title: Functional dissection of trigger factor and DnaK: interactions with nascent polypeptides and thermally denatured proteins.
abstract: In Escherichia coli, the ribosome-associated Trigger Factor (TF) cooperates with the DnaK system in the folding of newly synthesized cytosolic polypeptides. Here we investigated the functional relationship of TF and DnaK by comparing various functional properties of both chaperones. First, we analyzed the ability of TF and DnaK to associate with nascent polypeptides and full-length proteins released from the ribosome. Toward this end, we established an E. coli based transcription/translation system containing physiological ratios of TF, DnaK and ribosomes. In this system, TF can be crosslinked to nascent polypeptides of sigma32. No TF crosslink was found to full-length sigma32, which is known to be a DnaK substrate. In contrast, DnaK crosslinked to both nascent and full-length sigma32. DnaK crosslinks critically depended on the type of chemical crosslinker. Crosslinks represent specific substrate-chaperone interactions since they relied on the association of the nascent polypeptides with the substrate binding pocket of DnaK. While DnaK is known to be the major chaperone to prevent protein aggregation under heat shock conditions, we found that TF did not prevent aggregation of thermally unfolded proteins in vitro and was not able to complement the heat-sensitive phenotype of a deltadnaK52 mutant in vivo. These data indicate that TF and DnaK show strong differences in their ability to prevent aggregation of denatured proteins and to associate with native like substrates, but share the ability to associate with nascent polypeptides.
authors.affiliation: Institut für Biochemie und Molekularbiologie, Universität Freiburg, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Schaffitzel
authors.forename: S
authors.initials: S
authors.lastname: Rüdiger
authors.forename: B
authors.initials: B
authors.lastname: Bukau
authors.forename: E
authors.initials: E
authors.lastname: Deuerling
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D002474:Cell-Free System; D003432:Cross-Linking Reagents / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000031:analogs & derivatives* / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D018869:Heat-Shock Response; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011489:Protein Denaturation; D012270:Ribosomes / Q000378:metabolism; D012808:Sigma Factor; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32; C078167:OMPA outer membrane proteins; C041829:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; C063842:dnaK protein, E coli; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase; D005022:Ethyldimethylaminopropyl Carbodiimide