Peptidyl-prolyl Cis-trans Isomerization Research Articles

Peptidyl-prolyl isomerase Cyclophilin71 promotes SERRATE phase separation and miRNA processing in Arabidopsis

MicroRNAs (miRNAs) play an important role in gene regulation. In Arabidopsis, mature miRNAs are processed from primary miRNA transcripts by the Dicing complex that contains Dicer-like 1 (DCL1), SERRATE (SE), and Hyponastic Leaves 1 (HYL1). The Dicing complex can form nuclear dicing bodies (D-bodies) through SE phase separation. Here, we report that Cyclophilin71 (CYP71), a peptidyl-prolyl isomerase (PPIase), positively regulates miRNA processing. We show that CYP71 directly interacts with SE and enhances its phase separation, thereby promoting the formation of D-body and increasing the activity of the Dicing complex. We further show that the PPIase activity is important for the function of CYP71 in miRNA production. Our findings reveal orchestration of miRNA processing by a cyclophilin protein and suggest the involvement of peptidyl-prolyl cis-trans isomerization, a structural mechanism, in SE phase separation and miRNA processing.

Conserved Conformational Dynamics Reveal a Key Dynamic Residue in the Gatekeeper Loop of Human Cyclophilins.

Cyclophilins are ubiquitous human enzymes that catalyze peptidyl-prolyl cis-trans isomerization in protein substrates. Of the 17 unique isoforms, five closely related isoforms (CypA-E) are found in various environments and participate in diverse cellular processes, yet all have similar structures and the same core catalytic function. The question is what key residues are behind the conserved function of these enzymes. Here, conformational dynamics are compared across these isoforms to detect conserved dynamics essential for the catalytic activity of cyclophilins. A set of key dynamic residues, defined by the most dynamically conserved positions, are identified in the gatekeeper 2 region. The highly conserved glycine (Gly80) in this region is predicted to underlie the local flexibility, which is further tested by molecular dynamics simulations performed on mutants (G80A) of CypE and CypA. The mutation leads to decreased flexibility of CypE and CypA during substrate binding but increased flexibility during catalysis. Dynamical changes occur in the mutated region and a distal loop downstream of the mutation site in sequence. Examinations of the mutational effect on catalysis show that both mutated CypE and CypA exhibit shifted binding free energies of the substrate under distinct isomer conformations. The results suggest a loss of function in the mutated CypE and CypA. These catalytic changes by the mutation are likely independent of the substrate sequence, at least in CypA. Our work presents a method to identify function-related key residues in proteins.

Structural features of chloroplast trigger factor determined at 2.6 Å resolution.

The folding of newly synthesized polypeptides requires the coordinated action of molecular chaperones. Prokaryotic cells and the chloroplasts of plant cells possess the ribosome-associated chaperone trigger factor, which binds nascent polypeptides at their exit stage from the ribosomal tunnel. The structure of bacterial trigger factor has been well characterized and it has a dragon-shaped conformation, with flexible domains responsible for ribosome binding, peptidyl-prolyl cis-trans isomerization (PPIase) activity and substrate protein binding. Chloroplast trigger-factor sequences have diversified from those of their bacterial orthologs and their molecular mechanism in plant organelles has been little investigated to date. Here, the crystal structure of the plastidic trigger factor from the green alga Chlamydomonas reinhardtii is presented at 2.6 Å resolution. Due to the high intramolecular flexibility of the protein, diffraction to this resolution was only achieved using a protein that lacked the N-terminal ribosome-binding domain. The eukaryotic trigger factor from C. reinhardtii exhibits a comparable dragon-shaped conformation to its bacterial counterpart. However, the C-terminal chaperone domain displays distinct charge distributions, with altered positioning of the helical arms and a specifically altered charge distribution along the surface responsible for substrate binding. While the PPIase domain shows a highly conserved structure compared with other PPIases, its rather weak activity and an unusual orientation towards the C-terminal domain points to specific adaptations of eukaryotic trigger factor for function in chloroplasts.

Regulation of p53 Function by Formation of Non-Nuclear Heterologous Protein Complexes.

A transcription factor p53 is activated upon cellular exposure to endogenous and exogenous stresses, triggering either homeostatic correction or cell death. Depending on the stress level, often measurable as DNA damage, the dual outcome is supported by p53 binding to a number of regulatory and metabolic proteins. Apart from the nucleus, p53 localizes to mitochondria, endoplasmic reticulum and cytosol. We consider non-nuclear heterologous protein complexes of p53, their structural determinants, regulatory post-translational modifications and the role in intricate p53 functions. The p53 heterologous complexes regulate the folding, trafficking and/or action of interacting partners in cellular compartments. Some of them mainly sequester p53 (HSP proteins, G6PD, LONP1) or its partners (RRM2B, PRKN) in specific locations. Formation of other complexes (with ATP2A2, ATP5PO, BAX, BCL2L1, CHCHD4, PPIF, POLG, SOD2, SSBP1, TFAM) depends on p53 upregulation according to the stress level. The p53 complexes with SIRT2, MUL1, USP7, TXN, PIN1 and PPIF control regulation of p53 function through post-translational modifications, such as lysine acetylation or ubiquitination, cysteine/cystine redox transformation and peptidyl-prolyl cis-trans isomerization. Redox sensitivity of p53 functions is supported by (i) thioredoxin-dependent reduction of p53 disulfides, (ii) inhibition of the thioredoxin-dependent deoxyribonucleotide synthesis by p53 binding to RRM2B and (iii) changed intracellular distribution of p53 through its oxidation by CHCHD4 in the mitochondrial intermembrane space. Increasing knowledge on the structure, function and (patho)physiological significance of the p53 heterologous complexes will enable a fine tuning of the settings-dependent p53 programs, using small molecule regulators of specific protein–protein interactions of p53.

Conformational Insights into the Control of CNF1 Toxin Activity by Peptidyl-Prolyl Isomerization: A Molecular Dynamics Perspective.

The cytotoxic necrotizing factor 1 (CNF1) toxin from uropathogenic Escherichia coli constitutively activates Rho GTPases by catalyzing the deamidation of a critical glutamine residue located in the switch II (SWII). In crystallographic structures of the CNF1 catalytic domain (CNF1CD), surface-exposed P768 and P968 peptidyl-prolyl imide bonds (X-Pro) adopt an unusual cis conformation. Here, we show that mutation of each proline residue into glycine abrogates CNF1CD in vitro deamidase activity, while mutant forms of CNF1 remain functional on RhoA in cells. Using molecular dynamics simulations coupled to protein-peptide docking, we highlight the long-distance impact of peptidyl-prolyl cis-trans isomerization on the network of interactions between the loops bordering the entrance of the catalytic cleft. The energetically favorable isomerization of P768 compared with P968, induces an enlargement of loop L1 that fosters the invasion of CNF1CD catalytic cleft by a peptide encompassing SWII of RhoA. The connection of the P968 cis isomer to the catalytic cysteine C866 via a ladder of stacking interactions is alleviated along the cis-trans isomerization. Finally, the cis-trans conversion of P768 favors a switch of the thiol side chain of C866 from a resting to an active orientation. The long-distance impact of peptidyl-prolyl cis-trans isomerizations is expected to have implications for target modification.

An Alternative Pin1 Binding and Isomerization Site in the N-Terminus Domain of PSD-95.

Phosphorylation-dependent peptidyl-prolyl cis-trans isomerization plays key roles in cell cycle progression, the pathogenesis of cancer, and age-related neurodegeneration. Most of our knowledge about the role of phosphorylation-dependent peptidyl-prolyl cis-trans isomerization and the enzyme catalyzing this reaction, the peptidyl-prolyl isomerase (Pin1), is largely limited to proteins not present in neurons. Only a handful of examples have shown that phosphorylation-dependent peptidyl-prolyl cis-trans isomerization, Pin1 binding, or Pin1-mediated peptidyl-prolyl cis-trans isomerization regulate proteins present at excitatory synapses. In this work, I confirm previous findings showing that Pin1 binds postsynaptic density protein-95 (PSD-95) and identify an alternative binding site in the phosphorylated N-terminus of the PSD-95. Pin1 associates via its WW domain with phosphorylated threonine (T19) and serine (S25) in the N-terminus domain of PSD-95 and this association alters the local conformation of PSD-95. Most importantly, I show that proline-directed phosphorylation of the N-terminus domain of PSD-95 alters the local conformation of this region. Therefore, proline-directed phosphorylation of the N-terminus of PSD-95, Pin1 association, and peptidyl-prolyl cis-trans isomerization may all play a role in excitatory synaptic function and synapse development.

The Effect of Proline cis-trans Isomerization on the Folding of the C-Terminal SH2 Domain from p85.

SH2 domains are protein domains that modulate protein–protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the βE and βF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.

Unraveling Allosteric Mechanisms of Enzymatic Catalysis with an Evolutionary Analysis of Residue–Residue Contact Dynamical Changes

The evolution of protein conformational dynamics contains important information about protein function and regulation. Here, we describe an approach to dynamical-evolution analysis based on multiple microsecond molecular dynamics simulations and residue–residue contact analysis. We illustrate our approach by comparing three human cyclophilin isoforms, cyclophilin A, D, and E, which belong to a family of enzymes catalyzing peptidyl-prolyl cis–trans isomerization. Our results reveal that despite distinct overall equilibrium conformations between cyclophilins under substrate-free conditions, functional dynamical changes resembling substrate-binding and catalytic processes tend to be conserved. Key residues displaying either concerted or specific dynamical changes among isoforms during the reactions are identified, which delineate two distinct allosteric pathways for cyclophilin function consistent with recent nuclear magnetic resonance experiments. A sequence-based coevolution analysis is also employed for f...

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Cyclophilin catalyzes the ubiquitous process “peptidyl-prolyl cis-trans isomerization,” which plays a key role in protein folding, regulation, and function. Here, we present a detailed characterization of the unfolding of yeast mitochondrial cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and I2. The I1 state has native-like secondary structure and shows strong anilino-8-naphthalenesulphonate binding due to increased exposure of the solvent-accessible cluster of non-polar groups. Thus, it has some features of a molten globule. The I2 state is more unfolded, but it retains some residual secondary structure, and shows weak anilino-8-naphthalenesulphonate binding. Chemical shift perturbation analysis by 1H-15N heteronuclear single quantum coherence spectra reveals disruption of the tertiary contacts among the regions close to the active site in the first step of unfolding, i.e., the N-I1 transition. Both of the intermediates, I1 and I2, showed a propensity to self-associate under stirring conditions, but their kinetic profiles are different; the native protein did not show any such tendency under the same conditions. All these observations could have significant implications for the function of the protein.

Dissection of the contributions of cyclophilin genes to development and virulence in a fungal insect pathogen.

Cyclophilins are ubiquitous proteins found in all domains of life, catalyzing peptidyl-prolyl cis-trans isomerization (PPIase activity) and functioning in diverse cellular processes. The filamentous insect pathogenic fungus, Beauveria bassiana, contains 11 cyclophilin genes whose roles were probed via individual gene knockouts, construction of over-expression strains, and a simultaneous gene knockdown strategy using tandem SiRNA. Mutants were examined for effects on conidiation, hyphal growth, cyclosporine and stress resistance, and insect virulence. BbCypA was found to be the most highly expressed cyclophilin during growth and purified recombinant BbCypA displayed cyclosporine sensitive PPIase activity. Except for ΔBbCypA, targeted gene knockouts or overexpression of any cyclophilin resulted in temperature sensitivity (TS). Specific cyclophilin mutants showed impaired hyphal growth and differential effects on conidiation and cyclosporine resistance. Insect bioassays revealed decreased virulence for two cyclophilins (ΔBbCypE and ΔBbCyp6) and the simultaneous gene knockdown mutant constructs (SiRNA30). The BbSiRNA30 strains were unaffected in growth, conidiation, or under osmotic or cell wall perturbing stress, but did show increased resistance to cyclosporine and a TS phenotype. These results revealed common and unique roles for cyclophilins in B. bassiana and validate a method for examining the effects of multi-gene families via simultaneous gene knockdown.

Dynamical role of phosphorylation on serine/threonine-proline Pin1 substrates from constant force molecular dynamics simulations.

Cis-trans isomerization of peptidyl-prolyl bonds of the protein backbone plays an important role in numerous biological processes. Cis-trans isomerization can be the rate-limiting step due its extremely slow dynamics, compared to the millisecond time scale of many processes, and is catalyzed by a widely studied family of peptidyl-prolyl cis-trans isomerase enzymes. Also, mechanical forces along the peptide chain can speed up the rate of isomerization, resulting in "mechanical catalysis," and have been used to study peptidyl-prolyl cis-trans isomerization and other mechanical properties of proteins. Here, we use constant force molecular dynamics simulations to study the dynamical effects of phosphorylation on serine/threonine-proline protein motifs that are involved in the function of many proteins and have been implicated in many aberrant biological processes. We show that the rate of cis-trans isomerization is slowed down by phosphorylation, in excellent agreement with experiments. We use a well-grounded theory to describe the force dependent rate of isomerization. The calculated rates at zero force are also in excellent agreement with experimentally measured rates, providing additional validation of the models and force field parameters. Our results suggest that the slowdown in the rate upon phosphorylation is mainly due to an increase in the friction along the peptidyl-prolyl bond angle during isomerization. Our results provide a microscopic description of the dynamical effects of post-translational phosphorylation on cis-trans isomerization and insights into the properties of proteins under tension.

Complete Thermodynamic and Kinetic Characterization of the Isomer-Specific Interaction between Pin1-WW Domain and the Amyloid Precursor Protein Cytoplasmic Tail Phosphorylated at Thr668

Peptidyl prolyl cis-trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis-trans isomerization, accelerating the otherwise slow isomerization rate into time scales relevant for cellular signaling. Here we have combined NMR line shape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis-trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ∼22 μM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis-trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis-trans molecular switches and isomer-specific interactions involved in various biological processes.

Secreted Cyclophilin A, a Peptidylprolyl cis-trans Isomerase, Mediates Matrix Assembly of Hensin, a Protein Implicated in Epithelial Differentiation

Hensin is a rabbit ortholog of DMBT1, a multifunctional, multidomain protein implicated in the regulation of epithelial differentiation, innate immunity, and tumorigenesis. Hensin in the extracellular matrix (ECM) induced morphological changes characteristic of terminal differentiation in a clonal cell line (clone C) of rabbit kidney intercalated cells. Although hensin is secreted in monomeric and various oligomeric forms, only the polymerized ECM form is able to induce these phenotypic changes. Here we report that hensin secretion and matrix assembly were inhibited by the peptidylprolyl cis-trans isomerase (PPIase) inhibitors cyclosporin A (CsA) and a derivative of cyclosporin A with modifications in the d-Ser side chain (Cs9) but not by the calcineurin pathway inhibitor FK506. PPIase inhibition led to failure of hensin polymerization in the medium and ECM, plus the loss of apical cytoskeleton, apical microvilli, and the columnar epithelial shape of clone C cells. Cyclophilin A was produced and secreted into the media to a much greater extent than cyclophilins B and C. Our results also identified the direct CsA-sensitive interaction of cyclophilin A with hensin, suggesting that cyclophilin A is the PPIase that mediates the polymerization and matrix assembly of hensin. These results are significant because this is the first time a direct role of peptidylprolyl cis-trans isomerase activity has been implicated in the process of epithelial differentiation.

Effect of Cu2+ on the Oxidative Folding of Synthetic Maurotoxin In Vitro

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K+ channel types. It folds according to an α/β scaffold, i.e., a helix connected to a two stranded β-sheet by two disulphide bridges. In a former study, various parameters that affect the oxidation and folding of the reduced form of synthetic MTX were investigated in vitro. It was found that MTX achieves its final 3-D structure by evolving over time through a series of oxidation intermediates, from the least to the most oxidized species. MTX oxidative intermediates can be studied by iodoacetamide alkylation of free cysteine residues followed by mass spectrometry analysis. Here, we have analysed the effect of Cu2+ (0.1 to 50 mM) on the kinetics of MTX oxidative folding and found that it dramatically speeds up the formation of the four-disulphide bridged, native-like, MTX (maximal production within 30 minutes instead of > 60 hours). This catalysing effect of Cu2+ was found to be concentration-dependent, reaching a plateau at 10 mM copper ions. Cu2+ was also found to prevent the slow transition of a three disulphide-bridged MTX intermediate towards the final four disulphide-bridged product (12% of total MTX). The data are discussed in light of the potential effects of Cu2+ on MTX secondary structure formation, disulphide bridging and peptidyl prolyl cis-trans isomerization.

Force-Induced Prolyl Cis−Trans Isomerization in Elastin-like Polypeptides

Elastin-like polypeptides (ELPs) are stimulus-responsive polymers that contain repeats of five amino acids, Val-Pro-Gly-Xaa-Gly (VPGXG), where Xaa is a guest residue that can be any amino acid with the exception of proline. While studying the conformational mechanics of ELPs over a range of solvent conditions by single-molecule force spectroscopy, we noticed that some force-extension curves showed temperature-independent, extensional transitions that could not be fitted with a freely jointed chain or worm-like chain model. Here we show that the observed molecular elongation results from the force-induced peptidyl-prolyl cis-trans isomerization in prolines, which are repeated every fifth residue in the main chain of ELPs. Control experiments with poly(L-proline) demonstrate the similarity of the conformational transition between poly(L-proline) and ELPs. In contrast, the force-extension behavior of poly(L-lysine) showed no deviation in the relevant force range. Force-extension curves in hysteresis experiments showed an elongational difference between extension and relaxation pathways that suggests that the cis conformational state of the prolines could be exhausted on the time scale of the experiment. We present further computational evidence for this mechanism by Monte Carlo simulation of the force-extension behavior using an elastically coupled, two-state model. We believe ours is the first demonstration of force-induced prolyl cis-trans isomerization in proline-containing polypeptides. Our results suggest that single-molecule force spectroscopy could provide an alternate means to assay this important conformational transition in polypeptides.

Loss of Secretory Pathway FK506-binding Proteins Results in Cold-sensitive Lethality and Associate Extracellular Matrix Defects in the Nematode Caenorhabditis elegans

The FK506-binding proteins (FKBs) represent ubiquitous enzymes that catalyze the rate-limiting peptidyl prolyl cis-trans isomerization step in protein folding. The nematode Caenorhabditis elegans has eight FKBs, three of which (FKB-3, -4, and -5) have dual peptidyl prolyl cis-trans isomerase (PPIase) domains, signal peptides and ER retention signals. PPIase activity has been detected for recombinant FKB-3. Both FKB-3 and -5 are expressed in the exoskeleton-synthesizing hypodermis with transcript peaks that correspond to the molting and collagen synthesis cycles. FKB-4 is expressed at a low level throughout development. No phenotypes were observed in deletion mutants in each of the secretory pathway FKBs. Combined triple and fkb-4, -5 double deletion mutants were however found to arrest at 12 degrees C, but developed normally at 15-25 degrees C. This cold-sensitive larval lethal effect was not maternally derived, occurred during embryogenesis, and could be rescued following the transgenic introduction of a wild type copy of either fkb-4 or fkb-5. The temperature-sensitive defects also affected molting, cuticle collagen expression, hypodermal seam cell morphology, and the structural integrity of the cuticular extracellular matrix. This study establishes that the secretory pathway FK506-binding PPIase enzymes are essential for normal nematode development, collagen biogenesis, and the formation of an intact exoskeleton under adverse physiological conditions.

Prolyl cis- trans Isomerization as a Molecular Timer in Crk Signaling

In a recent issue of Molecular Cell, use NMR to identify peptidyl-prolyl cis-trans isomerization as an autoinhibitory switch in the adaptor protein Crk, suggesting a new mechanism for controlling the rate of formation of signaling complexes.

The Crystal Structure of Aspergillus fumigatus Cyclophilin Reveals 3D Domain Swapping of a Central Element

The crystal structure of Aspergillus fumigatus cyclophilin (Asp f 11) was solved by the multiwavelength anomalous dispersion method and was refined to a resolution of 1.85 A with R and R(free) values of 18.9% and 21.4%, respectively. Many cyclophilin structures have been solved to date, all showing the same monomeric conformation. In contrast, the structure of A. fumigatus cyclophilin reveals dimerization by 3D domain swapping and represents one of the first proteins with a swapped central domain. The domain-swapped element consists of two beta strands and a subsequent loop carrying a conserved tryptophan. The tryptophan binds into the active site, inactivating cis-trans isomerization. This might be a means of biological regulation. The two hinge loops leave the protein prone to misfolding. In this context, alternative forms of 3D domain swapping that can lead to N- or C-terminally swapped dimers, oligomers, and aggregates are discussed.

Role of Protein Dynamics in Reaction Rate Enhancement by Enzymes

An integrated view of protein structure, dynamics, and function is emerging, where proteins are considered as dynamically active assemblies and internal motions are closely linked to function such as enzyme catalysis. Further, the motion of solvent bound to external regions of protein impacts internal motions and, therefore, protein function. Recently, we discovered a network of protein vibrations in enzyme cyclophilin A, coupled to its catalytic activity of peptidyl-prolyl cis-trans isomerization. Detailed studies suggest that this network, extending from surface regions to active site, is a conserved part of enzyme structure and has a role in promoting catalysis. In this report, theoretical investigations of concerted conformational fluctuations occurring on microsecond and longer time scales within the discovered network are presented. Using a new technique, kinetic energy was added to protein vibrational modes corresponding to conformational fluctuations in the network. The results reveal that protein dynamics promotes catalysis by altering transition state barrier crossing behavior of reaction trajectories. An increase in transmission coefficient and number of productive trajectories with increasing amounts of kinetic energy in vibrational modes is observed. Variations in active site enzyme-substrate interactions near transition state are found to be correlated with barrier recrossings. Simulations also showed that energy transferred from first solvation shell to surface residues impacts catalysis through network fluctuations. The detailed characterization of network presented here indicates that protein dynamics plays a role in rate enhancement by enzymes. Therefore, coupled networks in enzymes have wide implications in understanding allostericity and cooperative effects, as well as protein engineering and drug design.

Plant immunophilins: functional versatility beyond protein maturation

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.