Figure 3.

Comparison of the kinetics and efficiency of GTP binding between wild-type (wt) Spi1p and mutant Spi1-25p. His-tagged Spi1p, his-tagged Spi1-25p, or UBC4 as a negative control were incubated with [α-³²P]GTP as described in Materials and Methods and the amount of bound nucleotide was quantitated. The initial binding kinetics of wild-type and mutant Spi1p are similar, but only 30% of Spi1-25p is competent to bind nucleotide. Data for each experiment were normalized for protein amount and calculated as percentage of maximal wild-type binding. Values from four independent experiments were averaged and graphed as percent of wild-type βmax using SigmaPlot (Jande Scientific) and curves were fit using the sigmoidal three parameter equation.