title: HSP90 functions in the circadian clock through stabilization of the client F-box protein ZEITLUPE.
abstract: The autoregulatory loops of the circadian clock consist of feedback regulation of transcription/translation circuits but also require finely coordinated cytoplasmic and nuclear proteostasis. Although protein degradation is important to establish steady-state levels, maturation into their active conformation also factors into protein homeostasis. HSP90 facilitates the maturation of a wide range of client proteins, and studies in metazoan clocks implicate HSP90 as an integrator of input or output. Here we show that the Arabidopsis circadian clock-associated F-box protein ZEITLUPE (ZTL) is a unique client for cytoplasmic HSP90. The HSP90-specific inhibitor geldanamycin and RNAi-mediated depletion of cytoplasmic HSP90 reduces levels of ZTL and lengthens circadian period, consistent with ztl loss-of-function alleles. Transient transfection of artificial microRNA targeting cytoplasmic HSP90 genes similarly lengthens period. Proteolytic targets of SCF(ZTL), TOC1 and PRR5, are stabilized in geldanamycin-treated seedlings, whereas the levels of closely related clock proteins, PRR3 and PRR7, are unchanged. An in vitro holdase assay, typically used to demonstrate chaperone activity, shows that ZTL can be effectively bound, and aggregation prevented, by HSP90. GIGANTEA, a unique stabilizer of ZTL, may act in the same pathway as HSP90, possibly linking these two proteins to a similar mechanism. Our findings establish maturation of ZTL by HSP90 as essential for proper function of the Arabidopsis circadian clock. Unlike metazoan systems, HSP90 functions here within the core oscillator. Additionally, F-box proteins as clients may place HSP90 in a unique and more central role in proteostasis.
authors.affiliation: Department of Molecular Genetics, Ohio State University, Columbus, OH 43210, USA.
authors.forename: Tae-sung
authors.initials: TS
authors.lastname: Kim
authors.forename: Woe Yeon
authors.initials: WY
authors.lastname: Kim
authors.forename: Sumire
authors.initials: S
authors.lastname: Fujiwara
authors.forename: Jeongsik
authors.initials: J
authors.lastname: Kim
authors.forename: Joon-Yung
authors.initials: JY
authors.lastname: Cha
authors.forename: Jin Ho
authors.initials: JH
authors.lastname: Park
authors.forename: Sang Yeol
authors.initials: SY
authors.lastname: Lee
authors.forename: David E
authors.initials: DE
authors.lastname: Somers
mesh_terms: D017360:Arabidopsis / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D016227:Benzoquinones / Q000494:pharmacology; D002940:Circadian Rhythm / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D059748:Proteolysis / Q000187:drug effects; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; D016227:Benzoquinones; D017931:DNA Primers; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; C410503:ZTL protein, Arabidopsis; C001277:geldanamycin