Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL

Abstract

In a newly isolated temperature-sensitive lethal Escherichia coli mutant affecting the chaperonin GroEL, we observed wholesale aggregation of newly translated proteins. After temperature shift, transcription, translation, and growth slowed over two to three generations, accompanied by filamentation and accretion (in approximately 2% of cells) of paracrystalline arrays containing mutant chaperonin complex. A biochemically isolated inclusion body fraction contained the collective of abundant proteins of the bacterial cytoplasm as determined by SDS/PAGE and proteolysis/MS analyses. Pulse-chase experiments revealed that newly made proteins, but not preexistent ones, were recruited to this insoluble fraction. Although aggregation of "stringent" GroEL/GroES-dependent substrates may secondarily produce an "avalanche" of aggregation, the observations raise the possibility, supported by in vitro refolding experiments, that the widespread aggregation reflects that GroEL function supports the proper folding of a majority of newly translated polypeptides, not just the limited number indicated by interaction studies and in vitro experiments.

Fig. 1.

Construction and temperature-sensitive behavior of the GroEL-deficient 461 strain. (A) Genetic configuration of strains and growth in liquid LB/ampicillin/0.02% arabinose of pBAD-EL/AI90, which serves as the wild-type (wt) strain, and the derived 461 cells. “Shift” indicates the point when the cultures were moved from 20°C to 37°C. Note that the wild-type cells exhibit a doubling time of ≈30–45 min, slightly slower than nonmanipulated wild-type E. coli strains. Note also that the same growth curves were obtained in the absence of ampicillin (data not shown). (B) Immuno-EM of paracrystalline inclusion in a 461 cell. (C) Early onset of deficient function of GroEL in 461 cells after temperature shift. The stringent GroEL substrate protein Rubisco fails to reach a soluble form in 461 early after shift to 37°C (third and fourth lanes). By contrast, ≈30% of newly translated Rubisco reaches the soluble fraction in wild-type cells (first and second lanes). T7-Rubisco-His₆, encoded by a second plasmid introduced into wild-type and 461 cells, was induced 15 min after temperature shift by infection with a CE6 λ phage bearing the T7 RNA polymerase gene. Cells were simultaneously radiolabeled with ³⁵S-Translabel. After 1 h, cells were harvested and separated into soluble and insoluble fractions as described in Inclusion Body Preparation in Materials and Methods to assess the relative amounts of newly synthesized soluble, presumably native Rubisco-His₆ and insoluble, presumably misfolded/aggregated Rubisco-His₆. Rubisco-His₆ was collected from the respective fractions by Talon affinity chromatography. Identical portions of eluted material were then analyzed by SDS/PAGE.

Fig. 2.

Translation in the mutant cells after temperature shift is initially the same as wild type but progressively slows. Additionally, MetE and GroEL are strongly expressed after shift (see text). For the translation experiment, wild-type or 461 cells grown in LB/ampicillin/arabinose at 20°C to the same OD (0.04) were shifted to 37°C, and at the indicated time points 0.4 ml of cells was labeled with 100 μCi of ³⁵S-Translabel for 10 min. Cells were immediately recovered by centrifugation and directly solubilized in SDS sample buffer. The solubilized material from an identical OD equivalent of cells for each strain was analyzed by SDS/PAGE and autoradiographed. Note that wild-type cells were not harvested at 4 h after shift because they had progressed beyond an OD of 1.5.

Fig. 3.

A large number of protein species are present in inclusion bodies prepared from 461 cells but not wild-type cells or GroE plasmid-rescued 461 cells. (A) Cell fractionation was carried out as described in Materials and Methods by using lysozyme treatment, sonication, Triton X-100 solubilization, and centrifugation. A Coomassie-stained SDS/PAGE gel shows proteins from soluble (S) and inclusion body (P) fractions prepared from equal OD amounts of cells of wild type harvested in log phase and 461 harvested after 3 h at 37°C (see Supporting Text). (B) Time course of aggregation in 461 cells. Fractionation of 461 cells at permissive temperature (20°C) and at various times after shift to 37°C into soluble (S) and inclusion body (P) fractions was carried out as described in A. Total protein in the respective fractions was displayed in SDS/PAGE.

Fig. 4.

MetE is oxidized in the insoluble fraction of 461 cells as revealed by in vivo thiol trapping (A), and aconitase can be refolded in vitro by GroEL alone (B). (A) Thiol trapping experiments were performed to ascertain the oxidation status of MetE in vivo in 461 cells (wild type does not express MetE in LB medium). Cell fractions were examined by isoelectric focusing and Western blotting (22) (see Supporting Methods in Supporting Text). The first two lanes show controls with purified MetE exposed in vitro to reducing (first lane) and oxidizing (second lane) conditions, then alkylated in the same order as for the cells; the third lane shows the supernatant fraction of alkylated 461 cells; and the fourth lane shows the inclusion body fraction of alkylated 461 cells (20-fold more total protein loaded vs. the third lane). Note that if oxidation or alkylation of MetE within inclusion bodies were different from that of the purified glutathionylated protein, then multiple bands with various isoelectric points would be expected; however, the close correspondence between the in vitro and in vivo thiol trapping samples strongly suggests that MetE is oxidized in the inclusion body fraction of the 461 cells similarly to the purified protein. (B) E. coli and yeast mitochondrial aconitase refolding in vitro: the E. coli enzyme is assisted by GroEL alone whereas the yeast enzyme requires both GroEL and GroES. Acid-unfolded aconitase B was diluted into neutralizing buffer containing the respective components, and refolding was carried out for 1 h. Iron–sulfur cluster formation was then carried out, followed by enzyme assay, as described in ref. . Activity is expressed as the percentage recovery of the input material.

Fig. 5.

Newly translated proteins, not preexistent ones, are subject to misfolding and aggregation in 461 cells. Shown are pulse–chase analyses measuring the partitioning of ³⁵S-labeled newly synthesized proteins into soluble and insoluble fractions. (A) Pulse-labeling of 461 (Left) and wild-type (Right) cells at 37°C followed by chase of 461 cells at 37°C in the presence of 200 μg/ml chloramphenicol to block further translation. (B) Pulse-labeling of 461 cells at 20°C followed by chase in chloramphenicol at 37°C. Thirty-milliliter cultures of 461 cells growing at 20°C (at an OD of ≈0.5) or that had been shifted to 37°C for 2 h were labeled for 20 and 10 min, respectively, with 3 mCi of ³⁵S-Translabel. At the various time points, 10-ml aliquots were removed from the culture and fractionated as in Materials and Methods.