Bro1 binding to Snf7 inhibits in vitro disassembly of ESCRT-III. (A) 0.1 µM purified Vps4, ATP, and an ATP regeneration system were incubated with membranes isolated from vps4Δ yeast cells; overexpression of full-length Bro1 or the Bro1 domain (BOD) is indicated. The amount of membrane-associated Snf7 remaining after incubation at 30°C for 10 min was determined by quantitative Western blot analysis. P < 0.001 for both samples. (B) In vitro ESCRT-III release performed in the presence or absence of ATP, 0.1 µM Vps4, and 0.1 µM GST-Bro1 domain (GST-BOD). (C) In vitro ESCRT-III release performed as in B except that the reaction was allowed to proceed for 5 min in the absence of GST-BOD or for 20 min in the absence or presence of 1 µM GST-BOD that was added either at the same time as purified Vps4 or 5 min after the addition of Vps4. (D) In vitro ESCRT-III release performed with membranes from yeast expressing wild-type Snf7 or the Snf7^Vps2MIM1 chimera in the presence or absence of ATP, 0.1 µM Vps4, and 0.1 µM GST-BOD. Error bars show the standard deviation.