title: 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.
authors.affiliation: Department of Biochemistry, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland.
authors.forename: Ewa
authors.initials: E
authors.lastname: Laskowska
authors.affiliation: Department of Genetics, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland.
authors.forename: Jerzy
authors.initials: J
authors.lastname: Bohdanowicz
authors.affiliation: Department of Biochemistry, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland.
authors.forename: Dorota
authors.initials: D
authors.lastname: Kuczyńska-Wiśnik
authors.affiliation: Department of Biochemistry, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Matuszewska
authors.affiliation: Department of Biochemistry, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland.
authors.forename: Sabina
authors.initials: S
authors.lastname: Kędzierska
authors.affiliation: Department of Molecular Biology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland.
authors.forename: Alina
authors.initials: A
authors.lastname: Taylor
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D009154:Mutation; D011489:Protein Denaturation
chemical_list: D001426:Bacterial Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C097578:IbpA protein, E coli; C097579:IbpB protein, E coli; D046911:Macromolecular Substances; C063842:dnaK protein, E coli