(A–B) Quantitative MVB sorting data for (A) vps20Δ vps27Δ, vps25Δ vps27Δ, and vps23Δ vps27Δ yeast exogenously expressing VPS20/VPS25/VPS23 and VPS27, snf7*** and VPS27, and snf7*** and vps27S270D S313D(vps27UIM), and vps20Δ bro1Δ, vps25Δ bro1Δ, and vps23Δ bro1Δ yeast exogenously expressing VPS20/VPS25/VPS23 and BRO1, snf7*** and BRO1, and snf7*** and bro1I377R L386R(bro1UBD), respectively, and for (B) vps20Δ bro1Δ, vps25Δ bro1, and vps23Δ bro1Δ yeast exogenously expressing snf7*** and BRO1, snf7*** L231K L234K (snf7*** BRO1) and BRO1, and snf7***and bro1I144D L336D(bro1SNF7), respectively. Error bars represent standard deviations from 3–5 independent experiments. (C–D) Representative TEM images of (C) Snf7WT, Bro1, and Snf7WT with Bro1 (1:1), and (D) Snf71-225 and Snf7R52E. Scale bars 100 nm. Cartoon diagrams of Snf7 activation. (E) Cartoon diagrams of closed and open Snf7, with the locations of Vps20-dependent activation sites, Arg52, Gln90, and Asn100, and Bro1-dependent activation region, α6. (F) Conceptual models of parallel ESCRT-III Snf7 activation pathways in MVB biogenesis of wild-type (left) and the core domain auto-activated Snf7 mutant, Snf7* (right).