The AAA+ ATPase Vps4 disassembles ESCRT-III and is essential for HIV-1 budding and other pathways. Vps4 is a paradigmatic member of a class of hexameric AAA+ ATPases that disassemble protein complexes without degradation. To distinguish between local displacement versus global unfolding mechanisms for complex disassembly, we carried out hydrogen/deuterium exchange during Saccharomyces cerevisiae Vps4 disassembly of a chimeric Vps24-2 ESCRT-III filament. EX1 exchange behavior shows that Vps4 completely unfolds ESCRT-III substrates on a time scale consistent with the disassembly reaction. The established unfoldase ClpX showed the same pattern, thus demonstrating a common unfolding mechanism. Vps4 hexamers containing a single cysteine residue in the pore loops were cross-linked to ESCRT-III subunits containing unique cysteines within the folded core domain. These data support a mechanism in which Vps4 disassembles its substrates by completely unfolding them and threading them through the central pore.
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We thank K. Nyquist (University of California, Berkeley) for samples of Escherichia coli ClpX and ClpP. This work was supported by grants R01AI112442 (J.H.H.) and R01GM094497 (A.M.) from the US National Institutes of Health.
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Integrated supplementary information
(a) Electron micrograph of negatively stained filaments of Vps24-2. Scale bar = 50 nm. (b) Sedimentation of Vps24-2 and MBP-Vps24-2-ssrA. Vps24-2 and MBP-Vps24-2-ssrA were concentrated to above 200 μM and incubated at 4 oC overnight before dilution to 5 μM and ultracentrifugation. The resulting pellet (P) and supernatant (S) fractions were analyzed by SDS-PAGE.
Sequence coverage map for (a) Vps24-2 and (b) MBP-Vps24-2-ssrA. Solid green lines above the protein sequence denote the pepsin digest fragments identified in the study. The two residues mutated to cysteine for crosslinking experiments are labeled with asterisks.
(a) Total deuteron incorporation into undigested Vps24-2 monomers (triangles) and filaments (squares) over time. (b) Deuteron incorporation over time for the Vps24-2 part of MBP-Vps24-2-ssrA, mapped onto the Vps24-2 structural model. (c) and (d) Deuteron incorporation at 5 sec, 20 sec, 2min and 5 min for Vps24-2 monomers (c) and Vps24-2 filaments (d), mapped onto the Vps24-2 structural model. The color-coding for different percentages of deuteron incorporation is the same as in Fig. 2b.
(a), (c) and (e) Mass spectra of the indicated peptides from helices α1, α2, and α3 of Vps24-2 monomers (left), filaments (middle) or filaments plus Vps4 (right). Controls and time points are indicated. Arrows above the spectra indicate regions of the spectrum representing EX2 and EX1 behavior. (b), (d) and (f) Peak-width analysis of the selected peptides at 5, 10, 20, 30, 40 and 60 sec. Open circles, Vps24-2 monomers; filled squares, Vps24-2 filaments; filled triangles, Vps24-2 filaments plus Vps4. The grey bar denotes the 2 Da peak-width change allowance for peptides undergoing EX2 kinetics 41. Each peptide is color coded as in Fig. 2a.
(a-d) Mass spectra of four additional peptides from the MBP portion of MBP-Vps24-2-ssrA prior to incubation in D2O (top row), alone (second row), and in the presence of ClpX (third row) or Vps4 (fourth row), or after unfolding in 6 M GdnHCl followed by complete deuteration (bottom row). Each peptide is color coded as in Fig. 4a.
(a) SDS-PAGE gel of Vps4CF-mediated disassembly of Vps24-2 filament. (b) Bar graph showing the ATPase activity of Vps4 constructs in the absence or presence of substrate Vps24-2. (c) Structural model of Vps24 with position of I92C and M136C mutation highlighted in yellow.
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Yang, B., Stjepanovic, G., Shen, Q. et al. Vps4 disassembles an ESCRT-III filament by global unfolding and processive translocation. Nat Struct Mol Biol 22, 492–498 (2015). https://doi.org/10.1038/nsmb.3015
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