Immunophilin AtFKBP13 sustains all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana deficient in AtCYP20-2

The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.     

Structural comparison of oxidized and reduced FKBP13 from Arabidopsis thaliana

AtFKBP13, an immunophilin in the chloroplast thylakoid lumen, participates in redox-regulatory processes via a pair of conserved disulfide bonds that are present at the N- and C-termini of the protein. Characterization of this protein by structural and biochemical analysis has revealed a novel mechanism of redox regulation in the thylakoid lumen. The protein is active in its oxidized form but is inactivated after reduction by the thioredoxin system. This is in sharp contrast with the regulation of biosynthetic enzymes in the stroma of the chloroplast, where reduction of enzymes by thioredoxin activates their function. To understand how the reduced form of AtFKBP13 is stabilized and how reduction of the cysteine residues affects the molecular properties of the enzyme, we determined the crystal structure of reduced AtFKBP13 at 1.88 A. Comparison of the reduced structure and the oxidized form that we published earlier shows rearrangements in redox site regions, readjustments of hydrogen-bonding interactions and the secondary structure of the active site residues 50-53, and reduced accessibility of the catalytic residues involved in the peptidyl proline isomerase (PPIase) activity of this enzyme. We propose that redox-linked changes in the secondary structure of the PPIase domain are responsible for significant functional differences in this protein in the reduced and oxidized states.     

PPIase activities and interaction partners of FK506-binding proteins in the wheat thylakoid

FK506-binding proteins (FKBPs) and cyclophilins, collectively called immunophilins, conserve peptidyl-prolyl cis/trans isomerase (PPIase) active sites, although many lack PPIase activity. The chloroplast thylakoid contains a large proportion of the plant immunophilin family, but their functions within this compartment are unclear. Some lumenal immunophilins are important for assembly of photosynthetic complexes, implicating them in the maintenance and turnover of the photosynthetic apparatus during acclimation processes. In this investigation into the functions of three FKBPs localized to the thylakoid of Triticum aestivum (wheat), we present the first evidence of PPIase activity in the thylakoid of a cereal plant, and also show that PPIase activity is not conserved in all lumenal FKBPs. Using yeast two-hybrid analysis we found that the PPIase-active FKBP13 interacts with the globular domain of the wheat Rieske protein, with potential impact on photosynthetic electron transfer. Specific interaction partners for PPIase-deficient FKBP16-1 and FKBP16-3 link these isoforms to photosystem assembly.     

AtCYP20-2 is an auxiliary protein of the chloroplast NAD(P)H dehydrogenase complex

AtCYP20-2 is one of 16 immunophilins in thylakoid lumen. The presence of the isomerase domain in AtCYP20-2, an enrichment of AtCYP20-2 in the stroma membranes and it’s co-migration with NAD(P)H dehydrogenase (NDH) in native gels provide evidence that AtCYP20-2 is an auxiliary protein of NDH. When different NDH mutants were studied, AtCYP20-2 was found to be strongly reduced especially in mutants deficient in the membrane domain of NDH, thus suggesting a role in the assembly of NDH hydrophobic domain. Lack of AtCYP20-2, however, did not lead to severe malfunction of NDH, indicating redundancy in the function of lumenal immunophilins.     

A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen.

We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single-copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin-like C-terminal segment of 20 kDa, a predicted N-terminal leucine zipper and a potential phosphatase-binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non-appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl-prolyl cis-trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light-mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated.     

The major peptidyl-prolyl isomerase activity in thylakoid lumen of plant chloroplasts belongs to a novel cyclophilin TLP20

Fractionation of proteins from the thylakoid lumen of spinach chloroplasts combined with peptidyl-prolyl cis/trans isomerase (PPIase) measurements revealed a major isomerase activity that was ascribed to a novel enzyme TLP20 (thylakoid lumen PPIase of 20 kDa). TLP20 was inhibited by cyclosporin A and mass spectrometric sequencing of tryptic peptides confirmed its classification as a cyclophilin. Genes encoding similar putative thylakoid cyclophilins with a unique insert of three amino acids NPV in their N-termini were found in chromosome 5 of both Arabidopsis and rice. TLP20 is suggested to be the major PPIase and protein folding catalyst in the thylakoid lumen of plant chloroplasts.     

Immobility of phycobilins in the thylakoid lumen of a cryptophyte suggests that protein diffusion in the lumen is very restricted

The thylakoid lumen is an important photosynthetic compartment which is the site of key steps in photosynthetic electron transport. The fluidity of the lumen could be a major constraint on photosynthetic electron transport rates. We used Fluorescence Recovery After Photobleaching in cells of the cryptophyte alga Rhodomonas salina to probe the diffusion of phycoerythrin in the lumen and chlorophyll complexes in the thylakoid membrane. In neither case was there any detectable diffusion over a timescale of several minutes. This indicates very restricted phycoerythrin mobility. This may be a general feature of protein diffusion in the thylakoid lumen.     

FKBP22 is part of chaperone/folding catalyst complexes in the endoplasmic reticulum of Neurospora crassa

FKBP22 is a dimeric protein in the lumen of the endoplasmic reticulum, which exhibits a chaperone as well as a PPIase activity. It binds via its FK506 binding protein (FKBP) domain directly to the Hsp70 chaperone BiP that stimulates the chaperone activity of FKBP22. Here we demonstrate additionally the association of FKBP22 with the molecular chaperones and folding catalysts Grp170, alpha-subunit of glucosidase II, PDI, ERp38, and CyP23. These proteins are associated with FKBP22 in at least two protein complexes. Furthermore, we report an essential role for FKBP22 in the development of microconidiophores in Neurospora crassa.     

All of the protein interactions that link steroid receptor.hsp90.immunophilin heterocomplexes to cytoplasmic dynein are common to plant and animal cells

Both plant and animal cells contain high molecular weight immunophilins that bind via tetratricopeptide repeat (TPR) domains to a TPR acceptor site on the ubiquitous and essential protein chaperone hsp90. These hsp90-binding immunophilins possess the signature peptidylprolyl isomerase (PPIase) domain, but no role for their PPIase activity in protein folding has been demonstrated. From the study of glucocorticoid receptor (GR).hsp90.immunophilin complexes in mammalian cells, there is considerable evidence that both hsp90 and the FK506-binding immunophilin FKBP52 play a role in receptor movement from the cytoplasm to the nucleus. The role of FKBP52 is to target the GR.hsp90 complex to the nucleus by binding via its PPIase domain to cytoplasmic dynein, the motor protein responsible for retrograde movement along microtubules. Here, we use rabbit cytoplasmic dynein as a surrogate for the plant homologue to show that two hsp90-binding immunophilins of wheat, wFKBP73 and wFKBP77, bind to dynein. Binding to dynein is blocked by competition with a purified FKBP52 fragment comprising its PPIase domain but is not affected by the immunosuppressant drug FK506, suggesting that the PPIase domain but not PPIase activity is involved in dynein binding. The hsp90/hsp70-based chaperone system of wheat germ lysate assembles complexes between mouse GR and wheat hsp90. These receptor heterocomplexes contain wheat FKBPs, and they bind rabbit cytoplasmic dynein in a PPIase domain-specific manner. Retention by plants of the entire heterocomplex assembly machinery for linking the GR to dynein implies a fundamental role for this process in the biology of the eukaryotic cell.     

叶绿体类囊体腔蛋白功能研究进展

【目的】类囊体是叶绿体光合作用中光反应进行的重要场所。类囊体腔是由类囊体膜包围形成的一个狭小空间。在类囊体腔中存在多种不同的蛋白家族,包括高叶绿素荧光(high chlorophyll fluorescence, HCF)蛋白、亲免蛋白、放氧复合物(oxygen-evolving complex, OEC)蛋白、PsbP类蛋白等,它们对植物的光合作用、核酸代谢以及氧化还原反应等都起着重要作用。【评论】文章分类综述了参与光合作用调控的类囊体腔蛋白在光系统组装、植物生长发育调节和高光逆境响应等生理活动中发挥的重要作用。【展望】文章可为未来研究类囊体腔蛋白的生理功能提供理论参考。     

Comparative analysis of enzyme activities and mRNA levels of peptidyl prolyl cis/trans isomerases in various organs of wild type and Pin1-/- mice

We investigated the enzyme activity of peptidyl prolyl cis/trans isomerases (PPIases) in brain, testis, lung, liver, and mouse embryonic fibroblasts (MEF) of Pin1+/+ and Pin1-/- mice. The aim of this study is to determine if other PPIases can substitute for the loss of Pin1 activity in Pin1-/- mice and what influence Pin1 depletion has on the activities of other PPIases members. The results show that high PPIase activities of Pin1 are found in organs that have the tendency to develop Pin1 knockout phenotypes and, therefore, provide for the first time an enzymological basis for these observations. Furthermore we determined the specific activity (k(cat)/K(M)) of endogenous Pin1 and found that it is strongly reduced as compared with the recombinant protein in all investigated organs. These results suggest that posttranslational modifications may influence the PPIase activity in vivo. The activities originating from cyclophilin and FKBP are not influenced by the Pin1 knockout, but a basal enzymatic activity towards phosphorylated substrates could be found in Pin1-/- lysates. Real time PCR experiments of all PPIases in different mouse organs and MEF of Pin1+/+ and Pin1-/- mice support the finding and reveal the specific expression profiles of PPIases in mice.     

Oxygen reduction in chloroplast thylakoids results in production of hydrogen peroxide inside the membrane

Hydrogen peroxide production in isolated pea thylakoids was studied in the presence of cytochrome c to prevent disproportionation of superoxide radicals outside of the thylakoid membranes. The comparison of cytochrome c reduction with accompanying oxygen uptake revealed that hydrogen peroxide was produced within the thylakoid. The proportion of electrons from water oxidation participating in this hydrogen peroxide production increased with increasing light intensity, and at a light intensity of 630 μmol quanta m^− 2 s^− 1 it reached 60% of all electrons entering the electron transport chain. Neither the presence of a superoxide dismutase inhibitor, potassium cyanide or sodium azide, in the thylakoid suspension, nor unstacking of the thylakoids appreciably affected the partitioning of electrons to hydrogen peroxide production. Also, osmolarity-induced changes in the thylakoid lumen volume, as well as variation of the lumen pH induced by the presence of Gramicidin D, had negligible effects on such partitioning. The flow of electrons participating in lumen hydrogen peroxide production was found to be near 10% of the total electron flow from water. It is concluded that a considerable amount of hydrogen peroxide is generated inside thylakoid membranes, and a possible mechanism, as well as the significance, of this process are discussed.     

Characterization of Arabidopsis thaliana AtFKBP42 that is membrane-bound and interacts with Hsp90

The twisted dwarf1 (twd1) mutant from Arabidopsis thaliana was identified in a screen for plant architecture mutants. The TWD1 gene encodes a 42 kDa FK506-binding protein (AtFKBP42) that possesses similarity to multidomain PPIases such as mammalian FKBP51 and FKBP52, which are known to be components of mammalian steroid hormone receptor complexes. We report here for the first time the stoichiometry and dissociation constant of a protein complex from Arabidopsis that consists of AtHsp90 and AtFKBP42. Recombinant AtFKBP42 prevents aggregation of citrate synthase in almost equimolar concentrations, and can be cross-linked to calmodulin. In comparison to one active and one inactive FKBP domain in FKBP52, AtFKBP42 lacks the PPIase active FKBP domain. While FKBP52 is found in the cytosol and translocates to the nucleus, AtFKBP42 was predicted to be membrane-localized, as shown by electron microscopy.     

Structure of cytochrome c6A, a novel dithio-cytochrome of Arabidopsis thaliana, and its reactivity with plastocyanin: implications for function

Cytochrome c6A is a unique dithio-cytochrome present in land plants and some green algae. Its sequence and occurrence in the thylakoid lumen suggest that it is derived from cytochrome c6, which functions in photosynthetic electron transfer between the cytochrome b6f complex and photosystem I. Its known properties, however, and a strong indication that the disulfide group is not purely structural, indicate that it has a different, unidentified function. To help in the elucidation of this function the crystal structure of cytochrome c6A from Arabidopsis thaliana has been determined in the two redox states of the heme group, at resolutions of 1.2 A (ferric) and 1.4 A (ferrous). These two structures were virtually identical, leading to the functionally important conclusion that the heme and disulfide groups do not communicate by conformational change. They also show, however, that electron transfer between the reduced disulfide and the heme is feasible. We therefore suggest that the role of cytochrome c6A is to use its disulfide group to oxidize dithiol/disulfide groups of other proteins of the thylakoid lumen, followed by internal electron transfer from the dithiol to the heme, and re-oxidation of the heme by another thylakoid oxidant. Consistent with this model, we found a rapid electron transfer between ferro-cytochrome c6A and plastocyanin, with a second-order rate constant, k2=1.2 x 10(7) M(-1) s(-1).     

S-sulfocysteine synthase function in sensing chloroplast redox status

The minor chloroplastic O-acetylserine(thiol)lyase isoform encoded by the CS26 gene in Arabidopsis thaliana has been described as an S-sulfocysteine synthase enzyme that plays an important role in chloroplast function. This enzyme is located in the thylakoid lumen, and its S-sulfocysteine activity is essential for the proper photosynthetic performance of the chloroplast under long-day growth conditions. Based on the present knowledge of this enzyme, we suggest that S-sulfocysteine synthase functions as a protein sensor to detect the accumulation of thiosulfate as a result of the inadequate detoxification of reactive oxygen species generated under conditions of excess light to produce the S-sulfocysteine molecule that triggers protection mechanisms of the photosynthetic apparatus.     

The binding of FKBP23 to BiP modulates BiP's ATPase activity with its PPIase activity

Peptidyl-prolyl cis-trans-isomerases (PPIases) are enzymes that can cis-trans-isomerize a Xaa-Pro peptide bond. Three families of PPIases are known: cyclophilins, FKBPs, and parvulins. The physiological functions of the PPIases are only poorly understood. In previous work, we reported that the mouse FK506-binding protein 23 (mFKBP23), which comprises an N-terminal PPIase domain and a C-terminal domain with Ca(2+)-binding sites, binds to mBiP in the endoplasmic reticulum (ER) and this binding is affected by the Ca(2+) concentration. In this study, we demonstrate the ability of mFKBP23 to modulate the ATPase activity of BiP, and that the bound mFKBP23, but not the free mFKBP23, can suppress the ATPase activity of mBiP through its PPIase activity.