Characterization of the Escherichia coli YedU protein as a molecular chaperone

Abstract

We have cloned, purified to homogeneity, and characterized as a molecular chaperone the Escherichia coli YedU protein. The purified protein shows a single band at 31 kDa on SDS–polyacrylamide gels and forms dimers in solution. Like other chaperones, YedU interacts with unfolded and denatured proteins. It promotes the functional folding of citrate synthase and α-glucosidase after urea denaturation and prevents the aggregation of citrate synthase under heat shock conditions. YedU forms complexes with the permanently unfolded protein, reduced carboxymethyl α-lactalbumin. In contrast to DnaK/Hsp70, ATP does not stimulate YedU-dependent citrate synthase renaturation and does not affect the interaction between YedU and unfolded proteins, and YedU does not display any peptide-stimulated ATPase activity. We conclude that YedU is a novel chaperone which functions independently of an ATP/ADP cycle.

Every organism responds to a sudden increase in the environmental temperature by the overexpression of a set of highly conserved heat shock proteins [1]. Most of these heat shock proteins function either as molecular chaperones, assisting in protein folding and renaturation or as proteases degrading proteins that are beyond help. The explosion of genome sequencing has revealed a number of conserved genes without assigned functions (more than 30% of the Escherichia coli ORFs have no known function) [2]. We are trying to discover new functions that would help to understand the role of the heat shock response in the cell. A recent screen of the 4290 ORFs in E. coli by DNA array technology revealed 77 ORFs that are induced more than 5-fold at the RNA level upon temperature upshift [2]. The function of more than 25 of these proteins is unknown. We recently cloned and characterized the heat shock protein FtsJ (renamed RrmJ) as the 23S RNA Um2552 methyltransferase [3]. In the present study, we describe the cloning and purification of the YedU heat shock protein and show that it acts as a molecular chaperone in vitro.

Molecular chaperones are implicated in protein folding, protein targeting to membranes, protein renaturation or degradation after stress, and the control of protein–protein interactions. They can distinguish native proteins from their non-native forms, owing to the specificity of their peptide binding site, and they catalyze protein folding and renaturation in vitro (reviewed in [4] and [5]). The major classes of bacterial chaperones comprise DnaK/Hsp70 (and its assistants DnaJ and GrpE), GroEL/Hsp60 (and its assistant GroES), HtpG/Hsp90, and the small heat shock proteins [4] and [5]. In this study, we show that YedU increases the refolding of unfolded proteins, protects proteins against thermal denaturation, and forms stable complexes with the permanently unfolded protein R-CMLA.