Tla 120

The wild-type, monomeric human K349 construct was bacterially expressed and purified
as described (Kull et al., 1996 (link)), and 15%
(wt/vol) sucrose was added before snap freezing in liquid nitrogen and storing at
−80°C. A plasmid for the mutant N255K construct was generated from the
wild-type plasmid using the QuikChange site-directed mutagenesis kit (Agilent
Technologies; Santa Clara, CA). Thawed K349 (either wild-type or N255K) was exchanged
into EM buffer (25 mM PIPES, 25 mM KCl, 1 mM EGTA, 1 mM DTT) using three rounds of
dilution and concentration in a Microcon ultracentrifugal filter (EMD Millipore;
Billerica, MA). Microtubule batches were grown from 250 µg of lyophilized bovine
brain tubulin (Cytoskeleton; Denver, CO), resuspended in 25 µl EM buffer and
clarified (Beckman TLA 120.2, 100K RPM, 4°C) prior to incubation at 37°C.
Taxol (2 mM in DMSO) was added to equimolar levels with tubulin after 10 min of
incubation. Following ∼45 min of polymerization, microtubules were brought to
room temperature and a ∼twofold excess of K349 was added prior to pelleting
through a glycerol cushion (50 µl of EM buffer + 60% glycerol wt/vol +
200 µM taxol) in order to remove unbound motor and unpolymerized tubulin (20
min, Beckman TLA 120.2, 50K RPM, 24°C). The motor–microtubule complex was
resuspended in ∼10 µl of EM buffer plus 200 µM taxol.

Proteins were extracted from cells after homogenization in 2 mL lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% digitonin, 1 × protease inhibitor) using an electric homogenizer with an adapter pestle. Cell lysates were incubated on ice for 30 min before centrifugation for 17,000×g for one min at 4 °C to remove unbroken cells and debris. Proteins in the supernatant were quantified by Bradford Assay and used for succeeding experiments.
For membrane protein extraction, cells were homogenized in 1 mL of 0.1 M Na₂CO₃ pH 11.5 using an electric homogenizer with an adapter pestle followed by incubation on ice for 30 min. The homogenates were centrifuged at 435,400×g for 1 h using a Beckman rotor TLA 120.1. Pellet fractions were resuspended in alkaline buffer to achieve homogeneity. All fractions were kept in -80 °C until further processing.

Purified tubulin proteins (T240) were purchased from Cytoskeleton and diluted to a final concentration of 5 mg/ml in general tubulin buffer (80 mM PIPES, pH 7.2, 0.2 mM MgCl₂, and 0.5 mM EGTA including 1 mM GTP). Add 2 μl of Cushion Buffer (60% glycerol and 20 μM Taxol in general tubulin buffer) to one 20 μl aliquot of tubulin Protein and incubate at 35 °C for exactly 20 min. This step allows tubulin to polymerize to MTs, then add 2 μl of 2 mM Taxol stock solution. Taxol-stabilized MTs were mixed with affinity-purified FSD1 proteins from E. coli, or with cell extracts (1.5 mg protein) prepared from RPE-1 cells in general tubulin buffer including protease inhibitors. After 30 min of incubation at room temperature, MTs in samples were spun through a 100 μl or 1 ml Cushion Buffer at 100,000 × g for 40 min in a TLA 120.1 or MLA-150 rotor (Beckman). Both supernatants and pellets were collected and analyzed.