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J Biol Chem. 2015 May 22;290(21):13490-9. doi: 10.1074/jbc.M115.642355. Epub 2015 Apr 1.

Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs).

Author information

1
From the Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650.
2
From the Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650 wes@biochem.utah.edu.
3
From the Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650 chris@biochem.utah.edu.

Abstract

The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly.

KEYWORDS:

ATPases associated with diverse cellular activities (AAA); endosomal sorting complexes required for transport (ESCRT); enzyme mechanism; fluorescence anisotropy; protein-protein interaction

PMID:
25833946
PMCID:
PMC4505595
DOI:
10.1074/jbc.M115.642355
[Indexed for MEDLINE]
Free PMC Article

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