Fig. 2

Sec4p^V29 shows decrease sensitivity to the activation by Sec2p. a Intrinsic nucleotide dissociation in presence of 0.3 mM GDP. 0.3 μM of either Sec4p^wt (black), Sec4p^V29 (red) or Sec4p^E80A,R81A (blue) preloaded with mantGDP was excited at 360 nm and the emission signal was detected at 450 nm. The assays were done at room temperature with buffer containing 20 mM Tris HCl pH 7.5, 100 mM NaCl and 5 mM MgCl₂. The plots represent one of the three measurements acquired. b mantGDP dissociation from 0.3 μM of either Sec4p^wt (black) or Sec4p^V29 (red) was measured by the reduction in fluorescence intensity after mixing excess of unlabelled GDP (300 μM) plus 66nM Sec2p_51–182. Assays were performed at 16 °C with buffer containing 20 mM Tris HCl pH 7.5, 100 mM NaCl and 5 mM MgCl₂