Woe Yeon Kim

Two Enzymes in One: Two Yeast Peroxiredoxins Display Oxidative Stress-Dependent Switching from a Peroxidase to a Molecular Chaperone Function

Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function. We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones. The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes. Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes. This triggers a peroxidase-to-chaperone functional switch. These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient H(2)O(2)-sensor in the cells. The chaperone function of these proteins enhances yeast resistance to heat shock.

Heat-shock dependent oligomeric status alters the function of a plant-specific thioredoxin-like protein, AtTDX

We found that Arabidopsis AtTDX, a heat-stable and plant-specific thioredoxin (Trx)-like protein, exhibits multiple functions, acting as a disulfide reductase, foldase chaperone, and holdase chaperone. The activity of AtTDX, which contains 3 tetratricopeptide repeat (TPR) domains and a Trx motif, depends on its oligomeric status. The disulfide reductase and foldase chaperone functions predominate when AtTDX occurs in the low molecular weight (LMW) form, whereas the holdase chaperone function predominates in the high molecular weight (HMW) complexes. Because deletion of the TPR domains results in a significant enhancement of AtTDX disulfide reductase activity and complete loss of the holdase chaperone function, our data suggest that the TPR domains of AtTDX block the active site of Trx and play a critical role in promoting the holdase chaperone function. The oligomerization status of AtTDX is reversibly regulated by heat shock, which causes a transition from LMW to HMW complexes with concomitant functional switching from a disulfide reductase and foldase chaperone to a holdase chaperone. Overexpression of AtTDX in Arabidopsis conferred enhanced heat shock resistance to plants, primarily via its holdase chaperone activity.

Heat-Shock and Redox-Dependent Functional Switching of an h-Type Arabidopsis Thioredoxin from a Disulfide Reductase to a Molecular Chaperone

A large number of thioredoxins (Trxs), small redox proteins, have been identified from all living organisms. However, many of the physiological roles played by these proteins remain to be elucidated. We isolated a high Mr (HMW) form of h-type Trx from the heat-treated cytosolic extracts of Arabidopsis (Arabidopsis thaliana) suspension cells and designated it as AtTrx-h3. Using bacterially expressed recombinant AtTrx-h3, we find that it forms various protein structures ranging from low and oligomeric protein species to HMW complexes. And the AtTrx-h3 performs dual functions, acting as a disulfide reductase and as a molecular chaperone, which are closely associated with its molecular structures. The disulfide reductase function is observed predominantly in the low Mr forms, whereas the chaperone function predominates in the HMW complexes. The multimeric structures of AtTrx-h3 are regulated not only by heat shock but also by redox status. Two active cysteine residues in AtTrx-h3 are required for disulfide reductase activity, but not for chaperone function. AtTrx-h3 confers enhanced heat-shock tolerance in Arabidopsis, primarily through its chaperone function.

ELF4 Regulates GIGANTEA Chromatin Access through Subnuclear Sequestration

Many organisms, including plants, use the circadian clock to measure the duration of day and night. Daily rhythms in the plant circadian system are generated by multiple interlocked transcriptional/translational loops and also by spatial regulations such as nuclear translocation. GIGANTEA (GI), one of the key clock components in Arabidopsis, makes distinctive nuclear bodies like other nuclear-localized circadian regulators. However, little is known about the dynamics or roles of GI subnuclear localization. Here, we characterize GI subnuclear compartmentalization and identify unexpected dynamic changes under diurnal conditions. We further identify EARLY FLOWERING 4 (ELF4) as a regulator of GI nuclear distribution through a physical interaction. ELF4 sequesters GI from the nucleoplasm, where GI binds the promoter of CONSTANS (CO), to discrete nuclear bodies. We suggest that the subnuclear compartmentalization of GI by ELF4 contributes to the regulation of photoperiodic flowering.

HSP90 functions in the circadian clock through stabilization of the client F-box protein ZEITLUPE

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 SCFZTL, 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.

Thioredoxin Reductase Type C (NTRC) Orchestrates Enhanced Thermotolerance to Arabidopsis by Its Redox-Dependent Holdase Chaperone Function

Genevestigator analysis has indicated heat shock induction of transcripts for NADPH-thioredoxin reductase, type C (NTRC) in the light. Here we show overexpression of NTRC in Arabidopsis (NTRC°(E)) resulting in enhanced tolerance to heat shock, whereas NTRC knockout mutant plants (ntrc1) exhibit a temperature sensitive phenotype. To investigate the underlying mechanism of this phenotype, we analyzed the proteins biochemical properties and protein structure. NTRC assembles into homopolymeric structures of varying complexity with functions as a disulfide reductase, a foldase chaperone, and as a holdase chaperone. The multiple functions of NTRC are closely correlated with protein structure. Complexes of higher molecular weight (HMW) showed stronger activity as a holdase chaperone, while low molecular weight (LMW) species exhibited weaker holdase chaperone activity but stronger disulfide reductase and foldase chaperone activities. Heat shock converted LMW proteins into HMW complexes. Mutations of the two active site Cys residues of NTRC into Ser (C217/454S-NTRC) led to a complete inactivation of its disulfide reductase and foldase chaperone functions, but conferred only a slight decrease in its holdase chaperone function. The overexpression of the mutated C217/454S-NTRC provided Arabidopsis with a similar degree of thermotolerance compared with that of NTRC°(E) plants. However, after prolonged incubation under heat shock, NTRC°(E) plants tolerated the stress to a higher degree than C217/454S-NTRC°(E) plants. The results suggest that the heat shock-mediated holdase chaperone function of NTRC is responsible for the increased thermotolerance of Arabidopsis and the activity is significantly supported by NADPH.

Cloning and Sequencing Analysis of a Full-Length cDNA Encoding a G Protein [alpha] Subunit, SGA1, from Soybean

The superfamily of G proteins consists of several families including translational factors, tubulins, and ras-related small molecular weight and heterotrimeric G proteins (Kaziro et al., 1991). The membrane-bound signal-transducing G proteins are heterotrimers composed of a subunits (39-52 kD), P subunits (35-36 kD), and y subunits (7-10 kD) (Gilman, 1987). Different heterotrimeric G proteins have distinct a subunits that contain a high-affinity-binding site for guanine nucleotides and exhibit GTPase activity. In response to interaction with specific receptors, the a subunit of each G protein binds GTP and dissociates from the complex of the Py subunits. The free a subunit is then able to interact with a specific effector to convert its activity from inactive precursor to active form. After hydrolysis of bound GTP by its intrinsic GTPase activity, the a subunit makes a conformational change that favors re-association of a with Py (Conklin and Bourne, 1993). Because the specificity of the interaction of each heterotrimeric G protein with its effector resides in the a subunit, extensive studies have been carried out for the specific function of various G protein a subunits in nonplant organisms (Simon et al., 1991). Severa1 reports have also been presented for the possible function of plant G protein a subunits on light and auxin signal transduction, phytochrome-regulated gene expression, and ion-channel current regulations (Fairley-Grenot and Assmann, 1991; Romero and Lam, 1993). Nevertheless, the precise roles and characteristics for the a subunits of plant G proteins are largely unknown, and only a few cDNAs encoding a subunits of plant G proteins have been cloned (Terryn et al., 1993). Here we describe the identification of a soybean cDNA that encodes a heterotrimeric G protein a subunit by screening a soybean (Glycine max L.) AZAPII cDNA library (Table I). The clone of a G protein a subunit of Arabidopsis thaliana was used as a probe. From the 12 positive clones of 1 X 106 recombinant phage plaques, the longest cDNA insert was selected and sequenced. It contained a fulllength cDNA encoding a soybean G protein a subunit, which was designated SGAl . The soybean cDNA clone,

A Chaperone Function of NO CATALASE ACTIVITY1 Is Required to Maintain Catalase Activity and for Multiple Stress Responses in Arabidopsis

Arabidopsis protein NCA1 interacts with catalases in the cytosol and increases catalase activity through maintaining catalase folding state, which is required for stress responses. Catalases are key regulators of reactive oxygen species homeostasis in plant cells. However, the regulation of catalase activity is not well understood. In this study, we isolated an Arabidopsis thaliana mutant, no catalase activity1-3 (nca1-3) that is hypersensitive to many abiotic stress treatments. The mutated gene was identified by map-based cloning as NCA1, which encodes a protein containing an N-terminal RING-finger domain and a C-terminal tetratricopeptide repeat-like helical domain. NCA1 interacts with and increases catalase activity maximally in a 240-kD complex in planta. In vitro, NCA1 interacts with CATALASE2 (CAT2) in a 1:1 molar ratio, and the NCA1 C terminus is essential for this interaction. CAT2 activity increased 10-fold in the presence of NCA1, and zinc ion binding of the NCA1 N terminus is required for this increase. NCA1 has chaperone protein activity that may maintain the folding of catalase in a functional state. NCA1 is a cytosol-located protein. Expression of NCA1 in the mitochondrion of the nca1-3 mutant does not rescue the abiotic stress phenotypes of the mutant, while expression in the cytosol or peroxisome does. Our results suggest that NCA1 is essential for catalase activity.

The 1-Cys peroxiredoxin, a regulator of seed dormancy, functions as a molecular chaperone under oxidative stress conditions

Peroxiredoxins are antioxidative enzymes that catalyze the reduction of alkyl hydroperoxides to alcohols and hydrogen peroxide to water. 1-Cys peroxiredoxins (1-Cys Prxs) perform important roles during late seed development in plants. To characterize their biochemical functions in plants, a 1Cys-Prx gene was cloned from a Chinese cabbage cDNA library and designated as C1C-Prx. Glutamine synthetase (GS) protection and hydrogen peroxide reduction assays indicated that C1C-Prx was functionally active as a peroxidase. Also C1C-Prx prevented the thermal- or chemical-induced aggregation of malate dehydrogenase and insulin. Hydrogen peroxide treatment changed the mobility of C1C-Prx on a two-dimensional gel, which implies overoxidation of the conserved Cys residue. Furthermore, after overoxidation, the chaperone activity of C1C-Prx increased approximately two-fold, but its peroxidase activity decreased to the basal level of the reaction mixture without enzyme. However, according to the structural analysis using far-UV circular dichroism spectra, intrinsic tryptophan fluorescence spectra, and native-PAGE, overoxidation did not lead to a conformational change in C1C-Prx. Therefore, our results suggest that 1-Cys Prxs function not only to relieve mild oxidative stresses but also as molecular chaperones under severe conditions during seed germination and plant development, and that overoxidation controls the switch in function of 1-Cys-Prxs from peroxidases to molecular chaperones.

Characterization of two fungal-elicitor-induced rice cDNAs encoding functional homologues of the rab-specific GDP-dissociation inhibitor

Abstract. By using the mRNA differential display approach to isolate defense signaling genes active at the early stage of fungal infection two cDNA fragments with high sequence homology to rab-specific GDP-dissociation inhibitors (GDIs) were identified in rice (Oryza sativa L.) suspension cells. Using polymerase-chain-reaction products as probes, two full-length cDNA clones were isolated from a cDNA library of fungal-elicitor-treated rice, and designated as OsGDI1 and OsGDI2. The deduced amino acid sequences of the isolated cDNAs exhibited substantial homology to Arabidopsis rab-GDIs. Northern analysis revealed that transcripts detected with the 3′-gene-specific DNA probes accumulated to high levels within 30u2009min after treatment with a fungal elicitor derived from Magnaporthe grisea. The functionality of the OsGDIs was demonstrated by their ability to rescue the Sec19 mutant of Saccharomyces cerevisiae which is defective in vesicle transport. The proteins, expressed in Escherchia coli, cross-reacted with a polyclonal antibody prepared against bovine rab-GDI. Like bovine rab-GDI, the OsGDI proteins efficiently dissociated rab3A from bovine synaptic membranes. Using the two-hybrid system, it was shown that the OsGDIs specifically interact with the small GTP-binding proteins belonging to the rab subfamily. The specific interaction was also demonstrated in vitro by glutathione S-transferase resin pull-down assay.