Vanadate

The following chemicals were purchased from Sigma and used at the indicated concentrations and pre-treatment periods: Genistein (100 μM) for 10min, Vanadate (1 mM) for 30 min, PP1 (10 μM) for 5 min, PP2 (25 μM) for 5 min, K252a (100 nM) for 5 min, SU5402 (40 μM) for 60 min. Herbimycin A (5 μM) for 15 min and Lavendustin A (50 μM) for 15 min were purchased from Tocris. Bisindolylmaleimide (200 nM) for 30 min, was purchased from EMD. Forskolin (100 μM) was purchased from Sigma and Sf9 cells were treated with it for 6 min. The antigen and purification strategy for the anti-PKC Apl II antibody was previously described [22 (link)].

The ATPase assays were carried out in DAB (50 mM K‐Ac, 30 mM HEPES, pH 7.4, 2 mM Mg(Ac)₂, 1 mM EGTA) as follows. We mixed dynein (monomeric for all constructs) to a final concentration of 10–20 nM with 2 mM Mg‐ATP (Sigma), 0.2 mM NADH (Sigma), 1 mM phosphoenolpyruvate (Sigma), 0.01 U pyruvate kinase (Sigma), 0.03 U lactate dehydrogenase (Sigma), 10 μM Taxol, 1 mM DTT, and 0–5 μM microtubules in DAB. Absorbance at 340 nm was continuously measured in an Eppendorf Spectrophotometer (UV‐Vis BioSpectrometer), and the data were fit to the following equation (Bhabha et al, 2014) using an excel curve fitting routine: $k_{\mathrm{obs}}=\left(k_{\mathrm{cat}}-k_{\mathrm{basal}}\right)\frac{[\text{MT}]}{K_{\mathrm{M}}+[\text{MT}]}+k_{\mathrm{basal}}.$
The vanadate inhibition of dynein ATPase activity was performed as previously described (Höök et al, 2005). Briefly, we mixed dynein [monomeric of wild‐type and mutant 5 (from the same batch that was used to solve the structure of mutant 5 in the presence of ADP‐vi)] to a final concentration of 20 nM with 1 mM Mg‐ATP (Sigma), 0.2 mM NADH (Sigma), 1 mM phosphoenolpyruvate (Sigma), 0.01 U pyruvate kinase (Sigma), 0.03 U lactate dehydrogenase (Sigma), 10 μM Taxol, 1 mM DTT, 6 μM microtubules, and 0–100 μM vanadate (Sigma) in DAB. The vanadate was boiled for 10 min before usage. Absorbance at 340 nm was continuously measured in an Eppendorf Spectrophotometer (UV‐Vis BioSpectrometer), and the turnover rate was calculated as described above.

[6,7-³H(N)]-estradiol-17β-glucuronide (E17βG, 41.8 Ci/mmol), [6,7-³H(N)]-estrone-3-sulfate (E3S, 45.6 Ci/mmol), [³H(G)]-taurocholic acid (TC, 5 Ci/mmol) as well as the liquid scintillation fluid Filter Count were purchased from Perkin-Elmer (Schwerzenbach, Switzerland). Unlabelled substrates including E3S, E17βG, and TC as well as valinomycin, puromycin, and ATP were purchased from Sigma Aldrich (Buchs, Switzerland). Compounds used for inhibition studies, including UDP-glucuronic acid (UDPGA), Stx64, and vanadate were purchased from Sigma Aldrich (Buchs Switzerland). Glucose-6-phosphate was purchased from Roche (Basel, Switzerland) and bovine serum albumin was purchased from Interchim (Montluçon, France). All chemicals used were of the highest purity available. HPTLC plates and solvents used for HPTLC analysis were purchased from Merck (Darmstadt, Germany).

Strains were tested for susceptibility to cell wall perturbing agents using a microdilution method as described (Bates et al., 2005 (link)). Briefly, cells from overnight culture in medium containing 2 units/mL chitinase were washed with water, passaged through a syringe with a 32-gauge needle, and suspended at an OD₆₀₀ = 1. These cells were then inoculated into fresh medium at an OD₆₀₀ of 0.01 and 95 μL of this suspension were dispensed into 96-well plates. A 5 μL volume of the cell wall perturbing agents was added to each well by duplicate across a range of double dilutions. The final OD₆₀₀ was determined after 16 h of incubation at 30°C. The maximum concentrations tested for each agent were Calcofluor White (Sigma), Congo Red (Sigma) and tunicamycin (Sigma, 100 μ g/mL, each), SDS (BioRad, 0.1%, w/v), hygromycin B (Sigma, 500 μ g/mL), NaCl₂ and KCl (1M, each), caffeine (Sigma; 50 mM) and vanadate (Sigma, 80 mM). Cells were grown in medium with no stressor added to normalize data. Growth data were normalized as percentage of those generated with the same strains without treatment.

Protein from the same batch that was used to solve the structure of mutant 5 in the presence of ADP‐Vi was used in the vanadate‐mediated UV photo‐cleavage. The assay was performed in a similar way as previously described (Schmidt et al, 2015). Briefly, mutant 5 monomer was mixed with 2 mM Mg‐ATP (Sigma) and 2 mM vanadate (Sigma) and either exposed to UV‐light (365 nm) or kept in the dark for 90 min. The vanadate was boiled for 10 min before usage. Afterward, the samples were analyzed by SDS–PAGE.

RanGTP, Paclitaxel (T7402, Sigma), and DMSO (D4540, Sigma) MT assembly reactions were prepared on ice adding 11 μM RanGTP, 0.1 μM Paclitaxel, or 0.5% DMSO, respectively. Sodium orthovanadate (vanadate; 450243, Sigma) was used at 0.5 mM for RanGTP feather assembly and at 2.5 μM for the release from dynein inhibition experiments. Recombinant p50 was used at 1 mg/ml. 250–500 nM of fluorescently labeled tubulin was added to visualize MTs. The polyclonal anti-NEDD1 antibody was added at 0.5–1 μM. Reactions were incubated at 20°C or processed for live imaging at room temperature. When specified, RanGTP aster and feathers were preassembled for 15 min at 20°C and then studied under different experimental conditions. For dynein inhibition on preassembled structures, vanadate was added at 0.5 mM. For the release of feathers from dynein inhibition samples were fixed immediately before the release to check for feather shape. Feathers with comparable shape were used for the experiments.