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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.


Structural context of approaches to test Hsc70 disassembly mechanismsA: Cut-away view of clathrin cage (interior surface in cyan; exterior in yellow/orange) with auxilin (magenta; pdb 1XI523). C-termini of CHCs form a helical tripod (red) under each vertex. B: Expanded view of boxed region from A. Hsc70 (pdb 4B9Q3; blue) modeled into the clathrin:auxilin cage based on an Hsp70 NBD:auxilin J domain structure26 positions its protein binding domain (PBD) to bind the terminal tail (red circles with Hsc70 binding QLMLT sequence in green). C: Sequences of the termini of the CHCs used (#1 is WT), with helical segments in red, and Hsc70 binding and FLAG sites in magenta.
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Figure 1: Structural context of approaches to test Hsc70 disassembly mechanismsA: Cut-away view of clathrin cage (interior surface in cyan; exterior in yellow/orange) with auxilin (magenta; pdb 1XI523). C-termini of CHCs form a helical tripod (red) under each vertex. B: Expanded view of boxed region from A. Hsc70 (pdb 4B9Q3; blue) modeled into the clathrin:auxilin cage based on an Hsp70 NBD:auxilin J domain structure26 positions its protein binding domain (PBD) to bind the terminal tail (red circles with Hsc70 binding QLMLT sequence in green). C: Sequences of the termini of the CHCs used (#1 is WT), with helical segments in red, and Hsc70 binding and FLAG sites in magenta.

Mentions: Clathrin cage disassembly is driven by Hsc70 binding to a sequence (`QLMLT') in the flexible CHC C-terminal tail 10 residues downstream of each of the 3 helices at the center of the triskelion under each cage vertex (fig. 1A, B)23,27. Based on structural studies it was proposed that Hsc70 doesn't induce, but sterically locks in, fluctuations that loosen the clathrin lattice until they accumulate to a point that disassembly ensues23. This Brownian/steric wedge model can be tested by moving the Hsc70 binding site to relieve steric clashes between the Hsc70 and cage walls. If disassembly persists even as steric clashes are relieved, it would argue against this mechanism.


Clathrin Coat Disassembly Illuminates the Mechanisms of Hsp70 Force Generation
Structural context of approaches to test Hsc70 disassembly mechanismsA: Cut-away view of clathrin cage (interior surface in cyan; exterior in yellow/orange) with auxilin (magenta; pdb 1XI523). C-termini of CHCs form a helical tripod (red) under each vertex. B: Expanded view of boxed region from A. Hsc70 (pdb 4B9Q3; blue) modeled into the clathrin:auxilin cage based on an Hsp70 NBD:auxilin J domain structure26 positions its protein binding domain (PBD) to bind the terminal tail (red circles with Hsc70 binding QLMLT sequence in green). C: Sequences of the termini of the CHCs used (#1 is WT), with helical segments in red, and Hsc70 binding and FLAG sites in magenta.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016234&req=5

Figure 1: Structural context of approaches to test Hsc70 disassembly mechanismsA: Cut-away view of clathrin cage (interior surface in cyan; exterior in yellow/orange) with auxilin (magenta; pdb 1XI523). C-termini of CHCs form a helical tripod (red) under each vertex. B: Expanded view of boxed region from A. Hsc70 (pdb 4B9Q3; blue) modeled into the clathrin:auxilin cage based on an Hsp70 NBD:auxilin J domain structure26 positions its protein binding domain (PBD) to bind the terminal tail (red circles with Hsc70 binding QLMLT sequence in green). C: Sequences of the termini of the CHCs used (#1 is WT), with helical segments in red, and Hsc70 binding and FLAG sites in magenta.
Mentions: Clathrin cage disassembly is driven by Hsc70 binding to a sequence (`QLMLT') in the flexible CHC C-terminal tail 10 residues downstream of each of the 3 helices at the center of the triskelion under each cage vertex (fig. 1A, B)23,27. Based on structural studies it was proposed that Hsc70 doesn't induce, but sterically locks in, fluctuations that loosen the clathrin lattice until they accumulate to a point that disassembly ensues23. This Brownian/steric wedge model can be tested by moving the Hsc70 binding site to relieve steric clashes between the Hsc70 and cage walls. If disassembly persists even as steric clashes are relieved, it would argue against this mechanism.

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.