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Guanosine triphosphatases (GTPases) function as molecular switches in signal transduction pathways that enable cells to respond to extracellular stimuli. Saccharomyces cerevisiae yeast protein two 1 protein (Ypt1p) is a monomeric small GTPase that is essential for endoplasmic reticulum-to-Golgi trafficking. By size-exclusion chromatography, SDS-PAGE, and native PAGE, followed by immunoblot analysis with an anti-Ypt1p antibody, we found that Ypt1p structurally changed from low-molecular-weight (LMW) forms to high-molecular-weight (HMW) complexes after heat shock. Based on our results, Ypt1p exhibited dual functions both as a GTPase and a molecular chaperone, and furthermore, heat shock induced a functional switch from that of a GTPase to a molecular chaperone driven by the structural change from LMW to HMW forms. Subsequently, we found, by using a galactose-inducible expression system, that conditional overexpression of YPT1 in yeast cells enhanced the thermotolerance of cells by increasing the survival rate at 55°C by ∼60%, compared with the control cells expressing YPT1 in the wild-type level. Altogether, our results suggest that Ypt1p is involved in the cellular protection process under heat stress conditions. Also, these findings provide new insight into the in vivo roles of small GTP-binding proteins and have an impact on research and the investigation of human diseases.

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.

2-Cys peroxiredoxins (Prxs) play important roles in the antioxidative defense systems of plant chloroplasts. In order to determine the interaction partner for these proteins in Arabidopsis, we used a yeast two-hybrid screening procedure with a C175S-mutant of Arabidopsis 2-Cys Prx-A as bait. A cDNA encoding an NADPH-dependent thioredoxin reductase (NTR) isotype C was identified and designated ANTR-C. We demonstrated that this protein effected efficient transfer of electrons from NADPH to the 2-Cys Prxs of chloroplasts. Interaction between 2-Cys Prx-A and ANTR-C was confirmed by a pull-down experiment. ANTR-C contained N-terminal TR and C-terminal Trx domains. It exhibited both TR and Trx activities and co-localized with 2-Cys Prx-A in chloroplasts. These results suggest that ANTR-C functions as an electron donor for plastidial 2-Cys Prxs and represents the NADPH-dependent TR/Trx system in chloroplasts.

Multiple isoforms of Arabidopsis thaliana h-type thioredoxins (AtTrx-hs) have distinct structural and functional specificities. AtTrx-h3 acts as both a disulfide reductase and as a molecular chaperone. We prepared five representative AtTrx-hs and compared their protein structures and disulfide reductase and molecular chaperone activities. AtTrx-h2 with an N-terminal extension exhibited distinct functional properties with respect to other AtTrx-hs. AtTrx-h2 formed low-molecular-mass structures and exhibited only disulfide reductase activity, whereas the other AtTrx-h isoforms formed high-molecular-mass complexes and displayed both disulfide reductase and molecular chaperone activities. The domains that determine the unique structural and functional properties of each AtTrx-hs protein were determined by constructing a domain-swap between the N- and C-terminal regions of AtTrx-h2 and AtTrx-h3 (designated AtTrx-h-2N3C and AtTrx-h-3N2C respectively), an N-terminal deletion mutant of AtTrx-h2 [AtTrx-h2-N(∆19)] and site-directed mutagenesis of AtTrx-h3. AtTrx-h2-N(∆19) and AtTrx-h-3N2C exhibited similar properties to those of AtTrx-h2, but AtTrx-h-2N3C behaved more like AtTrx-h3, suggesting that the structural and functional specificities of AtTrx-hs are determined by their C-terminal regions. Hydrophobicity profiling and molecular modelling revealed that Ala100 and Ala106 in AtTrx-h3 play critical roles in its structural and functional regulation. When these two residues in AtTrx-h3 were replaced with lysine, AtTrx-h3 functioned like AtTrx-h2. The chaperone function of AtTrx-hs conferred enhanced heat-shock-resistance on a thermosensitive trx1/2-null yeast mutant.

Based on the fact that the amino acid sequence of sulfiredoxin (Srx), already known as a redox-dependent sulfinic acid reductase, showed a high sequence homology with that of ParB, a nuclease enzyme, we examined the nucleic acid binding and hydrolyzing activity of the recombinant Srx in Arabidopsis (AtSrx). We found that AtSrx functions as a nuclease enzyme that can use single-stranded and double-stranded DNAs as substrates. The nuclease activity was enhanced by divalent cations. Particularly, by point-mutating the active site of sulfinate reductase, Cys (72) to Ser (AtSrx-C72S), we demonstrate that the active site of the reductase function of AtSrx is not involved in its nuclease function.

Peroxiredoxins (Prxs), which are classified into three isotypes in plants, play important roles in protection systems as peroxidases or molecular chaperones. The three Prx isotypes of Chinese cabbage, namely C1C-Prx, C2C-Prx, and C-PrxII, have recently been identified and characterized. The present study compares their molecular properties and biochemical functions to gain insights into their concerted roles in plants. The three Prx isotype genes were differentially expressed in tissue- and developmental stage-specific manners. The transcript level of the C1C-Prx gene was abundant at the seed stage, but rapidly decreased after imbibitions. In contrast, the C2C-Prx transcript was not detected in the seeds, but its expression level increased at germination and was maintained thereafter. The C-PrxII transcript level was mild at the seed stage, rapidly increased for 10 days after imbibitions, and gradually disappeared thereafter. In the localization analysis using GFP-fusion proteins, the three isotypes showed different cellular distributions. C1C-Prx was localized in the cytosol and nucleus, whereas C2C-Prx and C-Prx were found mainly in the chloroplast and cytosol, respectively. In vitro thiol-dependent antioxidant assays revealed that the relative peroxidase activities of the isotypes were C-PrxII > C2C-Prx > C1C-Prx. C1C-Prx and C2C-Prx, but not C-PrxII, prevented aggregation of malate dehydrogenase as a molecular chaperone. Taken together, these results suggest that the three isotypes of Prx play specific roles in the cells in timely and spatially different manners, but they also cooperate with each other to protect the plant.

A novel Arabidopsis thaliana inhibitor of apoptosis was identified by sequence homology to other known inhibitor of apoptosis (IAP) proteins. Arabidopsis IAP-like protein (AtILP) contained a C-terminal RING finger domain but lacked a baculovirus IAP repeat (BIR) domain, which is essential for anti-apoptotic activity in other IAP family members. The expression of AtILP in HeLa cells conferred resistance against tumor necrosis factor (TNF)-α/ActD-induced apoptosis through the inactivation of caspase activity. In contrast to the C-terminal RING domain of AtILP, which did not inhibit the activity of caspase-3, the N-terminal region, despite displaying no homology to known BIR domains, potently inhibited the activity of caspase-3 in vitro and blocked TNF-α/ActD-induced apoptosis. The anti-apoptotic activity of the AtILP N-terminal domain observed in plants was reproduced in an animal system. Transgenic Arabidopsis lines overexpressing AtILP exhibited anti-apoptotic activity when challenged with the fungal toxin fumonisin B1, an agent that induces apoptosis-like cell death in plants. In AtIPL transgenic plants, suppression of cell death was accompanied by inhibition of caspase activation and DNA fragmentation. Overexpression of AtILP also attenuated effector protein-induced cell death and increased the growth of an avirulent bacterial pathogen. The current results demonstrated the existence of a novel plant IAP-like protein that prevents caspase activation in Arabidopsis and showed that a plant anti-apoptosis gene functions similarly in plant and animal systems.

We have purified a novel antifungal protein from blast fungus (Magnaporthe grisea)-treated rice leaves using consecutive chromatographies on CM-Sepharose ion-change, Affi-gel blue, and HPLC gel filtration columns. We determined the N-terminal peptide sequence of the purified protein and subjected it to the NCBI/BLAST database and found the protein to be a partial fragment of the peroxisomal receptor protein in rice (OsPex5p). After cloning two cDNAs encoding OsPEX5L and OsPEX5S genes that are splice variants of OsPEX5 from a rice leaf cDNA library, we investigated their antifungal properties. The recombinant proteins were expressed in Escherichia coli and found to significantly inhibit cell growth of various pathogenic fungal strains. mRNA expression of the OsPEX5L gene was induced by diverse external stresses such as rice blast fungus, fungal elicitor, and other signaling molecules including H(2)O(2), abscisic acid, jasmonic acid, and salicylic acid. These results suggest that the peroxisomal receptor protein, OsPex5p, plays a critical role in the rice defense system against diverse external stresses including fungal pathogenic attack.