Figure 8.
Figure 8. Vps4 promotes the compaction of Vps32 spirals independently of its ATPase activity. (A) Coomassie-stained fractions of purified, recombinant C. elegans Vps4E219Q after size exclusion chromatography and SDS-PAGE analysis in the presence (bottom) or absence (top) of 10 mM ATP. MM, molecular mass. (B) A comparison of the elution profiles of Vps4E219Q in the absence and presence of 10 mM ATP after gel filtration chromatography highlights the ability of Vps4 to oligomerize in the presence of ATP. The data shown are from single representative experiments, which were repeated independently at least two times for each condition. (C) A representative EM image of recombinant C. elegans Vps4E219Q (10 µM), purified in the presence of 10 mM ATP. On the right are typical Vps4E219Q structures (end-on views) that were selected after low pass filtering. Bars: (left) 50 nm; (right) 10 nm. (D) A representative EM image of purified, recombinant C. elegans Vps4E219Q (10 µM), purified in the presence of 10 mM ATP, mixed with 4 µM Vps32. Bar, 50 nm. The inset shows a 2× zoomed image of a compacted Vps32 spiral after labeling using 1.8 nm Ni-NTA-Nanogold, which recognizes the polyhistidine tag on Vps4E219Q. A gold particle is highlighted (yellow arrowhead). The blue boxed regions highlight typical examples of Vps32 spirals that are compacted in the presence of Vps4E219Q. The red boxed region highlights a single-stranded ringlike structure, which is not affected by the presence of Vps4E219Q. Bar, 25 nm. (E) A single-stranded Vps32 ring (red boxed region in C) was straightened, and its width was measured (intensity after background subtraction was used to calculate the width of filaments). Bar, 5 nm. The data shown are from a single representative experiment, which was repeated independently two times (n = 15 for the experiment shown). (F and G) Analysis of the spacing between filaments (marked blue and red) in a negatively stained Vps32 spiral exposed to oligomerized Vps4E219Q was conducted. The average spacing between neighboring filaments was calculated to be 5.0 nm (G). Error bars represent means ± SEM. The difference in spacing between Vps32 filaments in the presence or absence of Vps4E219Q was statistically significant (P < 0.01, one-tail t test). The data shown are from a single representative experiment, which was repeated independently two times (n = 15 for the experiment shown). AU, arbitrary unit.

Vps4 promotes the compaction of Vps32 spirals independently of its ATPase activity. (A) Coomassie-stained fractions of purified, recombinant C. elegans Vps4E219Q after size exclusion chromatography and SDS-PAGE analysis in the presence (bottom) or absence (top) of 10 mM ATP. MM, molecular mass. (B) A comparison of the elution profiles of Vps4E219Q in the absence and presence of 10 mM ATP after gel filtration chromatography highlights the ability of Vps4 to oligomerize in the presence of ATP. The data shown are from single representative experiments, which were repeated independently at least two times for each condition. (C) A representative EM image of recombinant C. elegans Vps4E219Q (10 µM), purified in the presence of 10 mM ATP. On the right are typical Vps4E219Q structures (end-on views) that were selected after low pass filtering. Bars: (left) 50 nm; (right) 10 nm. (D) A representative EM image of purified, recombinant C. elegans Vps4E219Q (10 µM), purified in the presence of 10 mM ATP, mixed with 4 µM Vps32. Bar, 50 nm. The inset shows a 2× zoomed image of a compacted Vps32 spiral after labeling using 1.8 nm Ni-NTA-Nanogold, which recognizes the polyhistidine tag on Vps4E219Q. A gold particle is highlighted (yellow arrowhead). The blue boxed regions highlight typical examples of Vps32 spirals that are compacted in the presence of Vps4E219Q. The red boxed region highlights a single-stranded ringlike structure, which is not affected by the presence of Vps4E219Q. Bar, 25 nm. (E) A single-stranded Vps32 ring (red boxed region in C) was straightened, and its width was measured (intensity after background subtraction was used to calculate the width of filaments). Bar, 5 nm. The data shown are from a single representative experiment, which was repeated independently two times (n = 15 for the experiment shown). (F and G) Analysis of the spacing between filaments (marked blue and red) in a negatively stained Vps32 spiral exposed to oligomerized Vps4E219Q was conducted. The average spacing between neighboring filaments was calculated to be 5.0 nm (G). Error bars represent means ± SEM. The difference in spacing between Vps32 filaments in the presence or absence of Vps4E219Q was statistically significant (P < 0.01, one-tail t test). The data shown are from a single representative experiment, which was repeated independently two times (n = 15 for the experiment shown). AU, arbitrary unit.

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