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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.


Related in: MedlinePlus

Hsc70 binding makes cages less compressible but more prone to catastrophic deformationsA: Collision pressure model analogizes cages and cages+Hsc70 to balloons inflated to low and high pressure, respectively. Internal pressure generated by Hsc70s makes cages less deformable, but more prone to catastrophic deformation (bursting) especially as probing force is increased. B: Percent of cages +/− Hsc70 exhibiting indicated mean compressions with 100 pN force (mean compression for each cage is determined from 9–12 measurements obtained from probing each cage on a 3×3 or 4×4 grid, depending on cage size). C: As in B, but using a 200 pN tip force. D: Percent of cages exhibiting indicated maximum compressions during probing. Average max compressions for cage populations with max compressions <30 nm or >30nM are given (statistics in supplemental table 3).
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Figure 7: Hsc70 binding makes cages less compressible but more prone to catastrophic deformationsA: Collision pressure model analogizes cages and cages+Hsc70 to balloons inflated to low and high pressure, respectively. Internal pressure generated by Hsc70s makes cages less deformable, but more prone to catastrophic deformation (bursting) especially as probing force is increased. B: Percent of cages +/− Hsc70 exhibiting indicated mean compressions with 100 pN force (mean compression for each cage is determined from 9–12 measurements obtained from probing each cage on a 3×3 or 4×4 grid, depending on cage size). C: As in B, but using a 200 pN tip force. D: Percent of cages exhibiting indicated maximum compressions during probing. Average max compressions for cage populations with max compressions <30 nm or >30nM are given (statistics in supplemental table 3).

Mentions: The collision pressure model's predictions for Hsc70's effects on cage compressibility may be understood by analogy to balloons inflated to high vs. low pressure. Just as gas molecule collisions with a balloon's interior generate outwardly directed forces, bound Hsc70s may generate such forces on the interior walls of the cage. Cages with Hsc70s should therefore be less compressible than Hsc70-free cages when probed with a low force, just as high pressure balloons are less compressible than low pressure balloons (fig. 7A). However, a high pressure balloon is more prone to burst--to undergo catastrophic deformation--when probed with a strong force, while a low pressure balloon can deform more rather than burst (fig. 7A). To test this we used AFM to measure sizes, and mean and maximum deformations of cages, cages with auxilin, and cages with auxilin and Hsc70ΔC or Hsc70 at pH 6.0. In agreement with our cryoEM, the sizes of all of these were similar (supplemental figure 8; supplemental table 3). With a 100 pN tip force, Hsc70 reduced mean compressibility from 7.6±0.16 to 6.3±0.16 nm (fig. 7B), but with a 200 pN force, Hsc70 increased compressibility from 8.9±0.18 to 11.9±0.32 nm (fig. 7C). While the distribution of deformations was approximately Gaussian without Hsc70, addition of Hsc70 led to a non-Gaussian distribution and appearance of a population of large (>15 nm) deformations (fig. 7C), indicating that catastrophic deformation events were more frequent with Hsc70. To test this we measured the maximum deformation seen for each cage at a 100 pN force. Without Hsc70, 8% of cages exhibited max deformations of >30 nm with a mean of 46+2.5nm, but with Hsc70, 30% of the cages exhibited deformations >30 nm with a mean of 66+1.5nm (fig. 7D). With the smaller (<30 nm) deformations, Hsc70 had the opposite effect: mean max deformations for this group were 17+0.33 nm and 15+0.36 nm −/+ Hsc70, respectively. The effect of Hsc70 was therefore consistent with collision pressure predictions: Hsc70 made cages less compressible when probed at low force but markedly increased the frequency of catastrophic deformations.


Clathrin Coat Disassembly Illuminates the Mechanisms of Hsp70 Force Generation
Hsc70 binding makes cages less compressible but more prone to catastrophic deformationsA: Collision pressure model analogizes cages and cages+Hsc70 to balloons inflated to low and high pressure, respectively. Internal pressure generated by Hsc70s makes cages less deformable, but more prone to catastrophic deformation (bursting) especially as probing force is increased. B: Percent of cages +/− Hsc70 exhibiting indicated mean compressions with 100 pN force (mean compression for each cage is determined from 9–12 measurements obtained from probing each cage on a 3×3 or 4×4 grid, depending on cage size). C: As in B, but using a 200 pN tip force. D: Percent of cages exhibiting indicated maximum compressions during probing. Average max compressions for cage populations with max compressions <30 nm or >30nM are given (statistics in supplemental table 3).
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Figure 7: Hsc70 binding makes cages less compressible but more prone to catastrophic deformationsA: Collision pressure model analogizes cages and cages+Hsc70 to balloons inflated to low and high pressure, respectively. Internal pressure generated by Hsc70s makes cages less deformable, but more prone to catastrophic deformation (bursting) especially as probing force is increased. B: Percent of cages +/− Hsc70 exhibiting indicated mean compressions with 100 pN force (mean compression for each cage is determined from 9–12 measurements obtained from probing each cage on a 3×3 or 4×4 grid, depending on cage size). C: As in B, but using a 200 pN tip force. D: Percent of cages exhibiting indicated maximum compressions during probing. Average max compressions for cage populations with max compressions <30 nm or >30nM are given (statistics in supplemental table 3).
Mentions: The collision pressure model's predictions for Hsc70's effects on cage compressibility may be understood by analogy to balloons inflated to high vs. low pressure. Just as gas molecule collisions with a balloon's interior generate outwardly directed forces, bound Hsc70s may generate such forces on the interior walls of the cage. Cages with Hsc70s should therefore be less compressible than Hsc70-free cages when probed with a low force, just as high pressure balloons are less compressible than low pressure balloons (fig. 7A). However, a high pressure balloon is more prone to burst--to undergo catastrophic deformation--when probed with a strong force, while a low pressure balloon can deform more rather than burst (fig. 7A). To test this we used AFM to measure sizes, and mean and maximum deformations of cages, cages with auxilin, and cages with auxilin and Hsc70ΔC or Hsc70 at pH 6.0. In agreement with our cryoEM, the sizes of all of these were similar (supplemental figure 8; supplemental table 3). With a 100 pN tip force, Hsc70 reduced mean compressibility from 7.6±0.16 to 6.3±0.16 nm (fig. 7B), but with a 200 pN force, Hsc70 increased compressibility from 8.9±0.18 to 11.9±0.32 nm (fig. 7C). While the distribution of deformations was approximately Gaussian without Hsc70, addition of Hsc70 led to a non-Gaussian distribution and appearance of a population of large (>15 nm) deformations (fig. 7C), indicating that catastrophic deformation events were more frequent with Hsc70. To test this we measured the maximum deformation seen for each cage at a 100 pN force. Without Hsc70, 8% of cages exhibited max deformations of >30 nm with a mean of 46+2.5nm, but with Hsc70, 30% of the cages exhibited deformations >30 nm with a mean of 66+1.5nm (fig. 7D). With the smaller (<30 nm) deformations, Hsc70 had the opposite effect: mean max deformations for this group were 17+0.33 nm and 15+0.36 nm −/+ Hsc70, respectively. The effect of Hsc70 was therefore consistent with collision pressure predictions: Hsc70 made cages less compressible when probed at low force but markedly increased the frequency of catastrophic deformations.

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&mdash;Brownian ratchet, power-stroke and entropic pulling&mdash;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