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. 2017 Apr 5;6:e24487. doi: 10.7554/eLife.24487

Figure 1. Vps4101-437-Hcp1 is an active hexamer.

(A) Vps4 constructs and peptide-binding affinities assayed by fluorescence polarization. Peptide ‘20’ is a Vps2-derived 20-residue peptide C identified earlier (Han et al., 2015). Peptide ‘8’ is an 8-residue fragment (DEIVNKVL) of peptide ‘20’ that retains essentially full binding affinity. The relatively weak binding of full-length Vps4 reflects autoinhibition mediated by the MIT domains (Han et al., 2015). (B) Fluorescence polarization isotherms corresponding to values in panel A. Means and standard deviations are from four independent experiments. (C) Gel filtration of Vps4101-437-Hcp1 on a Superdex 200 column in 25 mM Tris/HCl pH 7.4, 100 mM NaCl and 1 mM DTT. The protein elutes as a symmetric peak with an apparent molecular mass of 290 kDa, in good agreement with the calculated molecular mass of a hexamer (330 kDa). (D) ATPase activities for Vps4 constructs: 1, Vps4 full-length; 2, Vps481-437; 3, Vps4101-437; 4, Vps4101-437-Hcp1. Vps4 subunit concentrations are indicated. Means and standard deviations from at least three independent measurements.

DOI: http://dx.doi.org/10.7554/eLife.24487.003

Figure 1—source data 1. Binding of fluorescently labeled ESCRT-III peptides to Vps4, related to Figure 1B.
DOI: http://dx.doi.org/10.7554/eLife.24487.004
elife-24487-fig1-data1.xlsx (24.6KB, xlsx)
DOI: 10.7554/eLife.24487.004
Figure 1—source data 2. ATPase activity of Vps4 constructs, related to Figure 1D.
DOI: http://dx.doi.org/10.7554/eLife.24487.005
elife-24487-fig1-data2.xlsx (63.8KB, xlsx)
DOI: 10.7554/eLife.24487.005

Figure 1.

Figure 1—figure supplement 1. Vps4101-437-Hcp1 is a hexamer.

Figure 1—figure supplement 1.

Equilibrium sedimentation of Vps4101-437-Hcp1. Absorbance is shown as a function of distance from the axis of rotation for three loading concentrations (open circles, 10 µM; open squares, 5 µM; open diamonds, 2.5 µM Vps4 subunits). The data were fit to a single species model where the molecular weight was allowed to float or held constant as indicated (black line), and residuals for each concentration are shown below. The molecular weight was fit to 329,402 Da in good agreement with the expected molecular weight for a hexamer of 334,223 Da. When the molecular weight was held constant, the data agree well with a hexamer model, whereas the residuals are clearly biased (and in opposite directions) when the molecular weight is set to a heptamer or pentamer.
DOI: http://dx.doi.org/10.7554/eLife.24487.006