title: Dissecting the heat stress response in Chlamydomonas by pharmaceutical and RNAi approaches reveals conserved and novel aspects.
abstract: To study how conserved fundamental concepts of the heat stress response (HSR) are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotolerance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenuation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell's entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP70B-antisense strains.
authors.affiliation: Molekulare Biotechnologie und Systembiologie, TU Kaiserslautern, Paul-Ehrlich-Str. 23, D-67663 Kaiserslautern, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Schmollinger
authors.forename: Miriam
authors.initials: M
authors.lastname: Schulz-Raffelt
authors.forename: Daniela
authors.initials: D
authors.lastname: Strenkert
authors.forename: Daniel
authors.initials: D
authors.lastname: Veyel
authors.forename: Olivier
authors.initials: O
authors.lastname: Vallon
authors.forename: Michael
authors.initials: M
authors.lastname: Schroda
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology; D016825:Chlamydomonas reinhardtii / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors; D018869:Heat-Shock Response; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D010766:Phosphorylation; D034622:RNA Interference; D013696:Temperature; D015533:Transcriptional Activation; D056811:Unfolded Protein Response
chemical_list: D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C001277:geldanamycin
keywords: Chlamydomonas reinhardtii; HSP70; HSP90; calcium; canavanine.; geldanamycin; heat shock factor; heat stress response; inhibitor feeding; protein folding; unfolded protein response