Linker extension impairs ESCRT-III flat spirals and the mediated membrane abscission

The endosomal sorting complex required for transport III (ESCRT-III) is conserved machinery that drives membrane abscission. While ESCRT-III flat spirals are proposed as a primed state, their essential role and regulation remain unclear. Leveraging our newly resolved architecture of yeast Snf7 flat spirals, we engineered a series of Snf7 mutants by inserting polyglycines into the linker between the helices α4 and α5. Our results demonstrate that extending the linker can transform Snf7 flat spirals into rings. Cryogenic electron microscopy analyses of these Snf7 rings reveal that the linker extension specifically relaxes α2/3 into a bent conformation while leaving other regions of Snf7 unaffected. Importantly, Snf7 rings are unable to mediate membrane abscission to form intraluminal vesicles, resulting in pronounced yeast sensitivity to extracellular canavanine. Our work identifies the linker as a critical regulator of ESCRT-III spiral assembly and establishes flat spirals as indispensable for membrane abscission, offering fundamental molecular insights into the membrane abscission mediated by ESCRT-III flat spirals.