Figure 7.
The Vps4 oligomer is functionally stable in vitro. (A) ATP hydrolysis reactions were initiated with 500 nM Vps4 and 1 mM ATP. After 30 min, reactions were diluted 10-fold in the absence of additional ATP (black, red) or with ATP maintained at 1 mM (blue). After an additional 30 min, ATP restoration (to 1 mM, red) was performed. The ADP generated per Vps4 was determined at 5-min intervals and plotted versus time. (B) The activities of ATP hydrolysis (ADP generated/Vps4/min) relative to the initial rate (I) are presented. (C) ESCRT-III release reactions were initiated with 30 nM Vps4 (red, blue) or with 30 nM Vps4 and 100 nM Vps4E233Q (black). After a 5-min incubation, Vps4 (30 nM, red) or Vps4E233Q (100 nM, blue) were added to the reactions initiated with Vps4 only. The fraction of Snf7 released into the soluble material was assessed at both 5 and 20 min.