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. 2004 Jun;186(12):3777-84.
doi: 10.1128/JB.186.12.3777-3784.2004.

Functional dissection of Escherichia coli trigger factor: unraveling the function of individual domains

G Kramer  1 , A Rutkowska, R D Wegrzyn, H Patzelt, T A Kurz, F Merz, T Rauch, S Vorderwülbecke, E Deuerling, B Bukau
Affiliations

Functional dissection of Escherichia coli trigger factor: unraveling the function of individual domains

G Kramer et al. J Bacteriol. 2004 Jun.

Abstract

In Escherichia coli, the ribosome-associated chaperone Trigger Factor (TF) promotes the folding of newly synthesized cytosolic proteins. TF is composed of three domains: an N-terminal domain (N), which mediates ribosome binding; a central domain (P), which has peptidyl-prolyl cis/trans isomerase activity and is involved in substrate binding in vitro; and a C-terminal domain (C) with unknown function. We investigated the contributions of individual domains (N, P, and C) or domain combinations (NP, PC, and NC) to the chaperone activity of TF in vivo and in vitro. All fragments comprising the N domain (N, NP, NC) complemented the synthetic lethality of Deltatig DeltadnaK in cells lacking TF and DnaK, prevented protein aggregation in these cells, and cross-linked to nascent polypeptides in vitro. However, DeltatigDeltadnaK cells expressing the N domain alone grew more slowly and showed less viability than DeltatigDeltadnaK cells synthesizing either NP, NC, or full-length TF, indicating beneficial contributions of the P and C domains to TF's chaperone activity. In an in vitro system with purified components, none of the TF fragments assisted the refolding of denatured d-glyceraldehyde-3-phosphate dehydrogenase in a manner comparable to that of wild-type TF, suggesting that the observed chaperone activity of TF fragments in vivo is dependent on their localization at the ribosome. These results indicate that the N domain, in addition to its function to promote binding to the ribosome, has a chaperone activity per se and is sufficient to substitute for TF in vivo.

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Figures

FIG. 1.
FIG. 1.
TF fragments complement synthetic lethality of the ΔtigΔdnaK double mutation. (Left panel) Schematic outline of the TF fragments synthesized in ΔdnaK cells based on the IPTG-regulated expression of the tig gene or tig fragments from the vector pTrc-99B (middle panel) (for details, see Materials and Methods). (Right panel) Cotransduction frequencies of ΔdnaK cells as an indicator of synthetic lethality at 30°C. P1 transduction was performed by using ΔdnaK cells synthesizing various TF fragments as recipient and a P1vir lysate prepared from E. coli Δtig::kan zba-3054::Tn10 (5). Transductants were selected at 30°C on tetracycline-containing LB plates and subsequently screened for deletion of the tig gene on kanamycin-containing LB plates.
FIG. 2.
FIG. 2.
Growth analysis of ΔtigΔdnaK cells expressing TF fragments. Growth of wild-type MC4100, ΔdnaK, and ΔtigΔdnaK cells expressing different TF fragments at different temperatures and in the presence of different amount of IPTG was analyzed. Cells grown overnight at 30°C in the presence of 50 μM IPTG were diluted (to concentrations corresponding to 104, 103, 102, or 10 cells/5 μl). Cells were spotted on LB plates and incubated for 24 h at the indicated temperatures.
FIG. 3.
FIG. 3.
Aggregation analyses. (A) Cells were grown at 30°C in LB media in the presence of different concentrations of IPTG. At log phase, cells were harvested and aggregates were isolated and analyzed by SDS-PAGE and Coomassie blue staining. Total lysates (left) and isolated aggregates (right) are shown. The asterisks indicate two aggregated proteins that were found exclusively in ΔtigΔdnaK cells expressing N, NP, or NC independent of the amount of IPTG inducer. The nature of these proteins is unknown. Note that the outer membrane proteins OmpF and OmpA copurified with aggregated cytoslic proteins. The reason for their partial disappearance in ΔtigΔdnaK cells synthesizing N, NP, or NC is unknown. (B) The Coomassie blue-stained SDS-PAGE shows controls. Aggregated proteins were isolated from ΔdnaK and ΔtigΔdnaK (pTrc-TF) cells grown in LB in the presence of 20 μM IPTG and ΔtigΔdnaK cells grown at 30°C in LB for a few generations (25). (C) Quantification of aggregates. Aggregated proteins were directly quantified from the Coomassie blue-stained SDS gel shown in panel A by use of the MacBasV2.5 program. The bar numbers correspond to the lanes in panel A. The average of the levels of aggregates isolated from ΔdnaK cells from three independent experiments (lanes 16, 21, and 26) was set as 1.
FIG. 4.
FIG. 4.
Interaction of nascent polypeptides with TF and TF fragments. We generated arrested 35S-labeled nascent polypeptides of IcdH (aa 1 to 173) (A) or PykF (aa 1 to 131) (B) in an in vitro TF-free transcription-translation system supplemented with TF or TF fragments. Chemical cross-linking of TF or TF fragments was performed by the addition of DSS (added where indicated) and sucrose cushion centrifugation; ribosome-nascent chain complexes were coimmunoprecipitated to identify cross-links of TF and TF variants. Cross-linking products are indicated by arrows and brackets. Antibodies raised against TF reach less efficiently with TF fragments.
FIG. 5.
FIG. 5.
Prevention of GAPDH aggregation and refolding of denatured GAPDH. (A) Aggregation of GAPDH is followed by an increase of the light-scattering signal at 620 nm after a 50-fold dilution of the denatured enzyme (final concentration, 2.5 μM). Addition of 2.5 μM TF (wt) or 20 μM TF fragment (nc) significantly inhibits aggregation. (B and C) Refolding of denatured GAPDH is monitored by measuring the enzymatic activity at different time points after a 50-fold dilution (final concentration, 2.5 μM) in the absence or the presence of TF or TF fragments. (B) Refolding activities of TF fragments N, NP, and NC in comparison to wild-type TF (wt). (C) By titration of TF and the NC fragment, activity of GAPDH was determined after 4 h.
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