2 mm DTE). The labeled DNA oligonucleotides were purified by denaturing polyacryamid gelelectrophoresis as previously described to remove non-conjugated dye molecules 1. Cell Culture MDCK cells (Madine-Darby Canine Kidney, ATCC number: CCL-34) were routinely maintained in MEM Eagle s supplemented with 10 % fetal bovine serum and penicillin (1000U/ml) and streptomycin (1g/ml) at 37ºC and 5% CO2. PC12 cells were cultured in neurobasal medium containing DMEM, 1% L-Glutamine, 1% NEAA, 5% FBS and 10% Horse serum. For live cell microscopy, cells were cultured on 35-mm glass bottom MatTek dishes (Nalgene Nunc International, Naperville, IL) and transferred to low bicarbonate DMEM without phenol red supplemented with 25 mm HEPES ph 7.4 (imaging medium). Transfections were performed with Effectene transfection reagent (Qiagen) for MDCK cells.

4 Fluorescence microscopy Confocal laser scanning microscopy was performed on a Leica TCS SP5 AOBS equipped with a 63X/1.3 NA oil immersion lens and a temperature controlled chamber at 37ºC, 5% CO2. mtfp, mcitrine and mcherry were excited by using the 458 nm, 514 nm Argon laser and 561nm DPSS laser lines respectively. Fluorophore emission bands were detected in the following range- CFP: nm, Citrine : nm, mcherry : nm. Time-domain FLIM Fluorescence lifetime images (FLIM) were acquired using a confocal laser scanning microscope (FV1000, Olympus, Germany) equipped with a time correlated single photon counting module (LSM Upgrade Kit, Picoquant, Germany). For detection of mtfp, the sample was excited with a 440 nm diode laser (LDH 440, Picoquant), spectrally filtered using a narrow band emission filter ((HQ 480/20, Chroma, USA)) and fluorescence was collected through an oil immersion objective (60X/1.35 UPlanSApo, Olympus). Photons were detected using a single photon avalanche photodiode (PDM Series, MPD, Italy) and timed using a single photon counting module (PicoHarp 300, Picoquant). For determining the average lifetime and interaction fraction see supplementary information. Supplementary References 1 Saccà, B., Meyer, R. & Niemeyer, C.M. Temperature-dependent FRET spectroscopy for the high-throughput analysis of self-assembled DNA from nanostructures in real time Nat Protoc 4, (2009).

5 Supplementary Results Supplementary Figure 1. Supp1ementary Figure 1. F-Rheb-peptide PDEδ structure and PDEδ electrostatic surface a) Ribbon representation of the PDEδ F-Rheb peptide complex showing four molecules in the asymmetric unit. Two of the molecules show farnesyl (in red) and the peptide (in blue) and two

6 molecules show farnesyl only b) Surface presentation of PDEδ, with transparency set at 40%, showing the farnesyl deeply penetrating PDEδ. Initial 2Fo-Fc electron density map around the F- Cys contoured at 1σ is shown separately c) Schematic representation of interacting residues between PDEδ (in grey) and the Rheb C terminus in green, black dotted lines mark polar interactions and the violet dotted lines mark hydrophobic interactions, residues in light brown are from the flexible loop of PDEδ d) The four molecules of the asymmetric unit superimposed, showing the movement of flexible PDEδ loop (residues 111 to 117) c) Electrostatic surface charge of PDEδ (generated by PyMol) showing the negative charge surrounding the opening of the farnesyl binding pocket (encircled with black dotted circle and the C-terminus peptide is in green) d) Electrostatic surface charge of RhoGDI shown as in c. Supplementary Figure 2. Supplementary Figure 2. Arl2GppNHp effect on PDEδ binding affinities a) Fluorescence polarization measurements where 0.25µM um fluorescein-labeled farnesylated Rheb peptide is titrated with wilde type or double mutant (94/98) PDEδ in the presence or absence of 10µM of full length Arl2 loaded with GppNHp (as indicated) b) Affinity constants obtained from a

7 Supplementary Figure 3. Supplementary Figure 3. Example micrographs of MDCK cells co-expressing mcitrine-pdeδ with either mcherry-rheb (a), mcherry-nras (b), mcherry-rheb and mtfp-arl2q70l (c) (d) Cells expressing mcitrine-pdeδ (F94A/I98A) and mtfp-arl2q70l.

8 Supplementary Table 1. Data collection and refinement statistics F-Rheb PDEδ F- Peptide PDEδ Data collection Space group P P 2 1 Cell dimensions a, b, c (Å) 47.9,57.4, , 70, α, β, γ ( ) 90, 90, 90 90,81.5,90 Resolution (Å) R sym or R merge 7.2 (39.0) 6.4 (35.2) I / σi (5.95) 17.25(5.63) Completeness (%) 97.2 (95.7) 99.6 (99.5) Redundancy 6.6 (6.7) 3.75 (3.9) Refinement Resolution (Å) No. reflections R work / R free 19.75/ /21.6 No. atoms Protein Ligand/ion Water B-factors Protein Ligand/ion Water R.m.s. deviations Bond lengths (Å) Bond angles ( )

9 Supplementary Movie Morphing Movie, made using the PDEδ bound to Arl2 or bound to Rheb as end conformers. The Arl2 interaction interface is colored in yellow and the Phe94 and Ile98 are shown as sticks. Example for residues that would clash with the farnesyl group (red) are shown in sticks and colored in cyan.