title: Assistance for a chaperone: Chlamydomonas HEP2 activates plastidic HSP70B for cochaperone binding.
abstract: Previous efforts aimed at the biochemical characterization of chloroplast HSP70B were hampered by the observation that recombinant HSP70B was inactive, i.e. incompetent of interacting with its nucleotide exchange factor CGE1. In addition, because heterologously expressed mitochondrial Hsp70 was inactive unless coexpressed with the escort protein Hep1, we wondered whether homologs of Hep1 existed in the chloroplast. Data base searches revealed that algae and higher plants indeed encode at least two HEP homologs, one predicted to be targeted to mitochondria, the others to chloroplasts. Using Chlamydomonas reinhardtii as plant model organism we demonstrate that this alga encodes an HEP homolog (termed HEP2) that is localized to the stroma. HEP2 is expressed constitutively as a low abundance protein with an apparent molecular mass of approximately 21 kDa. In cell extracts HEP2 interacts with HSP70B in an ATP-dependent fashion. Coexpression of HSP70B with HEP2 in Escherichia coli yielded high levels of CGE1-binding competent HSP70B, which also displayed ATPase activity. Inactive HSP70B was more prone to proteolysis than active HSP70B. Although inactive HSP70B interacted with HEP2, it could not be activated. Active HSP70B remained active for 48 h in the absence of HEP2, suggesting that HEP2 was not involved in maintaining HSP70B in an active state. However, some HSP70B expressed as a fusion protein with an N-terminal extension was activated when HEP2 was present during cleavage of the fusion protein, suggesting that in vivo HEP2 might be required for de novo folding of HSP70B after transit peptide cleavage.
authors.affiliation: Institute of Biology II, Plant Biochemistry, University of Freiburg, Schänzlestrasse 1, Freiburg D-79104, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Willmund
authors.forename: Manuela
authors.initials: M
authors.lastname: Hinnenberger
authors.forename: Sabine
authors.initials: S
authors.lastname: Nick
authors.forename: Miriam
authors.initials: M
authors.lastname: Schulz-Raffelt
authors.forename: Timo
authors.initials: T
authors.lastname: Mühlhaus
authors.forename: Michael
authors.initials: M
authors.lastname: Schroda
mesh_terms: D020418:Algal Proteins / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry*; D008928:Mitochondria / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins; D018087:Plastids / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
chemical_list: D020418:Algal Proteins; D018840:HSP70 Heat-Shock Proteins; C102156:HSP70B protein, Chlamydomonas; D018832:Molecular Chaperones; D010940:Plant Proteins; D015800:Protozoan Proteins