SecB is a bona fide generalized chaperone in Escherichia coli

Abstract

It is known that the DnaK and Trigger Factor (TF) chaperones cooperate in the folding of newly synthesized cytosolic proteins in Escherichia coli. We recently showed that despite a very narrow temperature range of growth and high levels of aggregated cytosolic proteins, E. coli can tolerate deletion of both chaperones, suggesting that other chaperones might be involved in this process. Here, we show that the secretion-dedicated chaperone SecB efficiently suppresses both the temperature sensitivity and the aggregation-prone phenotypes of a strain lacking both TF and DnaK. SecB suppression is independent of a productive interaction with the SecA subunit of the translocon. Furthermore, in vitro cross-linking experiments demonstrate that SecB can interact both co- and posttranslationally with short nascent chains of both secretory and cytosolic proteins. Finally, we show that such cotranslational substrate recognition by SecB is greatly suppressed in the presence of ribosome-bound TF, but not by DnaK. Taken together, our data demonstrate that SecB acts as a bona fide generalized chaperone.

Fig. 1.

Overproduction of SecB efficiently suppresses the growth defect of a Δtig ΔdnaKdnaJ strain. Shown is in vivo complementation of the ts phenotype of the MC4100 Δtig ΔdnaKdnaJ triple mutant by the p29SEN-based IPTG-inducible constructs expressing the various proteins indicated at the top. Fresh transformants were then grown for 24 h at 20°C in LB-ampicillin, serially diluted, and spotted on LB-ampicillin agar plates without (-) or with (+)2mM IPTG inducer at the indicated temperatures. Note that TF overproduction is toxic in the Δtig ΔdnaKdnaJ triple mutant, as described (9).

Fig. 2.

Overproduction of SecB rescues protein aggregation in the absence of TF and DnaK. Complementation of the aggregation-prone phenotype of the MC4100 Δtig ΔdnaKdnaJ triple mutant by the pSE380-based, IPTG-inducible constructs expressing SecB. The cultures were incubated for 3 h at permissive (30°C) and 2 h at nonpermissive (37°C) temperatures of growth. -, the absence of IPTG; + the presence of 1 mM IPTG. Identical densities of cells were pelleted, and aggregated proteins were isolated as described by Tomoyasu et al. (17). Aggregation samples were separated by SDS/12% PAGE and stained with Coomassie blue.

Fig. 3.

SecB and TF compete for nascent polypeptides of both secreted and cytosolic proteins. (A) Schematic representation of the 150prePhoE nascent chain with potential cross-linking sites depicted by asterisks. The signal sequence is represented by a thick line. (B) In vitro translation of 150prePhoE was carried out in cell- and membrane-free E. coli extracts from SecB-overproducing cells. After translation, samples were treated with 1 mM of the cross-linker BS³, and the ribosome-associated nascent chain complexes were purified over a high salt sucrose cushion. The pellet fractions were either separated directly by SDS/15% PAGE (lanes 1-4) or after immunoprecipitation with antiserum against TF, L23, or SecB (lanes 5-7, respectively). Left bracket, TF adducts; right brackets, L23 adducts; asterisk, SecB adducts. (C) TF and SecB cross-linking adducts from B (lanes 1-4) were quantified and corrected for efficiency of translation. The highest value for cross-linking efficiency was taken as 100%. (D) Schematic representation of 150RpoB nascent chain with potential cross-linking sites depicted by asterisks. (E) Nascent chains of 150RpoB were produced, cross-linked, and analyzed by SDS/PAGE and immunoprecipitation, as described in B. TF, L23, and SecB adducts are indicated as described in B. ⁁ or o, unknown adducts. (F) TF and SecB cross-linking adducts from E (lanes 1-4) were quantified as described in C. Error bars represent the SD of five independent experiments, using four different bifunctional cross-linking agents (see Results for more details).

Fig. 4.

SecB interaction with 150RpoB is ribosome-independent. In vitro translation of nascent 150RpoB nascent chains was carried out in cell- and membrane-free E. coli extracts prepared from SecB-overproducing cells. After translation, the samples were divided into equal aliquots and treated with 0.2 mM puromycin and 0.4 M KOAc (Puro/HS) or with 25 mM EDTA, or were mock-treated with incubation buffer. After 10 min incubation at 26°C, the samples were treated with 1 mM of the cross-linker BS³, and the ribosome-associated nascent chain complexes were purified over a high-salt sucrose cushion. Both the supernatant (s) and pellet (p) fractions were separated by SDS/15% PAGE. Left bracket, TF adducts; right bracket, L23 adducts; asterisk, SecB adducts. Columns at the bottom show the quantification of SecB cross-linking adducts on the corresponding above lanes, corrected for translational efficiency. The highest value for cross-linking efficiency was taken as 100%.