Biochemistry, Department of

 

Document Type

Article

Date of this Version

2004

Citation

The Journal of Biological Chemistry, Vol. 279, No. 2, Issue of January 9, pp. 1080–1089, 2004

Comments

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Abstract

Small heat shock proteins (sHSPs) are dynamic oligomeric

proteins that bind unfolding proteins and protect

them from irreversible aggregation. This binding results

in the formation of sHSP-substrate complexes from

which substrate can later be refolded. Interactions between

sHSP and substrate in sHSP-substrate complexes

and the mechanism by which substrate is transferred to

ATP-dependent chaperones for refolding are poorly defined.

We have established C-terminal affinity-tagged

sHSPs from a eukaryote (pea HSP18.1) and a prokaryote

(Synechocystis HSP16.6) as tools to investigate these issues.

We demonstrate that sHSP subunit exchange for

HSP18.1 and HSP16.6 is temperature-dependent and

rapid at the optimal growth temperature for the organism

of origin. Increasing the ratio of sHSP to substrate

during substrate denaturation decreased sHSP-substrate

complex size, and accordingly, addition of substrate

to pre-formed sHSP-substrate complexes increased

complex size. However, the size of pre-formed

sHSP-substrate complexes could not be reduced by addition

of more sHSP, and substrate could not be observed

to transfer to added sHSP, although added sHSP

subunits continued to exchange with subunits in sHSPsubstrate

complexes. Thus, although some number of

sHSP subunits within complexes remain dynamic and

may be important for complex structure/solubility, association

of substrate with the sHSP does not appear to be

similarly dynamic. These observations are consistent

with a model in which ATP-dependent chaperones associate

directly with sHSP-bound substrate to initiate

refolding.

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