Originally identified as the cellular targets of immunosuppressant drugs, the immunophilins encompass two ubiquitous protein families: the FK-506 binding proteins or FKBPs, and the cyclosporin-binding proteins or cyclophilins. Present in organisms ranging from bacteria to animals and plants, these proteins are characterized by their enzymatic activity; the peptidyl–prolyl cis–trans isomerization of polypeptides. Whilst this function is important for protein folding, it has formed the functional basis for more complex interactions between immunophilins and their target proteins. Beginning with a brief historical overview of the immunophilin family, and a representative illustration of the current state of knowledge that has accumulated for these proteins in diverse organisms, a detailed description is presented of the recent advances in the elucidation of the role of this ubiquitous protein family in plant biology. Though still in its infancy, investigation into the function of plant immunophilins has so far yielded interesting results – as a significant component of the chloroplast proteome, the abundance of immunophilins located in the thylakoid lumen suggests that these proteins may play important roles in this relatively uncharacterized subcellular compartment. Moreover, the importance of the complex multidomain immunophilins in functions pertaining to development is underscored by the strong phenotypes displayed by their corresponding mutants.
The story of this ubiquitous protein family begins in the early 1970s: as part of corporate tradition, employees of the Swiss pharmaceutical company Sandoz were encouraged to collect soil samples when on business trips and holidays. Among the recovered species, Tolypocladium inflatum was shown to synthesize a cyclic undecapeptide named cyclosporin A (CsA) which showed mild antifungal and antibiotic activity. Following this unpromising start, further screens revealed that CsA was able to reduce considerably the immune response in mice injected with sheep erythrocytes, with only minimal side effects. After more than a decade of clinical trials, cyclosporin was approved for prevention of organ transplant rejection by the US Food and Drug Administration agency. So began the race for determining the cellular receptor of this potent immunosuppressive drug. Fischer et al. (1984) successfully identified the cellular receptor of CsA in porcine kidney, a highly abundant and specific 18 kDa protein they named cyclophilin. In 1989, two independent groups found that cyclophilin was also responsible for cytosolic peptidyl–prolyl cis–trans isomerase (PPIase) activity (Fischer et al., 1989; Takahashi et al., 1989). At the same time, a fungal polyketide related to an antifungal agent called rapamycin was named FK-506 (Kino et al., 1987), and was found to possess similar pharmacological properties to cyclosporin. FK-506 and rapamycin were later found to bind to a functionally related protein named FK-506 binding protein (FKBP) (Harding et al., 1989; Siekierka et al., 1989; Standaert et al., 1990). As PPIases, FKBPs share the same enzymatic activity as cyclophilins although they show little primary sequence similarity: these two protein families are collectively known as immunophilins. To date, immunophilins have been identified in a wide range of organisms including plants, where they have been shown to carry out a variety of functions both individually and as functional components of multicomponent systems.
II. The mechanism of immunosuppression
Following the discovery of CsA and FK-506, the drug-binding properties of immunophilins triggered intensive research into their intracellular functions. Characterization of the enzymatic activity of these proteins led to the assumption that it was the drug-induced inhibition of the PPIase function that resulted in the observed suppression of the immune response. However, the finding that certain drug analogues could inhibit PPIase activity without suppressing the immune response (Bierer et al., 1990) revealed that the biochemical mechanism of these immunosuppressants was more complex than expected (Fig. 1).
The inhibitory mechanisms of CsA and FK-506 have been subject of a number of reviews (Schreiber and Crabtree, 1992, Rao et al., 1997), and a simplified version is described here. On perception of an antigenic stimulus by T-cell surface receptors, a complex signal transduction cascade is triggered: the antigen-receptor interaction results in activation of a tyrosine kinase, which phosphorylates and activates phospholipase C (PLC), which in turn cleaves phospahtidylinositol 4,5-bisphosphate into the second messengers inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglcerol. Binding of IP3 to an IP3 receptor located on the endoplasmic reticulum (ER) results in the release of Ca2+ from the ER into the cytoplasm, which in turn activates a plasma membrane localised Ca2+-release-activated Ca2+ channel, allowing influx of extracellular Ca2+. The increased Ca2+ levels then activate calcineurin, a calcium:calmodulin-dependent serine-threonine phosphatase (Klee et al., 1998). Calcium-activated calcineurin dephosphorylates the nuclear factor of activating T cells (NF-AT), which in turn is targeted to the nucleus where it up-regulates the transcription of early T-cell activation genes such as interleukin 2 and 4, granulocyte-macrophage colony-stimulating factor, and γ-interferon (Fig. 1A). CsA and FK-506 interrupt this calcium-triggered signaling cascade, resulting in the inhibition of the immune response. Following drug-binding by immunophilin, an active inhibitory ‘complex’ (FKBP-FK-506 or cyclophilin-CsA) is formed that blocks T-cell signaling by binding to and inhibiting calcineurin activity, thereby preventing dephosphorylation and nuclear targeting of NF-AT and up-regulation of immune responsive genes (Fig. 1B) (Schreiber & Crabtree, 1992; Rao et al., 1997). Residues of FKBP12 and cyclophilin A involved in calcineurin binding differ from the isomerase catalytic and drug-binding sites, in agreement with the lack of correlation between isomerase activity and calcineurin inhibition (Zydowsky et al., 1992; Etzkorn et al., 1994; Futer et al., 1995). The FKBP-rapamycin complex acts within T lymphocytes, where it binds FRAP (FKBP-rapamycin associated protein) or RAFT1 (rapamycin and FKBP targets), the homologue of TOR (Target Of Rapamycin) in yeast required for G1-S cell cycle progression (Kunz et al., 1993; Brown et al., 1994; Sabatini et al., 1994). The PPIase activity of both FKBP and cyclophilin has no physiological relevance in the immunosuppression, but elegantly demonstrates a unique gain of function mechanism for immunophilins in the presence of their drug ligands.
III. PPIase activity and protein folding
In order to perform their function within the cell, newly synthesized proteins are rapidly and efficiently converted from their primary linear sequence into their well-defined functionally competent tertiary structure. Whereas the folding of globular single-domain polypeptides occurs on a second–millisecond timescale, the isomerization of the imidic peptide bond preceding proline residues in an amino acid sequence, also known as the peptidyl–prolyl bond, constitutes a slower, rate-limiting step in the folding process (Kiefhaber et al., 1990). This is due to electron distribution and steric constraints intrinsic to prolyl bonds, which result in high rotational barriers (Odefey et al., 1995; Scherer et al., 1998). Most proteins possess peptide bonds connected in the trans conformation in both the newly synthesized unfolded form and in the folded native structure; however, prolyl bonds occur in both cis and trans conformations. Peptidyl–prolyl cis–trans isomerases (PPIase EC 220.127.116.11) catalyze the rapid isomerization of prolyl bonds from the cis to the trans configuration. Cis-prolyl bonds are uncommon, most likely due to unfavorable contacts between adjacent amino acid residues in this isomeric form, whereas the trans isomer usually takes preference (MacArthur & Thornton, 1991; Reimer & Fischer, 2002).
Whilst immunophilins are defined by their ability to bind immunosuppressant ligands, they share their PPIase activity with a third family of proteins known as the parvulins (Fischer & Aumüller, 2003; not discussed here) which show a higher degree of specificity for their substrates. Whilst few in vivo substrates of immunophilin PPIase activity have been identified, a number of in vitro protocols have been developed which allow the determination of the catalytic properties and substrate specificity of these enzymes. In a classic PPIase assay, a synthetic pentapeptide substrate, containing a proline residue, and a C-terminal paranitroaniline (pNA) group (Kofron et al., 1991) are incubated with the PPIase of interest and chymotrypsin, the latter cleaving the chromogenic pNA moiety upon PPIase-catalyzed cis–trans conversion of the substrate, resulting in emission at 390 nm. The in vitro assay described herein provides a rapid method by which the folding kinetics of PPIases can be ascertained with small peptides.
IV. Immunophilins are ubiquitous proteins
The isolation and characterization of immunophilins from phylogenetically diverse species suggests that the PPIase catalytic domain represents an important part of proteomes spanning the evolutionary divide. Although all organisms tested possess immunophilins containing a single catalytic domain (or single-domain immunophilins), FKBP or cyclophilin domains are also found in more complex proteins, often comprising more than one additional functional domain (multidomain immunophilins). In order to illustrate their functional diversity, a brief description of their targets is presented herein.
1. Bacterial immunophilins
Pathogenic bacteria possess a diverse complement of immunophilins and immunophilin-like proteins which have been shown to play varied roles in the biology of these organisms. Escherichia coli possesses at least six immunophilin-like proteins, including two cyclophilins, three FKBP like isoforms and the 48 kDa FKBP-like trigger factor. The distinctive FKBP isoforms include a conserved 12 kDa protein named SlpA (Bouvier & Stragier, 1991), a histidine-rich nickel-binding isoform called SlyD (Hottenrott et al., 1997) and a Mip (macrophage infectivity potentiator)-like FKBP called FkpA, which exists as a homodimer and possesses chaperone activity (Saul et al., 2004). SlyD and FkpA are 25–27 kDa bipartite proteins with the FKBP-like PPIase domain located in the C-terminus. Similar domain distribution is present in the Mip-like FKBP of the pathogenic bacteria Legionella pneumophila LpMIP (Fischer et al., 1992), Coxiella burnetii CbMIP (Mo et al., 1998) and Trypanosoma cruzi TcMIP (BarbosaPereira et al., 2002); these FKBPs are targeted to the bacterial cell surface, where they play an important role in macrophage infection by helping the pathogen to survive inside the cell (Wintermeyer et al., 1995).
2. Yeast immunophilins
The Saccharomyces cerevisiae proteome contains 12 immunophilin isoforms (encoded by eight CPR cyclophilin genes and four FPR FKBP genes) which are localized throughout the cell and have been shown, individually and collectively, to be dispensable for viability (Dolinski et al., 1997). By far the best-characterized yeast cyclophilin is Cyp40: possessing both PPIase and three-unit tetratricopeptide repeat (TPR) domains, it constitutes an integral component of the Hsp90 chaperone machinery. When heterologously expressed in the yeast CYP40 mutant cpr7, the activity of the Hsp90-dependent signaling components pp60v–src kinase and the mammalian glucocorticoid receptor is compromised, suggesting that CYP40 plays a general role in the Hsp90-mediated signal transduction pathways (Duina et al., 1996). The cytosolic protein FPR1 has been shown to interact with and regulate aspartokinase, possibly by modification of its structure upon product binding (Alarcon & Heitman, 1997). Also involved in metabolic control, CPR1 has been shown to mediate the import of fructose-1,6-bisphosphatase into intermediate transport vesicles (Randell Brown et al., 2001).
3. Metazoan immunophilins
The soil nematode Caenorhabditis elegans and the fruit-fly Drosophila melanogaster both possess in excess of 20 immunophilin isoforms, including the ubiquitous single-domain members and more complex isoforms consisting of multiple domains. The C. elegans cyclophilin CYP3 is highly expressed during embryonic development and is characterized by a ‘divergent loop’ motif, two neighbouring, unoxidized cysteine residues and two unique histidine and glutamic acid residues (Dornan et al., 1999). Based on the presence of the putative regulatory pair of cysteine residues and its expression pattern during development, CYP3 may function as both a redox-regulated stress responsive protein and as a foldase required to fold newly synthesized structural proteins synthesized during larval development. One of the earliest characterized immunophilins, the D. melanogaster cyclophilin NinaA forms a stable, specific complex with newly synthesized rhodopsin, thus acting as a putative molecular chaperone in the trafficking of rhodopsin from the endoplasmic reticulum to the photosensitive membranes (Colley et al., 1991; Baker et al., 1994). Moreover, Drosophila FKBP52 has been shown to interact in vivo with both the transient receptor potential-like (TRPL) cation channel and the Inactivation-no-afterpotential-D (INAD) scaffolding protein, which form a large multimeric signaling complex found in ocular photoreceptor cells. By interacting with leucyl–prolyl dipeptide bonds located at the cytoplasmic part of the channel, FKBP52 is thought to modulate channel activity by negatively regulating ion flux through TRPL2 (Goel et al., 2001).
3.1. FKBP12. In mammalian biology, immunophilin function has been a subject of intense research. Although such activities were initially due to their role as immunosuppressant binding proteins, later studies began to focus on their endogenous functions in the absence of drug ligands. A large number of cyclophilins and FKBPs have been isolated from diverse tissues, where they are likely to play distinct roles. Although significant evidence exists for mammalian immunophilin isoforms, the function of only a handful has been characterized in depth.
FKBP12 is an important single-domain immunophilin that associates with and modulates the activity of major intracellular Ca2+ release channels, including ryanodine receptors RyR1-3 and the inositol-1,4,5-triphosphate receptor (InsP3Rs). The ryanodine receptor (RyR), located in the endoplasmic reticulum of a variety of cells, functions as an intracellular Ca2+ release channel and is a crucial component of a number of fundamental cellular processes including muscle contraction and relaxation, fertilization, and apoptosis (Berridge et al., 2000). In skeletal muscle, stabilization of single-channel RyR1 by FKBP12 results in coupled activation of RyR1 clusters in transverse cisternae (Bers & Fill, 1998; Marx et al., 1998). RyR2, localized in cardiac muscle, interacts with the related immunophilin FKBP12.6, in an association which is dissolved upon phosphorylation by protein kinase A (Lam et al., 1995; Jeyakumar et al., 2001). Direct functional evidence for the function of FKBPs in calcium channel modulation came from earlier work in which mutant mice deficient in FKBP12.6 showed significant cardiac defects and altered RyR2 function (Shou et al., 1998). Most recently, a dual role for FKBP12 has been implicated in InsP3Rs function: the opening time of immobilized InsP3R channel pores is increased in the presence of FKBP12 and, in the presence of ATP, FKBP12 can coordinate gating with neighbouring receptors (Dargan et al., 2002). FKBP12 is also known to associate with the transforming growth factor-β (TGF-β) (Wang et al., 1994; Okadome et al., 1996), with which it acts as a physiologic regulator of cell cycle. In this complex interaction, FKBP12 inhibits basal signaling by TGF-β receptor I until TGF-βI is phosphorylated and released by TGF-β receptor II (Aghdasi et al., 2001). Cells from mice lacking FKBP12 show an arrest in cell cycle progression due to the reduced inhibitory effect of TGF-βI.
3.2. The steroid receptor complex immunophilins. Hsp90 is a highly abundant cytosolic protein which is important under both stress and physiological conditions: it acts as the central platform for the highly organized assembly of distinct multichaperone complexes by association with a well-defined protein complement. In higher eukaryotes, the functional foundations of the steroid receptor complex consist of Hsp90 associated with Hsp70, one of three immunophilins (FKBP51, FKBP52 or CYP40) and a number of additional cofactors including p23 and p50 (reviewed by Pratt & Toft, 1997). FKBP51 and FKBP52 are multidomain proteins composed of two consecutive N-terminal FKBP domains (FK1 and FK2) and a C-terminal, three-unit repeat of the TPR domain, the latter also possessed by CYP40 and required for Hsp90 binding. The three immunophilins compete for the unique binding site within Hsp90: in the mammalian system, FKBP52 shows the strongest affinity for Hsp90 binding, whereas FKBP51 and CYP40 are the more potent chaperones (Pirkl & Buchner, 2001). Preference binding of a specific immunophilin appears to be dictated by the nature of the substrate: the mature progesterone receptor favours FKBP51 binding over FKBP52 and CYP40, with enhanced affinity in mature glucocorticoid receptor (GR) complexes assembled in vitro, but not in estrogen receptor complexes (Nair et al., 1997; Barent et al., 1998). It has been suggested that binding of steroid hormones to receptor complexes may be actively regulated through selective interaction with distinct immunophilins; although 75% homologous, distinct residues within FKBP51 and FKBP52 may interact with one or more specific binding sites on the receptor, resulting in conformations with lower or higher affinity for GR, respectively (Riggs et al., 2003). The steroid receptor complex is only one of the clients of the Hsp90 multichaperone complex; whereas the previously mentioned immunophilins are known to mediate this physiological function, as one of the most abundant chaperone systems in the eukaryotic cytosol, the Hsp90 complex is also active under stress conditions.
V. Plant immunophilins
The discovery of the first immunophilins in plants dates to the early 1990s, the cloning era yielding the first plant cyclophilin-like cDNA sequences from tomato (Lycopersicon esculentum), maize (Zea mays) and oilseed rape (Brassica napus; Gasser et al., 1990). The Arabidopsis genome-sequencing project has allowed the identification of all the members of the immunophilin superfamily for a single plant species: comprising one of the largest such families identified to date, it consists of 29 cyclophilin isoforms and 23 FKBP isoforms (He et al., 2004; Romano et al., 2004a; Fig. 2). Extensive phylogenetic analysis has allowed thorough classification of all Arabidopsis isoforms and constitutes a sound foundation for the ongoing functional characterization of these proteins in plants. Most recently, sequence data obtained for the unicellular green alga Chlamydomonas reinhardtii suggests that this sharp increase in the number and diversity of immunophilin isoforms occurred following the divergence of red and green algae. Remarkably, the Chlamydomonas reinhardtii genome has been found to encode a total of 52 putative isoforms, including a high number of distinct orthologs of Arabidopsis immunophilins (Vallon, 2005). The plant immunophilin family consists of a wide variety of isoforms varying in form, function and cellular location.
1.1. Cyclophilins Twenty-one conserved, single-domain cyclophilin isoforms (i.e. those possessing only the PPIase domain) are encoded by the Arabidopsis genome: nine of these are predicted to be cytosolic, five are predicted to be targeted to the secretory pathway and two to the mitochondria. Although the precise localization of the majority of these isoforms awaits experimental corroboration, the subcellular location of the five single-domain chloroplast cyclophilins has been accurately ascertained in multiple studies (Peltier et al., 2002; Schubert et al., 2002; He et al., 2004; Romano et al., 2004b). Early studies purified the protein and cloned the gene for a cyclophilin localized in the chloroplast (Luan et al., 1994a; Luan & Schreiber, 1994b). In vitro import assays confirmed the location of one of two highly conserved chloroplast cyclophilins, AtCYP20-3 (previously known as ROC4), as being located in the stroma (Lippuner et al., 1994). More recently, the closely related AtCYP20-2 has been shown to be a lumenal protein (Edvardsson et al., 2003) that also partitions with thylakoid membranes enriched in photosystem II supercomplex particles (Romano et al., 2004b). The three remaining chloroplast isoforms (AtCYP26-2, AtCYP28 and AtCYP37) show little homology within the cyclophilin consensus sequence: AtCYP28 is a lumenal protein, whereas AtCYP37 has been detected in both lumenal and thylakoid membrane proteomes (Peltier et al., 2002; Schubert et al., 2002; Friso et al., 2004). AtCYP26-2 possesses a high-scoring N-terminal lumenal signal peptide but is yet to be isolated from chloroplast preparations (Table 1).
Table 1. Chloroplast localized members of the immunophilin superfamily
The multidomain cyclophilin isoforms encompass a set of proteins possessing unique domain arrangements: the smallest, the thylakoid lumenal multidomain isoform AtCYP38, is composed of an N-terminal leucine zipper domain, a central acidic region and the N-terminal catalytic PPIase domain (He et al., 2004; Romano et al., 2004a). A homologue of AtCYP38 was earlier identified from spinach as a lumenal protein called TLP40 (Fulgosi et al., 1998) that will be discussed in more detail later (Section VI, 1.1). Functionally implicated in vegetative development, Arabidopsis AtCYP40 shares its domain organization with CYP40 proteins identified in other eukaryotes, consisting of an N-terminal cyclophilin domain and a C-terminal TPR triplet separated by two putative nuclear targeting signals (Berardini et al., 2001).The existence of four multidomain cyclophilin isoforms harbouring RNA interaction domains suggests that the cyclophilin domain may have become an integral component of the nuclear RNA processing machinery. AtCYP57, AtCYP63 and AtCYP95 all possess RNA interaction motifs in the form of arginine/serine rich regions; AtCYP59 also contains this motif in addition to a zinc finger domain and a highly charged C-terminus (He et al., 2004; Romano et al., 2004a).
1.2. FKBPs Composed of 23 members, the Arabidopsis FKBP family is one of the largest FKBP families identified to date. Like their superfamily cyclophilin partners, the plant FKBPs can be categorized into single and multidomain isoforms, consisting of 16 and 7 members, respectively (He et al., 2004). The most striking finding which emerged from recent proteomic studies is the presence of 11 single-domain FKBP isoforms in the chloroplast lumen: the twin arginine N-terminal targeting motif suggests that these isoforms are targeted to this subcompartment by way of the well-characterized ΔpH pathway (Mori & Cline, 2001). Indeed, a study using in vitro import assay has shown that AtFKBP13 is translocated into the lumen through the ΔpH pathway (Gupta et al., 2002). Two single-domain FKBPs are predicted to be targeted to the secretory pathway, two contain nuclear localization signals and the remaining are predicted to be cytosolic; interestingly, the plant mitochondria is devoid of FKBP representatives (He et al., 2004).
As identified in their animal orthologues, four of the seven multidomain FKBPs possess multiple catalytic (FK) domains in addition to the TPR domain described earlier, although the four plant multidomain isoforms also have predicted calmodulin-binding domains: FKBP42 has one FK domain, whereas FKBP62, FKBP65 and FKBP72 contain three copies of the FK domain (He et al., 2004). Two of the remaining isoforms show an interesting parallel to multidomain cyclophilins in that they too possess RNA interaction motifs in the form of arginine/lysine rich N-terminal extensions, as well as multiple predicted nuclear localization signals. Consistent with its bacterial origin, the plant trigger factor is predicted to be localized in the chloroplast: the Arabidopsis trigger factor AtTIG is a poorly conserved FKBP-like protein with a central FKBP domain flanked by ribosome binding termini (He et al., 2004; see Section VI, 1.4).
VI. Specific functions of plant immunophilins
1. The chloroplast: a focal point of immunophilin function
Early studies using drug-affinity purification attempted to isolate immunophilins from distinct cellular compartments (Luan et al., 1994a). Perhaps the most striking finding was that several immunophilins, both cyclophilins and FKBPs, were produced specifically in the green tissues and induced by light when etiolated seedlings were illuminated. These immunophilin proteins were shown to be chloroplast residents (Luan et al., 1994a,b). More recently, the complementation of proteomic methodologies with extensive genome-sequencing efforts has allowed confident and accurate assignment of protein sequences to their coding cDNAs. Coupled with predicitive bioinformatics tools allowing, for example, the assignment of subcellular localization information to proteins expressed at low levels, this triple-pronged approach has yielded valuable and near-comprehensive data sets (Table 1). One such example is the in-depth characterization of the lumenal compartment of the chloroplast thylakoid membrane system (Peltier et al., 2002; Schubert et al., 2002), traditionally pigeon-holed as a proton sink required to drive photophosphorylation through ATP-synthase, housing the oxygen evolving complex, violaxanthin de-epoxidase and plastocyanin. The proteomic age has allowed the identification of up to 100 additional lumenal proteins, whose role in chloroplast biology is yet to be established. Among the newly identified proteins, the isomerases make up the largest component: in addition to a protein disulfide isomerase, as mentioned above (Section V.1.1–V.1.2), 15 immunophilin isoforms are localized within the thylakoid lumen.
1.1. TLP40 A number of photoprotective mechanisms have evolved to ensure damage limitation in photosynthetic membranes subjected to physiological stresses: these include physical separation of photosystem II (PSII) from light harvesting complex proteins (LHCII; Sundby et al., 1986, Pastenes and Horton, 1996), temperature-induced conformational changes (Havaux, 1994), and excess energy quenching by the xanthophyll cycle (Gilmore, 1997) and the PsbS protein (Li et al., 2000). Protein phosphorylation constitutes an important regulatory component of these photoprotective processes: elevated temperatures result in the activation of a membrane bound redox-sensitive kinase which phosphorylates up to 20 thylakoid membrane proteins of PSII. Due to its susceptibility to light-induced damage, the core PSII D1 protein shows the highest turnover rate of all thylakoid proteins, and thus is subject to a dynamic regenerative mechanism involving highly regulated degradation, removal, synthesis and integration (Aro et al., 1993). Proteolysis of damaged D1 only occurs following its dephosphorylation, a process in which the multidomain cyclophilin TLP40 appears to be intimately involved. The first lumenal immunophilin to be characterized in spinach, TLP40 is composed of an N-terminal leucine zipper, a putative phosphatase binding domain and a C-terminal cyclophilin-type PPIase domain and is predominantly localized in the thylakoid membrane (Fulgosi et al., 1998). TLP40 associates with and regulates the activity of a membrane phosphatase involved in PSII reaction centre protein dephosphorylation (Vener et al., 1999): following an abrupt elevation in temperature, TLP40 is released from the thylakoid membrane into the lumen, coinciding with phosphatase activation and dephosphorylation of PSII reaction centre proteins (Rokka et al., 2000).
1.2. Redox regulation of immunophilin function Redox reactions are at the heart of the complex combination of pathways which comprise photosynthesis, and as such constitute an integral part of chloroplast biology. As the foundation of the process encompassing the photochemical reactions involving both photosystem I and II and the cytochrome b6f complex, redox reactions drive the production of reducing equivalents used for carbohydrate synthesis in the Calvin cycle. In addition to its role in primary metabolism, redox has been found to be a key regulator in chloroplast biology at multiple levels. Through a set of specific signaling proteins called thioredoxins, redox can act as an indicator of the energy status of the chloroplast by regulating the activity of biosynthetic enzymes, such as members of the Calvin cycle, which are converted from a less active oxidized state in the dark to a more active reduced state by light. Regulation of both nuclear-encoded (Escoubas et al., 1995; Oswald et al., 2000) and chloroplast-encoded (Pfannschmidt, 2003) gene transcription is known to be regulated by redox state, as well as the translation of certain chloroplast encoded genes by a multiprotein complex (see the RB60 complex described below; Trebitsh et al., 2000; Trebitsh & Danon, 2001). Recent evidence suggests that immunophilins play an important role in redox regulation.
With 11 distinct isoforms ranging in size from 13 to 20 kDa, the FKBPs make up the largest portion of the lumenal immunophilins; as yet largely uncharacterized, sequence variation lying within the N-terminal region may confer substrate specificity to the different FKBPs. The smallest isoform, AtFKBP13, is the best characterized to date: a yeast 2-hybrid screen revealed the Rieske Fe–S subunit of the cytochrome b6f complex as an interacting partner of this FKBP protein. Interestingly, only the precursor (i.e. possessing both stromal and lumenal transit peptides) and partially processed (i.e. possessing only the lumenal transit peptide) forms of AtFKBP13 were able to interact with the precursor, partially processed, and lumenal (i.e. fully processed, lacking both chloroplast and stromal transit peptides) forms of the Rieske protein. Moreover, the transit peptide of AtFKBP13 alone also showed interaction, albeit reduced, with both the precursor and partially processed forms of the Rieske protein. This suggests that the mature region of the Rieske protein is sufficient for interaction, but that interaction between the two proteins can only occur in the presence of the AtFKBP13 transit peptide (Gupta et al., 2002). Coupled with the finding that mutants lacking the AtFKBP13 protein show a small increase in the levels of Rieske protein, this suggests that AtFKBP13 may be required for the regulation of cytochrome b6f levels. Further evidence for a post-transcriptional mode of Rieske regulation comes from early data showing that a 10-fold light-induced increase in Rieske transcripts only results in a 2- to 3-fold increase in protein levels (Palomares et al., 1993). A significant feature of the AtFKBP13 primary structure is the presence of two proximal pairs of cysteine residues which form two disulfide bridges. Recent evidence suggests that AtFKBP13 is likely to be oxidized under native conditions, and that disruption of either of the two disulfide bonds, or reduction of the protein by thioredoxin, leads to a reduction in PPIase activity (Gopalan et al., 2004). A key finding is that, in contrast to the stromal cyclophilin AtCYP20-3 and a number of other biosynthetic enzymes, AtFKBP13 is activated by oxidation (conversion of 2SH to S–S) rather than reduction (S–S to 2SH). These data suggest that the redox state of AtFKBP13 may play an important role in its function. One possibility we would like to propose is that AtFKBP13 has three functionally distinct forms which differ in localization and redox status (Fig. 3). Upon entry into the chloroplast, oxidized precursor AtFKBP13 may act as a chaperone for precursor Rieske protein, maintaining it in an inactive soluble form. Under conditions which up-regulate cytochrome b6f synthesis, AtFKBP13 may be reduced by thioredoxin and the AtFKBP13–Rieske complex may then be recruited to the membrane. Upon release of the Rieske subunit, AtFKBP13 may be processed to the mature form and targeted to the thylakoid lumen, where its PPIase activity may be activated by oxidation to form a catalytically active protein, which may function in folding other lumenal or thylakoid membrane proteins. A regulatory mechanism such as the one described would allow harmonization of photosynthetic activity with post-translational targeting. Redox regulation of the synthesis of photosystem components has been previously shown for the synthesis of photosystem II core protein psbA in Chlamydomonas reinhardtii, whose translation is finely tuned to electron flow by the RB60 complex, a target protein of thioredoxin (Trebitsh et al., 2000; Trebitsh & Danon, 2001). It is interesting to note that the PetC gene encoding the Rieske protein is the only nuclear encoded cytochrome b6f complex subunit; as well as being regulated at the transcriptional level (Knight et al., 2002), post-translational control of Rieske accumulation by AtFKBP13 sequestration may ensure an additional level of control, which is more directly linked to electron flow, providing ‘up to the minute’ information on cytochrome b6f requirements. The importance of the Rieske subunit is illustrated by the finding that a petC mutant of Lemna perpusilla is able to synthesize the additional, chloroplast-encoded subunits of the b6f complex, although they are rapidly degraded (Bruce & Malkin, 1991). The above hypothesis for AtFKBP13 not only provides important evidence as to the potential control mechanism for the Rieske subunit, but also suggests, for the first time, that a lumenal protein may be present in distinct functional formats which are operational in separate suborganellar compartments.
Redox regulation of chloroplast immunophilins is not only a feature of the lumenal AtFKBP13 but may also play a role in the function of one of two immunophilins present in the stroma, the conserved single-domain cyclophilin AtCYP20-3 (Motohashi et al., 2003). AtCYP20-3 may not be required in the chloroplast import process, given cyclosporin A failed to inhibit import of Rubsico small subunit protein in an in vitro import experiment (Lippuner et al., 1994). However, when compared to other plant single-domain cyclophilins, a unique feature of AtCYP20-3 is two pairs of cysteine residues, the potential importance of which has been uncovered in recent studies. AtCYP20-3 was initially identified as a putative substrate for chloroplast thioredoxin (Motohashi et al., 2001); subsequently, oxidized AtCYP20-3 was found to be catalytically inactive, and PPIase activity restored by incubation and reduction with chloroplast thioredoxin-m (Motohashi et al., 2003). AtCYP20-2, a conserved single-domain cyclophilin which partitions between the lumen and the inner thylakoid membrane, in association with photosystem II supercomplexes (Romano et al., 2004b), possesses three cysteine residues, two of which are also found in AtCYP20-3. It is tempting to speculate that, much like its stromal counterpart, the activity of this protein may be subject to redox-dependent regulation. An additional lumenal immunophilin AtFKBP16-2 shows significant homology to AtFKBP13 and shares its four cysteine residues (He et al., 2004), suggesting that it too may be a redox-regulated protein. Together with the data obtained for AtFKBP13, these findings suggest that a portion of the chloroplast immunophilins may comprise a novel target of the thioredoxin system: as cysteine-containing protein foldases, they may provide a link between protein tertiary structure formation and chloroplast redox state.
1.3. Evolutionary dynamics of the lumenal immunophilins Whereas recent proteomics studies have allowed the elucidation of the subcellular location of the majority of the remaining members of the chloroplast immunophilins (Table 1), there are still no clues as to their function in vivo. It is interesting to note that, with the exception of AtCYP20-2, the lumenal cyclophilin-like proteins possess an atypical, poorly conserved primary structure: AtCYP26-2, AtCYP28, AtCYP37 and AtCYP38 only show 10–40% similarity to human cyclophilin A (Romano et al., 2004a). Interestingly, with the exception of a number of insertions and/or deletions, which may confer substrate specificity, the lumenal FKBPs show little evolutionary diversion from the archetypal primary structure. One explanation for the sequence conservation present in the FKBP isoforms is the finding that AtFKBP13 remains catalytically active up to pH 5, whereas AtCYP20-2 is only active at pH 6 and above (P. Romano, unpublished results). It is likely that the catalytic site of FKBP-type PPIase may be more tolerant to a relatively acidic environment, such as the thylakoid lumen, whose pH is known to be between 4.5 and 6, under physiological conditions. In addition to explaining the sequence conservation of the lumenal FKBP isoforms, this may have contributed to the preferential multiplication of FKBP-type, rather than cyclophilin-type, lumenal immunophilins. The reduced pH tolerance noted for AtCYP20-2 PPIase activity may explain the reduced number and conservation of cyclophilin-type immunophilins: AtCYP26-2, AtCYP28, AtCYP37 and AtCYP38 may have forfeited their PPIase function in order to carry out novel roles within this catalytically suboptimal cellular environment. Given the localization of AtCYP20-2 and AtCYP38 (TLP40), one may surmise that the lumenal cyclophilins may have evolved functions related to membrane proteins. Conversely, being more tolerant to the soluble acidic environment, the FKBPs may have developed affinity for both membrane (e.g. AtFKBP13–Rieske) and lumenal substrates.
1.4. The trigger factor protein The phylogenetic outlier, and the only isoform which is present in eubacteria and plants, but not in other eukaryotes, is the trigger factor (TF) protein which, in comparison to archetypal FKBP and cyclophilin proteins, shows considerably enhanced (20- to 100-fold) folding activity (Stoller et al., 1995). Ironically, as the least conserved member of the FKBP family, at least in bacteria, TF function appears to be the most directly linked to protein folding, composed of a central FKBP-type PPIase domain flanked by an N-terminal ribosome binding/chaperone domain and a C-terminal chaperone domain (Zarnt et al., 1997). By binding to ribosomes at 1 : 1 stoichiometry, the 48 kDa bacterial TF is the first protein to interact with nascent polypeptide chains (Hesterkamp et al., 1996), these chains eventually interacting with the chaperones DnaK (an Hsp70 homologue), DnaJ (Deuerling et al., 1999) and the Hsp60 chaperonins GroEL/GroES (Kandror et al., 1995); these chaperones work in concert to prevent premature protein folding. Most recently, the TF has also been shown to associate with the E. coli ribosome–signal recognition particle (SRP) complex (Buskiewicz et al., 2004). Composed of the 48 kDa Ffh protein, a 114nt RNA and a 4.5S RNA, SRP associates with nascent chain ribosomes by recognition of an N-terminal SRP signature sequence present in proteins destined for the inner membrane (IM) of the bacterial cell. Once complexed with the SRP, ribosomes synthesizing IM proteins are targeted to the translocation pore of the membrane, and the whole complex then binds to the SRP receptor FtsY (Driessen et al., 2001). TF and SRP are capable of binding simultaneously to the ribosome but TF is displaced or excluded when the ribosome–SRP complex becomes bound to the FtsY (Buskiewicz et al., 2004).
As yet, no evidence exists to support the function of the chloroplast trigger factor; however, the function of the chloroplast post-translational chaperone machinery which includes heat-shock proteins and a unique SRP complex is well documented in plants. In contrast to its cytoplasmic counterpart, the chloroplast SRP (cpSRP) is composed of the Ffh homologue cpSRP54 and an additional protein cpSRP43, but lacks RNA. cpSRP demonstrates both post-translational interaction with its target, the chlorophyll a/b binding protein LHCII, as well as cotranslational transport of chloroplast-encoded thylakoid proteins such as D1 (Nilsson et al., 1999). Chloroplasts also contain a homologue of the SRP receptor FtsY; named cpFtsY, it is present both in the stroma and in association with the thylakoid membrane (Moore et al., 2003). As yet, no evidence exists for the precise function of the TF in the chloroplast. However, it may be surmised that TF may in some way cooperate with the protein maturation process either in collaboration with the Hsp70 chaperone system or as part of the SRP complex. cpSRP54 is known to exist in two pools in the chloroplast: (1) bound to spSRP43 in an LHCII transit complex formation, and (2) an spSRP43-less form associated with 70S ribosomes, which indiscriminately targets proteins possessing a hydrophobic domain composed of a minimum of 10 amino acids (Franklin & Hoffman, 1993; Schunemann et al., 1998). Given its critical function in the bacterial cell, it would be surprising if the chloroplast TF were not an integral component of the cotranslational machinery of this organelle, possibly as an additional component of the 70S ribosome-cpSRP54 complex described above.
2. The role of immunophilins in plant development
Although the role of immunophilins in chloroplast biology is still at an early stage, genetic analyses have allowed significant insight to be obtained into the role of the immunophilins in plant development. To date, three multidomain immunophilins have been shown to be potential players in signal transduction pathways analogous to those already characterized in the animal system. One such functional parallel is the previously described Hsp90 complex, which is known to form the functional scaffold of the steroid receptor complex in humans and has been shown to be present in plants (Stancato et al., 1996). In vitro experiments demonstrated that wheat germ lysate FKBP73 and FKBP77 are able to associate with mammalian p23 and plant Hsp90 via its TPR domain (Owens-Grillo et al., 1996), suggesting that the multiprotein complex components of this chaperoning mechanism are likely to be conserved between animal and plant kingdoms.
2.1. The pasticcino mutants More direct evidence illustrating the role of plant immunophilins in development comes from a screen for genes involved in cell division and differentiation: mutants showing hypertrophic growth of aerial tissue when grown on cytokinin-containing medium were selected for genetic analysis. One such class of mutants encompassing three members was named pasticcino or pas (1–3) in view of their excessive accumulation of sugars. pas mutants show a range of severe developmental defects throughout the growth stages: embryo formation is altered at the heart stage where cotyledon primordia are initiated; cotyledons do not form correctly, leading to a flat apex; seedlings possess short, thick hypocotyls and misshaped cotyledons; and mature plants are characterized by abnormal compact rosettes with multiple shoots similar to that observed in shooty teratomas. Irregular root development is also observed and consists of short primary root and no or very rare secondary root formation. pas mutants show altered response to exogenous cytokinin but are not affected in cytokinin biosynthesis (Faure et al., 1998). The complex phenotype observed in the pas1 mutant is caused by a mutation in the AtFKBP72 coding region: the AtFKBP72 protein possesses three FKBP-like domains and three TPR domains (Vittorioso et al., 1998). Expression of PAS1 increases in the presence of cytokinin and is altered in other pas mutants that show similar phenotypes to pas1. Moreover, the expression of meristematic homeobox genes KNAT2 and KNAT6 and SHOOT MERISTEMLESS is higher in pas1 mutants. Enhanced expression of these genes is consistent with an enlarged meristematic zone that can be mimicked by cytokinin addition. The cytokinin induction of primary cytokinin response markers ARR1 and ARR6 is enhanced and prolonged in pas mutants, suggesting that PAS functions to repress the cytokinin response. Finally, down-regulation of the primary auxin response genes IAA4 and IAA1 in pas mutants suggests an alteration in auxin response (Harrar et al., 2003).
2.2. Twisted dwarf and ultracurvata Additional evidence for the role of multidomain FKBP proteins in plant development comes from the characterization of mutants showing very marked developmental abnormalities: known as both twisted dwarf 1 (TWD) (Geisler et al., 2003) and ultracurvata 2 (UCU2) (Pérez-Pérez et al., 2004), the extreme phenotypes displayed by these mutants have been attributed to disruptions of the AtFKBP42 gene. Plants lacking AtFKBP42 display a pleiotropic phenotype which includes dwarfism, circinate leaves and helical rotation of a number of organs, this last effect visible at both whole plant and epidermal levels. These plants show severely distorted roots and stems, and have small flowers with occasional homeotic transformations, resulting in a partial reduction in fertility (Fig. 4). The AtFKBP42 protein consists of an N-terminal inactive PPIase domain, one TPR domain containing three motifs, a putative calmodulin-binding domain and a C-terminal transmembrane domain. Immunolocalization and in vivo epitope tagging experiments have shown AtFKBP42 protein to be a membrane-anchored protein localized to both the vacuolar and plasma membranes (Kamphausen et al., 2002; Geisler et al., 2003; Geisler et al., 2004). AtFKBP42 has been shown to interact and colocalise with the C-terminal domain of Arabidopsis p-glycoprotein ABC transporter AtPGP1 and its close homologue AtPGP19 (Geisler et al., 2003). Interestingly, although AtFKBP42 is known to be an inactive PPIase, interaction between the two proteins is mediated by the PPIase-like domain. More recently, AtFKBP42 has also been shown to interact with the closely related multidrug resistance-associated MRP/ABCC-like ABC transporters AtMRP1 and AtMRP2, which are residents of the vacuolar membranes (Liu et al., 2001; Geisler et al., 2004). In contrast to AtPGP1 and AtPGP19, interaction occurs via the C-terminal TPR domain of AtFKBP42 and the functional calmodulin-binding domain of AtMRP1, as shown by both yeast 2-hybrid analysis and coimmunoprecipitation. Additional functional evidence comes from the finding that AtFKBP42 binds to vacuolar membranes where it affects the uptake of previously characterized vacuolar transporter substrates of AtMRP1 and AtMRP2 (Geisler et al., 2004).
2.3. The FKBP12 interactor AtFIP37 In order to understand the role of immunophilins in plant development better, the in-depth characterization of signaling partners operating alongside cyclophilins and FKBPs is of critical importance. One such partner is AtFIP37, an interacting partner of the single-domain AtFKBP12 (Faure et al., 1998), whose function appears to be essential throughout development, but especially in the early stages of embryogenesis and endosperm formation (Vespa et al., 2004). AtFIP37 shows significant homology to animal proteins involved in splicing processes such as HsWTAP (a nuclear-localized interactor of the antitumoral protein WT1) (Little et al., 2000) and DmFL(2)D (of unknown subcellular distribution, but known to be involved in alternative splicing of female-specific premRNAs) (Penalva et al., 2000). Plants lacking AtFIP37 display a number of developmental complications, including a reduced rate of cell division in both the endosperm and embryo, resulting in a seed-lethal phenotype. Interestingly, this embryo-lethal phenotype is also observed in plants lacking the ‘target of rapamycin’ or TOR protein, a phosphoinositol kinase related kinase required for the regulation of protein synthesis, which is inactivated by formation of a ternary complex with FKBP12 and rapamycin. With no apparent effect on cell division, arrested seed development in tor mutants may be attributed to a deficiency in protein synthesis (Menand et al., 2002). The lack of any seed-related phenotype in an AtFKBP12 mutant suggests that AtFKBP12 is not the only target of AtFIP37.
Whilst clearly crucial for seed development, AtFIP37 mRNA can be detected throughout development, in all vegetative tissues – in particular, in lateral roots, in leaves including vascular bundles and trichomes, and in pollen grains of postanthetic flowers. An AtFIP37–yellow fluorescent protein fusion is targeted to the nucleus (Fig. 5a); in particular, the fluorescent signal appears to be localized to nuclear speckles, subnuclear structures enriched in premessenger RNA splicing factors. AtFIP37 overexpression has provided significant insight into the putative function of this protein in mature tissue: in addition to an increase in both the number and the branch number of trichomes, transgenic lines show an increase in endoreduplication (DNA replication without cell division) in trichome nuclei alone (Fig. 5b). The nature of the AtFIP37 overexpression phenotype suggests that the protein may only affect endoreduplication in cells, such as trichomes, that form early in development, and which stop dividing. One possibility is that AtFIP37 may regulate the activity of components involved in the initiation of DNA replication, such as the B-type cyclin CYCE, a crucial component of endoreduplication in fruit-fly (Richardson et al., 1995). The increased nuclear volume of polyploidal 35S::AtFIP37 plant trichomes also suggests that, unlike other cell-cycle component overexpressors such as KRP2 (De Veylder et al., 2001), ploidy and cell size are not uncoupled. AtFIP37 shows striking parallels with the nuclear splicing modulator AtSRp30, which, when overexpressed, results in multibranched trichomes, as well as alternative splicing of several genes. A strong possibility is that AtFIP37 may also be involved in mRNA splicing – in particular, that of genes involved in the endoreduplicative cycle.
VII. Concluding remarks
While, in some cases, considerable progress has been made in understanding immunophilin function, the significance of much preliminary data remains untapped. For example, while we know that immunophilin expression is up-regulated by both biotic and abiotic stresses (Luan et al., 1994b, 1996; Marivet et al., 1994; 1995; Chou & Gasser, 1997; Scholze et al., 1999; Godoy et al., 2000; Kong et al., 2001; Sharma & Singh, 2003), the precise significance of these responses is yet to be determined. It may be that the transcriptional response may be part of a general requirement for protein folding following synthesis of stress-responsive proteins; expressed sequence tag (EST) data indicates that it is likely that certain isoforms may be expressed at higher levels (He et al., 2004; Romano et al., 2004b) and therefore may act as housekeeping immunophilins, which may carry out basic folding functions. Additional hints at the putative function of a handful of immunophilins also exist: plants lacking AtCYP40 show a defect in the transition from the juvenile to adult stages of vegetative development (Berardini et al., 2001), a single-domain cyclophilin localized in and around the endoplasmic reticulum has been shown to interact with GNOM5 (a GTP exchange factor that regulates the trafficking of PIN1 auxin transporter required for the coordination of cell polarity along the apical–basal embryo axis) (Grebe et al., 2000) and several single-domain cyclophilins have been found to associate with the VirD protein (a component of the Agrobacterium tumefaciens Ti plasmid trafficking multicomplex) (Deng et al., 1998). The precise functional details of these cyclophilins are yet to be determined.
Functional characterization of immunophilins is still at an early stage, but it is clear that the chloroplast constitutes one of the chief intracellular sites of immunophilin function. While two of the four lumenal cyclophilins show close association with photosynthetic complexes, evidence for luminal and stromal immunophilins suggests that their function may comprise multiple roles in different redox states and ‘maturation’ states (i.e. precursor and mature, N-terminally cleaved forms) in both the stroma and lumen. The possibility that chloroplast immunophilins may have distinct functions dependent on their ‘maturation’ state raises the intriguing notion that proteins destined for the lumen may play distinct roles throughout their import pathway before reaching their final subcellular destination. The abundance of immunophilin isoforms in and around the thylakoid membrane may be a testament to the highly dynamic nature of this multicomponent system, which must be maintained in a functionally competent state in order to meet the metabolic demands of the cell and whole plant. While they may be functionally redundant with respect to their PPIase activity, their diverged sequences suggest that they may play additional highly specialized functions within this organelle.
The state of play for the Arabidopsis immunophilin family is one in which a large body of genomic and postgenomic data has so far been collected and analyzed; adding to this, proteomic studies have allowed accurate assignment of some immunophilins to their specific subcellular localization. Progress must now be made in establishing the function of these proteins through a multifaceted approach combining reverse genetics and physiological analysis of specific mutants with molecular interaction studies such as yeast 2-hybrid and pull-down assays. This approach may be highly valuable for distinct isoforms which show unique primary sequences, such as the multidomain FKBP and cyclophilin proteins. In particular, those isoforms which are predicted to be located in the nucleus which are known components of multiprotein complexes involved in mRNA processing would lend themselves to this kind of analysis. By contrast, interacting partners of the conserved single-domain immunophilins may be harder to identify accurately, given the high degree of homology and the likely functional redundancy existing between these proteins. Moreover, a number of immunophilins may well interact with membrane proteins, associations which would be difficult to identify using conventional screens. Attention must now be focused on the functional characterization by these means in order to understand the role of immunophilins in plant biology better.