Mechanistic Differences between Two Conserved Classes of Small Heat Shock Proteins Found in the Plant Cytosol

Christopher Jones,Eman Basha,Vicki Wysocki,Elizabeth Vierling

doi:10.1074/jbc.m109.074088

Abstract

The small heat shock proteins (sHSPs) and alpha-crystallins are highly effective, ATP-independent chaperones that can bind denaturing client proteins to prevent their irreversible aggregation. One model of sHSP function suggests that the oligomeric sHSPs are activated to the client-binding form by dissociation at elevated temperatures to dimers or other sub-oligomeric species. Here we examine this model in a comparison of the oligomeric structure and chaperone activity of two conserved classes of cytosolic sHSPs in plants, the class I (CI) and class II (CII) proteins. Like the CI sHSPs, recombinant CII sHSPs from three divergent plant species, pea, wheat, and Arabidopsis, are dodecamers as determined by nano-electrospray mass spectrometry. While at 35 to 45 degrees C, all three CI sHSPs reversibly dissociate to dimers, the CII sHSPs retain oligomeric structure at high temperature. The CII dodecamers are, however, dynamic and rapidly exchange subunits, but unlike CI sHSPs, the exchange unit appears larger than a dimer. Differences in dodecameric structure are also reflected in the fact that the CII proteins do not hetero-oligomerize with CI sHSPs. Binding of the hydrophobic probe bis-ANS and limited proteolysis demonstrate CII proteins undergo significant, reversible structural changes at high temperature. All three recombinant CII proteins more efficiently protect firefly luciferase from insolubilization during heating than do the CI proteins. The CI and CII proteins behave strictly additively in client protection. In total, the results demonstrate that different sHSPs can achieve effective protection of client proteins by varied mechanisms.

Highlights

class I” (CI) and class II (CII) proteins are on average only 33% percent identical within a species [10], and evolutionary analyses indicates that CI and II sHSPs diverged over 400 million years ago, as both families are found in the moss Funaria hygrometrica [13]
SHSP Mechanism ied proteins PsHsp17.7-CII from pea [2] and TaHsp17.8-CII from wheat [10], and in addition, AtHsp17.7-CII from A. thaliana, which had not been characterized before. In all experiments these CII proteins were directly compared with CI sHSPs from the same plant species, including the well-characterized wheat TaHsp16.9-CI and pea PsHsp18.1-CI [3, 9], along with AtHsp17.6-CI from Arabidopsis [21]
We have since found that urea destabilizes the CII oligomers, urea does not appear to have a similar effect on CI oligomers

Summary

Introduction

A large body of data defining the current model for sHSP chaperone action has been derived from studies of one family of plant cytosolic sHSPs, the “class I” (CI) proteins, represented by wheat TaHsp16.9-CI, for which the crystal structure of the native dodecamer has been solved [5], and pea PsHsp18.1-CI, a close homolog (65% identical/86% similar), which has been used extensively in biochemical experiments (2, 3, 5, 9 –12) Both of these plant CI sHSPs readily dissociate at high temperature to a dimeric form that is proposed to be the client-binding species [5]. The CII proteins exemplify how similar or better efficiency in client binding can be achieved in different ways by different sHSPs

Methods

Results

Conclusion