Biochemistry, Department of


Date of this Version



The Journal of Biological Chemistry, Vol. 279, No. 9, Issue of February 27, pp. 7566–7575, 2004


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


The small heat shock proteins (sHSPs) are a ubiquitous

class of ATP-independent chaperones believed to

prevent irreversible protein aggregation and to facilitate

subsequent protein renaturation in cooperation

with ATP-dependent chaperones. Although sHSP chaperone

activity has been studied extensively in vitro, understanding

the mechanism of sHSP function requires

identification of proteins that are sHSP substrates in

vivo. We have used both immunoprecipitation and affinity

chromatography to recover 42 proteins that specifically

interact with Synechocystis Hsp16.6 in vivo during

heat treatment. These proteins can all be released from

Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones

and are heat-labile. Thirteen of the putative

substrate proteins were identified by mass spectrometry

and reveal the potential for sHSPs to protect cellular

functions as diverse as transcription, translation, cell

signaling, and secondary metabolism. One of the putative

substrates, serine esterase, was purified and tested

directly for interaction with purified Hsp16.6. Hsp16.6

effectively formed soluble complexes with serine esterase

in a heat-dependent fashion, thereby preventing formation

of insoluble serine esterase aggregates. These

data offer critical insights into the characteristics of

native sHSP substrates and extend and provide in vivo

support for the chaperone model of sHSP function.