Relief of autoinhibition enhances Vta1 activation of Vps4 via the Vps4 stimulatory element

Abstract

The endosomal sorting complexes required for transport (ESCRTs) impact multiple cellular processes including multivesicular body sorting, abscission, and viral budding. The AAA-ATPase Vps4 is required for ESCRT function, and its full activity is dependent upon the co-factor Vta1. The Vta1 carboxyl-terminal Vta1 SBP1 Lip5 (VSL) domain stimulates Vps4 function by facilitating oligomerization of Vps4 into its active state. Here we report the identification of the Vps4 stimulatory element (VSE) within Vta1 that is required for additional stimulation of Vps4 activity in vitro and in vivo. VSE activity is autoinhibited in a manner dependent upon the unstructured linker region joining the amino-terminal microtubule interacting and trafficking domains and the carboxyl-terminal VSL domain. The VSE is also required for Vta1-mediated Vps4 stimulation by ESCRT-III subunits Vps60 and Did2. These results suggest that ESCRT-III binding to the Vta1 microtubule interacting and trafficking domains relieves linker region autoinhibition of the VSE to produce maximal activation of Vps4 during ESCRT function.

Keywords: AAA-ATPase; ATPases; ESCRT; Endosomes; Multivesicular Body; Protein Sorting; Protein Turnover; Receptor Endocytosis; Vps4; Vta1.

FIGURE 1.

ATPase stimulation by Vta1 truncations. A, schematic diagram of Vta1 and deletions summarizing Vps4 stimulatory activity. As indicated, the VSL domain mediates Vps4 binding, whereas the MIT domains mediate binding to the ESCRT-III subunits Vps60 and Did2. Minimal Vta1 stimulation of Vps4 ATPase activity is retained in the setting of amino-terminal deletions to the VSL(290–330) domain. A slightly larger fragment (267–330) retains or exceeds the stimulatory activity of the full-length Vta1. B, 500 nm Vps4-specific ATPase activity in the presence of 250 nm–10 μm Vta1 VSL domain, wild type Vta1, or Vta1(267–330). Specific activity is expressed as ADP generated per Vps4 molecule per min (ADP/Vps4/min). Error bars, S.E.

FIGURE 2.

Mapping of a stimulatory element in Vta1. A, schematic diagram of Vta1 illustrating the amino acid sequence 267–290. Regions A–D indicate groups of residues mutated to alanine for scanning mutagenesis. Amino acid residues that are acidic are denoted in blue, and basic residues are indicated in red. Residues 281–290 form an α-helix as indicated. B, ATPase activity of 500 nm Vps4 in the presence of 10 μm Vta1 or Vta1 truncations. C, ATPase activity of 500 nm Vps4 in the presence of 250 nm–10 μm Vta1 or Vta1(275–330). D, ATPase activity of 500 nm Vps4 in the presence of 10 μm Vta1, VSL(290–330), or Vta1 alanine-scanning mutants A–D. Error bars, S.E.

FIGURE 3.

Characterization of Vta1 VSE. A, structure of Vta1 carboxyl terminus. VSL domain residues participating in dimerization (blue) and Vps4 β-domain association (yellow) are shown. Residues implicated in VSE function (red) are indicated. Mutation of residues indicated in green did not impact activity. Leucine 284, isoleucine 287, and methionine 288 fall on the same surface of the helix (inset). B, ATPase activity of 500 nm Vps4 in the presence of 10 μm Vta1, Vta1 mutants, or Vta1 VSL domain. Error bars, S.E.

FIGURE 4.

Vps4 stimulations by Vta1-Vps60 or Vta1-Did2 are impacted by VSE. A, ATPase activity of 500 nm Vps4 in the presence of 2 μm Vta1 or Vta1 point mutants incubated with immobilized GST (−) or GST-Vps60 (+). B, ATPase activity of 500 nm Vps4 in the presence of 2 μm Vta1 or Vta1 point mutants incubated with immobilized GST (−) or GST-Did2 (+). C, ATPase activity of 500 nm Vps4 in the presence of 10 μm Vta1, Vta1(275–330), or Vta1(275–330) point mutants. Error bars, S.E.

FIGURE 5.

VSE contributes to Vta1 function in vivo. A, the MVB cargo GFP-CPS was visualized by fluorescence microscopy in living vta1Δ cells transformed with vector or plasmids expressing Vta1 or Vta1 point mutants. B, pulse-chase immunoprecipitation was performed on endogenous CPS in vta1Δ cells transformed with vector or plasmids expressing Vta1 or Vta1 point mutants. CPS maturation was quantitated using a PhosphorImager and plotted relative to time zero. CPS maturation kinetics in cells expressing Vta1(L284E) or Vta1(L284E,I287E,M288E) was significantly different (*, p < 0.05) from cells lacking Vta1 or cells expressing wild type Vta1. B, subcellular fractionation of the ESCRT-III subunit Snf7 was performed on vta1Δ cells transformed with vector or plasmids expressing Vta1 or Vta1 mutants. Snf7 fractionation in cells expressing Vta1(L284E) or Vta1(L284E,I287E,M288E) was significantly different (*, p < 0.05) from cells lacking Vta1 or cells expressing wild type Vta1. Error bars, S.E.

FIGURE 6.

Vta1 stimulates Vps4 via both the VSL domain and the VSE. A, the Vta1 VSL domain stimulates Vps4 ATPase activity via the Vps4 β-domain. In the absence of ESCRT-III binding, the VSE contributes to stimulation of Vps4 ATPase activity along with the VSL domain. However, linker region autoinhibition of the VSE curtails the extent of stimulation in the absence of ESCRT-III. B, ESCRT-III binding Vta1 relieves autoinhibition of the VSE, enhancing stimulation of Vps4. This VSE stimulation may occur through interaction with the Vps4 AAA domain rather than the β-domain.