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.

Fig. 1.

Reduction in HSP90 lengthens period in Arabidopsis. (A and B) Seedlings were grown in 12 h white light/12 h dark for 5–7 d before release into constant red light (17 μmol m⁻² s⁻¹) and imaged for LHY:LUC or CCA1:LUC activity for the indicated time. Free-running period of Arabidopsis seedlings expressing the CCA1:LUC (n = 12) or LHY:LUC (n = 12) reporters treated with 2 μM GDA or DMSO (0) during the imaging. **P < 0.01. (C and D) An RNAi-mediated HSP90-reduced line was crossed with CCR2:LUC, and F3 plants expressing the reporter and positive for the transgene were imaged for CCR2:LUC activity. Lines 19 (n = 46) and 83 (n = 52) displayed characteristic features of HSP90 reduction (multiple branches), whereas line 41 (n = 74) and WT (n = 71) did not. Seedlings grown and imaged as in A and B. **P < 0.01 between indicated comparisons according to Hsu's MCB (multiple comparisons with the best) (Fig. S2A). (E and F) Free-running period under constant red light of CCA1:LUC in Arabidopsis protoplasts simultaneously transiently transfected with CCA1:LUC and amiRNA designed to reduce HSP90 expression. Cytosolic HSP90 expression (HSP90.1–4) was reduced by one (HSP90.1–4) or four simultaneously transfected constructs (HSP90.1+2+3+4). Protoplasts imaged in constant red light for the time indicated. **P < 0.01 between indicated comparisons according to Hsu's MCB (Fig. S2B).

Fig. 2.

Reduction in HSP90 diminishes ZTL levels and stabilizes only SCF^ZTL targets. (A and B) Seedlings were grown 7–10 d in 12 h light/12 h dark (LD) cycles then treated at ZT 4 with 5 μM GDA or vehicle (DMSO), treated again 10 h later at ZT 14, and sampled every 4 h at the indicated times over an LD cycle. Lights on at ZT 0 and lights off at ZT 12 in all experiments. Immunoblot is representative of at least three trials; error bars in B represent ±SEM. Quantitation relative to Coomassie (Cms)-stained portion of gel and normalized to maximum expression level. *0.01 < P ≤ 0.05; **P ≤ 0.01. (C) Representative ZTL immunoblot (Upper) and average ZTL levels in three RNAi-mediated HSP90-reduced lines and untransformed WT (Lower). Samples were harvested at ZT 14 after 3 d of entrainment under LD from the seedlings used in Fig. 1 C and D. Identical letters above each line indicate not significantly different (Tukey's test). Quantitation relative to Cms-stained portion of gel and normalized to maximum expression level (in B) or nonspecific bands near the target band (in C). (D–K) Seedlings expressing TOC1:TOC1-YFP or PRRn:PRRn-GFP in four separate transgenic lines were grown 7–10 d in LD cycles then treated at ZT 4 with 5 μM GDA or vehicle (DMSO) and further treated and processed as described in A and B. (D, F, H, J) LD time course of ZTL and TOC1 or PRRn protein abundance in the corresponding backgrounds and treatment. (E, G, I, K) Quantitation of ZTL (Left) and TOC1 or PRRn protein (Right) for the time course shown. *0.01 < P ≤ 0.05; **P ≤ 0.01. All immunoblots are representative of at least three trials; error bars in all panels indicate ±SEM. Quantitation of the protein levels relative to Cms-stained portion of gel and normalized to maximum expression level.

Fig. 3.

HSP90 interacts with ZTL in vivo and exhibits holdase activity in vitro. (A) ZTL and HSP90 interact independent of light conditions. N. benthamiana were grown in LD, and protein extracts from leaves transiently and ectopically expressing ZTL and AtHSP90.1-HA were harvested at ZT 8 (light) and at the equivalent time in dark-adapted leaves (dark) and cross-linked with 1% formaldehyde. Anti-HA immunoprecipitates (IP) were probed with anti-ZTL polyclonal antibody (coIP). (B) ZTL and HSP90 interact in Arabidopsis at endogenous levels. Seedlings were grown 7–10 d in LD cycles, harvested at ZT 13, and cross-linked with 1% formaldehyde. ZTL immune complexes from protein extracts were probed for endogenous ZTL (IP) and coimmunopreciptated endogenous HSP90 (coIP). Adenosine kinase (ADK) was used as loading control. ztl-3 was used as negative control; ztl-1 is a ZTL protein-positive mutant allele. Arrows indicate migration position of ZTL or HSP90. (C) ZTL is stabilized in the presence of AtHsp90.2 under thermal denaturing conditions. Bacterial-expressed and HPLC-purified MBP-ZTL (1 μM) was tested in the absence or presence of AtHSP90.2 in various molar ratios in a chaperone holdase assay (denaturating conditions: 26.6 mM guanidine·HCl and 45 °C). Absorbance was measured over a time course of heat treatment. BSA was used as a negative control.

Fig. 4.

HSP90 and GI are linked in the posttranscriptional regulation of ZTL protein. (A) Seedlings of the indicated genotype were grown 7–10 d in LD cycles then treated with 5 μM GDA or DMSO 10 h before harvest at ZT 14. A second addition was immediately applied to the remaining tissues subsequently harvested at ZT 2. ZTL protein levels from WT, GI:GI-TAP, GI-OX, gi-2, gi-201, and ztl-3 lines under GDA or vehicle (DMSO) treatment at ZT 14 (Upper) and ZT 2 (Lower) were detected by immunoblot. Shorter (S.E.) or longer (L.E.) exposures are shown to visualize the wide range of signal intensities. (B) ZTL quantitations are based on L.E. at both time points in WT, GI:GI-TAP, gi-2, gi-201, and ztl-3 lines. (C) ZTL protein quantification based on S.E. at both time points for GI-OX. *0.01 < P ≤ 0.05; **P ≤ 0.01. GDA treatment significantly reduced ZTL abundance in all genotypes except in the gi-2 and gi-201 backgrounds (ZTL is undetectable in ztl-3). ZTL levels expressed relative to Coomassie and normalized to the maximum level at each time point. Mean is derived from at least three trials; error bars represent ±SEM.