Abstract
Intralumenal vesicle formation of the multivesicular body is a critical step in the delivery of endocytic cargoes to the lysosome for degradation. Endosomal sorting complex required for transport III (ESCRT-III) subunits polymerize on endosomal membranes to facilitate membrane budding away from the cytoplasm to generate these intralumenal vesicles. The ATPase Vps4 remodels and disassembles ESCRT-III, but the manner in which Vps4 activity is coordinated with ESCRT-III function remains unclear. Ist1 is structurally homologous to ESCRT-III subunits and has been reported to inhibit Vps4 function despite the presence of a microtubule-interacting and trafficking domain-interacting motif (MIM) capable of stimulating Vps4 in the context of other ESCRT-III subunits. Here we report that Ist1 inhibition of Vps4 ATPase activity involves two elements in Ist1: the MIM itself and a surface containing a conserved ELYC sequence. In contrast, the MIM interaction, in concert with a more open conformation of the Ist1 core, resulted in stimulation of Vps4. Addition of the ESCRT-III subunit binding partner of Ist1, Did2, also converted Ist1 from an inhibitor to a stimulator of Vps4 ATPase activity. Finally, distinct regulation of Vps4 by Ist1 corresponded with altered ESCRT-III disassembly in vitro. Together, these data support a model in which Ist1-Did2 interactions during ESCRT-III polymerization coordinate Vps4 activity with the timing of ESCRT-III disassembly.
Highlights
Monomeric ESCRT-III subunits in the cytoplasm are autoinhibited by intramolecular binding of their C termini (␣6) to the ␣1/2/5 groove (Fig. 1B, closed conformation), whereas recruitment into ESCRT-III polymers on membranes leads to displacement of ␣6 from the ␣1/2/5 groove (Fig. 1B, semi-open conformation) and additional conformational changes within the Ist1 core (␣1–5) (Fig. 1B, open conformation) [45,46,47,48,49, 60]
We predicted that Ist1 intramolecular interactions between the microtubule-interacting and trafficking domain-interacting motif (MIM)-containing ␣6 and ␣1/2/5 groove in a closed conformation may contribute to Vps4 inhibition, whereas a more open Ist1 conformation resulting in displacement of ␣6 or additional conformational changes in the Ist1 core (␣1-␣5) may generate Vps4 stimulation in a MIMdependent manner
How does the Ist1 MIM element contribute to inhibition of Vps4 activity whereas the MIM1 elements of Did2 and Vps2 stimulate Vps4 [20]? The Ist1 MIM element itself is functionally distinct from the Did2 MIM1 because the Did2 MIM1 could not replace the Ist1 MIM element in Vps4 binding and/or regulation (Figs. 6 and 7)
Summary
Conversion of Ist1 from an inhibitor to a stimulator of Vps4 ATPase activity in vitro has been observed upon addition of Did2, the ESCRT-III subunit to which Ist1 binds [20, 23, 26, 45, 60]. Two mutant forms of Ist1 hyperstimulated Vps4 ATPase activities (i.e. greater stimulation than observed in other ESCRT-III subunits [20]): Ist1(L168A,Y172A) (Fig. 2, B and C) and Ist1(K135A) (Fig. 2C).
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