Immunophilins are defined as receptors for immunosuppressive drugs including cyclosporin A, FK506, and rapamycin. The cyclosporin A receptors are referred to as cyclophilins (CYPs) and FK506-and rapamycin-binding proteins are abbreviated as FKBPs. These two groups of proteins (collectively called immunophilins) share little sequence homology, but both have peptidyl prolyl cis/trans isomerase (PPIase) activity that is involved in protein folding processes. Studies have identified immunophilins in all organisms examined including bacteria, fungi, animals, and plants. Nevertheless, the physiological function of immunophilins is poorly understood in any organism. In this study, we have surveyed the genes encoding immunophilins in Arabidopsis genome. A total of 52 genes have been found to encode putative immunophilins, among which 23 are putative FKBPs and 29 are putative CYPs. This is by far the largest immunophilin family identified in any organism. Both FKBPs and CYPs can be classified into single domain and multiple domain members. The single domain members contain a basic catalytic domain and some of them have signal sequences for targeting to a specific organelle. The multiple domain members contain not only the catalytic domain but also defined modules that are involved in protein-protein interaction or other functions. A striking feature of immunophilins in Arabidopsis is that a large fraction of FKBPs and CYPs are localized in the chloroplast, a possible explanation for why plants have a larger immunophilin family than animals. Parvulins represent another family of PPIases that are unrelated to immunophilins in protein sequences and drug binding properties. Three parvulin genes were found in Arabidopsis genome. The expression of many immunophilin and parvulin genes is ubiquitous except for those encoding chloroplast members that are often detected only in the green tissues. The large number of genes and diversity of structure domains and cellular localization make PPIases a versatile superfamily of proteins that clearly function in many cellular processes in plants.Immunosuppressive drugs cyclosporin A (CsA), FK506, and rapamycin are used clinically in transplantation to prevent graft rejection. During the course to understand the molecular mechanisms of immunosuppression by CsA, FK506, and rapamycin, the cellular receptors of these drugs have been purified and characterized (Schreiber, 1991;Fruman et al., 1994). CsA binds to a family of receptors named cyclophilins (CyPs), and FK506 and rapamycin bind to a distinct set of receptors called FKBPs (FK506 and rapamycin-binding proteins). Cyclophilins and FKBPs are collectively referred to as immunophilins (Schreiber, 1991).The complexes formed by immunophilins and their cognate ligands are the functional modules for immunosuppression. The FKBP12-FK506 and CyPCsA complexes, but not their separate components, bind to and inhibit the activity of calcineurin, a Ca 21 , calmodulin-dependent protein phosphatase (Liu et al., 1991). Studies have demonstrated that inhib...

We have identified a detoxifying efflux carrier from Arabidopsis using a functional cloning strategy. A bacterial mutant, KAM3, is deficient in multidrug resistance and does not survive on medium containing norfloxacin. After transformation of KAM3 cells with an Arabidopsis cDNA library, transformants were selected for restored growth on the toxic medium. One cDNA clone that complemented KAM3 encodes a novel protein with twelve putative transmembrane domains and contains limited sequence homology to a multidrug and toxin efflux carrier from bacteria. We named this Arabidopsis protein AtDTX1 (for Arabidopsis thaliana Detoxification 1). A large gene family of at least 56 members encoding related proteins was identified from the Arabidopsis genome. Further functional analysis of AtDTX1 protein in KAM3 mutant demonstrated that AtDTX1 serves as an efflux carrier for plant-derived alkaloids, antibiotics, and other toxic compounds. Interestingly, AtDTX1 was also capable of detoxifying Cd(2+), a heavy metal. Further experiments suggest that AtDTX1 is localized in the plasma membrane in plant cells thereby mediating the efflux of plant-derived or exogenous toxic compounds from the cytoplasm.

Photosynthetic light reactions rely on the proper function of large protein complexes (including photosystems I and II) that reside in the thylakoid membrane. Although their composition, structure, and function are known, the repertoire of assembly and maintenance factors is still being determined. Here we show that an immunophilin of the cyclophilin type, CYP38, plays a critical role in the assembly and maintenance of photosystem II (PSII) supercomplexes (SCs) in Arabidopsis. Mutant plants with the CYP38 gene interrupted by T-DNA insertion showed stunted growth and were hypersensitive to high light. Leaf chlorophyll fluorescence analysis and thylakoid membrane composition indicated that cyp38 mutant plants had defects in PSII SCs. Sucrose supplementation enabled the rescue of the mutant phenotype under low-light conditions, but failed to mitigate hypersensitivity to high-light stress. Protein radiolabeling assays showed that, although individual thylakoid proteins were synthesized equally in mutant and wild type, the assembly of the PSII SC was impaired in the mutant. In addition, the D1 and D2 components of the mutant PSII had a short half-life under high-light stress. The results provide evidence that CYP38 is necessary for the assembly and stabilization of PSII.thylakoid lumen ͉ immunophilin ͉ photosynthesis ͉ protein folding ͉ chaperone T he light reactions and attendant evolution of oxygen in photosynthesis are carried out by four multisubunit protein complexes residing in the chloroplast thylakoid membranes: photosystems I (PSI) and II (PSII), cytochrome b 6 f complex, and CF O -CF 1 complex (1-3). For a complete understanding of the photosynthetic process, it is essential to understand the biogenesis and maintenance of the participating complexes. Earlier studies on thylakoid protein supercomplex (SC) assembly, especially PSII, concentrated on the role of stromal factors, such as the translation and import machinery (4), because only a limited number of proteins were known to reside in the thylakoid lumen. However, recent proteomic findings suggest a population of 80-100 proteins in that compartment (5-7). The immunophilin family is one of the predominant groups identified.Immunophilins were originally discovered in their capacity as cellular receptors for immunosuppressive drugs: cyclosporin A and FK506 (8, 9). The receptors for cyclosporin A and FK506, named cyclophilins (CYPs) and FK506-binding proteins (FKBPs), respectively, were collectively designated as immunophilins. A common feature of most immunophilins is the associated peptidyl-prolyl cis-trans isomerase activity that catalyzes the cis-trans conversion of X-Pro peptide bonds, a rate-limiting step in protein folding (8). These proteins are now known to occur widely in organisms ranging from bacteria and fungi to animals and plants. Studies in animal and plant systems have uncovered diverse functions of immunophilins, such as protein foldases, chaperones, and scaffolding facilitators. They also possibly have unknown catalytic capabilities (10, ...

Cyclophilins belong to a large family of enzymes called “peptidyl prolyl isomerases” that assist protein folding and assembly. The cyclophilin CYP20–3 (also known as “ROC4”) is the only member of this group located in the stroma (soluble phase) of chloroplasts. In the present study we isolated mutant Arabidopsis plants defective in the CYP20–3 gene and found them to be hypersensitive to oxidative stress conditions created by high light levels, rose bengal, high salt levels, and osmotic shock. Chloroplast serine acetyltransferase (SAT1), a rate-limiting enzyme in cysteine biosynthesis, was identified as an interacting partner for CYP20–3 by protein interaction analyses. In the present experiments, SAT1 activity increased significantly under conditions of light and oxidative stress in concert with total thiols in wild-type plants. By contrast, these parameters changed only marginally in experiments with the cyp20–3 mutant, suggesting that CYP20–3 links light and stress to SAT1 activity and cysteine biosynthesis. In further support of this conclusion, our analyses showed that the salt-hypersensitive phenotype of the mutant developed under illumination and not in the dark. Together with the earlier report that CYP20–3 foldase activity is enhanced by thioredoxin-mediated reduction, our findings suggest that CYP20–3 links photosynthetic electron transport and redox regulation to the folding of SAT1, thereby enabling the cysteine-based thiol biosynthesis pathway to adjust to light and stress conditions.

Change in redox status has long been known to link light to the posttranslational regulation of chloroplast enzymes. So far, studies have been conducted primarily with thioredoxin-linked members of the stroma that function in a broad array of biosynthetic and degradatory processes. Consequently, little is known about the role of redox in regulating the growing number of enzymes found to occur in the lumen, the site of oxygen evolution in thylakoid membranes. To help fill this gap, we have studied AtFKBP13, an FKBP-type immunophilin earlier shown to interact with a redoxactive protein of the lumen, and found the enzyme to contain a pair of disulfide bonds in x-ray structural studies. These disulfides, which in protein mutagenesis experiments were shown to be essential for the associated peptidyl-prolyl isomerase activity, are unique to chloroplast FKBPs and are absent in animal and yeast counterparts. Both disulfide bonds were redox-active and were reduced by thioredoxin from either chloroplast or bacterial sources in a reaction that led to loss of enzyme activity. The results suggest a previously unrecognized paradigm for redox regulation in chloroplasts in which activation by light is achieved in concert with oxygen evolution by the oxidation of sulfhydryl groups (conversion of SH to SOS). Such a mechanism, occurring in the thylakoid lumen, is in direct contrast to regulation of enzymes in the stroma, where reduction of disulfides targeted by thioredoxin (SOS converted to SH) leads to an increase in activity in the light.