Effects of mutations in heat-shock genes groES and groEL on protein export in Escherichia coli.

Abstract

Escherichia coli heat‐shock proteins GroES and GroEL are essential cytoplasmic proteins, which have been termed ‘chaperonins’ because of their ability to assist protein assembly of bacteriophage capsids and multimeric enzymes of foreign origin. In this report we show that temperature‐sensitive mutations in groES and groEL genes cause defective export of the plasmid‐encoded beta‐lactamase (Bla) in vivo. Since efficient translocation of proteins across biological membranes is thought to be supported by cytoplasmic factors that protect presecretory molecules from being misfolded, these results suggest that both GroES and GroEL proteins possess a chaperone function by which they facilitate export of Bla. The translocation of other secretory proteins, however, appears to depend minimally on GroE, suggesting that GroE interacts only with a specific class of secreted proteins.

Molecular Characterization of β‐Lactamase Inclusion Bodies Produced in Escherichia coli. 1. Composition

“…It is possible that heat shock proteins recognize and bind to partially folded intermediates, forming a tight complex that does not dissociate and is eventually sequestered within an aggregate. GroEL, a cytoplasmic chaperonin, has been shown to be necessary for the secretion of 0-lactamase (Kusukawa et al, 1989). It has also been shown to interact with the 8-lactamase precursor in uitro (Laminet et al, 1990 the major E. coli chaperonins (GroEL/ES and DnaK) nor the lower molecular weight heat shock proteins discovered by Allen et al (1992) were found in the cytoplasmic inclusion bodies formed by A(-2O-l)-~-lactamase, indicating that such proteins are not necessarily involved in protein aggregation.…”

Heat Shock Proteins hsp60 and hsp70: Their Roles in Folding, Assembly and Membrane Translocation of Proteins

“…In contrast, no interaction of GroEL was seen with soluble cytoplasmic proteins or with mature secreted proteins. However, the significance of these in vitro experiments is not entirely clear: in temperature-sensitive GroEL and GroES mutants only the processing of ^-lactamase was slowed down; there was no effect on the secretion of other proteins, including proOmpA (KUSUKAWA et al 1989). …”

“…Only in the presence of GroEL was the export competence of newly synthesized ^-lactamase conserved during preincubation. Therefore, the GroEL protein was proposed to stabilize a secretion-competent conformation and exert a chaperone function analogous to SecB and trigger factor (BOCHKAREVAet al 1988;KUSUKAWA et al 1989). As shown recently, the overproduction of GroEL in E. coli facilitates the export of a hybrid protein consisting of the signal sequence and the amino terminal region of the maltose-binding protein fused to /?-galactosidase (PHILLIPS and SILHAVY 1990).…”

“…Since the interaction of GroEL and GroES was only observed in the presence of ATP (CHANDRASEKHAR et al 1986), a role of the GroES protein in the release of GroEL-associated proteins was assumed (KUSUKAWAet al 1989). Among different GroES mutants analyzed in respect of processing kinetics in vivo only one affected the export of ß-lactamase, suggesting that a specific domain is important for the function of GroES (KUSUKAWA et al 1989).…”

Folding intermediates of β‐lactamase recognized by GroEL

“…While in the E. coli cell only the precursor may interact with GroEL before it is transported to the periplasm [8,9], this model system offers unique insight into chaperone guidance of protein folding because of differences in interaction between GroEL and various forms of ß-lase. The precursor of ß-lase is known to bind very tightly [10], while the mature form binds much more weakly [7], and differences between reduced and oxidized ß-lase are also apparent (this study).…”