Glutathione S-sepharose resin, pGEX6P2, [3H]GDP and [35S]GTPγS came from GE Healthcare UK (Little Chalfont, Buckinghamshire, UK).
(b) Purification of Rab proteins
Cells transformed with pGEX6P2 containing cDNAs of Rab14 were pre-incubated overnight in Luria broth medium, after which the medium was diluted 1 : 100 and incubated for a further 3 h at 37°C. Expression of the glutathione S-transferase (GST)-fusion protein was then induced by adding 0.1 mM isopropyl 1-thio-β
-D-galactoside; this was followed by an additional incubation for 24 h at 16°C. Cells were collected by centrifugation and suspended in Tris-buffered saline (TBS; 140 mM NaCl and 50 mM Tris–HCl (pH 7.3)), disrupted by sonication, and then cleared by centrifugation. Aliquots of supernatant were applied to a glutathione S-sepharose column equilibrated with TBS. The column was washed with TBS, and proteins were eluted with 50 mM Tris–HCl (pH 8.0) containing 0.1 M NaCl and 10 mM glutathione. Eluted proteins were dialysed against 50 mM Tris–HCl and 1 mM ethylenediaminetetraacetic acid. The protein concentrations of the eluates were determined in accordance with the method of Lowry et al. (1951)
. The cDNA fragments containing the entire coding sequences of human Rab14 were amplified by PCR. The protein was expressed and purified as described above. Dominant positive mutant of Rab14 (Q70L) was constructed using site-directed mutagenesis (Kyei et al. 2006
(c) Determination of [3H]GDP and [35S]GTPγS binding activities
Rab14 (5 µg) was incubated for 2 h at 25°C in a 100 µl mixture containing 50 mM Tris–HCl (pH 8.0), various concentrations of [3H]GDP or [35S]GTPγS, and 5 mM MgCl2. The reaction was stopped by the addition of 1 ml of wash buffer (50 mM Tris–HCl (pH 8.0), 25 mM MgCl2, 0.1 M NaCl and 0.025% bovine serum albumin), followed by rapid transfer to a nitrocellulose membrane (Advantec Toyo, Tokyo, Japan). The nitrocellulose membrane was washed twice with wash buffer and dried. The radioactivity on the nitrocellulose membrane was counted by a liquid scintillation counter (LSC-5100, Aloka, Tokyo, Japan).
(d) Competition for GTPγS binding to GST-Rab14 by nucleotides
A reaction mixture containing 5.0 µg GST-Rab14 and 50 mM Tris–HCl (pH 8.0), was incubated at 25°C for 20 min with 20 µM [35S] GTPγS in the absence or presence of individual nucleotides (GTPγS, GTP, GDP, ATP and ADP) at 20 mM concentrations. The reaction was stopped by the addition of 1 ml of wash buffer, followed by rapid transfer to a nitrocellulose membrane. The radioactivity on the nitrocellulose membrane was counted by an LSC as described above.
(e) Measurement of GTPase or ATPase activity
GST-Rab14 (0.5 µg) was incubated at 25°C in 100 µl of reaction mixture (50 mM Tris–HCl (pH 7.8), 1 mM dithiothreitol, 50 µM GTP (or ATP) and 5 mM MgCl2). The reaction was stopped by the addition of 200 µl of Biomol Green Reagent (Biomol Research Laboratories, Pennsylvania, USA). After incubation of the mixture at room temperature for 30 min, the absorbance at a wavelength of 620 nm was measured. The amount of inorganic phosphate liberated was determined by using Na2HPO4 as the standard. As a control, GST was substituted for the GST-Rab14. The amounts of phosphate liberated were calculated by subtracting the values for GST alone from those for GST-Rab14 ().
Lineweaver-Burk plots of GTPase activity (filled triangles) and ATPase activity (filled squares).
(f) Exchange of bound [3H]-GDP with nucleotides
[3H]-GDP-bound GST-Rab14 protein was made by incubating GST-Rab14 (5.0 µg) for 1 h at 25°C in a reaction mixture containing 50 mM Tris–HCl (pH 8.0), 5 mM MgCl2 and 20 µM [3H]-GDP. Exchange of [3H]-GDP on GST-Rab14 was initiated by adding a 1000-fold excess of either unlabelled GTP or ATP.
After incubation at 25°C, bound [3H]-GDP was trapped on a nitrocellulose membrane filter and its radioactivity was measured as described for the [3H]-GDP binding assay.