Figure 3.

The DCB-HUS domains potentiates BIG1 activity on membranes. (A) BIG1^DCBHUSSec7 binds to liposomes containing 40% PC, 35% DOPC, 10% PE, 10% DOPE and 5% PI(4)P in a co-sedimentation assay; S = supernatant, P = pellet (n = 2, error bars = SD). (B) BIG1^DCBHUSSec7 is 32 times more active on membranes. Representative nucleotide exchange kinetics traces for BIG1^DCBHUSSec7 (200 nM) activation of Δ17Arf1 in solution (top) or of ^myrArf1 in the presence of liposomes containing 38% PC, 10% DOPC, 10% PE, 10% DOPE, 5% PS, 2% PI(4)P, 10% PI and 15% cholesterol (bottom). Nucleotide exchange was monitored by tryptophan fluorescence. Insets show k_cat/K_m determination of BIG1^DCBHUSSec7 for each substrate (n = 2). Values are given in Table 1.