Gtpγs

Recombinant GST and GST-Arl1QL were expressed in BL21 cells and immobilized on glutathione-sepharose resin (GE Healthcare). The immobilized GST fusion proteins were exchanged for GTPγS (Roche, Mannheim, Germany) according to the protocol described in Lu et al. [36 (link)]. Briefly, the GST fusion proteins were equilibrated in NE buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 10 mM EDTA, 5 mM MgCl₂, and 1 mM DTT) with 0.1% (w/v) sodium cholate and 10 μM GTPγS. After equilibration, the resin was incubated with NE buffer containing 1 mM GTPγS, 0.1% sodium cholate and 3 mM L-α-dimyristoylphosphatidylcholine for 1.5 h at RT. The resins were then washed and equilibrated in NS buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, and 1 mM DTT) with 10 μM GTPγS. HeLa cells were transfected with arfaptin-1b-myc or arfaptin-1b-F317A-myc for 48 h. The transfected cells were then lysed, and the lysates were suspended in NS buffer and precleared by incubating with immobilized GST resin (1 mg HeLa cell lysates/10 μg GST proteins) at 4°C for 2 h. The resulting unbound fractions were collected and incubated with immobilized GST fusion proteins (2.5 mg HeLa cell lysates/5 μg GST fusion proteins) at 4°C overnight. After washing, the bound proteins were resolved on a 12.5% SDS-PAGE gel and analyzed by western blot assays.

CP55940 [(-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol] was purchased from Tocris Bioscience (Bristol, United Kingdom). AEA and indomethacin were purchased from Sigma-Aldrich (Poole, Dorset, United Kingdom). [³H]CP55940 (174.6 Ci/mmol) and [³⁵S]GTPγS (1250 Ci/mmol) were obtained from PerkinElmer (Seer Green, Buckinghamshire, United Kingdom), GTPγS from Roche Diagnostic (Burgess Hill, West Sussex, United Kingdom), and GDP from Sigma-Aldrich. Compounds were dissolved in DMSO (final concentration of 0.1% in assay media for all assays) and added directly to the media at the concentrations and times indicated.

In order to analyse the interaction of GST-Rab27a and SPIRE1 depending on its nucleotide loading (GTP versus GDP), GST-Rab27a proteins were loaded with the non-hydrolysable GDP (GDPβS) and GTP (GTPγS) analogues (both from Sigma-Aldrich), respectively, prior to GST pull-down assays. For each nucleotide exchange 5.8 mg of purified GST-Rab27a fusion protein was used, corresponding to 2.5 mg pure Rab27a protein, and mixed with a fourfold molar excess of GDPβS and GTPγS (1.13 mM each), respectively, 12.5 units alkaline phosphatase (CIAP; Roche, Penzberg, Germany) and CIAP buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM MgCl₂, 2 mM DTE, 200 mM (NH₄)₂SO₄, 100 µM ZnCl₂) in a total volume of 800 µl. The mixture was incubated for 17 h at 4 °C on a rotating wheel. On the next day, CIAP buffer was exchanged by RAB polarisation buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 5 mM MgCl₂, 1 mM DTE, 5 µM GDPβS/GTPγS) using NAP-10 columns (GE Healthcare Lifesciences) according to manual.

Reagents were obtained from the following sources: Ni Sepharose 6 Fast Flow (Cytiva, 17531802), Glutathione Sepharose 4 Fast Flow (Cytiva, 17513201), tris(2-carboxyethyl)phosphine (Thermo, 75259), EDTA-free protease inhibitor cocktail (Roche, 11873580001), PreScission Protease (Absin, abs01243), Anapoe-X-100 (Anatrace, 9002-93-1), ATP (Roche, 11140965001), MgOAc (Sigma-Aldrich, M0631), L-glutamic acid potassium (Sigma-Aldrich, G1501), biotinylated anti-His antibody (Bioss Antibodies, bs-0287R-bio), streptavidin (Sangon Biotech, A610492), LD555-MAL (Lumidyne, 04), LD655-MAL (Lumidyne, 10), GDP (Sigma-Aldrich, G7127), GTPγS (Roche, 10220647001), GMppNHp (Sigma-Aldrich, G0635), mPEG-SVA-5000 (Laysan Bio, 170-106), Biotin-PEG-SVA-5000 (Laysan Bio, 170-124), benzoic acid (Sigma-Aldrich, 242381), protocatechuate 3,4-dioxygenase (Sigma-Aldrich, P8279), and n-dodecyl-β-D-maltoside (Avanti Polar Lipids, 850520). All lipids were obtained from Avanti Polar Lipids: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, 850457); 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS, 840034); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE, 810144); and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhodamine-PE, 810150).