title: In vitro characterization of bacterial and chloroplast Hsp70 systems reveals an evolutionary optimization of the co-chaperones for their Hsp70 partner.
abstract: The chloroplast Hsp70 (heat-shock protein of 70 kDa) system involved in protein folding in Chlamydomonas reinhardtii consists of HSP70B, the DnaJ homologue CDJ1 and the GrpE-type nucleotide-exchange factor CGE1. The finding that HSP70B needs to be co-expressed with HEP2 (Hsp70 escort protein 2) to become functional allowed the reconstitution of the chloroplast Hsp70 system in vitro and comparison with the homologous Escherichia coli system. Both systems support luciferase refolding and display ATPase and holdase activities. Steady-state activities are low and strongly stimulated by the co-chaperones, whose concentrations need to be balanced to optimally support luciferase refolding. Although the co-chaperones of either system generally stimulate ATPase and folding-assistance activities of the other, luciferase refolding is reduced ~10-fold and <2-fold if either Hsp70 is supplemented with the foreign DnaJ and GrpE protein respectively, suggesting an evolutionary specialization of the co-chaperones for their Hsp70 partner. Distinct features are that HSP70B's steady-state ATPase exhibits ~20-fold higher values for Vmax and Km and that the HSP70B system displays a ~6-fold higher folding assistance on denatured luciferase. Although truncating up to 16 N-terminal amino acids of CGE1 does not affect HSP70B's general ATPase and folding-assistance activities in the physiological temperature range, further deletions hampering dimerization of CGE1 via its N-terminal coiled coil do.
authors.forename: Daniel
authors.initials: D
authors.lastname: Veyel
authors.affiliation: *Molekulare Biotechnologie and Systembiologie, TU Kaiserslautern, Paul-Ehrlich-Strasse 23, D-67663 Kaiserslautern, Germany.
authors.forename: Frederik
authors.initials: F
authors.lastname: Sommer
authors.affiliation: *Molekulare Biotechnologie and Systembiologie, TU Kaiserslautern, Paul-Ehrlich-Strasse 23, D-67663 Kaiserslautern, Germany.
authors.forename: Ligia Segatto
authors.initials: LS
authors.lastname: Muranaka
authors.affiliation: *Molekulare Biotechnologie and Systembiologie, TU Kaiserslautern, Paul-Ehrlich-Strasse 23, D-67663 Kaiserslautern, Germany.
authors.forename: Mark
authors.initials: M
authors.lastname: Rütgers
authors.affiliation: ‡Laboratoire de Biologie Moléculaire et Cellulaire des Eucaryotes, FRE3354, Centre National de la Recherche Scientifique, Université Pierre et Marie Curie, Institut de Biologie Physico-Chimique, 75005 Paris, France.
authors.forename: Stéphane D
authors.initials: SD
authors.lastname: Lemaire
authors.affiliation: *Molekulare Biotechnologie and Systembiologie, TU Kaiserslautern, Paul-Ehrlich-Strasse 23, D-67663 Kaiserslautern, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schroda
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D019143:Evolution, Molecular; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D040681:Structural Homology, Protein; D013696:Temperature
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D010940:Plant Proteins; D000251:Adenosine Triphosphatases