The proper ratio of GrpE to DnaK is important for protein quality control by the DnaK–DnaJ–GrpE chaperone system and for cell division

Shinya Sugimoto,Kenji Sonomoto,Kozue Saruwatari,Chihana Higashi

doi:10.1099/mic.0.2008/017376-0

Abstract

A balance of the intracellular concentrations of molecular chaperones in response to environmental conditions is of considerable importance for cellular homeostasis. Here, the physiological consequences of overexpression of GrpE in wild-type Escherichia coli MC4100 were examined. Overexpression of GrpE resulted in defects in cell division and growth, but overexpression of GrpE-G122D, which carries the G122D point mutation resulting in impaired interaction with DnaK, did not; this indicated that the effect of GrpE overexpression could be related to the DnaK chaperone function. Phase-contrast and fluorescence micrographs suggested that the N-terminal GFP-fused GrpE was colocalized with DnaK on the surface of inclusion bodies. An in vitro luciferase-refolding activity assay using purified DnaK, DnaJ and GrpE proteins demonstrated that high concentrations of GrpE significantly inhibited DnaK-mediated refolding. Furthermore, cell-free extracts from wild-type cells and GrpE-G122D-overexpressing cells significantly enhanced the refolding of luciferase. In the GrpE-overexpressing cells, abnormal localization of the cell-division protein FtsZ was observed by indirect immunofluorescence microscopy. In conclusion, the overexpression of GrpE caused a defect in the functionality of the DnaK chaperone system; this would result in filamentous morphology via abnormalities in the cell-division machinery.

Highlights

The GrpE protein of Escherichia coli is a known nucleotideexchange factor for the molecular chaperone DnaK, which functions cotranslationally and post-translationally to promote protein folding and disaggregation in cells; this occurs via nucleotide-regulated binding and release cycles (McCarty et al, 1995; Russell et al, 1998; Szabo et al, 1994)
E. coli MC4100 cells were transformed with the self-ligated pGEM-T vector, pGRPE(T7), pG122D, pGreen and pGFP-N-GRPE, and the resulting transformants were named MC4100GEM, MC4100GRPE, MC4100G122D, MC4100GFP and MC4100GFP-N-GRPE, respectively (Table 1)
It has been reported that a mutation in the grpE gene results in an abnormal filamentous morphology of E. coli cells similar to that observed in the case of an E. coli dnaK deletion mutant (Bukau & Walker, 1989; Kang & Craig, 1990)

Summary

Introduction

The GrpE protein of Escherichia coli is a known nucleotideexchange factor for the molecular chaperone DnaK, which functions cotranslationally and post-translationally to promote protein folding and disaggregation in cells; this occurs via nucleotide-regulated binding and release cycles (McCarty et al, 1995; Russell et al, 1998; Szabo et al, 1994). Excess amounts of GrpE inhibited the chaperone activity of DnaK, leading to Physiological consequences of GrpE overexpression accumulation of protein aggregates in cells and irregular localization of FtsZ. E. coli MC4100 cells were transformed with the self-ligated pGEM-T vector (pGEM), pGRPE(T7), pG122D, pGreen and pGFP-N-GRPE, and the resulting transformants were named MC4100GEM, MC4100GRPE, MC4100G122D, MC4100GFP and MC4100GFP-N-GRPE, respectively (Table 1).

Results

Conclusion