Relief of Autoinhibition Enhances Vta1 Activation of Vps4 via the Vps4 Stimulatory Element

Abstract

Background: Vta1 promotes Vps4 ATPase activity and facilitates ESCRT-III stimulation of Vps4. Results: The Vta1 VSE (Vps4 stimulatory element) mediates ESCRT-III-enhanced activation of Vps4 and contributes to Vta1 function in vivo. Conclusion: ESCRT-III binding Vta1 relieves autoinhibition of the VSE to promote activation of Vps4. Significance: These studies identify a novel mechanism whereby ESCRT-III and Vta1 regulate Vps4 activity.

Vps4 Stimulatory Element of the Cofactor Vta1 Contacts the ATPase Vps4 α7 and α9 to Stimulate ATP Hydrolysis

“…All cloned PCR products and mutant plasmids were sequenced to exclude unexpected mutations. The generation of pGST-Vta1, pET28-Vta1, Vta1 fragments, Vta1(VSE⌬) mutant, and pET28-Ist1 (WT and L168A,Y172A) expression plasmids have been previously described (26,40,53 Fig. 2A.…”

“…The significances of differences in rates were assessed by T tests using Prism5 (GraphPad). An example of this analysis including images of TLC plates, and determination of rates has been presented in Norgan et al (53).…”

“…Second, ESCRT-III interactions with Vps4 enhance ATP hydrolysis by relieving autoinhibition of the AAA domain; this occurs via MIM-MIT interactions and ESCRT-III acidic region interactions with the Vps4 linker region (31,51,52). Third, Vta1 activation of Vps4 occurs through both promoting Vps4 oligomerization and enhancing ATP hydrolysis by the Vps4 oligomer (40,53). The Vta1 carboxyl-terminal Vta1/SBP1/Lip5 (VSL) domain is responsible for dimerization of Vta1 and interacts with the Vps4 ␤-domain (an insert within the AAA domain) to promote Vps4 oligomerization (39,40,43,54,55) (see Fig.…”

“…The Vps4 stimulatory element (VSE), an element just upstream from the Vta1 VSL domain, mediates Vta1 enhancement of Vps4 oligomer ATP hydrolysis (53). Moreover, binding of ESCRT-III subunits Did2 or Vps60 to the amino-terminal Vta1 MIT domains relieve autoinhibition within Vta1 to further enhance stimulation of Vps4 ATP hydrolysis via the Vta1 VSE (31,43,46,47,53). These observations indicate a complex orchestration of inputs from ESCRT-III and Vta1 that regulate Vps4 AAA domain ATP hydrolysis via the Vps4 MIT domain, linker region, and ␤-domain.…”

“…The Vta1 VSE has been implicated in mediating ESCRT-IIIenhanced Vta1 stimulation of Vps4 (53), but the surface of Vps4 that is contacted by the VSE to effect increased ATP hydrolysis is unknown. Structures of the Vta1 carboxyl terminus dimer, including both the VSL and VSE (43), and the VSL dimer in complex with a Vps4 fragment containing the ␤-domain and part of the small AAA domain (55) have been determined.…”