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Clathrin Coat Disassembly Illuminates the Mechanisms of Hsp70 Force Generation

View Article: PubMed Central - PubMed

ABSTRACT

Hsp70s use ATP hydrolysis to disrupt protein:protein associations or move macromolecules. One example is Hsc70-mediated disassembly of clathrin coats that form on vesicles during endocytosis. We exploit the exceptional features of these coats to test three models—Brownian ratchet, power-stroke and entropic pulling—proposed to explain how Hsp70s transform their substrates. Our data rule out the ratchet and power-stroke models, and instead support a collision pressure mechanism whereby collisions between clathrin coat walls and Hsc70s drive coats apart. Collision pressure is the complement to the pulling force described in the entropic pulling model. We also find that self-association can augment collision pressure to allow disassembly of clathrin lattices predicted to resist disassembly. These results illuminate how Hsp70s generate the forces that transform their substrates.

No MeSH data available.


Scattering increases due to Hsc70 binding reflect multiple Hsc70s binding per CHC, not cage expansionA. SDS PAGE of pellets of the indicated cages incubated with 1, 3, or 9 μM Hsc70 (lanes 1–9) or Hsc70ΔC (lanes 10–18; [Hsc70] and [Hsc70ΔC] increase from left to right as indicated). Lanes 19–24 show experiments without cages. B: As in A, but using FLAG cages and Fab (lanes 10–12 are no cage control). C: Hsc70/CHC or Hsc70ΔC/CHC ratios plotted vs. [Hsc70] or [Hsc70ΔC], as indicated. D: As in C, but using FLAG cages and Fab.
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Figure 5: Scattering increases due to Hsc70 binding reflect multiple Hsc70s binding per CHC, not cage expansionA. SDS PAGE of pellets of the indicated cages incubated with 1, 3, or 9 μM Hsc70 (lanes 1–9) or Hsc70ΔC (lanes 10–18; [Hsc70] and [Hsc70ΔC] increase from left to right as indicated). Lanes 19–24 show experiments without cages. B: As in A, but using FLAG cages and Fab (lanes 10–12 are no cage control). C: Hsc70/CHC or Hsc70ΔC/CHC ratios plotted vs. [Hsc70] or [Hsc70ΔC], as indicated. D: As in C, but using FLAG cages and Fab.

Mentions: Since neither cage expansion, aggregation, nor binding of one Hsc70 to each CHC in a cage explained the Hsc70 induced scattering increases at pH 6, we determined if multiple Hsc70s might be binding each CHC by reacting Hsc70, Hsc70ΔC or Fab with cages at pH 6 and then pelleting the cages and their associated proteins (figs. 5A, B). In agreement with the observed scattering and our model predictions (fig. 3I), ~1 Fab per CHC was bound to cages at all concentrations tested (fig. 5D), while ~0.5, ~0.8, and ~2 Hsc70ΔCs were bound at 1, 3, and 9 μM, respectively (fig. 5C). However, Hsc70 was bound in molar excess of CHC, with ~10 Hsc70s per CHC at 9 μM Hsc70 (fig. 5C). Binding of such a large number of Hsc70s was dependent on auxilin, ATP and, critically, the presence of an Hsc70 binding site in the CHC (supplemental figure 5). We conclude that the large scattering increases seen when Hsc70 is reacted with pH 6.0 cages reflect multiple Hsc70s binding per CHC, which is likely due to Hsc70 self-association since it is not seen with Hsc70ΔC.


Clathrin Coat Disassembly Illuminates the Mechanisms of Hsp70 Force Generation
Scattering increases due to Hsc70 binding reflect multiple Hsc70s binding per CHC, not cage expansionA. SDS PAGE of pellets of the indicated cages incubated with 1, 3, or 9 μM Hsc70 (lanes 1–9) or Hsc70ΔC (lanes 10–18; [Hsc70] and [Hsc70ΔC] increase from left to right as indicated). Lanes 19–24 show experiments without cages. B: As in A, but using FLAG cages and Fab (lanes 10–12 are no cage control). C: Hsc70/CHC or Hsc70ΔC/CHC ratios plotted vs. [Hsc70] or [Hsc70ΔC], as indicated. D: As in C, but using FLAG cages and Fab.
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Related In: Results  -  Collection

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Figure 5: Scattering increases due to Hsc70 binding reflect multiple Hsc70s binding per CHC, not cage expansionA. SDS PAGE of pellets of the indicated cages incubated with 1, 3, or 9 μM Hsc70 (lanes 1–9) or Hsc70ΔC (lanes 10–18; [Hsc70] and [Hsc70ΔC] increase from left to right as indicated). Lanes 19–24 show experiments without cages. B: As in A, but using FLAG cages and Fab (lanes 10–12 are no cage control). C: Hsc70/CHC or Hsc70ΔC/CHC ratios plotted vs. [Hsc70] or [Hsc70ΔC], as indicated. D: As in C, but using FLAG cages and Fab.
Mentions: Since neither cage expansion, aggregation, nor binding of one Hsc70 to each CHC in a cage explained the Hsc70 induced scattering increases at pH 6, we determined if multiple Hsc70s might be binding each CHC by reacting Hsc70, Hsc70ΔC or Fab with cages at pH 6 and then pelleting the cages and their associated proteins (figs. 5A, B). In agreement with the observed scattering and our model predictions (fig. 3I), ~1 Fab per CHC was bound to cages at all concentrations tested (fig. 5D), while ~0.5, ~0.8, and ~2 Hsc70ΔCs were bound at 1, 3, and 9 μM, respectively (fig. 5C). However, Hsc70 was bound in molar excess of CHC, with ~10 Hsc70s per CHC at 9 μM Hsc70 (fig. 5C). Binding of such a large number of Hsc70s was dependent on auxilin, ATP and, critically, the presence of an Hsc70 binding site in the CHC (supplemental figure 5). We conclude that the large scattering increases seen when Hsc70 is reacted with pH 6.0 cages reflect multiple Hsc70s binding per CHC, which is likely due to Hsc70 self-association since it is not seen with Hsc70ΔC.

View Article: PubMed Central - PubMed

ABSTRACT

Hsp70s use ATP hydrolysis to disrupt protein:protein associations or move macromolecules. One example is Hsc70-mediated disassembly of clathrin coats that form on vesicles during endocytosis. We exploit the exceptional features of these coats to test three models—Brownian ratchet, power-stroke and entropic pulling—proposed to explain how Hsp70s transform their substrates. Our data rule out the ratchet and power-stroke models, and instead support a collision pressure mechanism whereby collisions between clathrin coat walls and Hsc70s drive coats apart. Collision pressure is the complement to the pulling force described in the entropic pulling model. We also find that self-association can augment collision pressure to allow disassembly of clathrin lattices predicted to resist disassembly. These results illuminate how Hsp70s generate the forces that transform their substrates.

No MeSH data available.