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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.


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Moving the Hsc70 binding site slows disassembly and reveals a reaction intermediate of large scattering amplitude, and replacing it with a FLAG-tag allows disassembly by anti-FLAG FabsA: Scattering (normalized to starting value of 1) vs. time for reactions with WT (+0AA) cages reacted with 0.25 (magenta), 0.5 (blue), 1.0 (red) or 2.0 (black) μM Hsc70. B: As in A, but with Hsc70 binding site moved 10 AA downstream (+10AA). C: As in A, but with site moved 25 AA (+25AA). D: Hyperbolic fits of WT cage disassembly rates vs. [Hsc70]. E: As in D, but for +10AA cages. F: As in D, but for +25AA cages. G: As in A, but using Hsc70ΔC. H: As in G, but with +10AA cages. I: As in G, but with +25AA cages. J: As in A, but using anti-FLAG Fab and cages with Hsc70 binding site replaced with a FLAG tag. K: As in J, but with tag shifted 10AA. L: As in J, but with tag moved 25 AA. In J–L, insets show initial 10 sec. of reactions to resolve Fab binding phase. For all plots, trace thickness, error bars and fitted values in figs 2, 3, 5 and 6 reflect +/− sem. The number of replicates for each experimental condition in these figures is specified in Supplemental Table 2.
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Figure 2: Moving the Hsc70 binding site slows disassembly and reveals a reaction intermediate of large scattering amplitude, and replacing it with a FLAG-tag allows disassembly by anti-FLAG FabsA: Scattering (normalized to starting value of 1) vs. time for reactions with WT (+0AA) cages reacted with 0.25 (magenta), 0.5 (blue), 1.0 (red) or 2.0 (black) μM Hsc70. B: As in A, but with Hsc70 binding site moved 10 AA downstream (+10AA). C: As in A, but with site moved 25 AA (+25AA). D: Hyperbolic fits of WT cage disassembly rates vs. [Hsc70]. E: As in D, but for +10AA cages. F: As in D, but for +25AA cages. G: As in A, but using Hsc70ΔC. H: As in G, but with +10AA cages. I: As in G, but with +25AA cages. J: As in A, but using anti-FLAG Fab and cages with Hsc70 binding site replaced with a FLAG tag. K: As in J, but with tag shifted 10AA. L: As in J, but with tag moved 25 AA. In J–L, insets show initial 10 sec. of reactions to resolve Fab binding phase. For all plots, trace thickness, error bars and fitted values in figs 2, 3, 5 and 6 reflect +/− sem. The number of replicates for each experimental condition in these figures is specified in Supplemental Table 2.

Mentions: Disassembly was measured by light-scattering in a stopped-flow fluorometer (supplemental figure 1) with cages reacted with varying concentrations of Hsc70. With +0AA cages, reaction profiles (fig. 2A) were like those seen by Rothnie et al. who used a similar approach21. Reaction of cages with 2 μM Hsc70 resulted in an initial ~15% increase in scattering, followed by a drop to ~20% of the initial level. The large drop is due to disassembly21,24. The nature of the initial smaller increase is unknown but was assigned by Rothnie et al., to binding of the 1st Hsc70 to one of the 3 CHC termini in their sequential mechanism, in which 3 Hsc70s must bind all 3 sites in a triskelion before that triskelion is released from the cage. A subsequent single molecule study cast doubt on this mechanism as it showed that disassembly began when only one (or even fewer20) Hsc70(s) were bound for every 2 triskelia, and that Hsc70s continued to bind and accelerate disassembly even after it had begun19.


Clathrin Coat Disassembly Illuminates the Mechanisms of Hsp70 Force Generation
Moving the Hsc70 binding site slows disassembly and reveals a reaction intermediate of large scattering amplitude, and replacing it with a FLAG-tag allows disassembly by anti-FLAG FabsA: Scattering (normalized to starting value of 1) vs. time for reactions with WT (+0AA) cages reacted with 0.25 (magenta), 0.5 (blue), 1.0 (red) or 2.0 (black) μM Hsc70. B: As in A, but with Hsc70 binding site moved 10 AA downstream (+10AA). C: As in A, but with site moved 25 AA (+25AA). D: Hyperbolic fits of WT cage disassembly rates vs. [Hsc70]. E: As in D, but for +10AA cages. F: As in D, but for +25AA cages. G: As in A, but using Hsc70ΔC. H: As in G, but with +10AA cages. I: As in G, but with +25AA cages. J: As in A, but using anti-FLAG Fab and cages with Hsc70 binding site replaced with a FLAG tag. K: As in J, but with tag shifted 10AA. L: As in J, but with tag moved 25 AA. In J–L, insets show initial 10 sec. of reactions to resolve Fab binding phase. For all plots, trace thickness, error bars and fitted values in figs 2, 3, 5 and 6 reflect +/− sem. The number of replicates for each experimental condition in these figures is specified in Supplemental Table 2.
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Figure 2: Moving the Hsc70 binding site slows disassembly and reveals a reaction intermediate of large scattering amplitude, and replacing it with a FLAG-tag allows disassembly by anti-FLAG FabsA: Scattering (normalized to starting value of 1) vs. time for reactions with WT (+0AA) cages reacted with 0.25 (magenta), 0.5 (blue), 1.0 (red) or 2.0 (black) μM Hsc70. B: As in A, but with Hsc70 binding site moved 10 AA downstream (+10AA). C: As in A, but with site moved 25 AA (+25AA). D: Hyperbolic fits of WT cage disassembly rates vs. [Hsc70]. E: As in D, but for +10AA cages. F: As in D, but for +25AA cages. G: As in A, but using Hsc70ΔC. H: As in G, but with +10AA cages. I: As in G, but with +25AA cages. J: As in A, but using anti-FLAG Fab and cages with Hsc70 binding site replaced with a FLAG tag. K: As in J, but with tag shifted 10AA. L: As in J, but with tag moved 25 AA. In J–L, insets show initial 10 sec. of reactions to resolve Fab binding phase. For all plots, trace thickness, error bars and fitted values in figs 2, 3, 5 and 6 reflect +/− sem. The number of replicates for each experimental condition in these figures is specified in Supplemental Table 2.
Mentions: Disassembly was measured by light-scattering in a stopped-flow fluorometer (supplemental figure 1) with cages reacted with varying concentrations of Hsc70. With +0AA cages, reaction profiles (fig. 2A) were like those seen by Rothnie et al. who used a similar approach21. Reaction of cages with 2 μM Hsc70 resulted in an initial ~15% increase in scattering, followed by a drop to ~20% of the initial level. The large drop is due to disassembly21,24. The nature of the initial smaller increase is unknown but was assigned by Rothnie et al., to binding of the 1st Hsc70 to one of the 3 CHC termini in their sequential mechanism, in which 3 Hsc70s must bind all 3 sites in a triskelion before that triskelion is released from the cage. A subsequent single molecule study cast doubt on this mechanism as it showed that disassembly began when only one (or even fewer20) Hsc70(s) were bound for every 2 triskelia, and that Hsc70s continued to bind and accelerate disassembly even after it had begun19.

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.


Related in: MedlinePlus