
The circadian clock is an internal system that is synchronized by external stimuli, such as light and temperature, and influences various physiological and developmental processes in living organisms. In the model plant Arabidopsis, transcriptional, translational and post-translational processes are interlocked by feedback loops among morning- and evening-phased genes. In a post-translational loop, plant-specific single-gene encoded GIGANTEA (GI) stabilize the F-box protein ZEITLUPE (ZTL), driving the targeted-proteasomal degradation of TIMING OF CAB EXPRESSION 1 (TOC1) and PSEUDO-RESPONSE REGULATOR 5 (PRR5). Inherent to this, we demonstrate the novel biochemical function of GI as a chaperone and/or co-chaperone of Heat-Shock Protein 90 (HSP90). GI prevents ZTL degradation as a chaperone and facilitates ZTL maturation together with HSP90/HSP70, enhancing ZTL activity in vitro and in planta. GI is known to be involved in a wide range of physiology and development as well as abiotic stress responses in plants, but it could also interact with diverse client proteins to increase protein maturation. Our results provide evidence that GI helps proteostasis of ZTL by acting as a chaperone and a co-chaperone of HSP90 for proper functioning of the Arabidopsis circadian clock.
Circadian clock rhythms regulate numerous metabolic, physiological, and developmental responses in living organisms in relation to the approximately 24-hour light-dark cycle of the Earth. In plants, diurnal and seasonal variations are coupled to changes in day-length and temperature, which serve as input signals. These are transduced to central circadian oscillators in Arabidopsis plants, such as
Molecular chaperones not only inhibit protein denaturation and aggregation but also facilitate protein folding and refolding. HSP90 (HEAT-SHOCK PROTEIN 90)-associated heteromeric chaperone machinery is well characterized in animal cells, and is composed by HSP90, HSP70, HOP (HSP90/HSP70 ORGANIZING PROTEIN), HSP40 and p23. This captures denatured substrates, and refolds the substrates with correctly folded states, thereby activating enzyme activity. We have reported that the diurnal rhythmicity of Arabidopsis circadian clocks is lengthened and that ZTL protein levels are diminished when cytosolic HSP90s are absent or deactivated by the Hsp90 inhibitor geldanamycin (Kim et al (2011) Proc Natl Acad Sci U S A 108, 16843–16848). Our results showed that the molecular chaperone activity of HSP90 is necessary for proper folding of ZTL proteins. Based on these findings, it has been questioned whether the possible function of GI as a chaperone that maintains the circadian clock in Arabidopsis is to protect ZTL proteins or to enhance the folding of ZTL with HSP90.
Our study demonstrates that GI exhibits novel chaperone activity that protects and enhances the refolding of model substrates (such as malate dehydrogenase and glucose-6-phosphate dehydrogenase) as well as that of real substrate ZTL,
GI is a large and unique plant protein that is not known to contain a conserved domain, but that is involved in diverse physiological and developmental processes such as flowering (FKF1, CDF1, SVP, TEM1, TEM2, COP1 and ELF3 as partner proteins), circadian clock (ZTL and TOC1), light signaling, hypocotyl elongation (SPY), sucrose signaling, starch accumulation, chlorophyll accumulation, transpiration, and salt stress response (SOS2) (Mishra and Paniqrahi (2015) Front Plant Sci 6, 8). However, the ways that GI regulates this wide range of physiological processes in plants still needs to be elucidated. Our findings reveal the novel GI biochemical functions of acting as a chaperone and/or co-chaperone that can affect the maturation of large numbers of client proteins that regulate diverse physiological processes.
This work was supported by a grant from the Cooperative Research Program for Agriculture, Science, & Technology Development (project no. PJ010953042017), Rural Development Administration, Republic of Korea (WYK).