Aggregation of heat-shock-denatured, endogenous proteins and distribution of the IbpA/B and Fda marker-proteins in Escherichia coli WT and grpE280 cells

Abstract

Submission of wild-type Escherichia coli to heat shock causes an aggregation of cellular proteins. The aggregates (S fraction) are separable from membrane fractions by ultracentrifugation in a sucrose density gradient. In contrast, no protein aggregation was detectable in an E. coli grpE280 mutant either by this technique or by electron microscopy. In search of an explanation for this observation at a molecular level, two kinds of marker proteins were used: Fda (fructose-1,6-biphosphate aldolase), the previously identified S fraction component, and IbpA/B, small heat-shock proteins abundantly associated with the S fraction proteins. Both types of marker proteins, normally never found in the outer-membrane (OM) fraction of WT cells, were present in the OM fraction from grpE cells after heat shock. This pointed to the presence of aggregates smaller than those in WT cells that cosedimented with the OM fraction. The OM fraction was enlarged in grpE cells. Although not proven directly, the presence of still smaller aggregates, not exceeding the solubility level and containing inactive Fda, was noted in the soluble CP fraction containing the cytoplasmic and periplasmic proteins. Therefore, aggregation occurred in both strains, but in a different way. The autoregulation of the heat-shock response causes a greater increase of DnaK/DnaJ and IbpAB levels in grpE cells than in WT after temperature elevation. This may explain the prevalence of the small-sized aggregates in the grpE cells. Estimation of total Fda protein before and after heat shock did not show any loss. This indicated that renaturation rather than proteolysis underlies the final disappearance of the aggregates. Though surprising at first, this is not contradictory with the participation of heat-shock proteases in removal of protein components of the S fraction as shown before, since proteins that are irreversibly denatured are probably substrates for the proteases.

Asymmetric segregation of protein aggregates is associated with cellular aging and rejuvenation

“…To this end, the endogenous ibpA was replaced by a chromosomal gene fusion to the yellow fluorescent protein (YFP) in the MG1655 sequenced wild-type E. coli strain. IbpA was previously shown to be present in the insoluble cellular fraction of heat-stressed cells (20). To check whether IbpA-YFP can serve as detector for protein aggregates, we exposed the strain expressing the fluorescent gene construct (MGAY) to various aggregating conditions.…”

Effects of SecE Depletion on the Inner and Outer Membrane Proteomes of Escherichia coli

“…5B). We found that the outer membrane fraction of SecE-depleted cells was contaminated with aggregates that can cosediment with outer membranes during density gradient centrifugation (35,39). The spots corresponding to aggregated proteins were identified and removed from the analysis set as described in Materials and Methods (see Table S3 and Fig.…”

“…Spot quantities were normalized using the "total intensity of valid spots" method to compensate for non-expression-related variations in spot quantities between gels (there were no significant variations in the total spot quantity between the two groups [SecE depletion and control]). Since protein aggregates can cosediment with outer membranes during density gradient centrifugation, an additional normalization step was required for analysis of the outer membrane gels (35,39). First, to distinguish between outer membrane FIG.…”

“…This study provides several testable hypotheses and new substrates that can be used to further discover guiding principles for protein translocation and insertion. 35 S]methionine (60 Ci/ml [1 Ci ϭ 37 GBq]) for 30 s, and this was followed by precipitation in 10% trichloroacetic acid either immediately or after a chase with cold methionine (final concentration, 0.5 mg/ml) for 3 and 10 min. Trichloroacetic acid-precipitated samples were washed with acetone, resuspended in 10 mM Tris-HCl (pH 7.5), 2% SDS, and immunoprecipitated with antiserum to OmpA.…”

The Small Heat-shock Protein HspL Is a VirB8 Chaperone Promoting Type IV Secretion-mediated DNA Transfer

“…Despite the lack of any obvious transmembrane domain or Sec-dependent signal peptide, ␣-Hsp associations with membranes have been well documented (2). E. coli IbpA/B localized in the outer membrane and functioned in protecting cells from heat and oxidative stress (51)(52)(53). M. tuberculosis Hsp16.3, a major immunodominant protein, was found to be a major membrane protein (54).…”