Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly

Anna Young; Svetla Stoilova-McPhie; Alice Rothnie; Yvonne Vallis; Phillip Harvey-Smith; Neil Ranson; Helen Kent; Frances M. Brodsky; Barbara M.F. Pearse; Alan Roseman; Corinne J. Smith

doi:10.1111/tra.12085

Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly

Anna Young, Svetla Stoilova-McPhie, Alice Rothnie, Yvonne Vallis, Phillip Harvey-Smith, Neil Ranson, Helen Kent, Frances M. Brodsky, Barbara M.F. Pearse, Alan Roseman, Corinne J. Smith^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

The molecular chaperone, Hsc70, together with its cofactor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin^401-910 and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map,we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Original language	English
Pages (from-to)	987-996
Number of pages	10
Journal	Traffic
Volume	14
Issue number	9
Early online date	25 May 2013
DOIs	https://doi.org/10.1111/tra.12085
Publication status	Published - Sept 2013

Bibliographical note

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Funding: Royal Society; Medical Research Council (MRC grant) [G120/488, G0601125]; BBSRC; Royal Society Research Grant; NIH grant [GM038093]; Wellcome Trust [055663/Z/98/Z]; NIH [P41 RR001081]

Additional Supporting Information may be found in the online version of
this article.

Keywords

3D structure
cryo-electron microscopy (cryo-EM)
endocytosis
molecular chaperone
vesicle uncoating

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10.1111/tra.12085

Hsc70-induced changes in clathrin-auxilin cage structure
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Accepted author manuscript, 14.5 MBLicence: CC BY 3.0

http://onlinelibrary.wiley.com/doi/10.1111/tra.12085/abstract

Cite this

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title = "Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly",

abstract = "The molecular chaperone, Hsc70, together with its cofactor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin401-910 and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map,we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly. {\textcopyright} 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.",

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author = "Anna Young and Svetla Stoilova-McPhie and Alice Rothnie and Yvonne Vallis and Phillip Harvey-Smith and Neil Ranson and Helen Kent and Brodsky, {Frances M.} and Pearse, {Barbara M.F.} and Alan Roseman and Smith, {Corinne J.}",

note = "This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Funding: Royal Society; Medical Research Council (MRC grant) [G120/488, G0601125]; BBSRC; Royal Society Research Grant; NIH grant [GM038093]; Wellcome Trust [055663/Z/98/Z]; NIH [P41 RR001081] Additional Supporting Information may be found in the online version of this article.",

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AU - Young, Anna

AU - Stoilova-McPhie, Svetla

AU - Rothnie, Alice

AU - Vallis, Yvonne

AU - Harvey-Smith, Phillip

AU - Ranson, Neil

AU - Kent, Helen

AU - Brodsky, Frances M.

AU - Pearse, Barbara M.F.

AU - Roseman, Alan

AU - Smith, Corinne J.

N1 - This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Funding: Royal Society; Medical Research Council (MRC grant) [G120/488, G0601125]; BBSRC; Royal Society Research Grant; NIH grant [GM038093]; Wellcome Trust [055663/Z/98/Z]; NIH [P41 RR001081] Additional Supporting Information may be found in the online version of this article.

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N2 - The molecular chaperone, Hsc70, together with its cofactor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin401-910 and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map,we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

AB - The molecular chaperone, Hsc70, together with its cofactor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin401-910 and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map,we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

KW - 3D structure

KW - cryo-electron microscopy (cryo-EM)

KW - endocytosis

KW - molecular chaperone

KW - vesicle uncoating

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DO - 10.1111/tra.12085

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C2 - 23710728

SN - 1398-9219

VL - 14

SP - 987

EP - 996

JO - Traffic

JF - Traffic

IS - 9

ER -

Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly

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Keywords

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