ESCRT-III and Vps4 bind to GUV membranes. The GUV membrane is stained red with rhodamine-PE, and selected proteins are labeled green with Alexa-488. a, Vps20 and b, Vps20ΔC are recruited to membranes containing 10% POPS and 3% PI(3)P. c, Vps20ΔC is not recruited to membranes lacking POPS. d, Vps4 colocalizes with GUVs in the presence of Vps20ΔC, Snf7, Vps2, and Vps24. e, Vps4 is not recruited to GUVs if Vps2 and Vps24 are omitted. f, Five rounds of buffer addition to GUVs leads to no ILV formation. g, Vps20ΔC, Snf7, Vps24, Vps2 and Vps4 generate ILVs. h, ILVs were counted in a total 100 GUVs for each condition shown. Error bars were calculated using two independent experiments and calculating the difference between them. “All” refers to Snf7, Vps24, Vps2 and Vps4. Scale bar = 10 μm.