Figure 1.

Bet3-associated Ypt1-GEF activity. GST-Bet3 (▴) and GST (●) were expressed in yeast cells and purified using GA. The purified fractions were tested for Ypt1-GEF activity using GDP-release and GTP-uptake filtration assays, with recombinant Ypt1 protein as a substrate. (a) Time course of GDP release. Ypt1p-[³H]GDP was incubated in the presence of 30 μl of GST-Bet3p or GST in a 50 μl reaction. (b) Time course of GTP uptake, in the presence (filled symbols) or absence (open symbols, dashed lines) of 0.2 μM Ypt1p. Reactions were performed in the presence of [α-³²P]GTP. (c) GTP uptake by Ypt1p shows a concentration-dependent stimulation by GST-Bet3p, but not by GST. As described in panel b legend, 10 or 30 μl of GST-Bet3 or GST were assayed in a 50-μl reaction; the 30-min time points are shown. Results shown in this figure are the average of duplicate measurements and are representative of 2–6 experiments. Error bars represent range divided by 2.