Present address: ZMBP-Pflanzenphysiologie, Auf der Morgenstelle 5, 72076 Tübingen, Germany.
Characterization of Arabidopsis thaliana AtFKBP42 that is membrane-bound and interacts with Hsp90
Article first published online: 31 OCT 2002
The Plant Journal
Volume 32, Issue 3, pages 263–276, November 2002
How to Cite
Kamphausen, T., Fanghänel, J., Neumann, D., Schulz, B. and Rahfeld, J.-U. (2002), Characterization of Arabidopsis thaliana AtFKBP42 that is membrane-bound and interacts with Hsp90. The Plant Journal, 32: 263–276. doi: 10.1046/j.1365-313X.2002.01420.x
- Issue published online: 31 OCT 2002
- Article first published online: 31 OCT 2002
- Received 5 February 2002; revised 11 June 2002; accepted 23 June 2002.
- steroid hormone receptor;
- plasma membrane;
- signal transduction
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.
FK506-binding proteins (FKBP) are ubiquitously expressed. Phenotypes of gene deletion mutants were analysed to gain insight into the function of FKBPs. Few examples have been reported in higher eukaryotes. The deletion of the murine FKBP12 gene results in cardiac defects and increased lethality due to the arrest of cells in the G1 phase of the cell cycle (Aghdasi et al., 2001; Shou et al., 1998). In Arabidopsis, two different FKBP mutants were identified. (i) The pasticcino1 (pas1) mutant shows ectopic cell division and abnormal cell differentiation, which results in disorganized seedlings. The Pas1 protein (AtFKBP72; Fischer, 1994) was found to be localized in the nucleus and exhibits low PPIase-activity (Carol et al., 2001; Faure et al., 1998b; Vittorioso et al., 1998). (ii) The TWD1 gene encodes the AtFKBP42 protein. Gene deletion mutants are reduced in size, exhibit disorientated growth of all organs, but develop fertile flowers and seeds. Due to its reduced height and twisted appearance, this mutant was termed twisted dwarf1 (twd1) (B.S. and co-workers, unpublished results). The ucu2 mutation published by Perez-Perez et al. (2001a) is allelic to twd1. Gene analysis of TWD1 predicted a multidomain FKBP of 42 kDa.
The FKBPs belong to the enzyme class of peptidyl-prolyl cis/trans isomerases (PPIases, EC 220.127.116.11). Members of this enzyme class specifically catalyse the peptidyl-bond isomerization preceding a proline (Fischer et al., 1984; Fischer et al., 1989). PPIases are involved in de novo protein biosynthesis and in the restructuring of proteins (Schiene and Fischer, 2000). Thus far, three families of PPIases have been identified: cyclophilins, FKBPs and parvulins (for review see Fischer, 1994; Vener, 2001). Cyclophilins and FKBPs are specifically inhibited by immunosuppressive drugs such as cyclosporin A and FK506 or rapamycin, respectively (Fischer, 1994; Ivery, 2000). PPIases contain at least one PPIase domain that classifies the proteins as a member of the corresponding family, represented by the prototypes Cyp18, FKBP12 and Escherichia coli parvulin10 (EcPar10). In addition, NH2- and COOH-terminal extensions of the PPIase domains are often involved in protein–protein interaction, such as the WW-domain of hPin1 (hPar18) or the tetratricopeptide repeat (TPR) domain of Cyp40, FKBP51 and FKBP52. Whereas WW-domains recognize proline-rich motifs in large proteins (Lu et al., 1999), the TPR domain mediates interaction with the COOH-terminal region of the heat-shock protein Hsp90 (Owens-Grillo et al., 1996; Young et al., 1998).
The PPIase–Hsp90 interaction is well characterized for the mammalian system. It is necessary for the maturation cycle of non-activated steroid hormone receptor (SHR) complexes. PPIases with a TPR domain are part of different soluble, cytosolic SHR complexes. Steroid binding results in the translocation of the hormone-activated SHR into the nucleus (for review see Pratt, 1998; Pratt and Toft, 1997; Richter and Buchner, 2001; Schiene-Fischer and Yu, 2001).
The homologous proteins of mammalian Cyp18, Cyp40, FKBP12 and Par14, identified in the plant system, do not differ in their domain composition (Berardini et al., 2001; Faure et al., 1998a). In contrast, Digitalis lanata DlPar13 and AtPar13 have hPar18-like substrate specificity, but lack the WW-domain (Landrieu et al., 2000; Metzner et al., 2001). Most TPR-containing FKBPs in plants vary from their mammalian counterparts by one additional FKBP domain, generally resulting in a higher molecular mass (Harrar et al., 2001). In Arabidopsis, wheat and maize, at least one PPIase has been identified with three FKBP and one TPR domain. (Aviezer et al., 1998; Faure et al., 1998b; Hueros et al., 1998; Kurek et al., 1999; Vittorioso et al., 1998; Vucich and Gasser, 1996).
In wheat germ lysate it is, in principle, possible to assemble a functional SHR complex with immunopurified mammalian glucocorticoid receptor (Owens-Grillo et al., 1996; Pratt et al., 2001; Stancato et al., 1996). This implies the presence of homologous proteins of all required mammalian factors including a TPR-containing PPIase and Hsp90.
In contrast to the mammalian system, the Arabidopsis thaliana genome does not harbour any gene encoding soluble SHR (Becraft, 2001). The plant steroid receptors BRI1 (brassinolide insensitive 1) from Arabidopsis and rice (Oryza sativa) are plasma membrane-spanning proteins of the leucine-rich repeat (LRR) type with an intracellular serine/threonine kinase domain. The plant steroid brassinolide (BL) is perceived extracellularly (Wang et al., 2001). Brassinolide binding induces autophosphorylation of the kinase domain (Oh et al., 2000). The following signal transduction pathway is not well understood. There are still open questions, such as phosphorylation targets or the oligomerization state of BRI1 (for review see Friedrichsen and Chory, 2001; Müssig and Altmann, 2001). Two downstream proteins of the BL-induced signalling cascade have been analysed in more detail. The BIN2/UCU1 kinase of a glycogen synthase–kinase 3/shaggy-like type is one phosphorylation target of BRI1 that functions as a negative regulator (Li and Nam, 2002; Li et al., 2001). BIN2/UCU1 mutants show a dwarf phenotype with curled leaves due to reduced cell expansion (Li and Nam, 2002; Perez-Perez et al., 2001b; Perez-Perez et al., 2002). Brassinolide-induced gene expression is regulated by the nuclear protein BES1, which appears to be destabilized by BIN2/UCU1 (Yin et al., 2002).
Mutation of AtFKBP42 leads to plants that do not respond to exogenous BL application, and display a dwarf phenotype with additional disorientated growth of all organs. The TWD1 gene encodes a 42 kDa FKBP with a TPR domain. In this report we describe the biochemical characterization and localization analysis of AtFKBP42. The precise domain structure of AtFKBP42, as well as orthologous proteins from other species, were identified. Important protein properties and the cellular localization were examined. Further analysis regards the protein–protein interactions of AtFKBP42 with calmodulin (CaM) and AtHsp90, employing cross-linking experiments or isothermal titration calorimetry approaches.
Analysis of the domain structure of AtFKBP42
The amino acid (aa) sequence of AtFKBP42 was analysed for domain structures using the internet-based tool SMART (http://smart.embl-heidelberg.de/) (Schultz et al., 1998; Schultz et al., 2000). The program predicted one FKBP12 domain, one TPR domain containing three motifs, and a COOH-terminal transmembrane region. Sequence alignment of the FKBP domain (aa residues 50–159) with hFKBP12 showed 30% aa identity and 53% similarity. Nevertheless, 10 of 14 aa residues thought to be important for catalysis and FK506 binding differ from those of hFKBP12 (for review see Kay, 1996) (Figure 1). Variants of three hFKBP12 aa residues were published, with changes to the same aa residue as found in AtFKBP42 wild type. The residual PPIase activities of the hFKBP12 variants F48L, W59L and F99Y were determined to be 25, 13 and 0–5%, respectively (DeCenzo et al., 1996; Timerman et al., 1995; Tradler et al., 1997). The corresponding aa residues of AtFKBP42 are Leucine97, Leucine109 and Tyrosine151.
A TPR domain typical of FKBPs consists of three degenerated 34 aa repeats (Lamb et al., 1995). The first repeat comprises residues 179–212; the second and third repeats are directly connected from residues 230–297 (Figure 1). CaM binding was published for several multidomain FKBPs (Carol et al., 2001; Hueros et al., 1998; Massol et al., 1992). The sequence similarity of AtFKBP42 to these proteins suggests CaM binding as well. The very COOH-terminal region (residues 340–365) of AtFKBP42 is predicted to be transmembrane and could function as membrane anchor.
Extensive homology searches revealed three eukaryotic homologous proteins with identical domain arrangement, including the predicted membrane localization. The 38 kDa (FKBP38) representatives from human and mouse are described (Lam et al., 1995; Pedersen et al., 1999). A predicted 44.8 kDa protein from Drosophila melanogaster (DmFKBP45; accession number AAF57662; Adams et al., 2000) was identified as an AtFKBP42 homologous protein, by a homology search at the NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al., 1990). The characteristic domain arrangement – one FKBP domain, one TPR domain comprised of three repeats and the transmembrane segment – were predicted for all four proteins using the SMART analysis tool (Figure 1). Putative CaM binding was published for hFKBP38 and muFKBP38 (Lam et al., 1995; Pedersen et al., 1999). The membrane anchor was also predicted for AtFKBP42, hFKBP38, muFKBP38 and DmFKBP45 with the public analysis tools TMpred (http://www.ch.embnet.org/software/TMPRED_form.html) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0).
Circular dichroism spectroscopy of AtFKBP42
Protein fragments of AtFKBP42 were expressed and purified from E. coli extracts. The smaller fragment, (1–180)AtFKBP42, contains residues 1–180, which represent the FKBP domain. The larger fragment, (1–339)AtFKBP42, contains residues 1–339. This fragment lacks the predicted membrane anchor. The method of circular dichroism (CD) spectroscopy can be used to determine the secondary structure of proteins (for review see Greenfield, 1996; Woody, 1995). To test the existence of secondary structure elements of the recombinant AtFKBP42 proteins, thermal stability was analysed by CD spectroscopy. The spectra were recorded at 20 and 80°C for (1–180)AtFKBP42, and at 20 and 65°C for (1–339)AtFKBP42 (Figure 2a). CD spectra of the proteins were altered at elevated temperatures (data not shown). Therefore the CD signal change was followed over time at a wavelength of 205 nm with increasing temperature from 20 to 80°C for (1–180)AtFKBP42, and at 222 nm from 20 to 65°C for (1–339)AtFKBP42 (Figure 2b).
The CD spectrum of the (1–180)AtFKBP42 at 20°C shows an absolute minimum at 208 nm, indicating a high content of β-sheet, which is typical for FKBP domains. The structure of hFKBP12 contains a β-sheet of five antiparallel strands, a short helix and unstructured elements (Michnick et al., 1991). The (1–339)AtFKBP42 CD spectrum, recorded at 20°C, displays a double minimum at 222 and 209 nm, indicating higher α-helical contents of the protein caused by the additional TPR domain. TPR domains are mostly composed of α-helical structures (Blatch and Lässle, 1999; Taylor et al., 2001). The α-helical content of a protein can be calculated from its CD spectra with the cdnn software (free download: http://bioinformatic.biochemtech.uni-halle.de/cdnn). cdnn analysis predicted 18% α-helical content for (1–180)AtFKBP42 und 37% for (1–339)AtFKBP42, which confirms the higher α-helical content of (1–339)AtFKBP42.
The change in the CD signal followed over time with increasing temperature remains constant for (1–180)AtFKBP42 up to ≈48°C. The mid-point of the CD signal change (Tm) was determined at ≈59°C. The signal of (1–339)AtFKBP42 changes little in the range from 25 to 40°C. Above 40°C, the signal changes more rapidly. Tm was determined at ≈43°C. Thus the CD signal of (1–180)AtFKBP42 remains constant up to higher temperatures than that of (1–339)AtFKBP42.
Interaction of the AtFKBP42 TPR domain with the COOH-terminal region of AtHsp90.1
Analysis of the primary amino acid composition of AtFKBP42 predicted a TPR domain adjacent to the FKBP domain. The TPR domain interaction with Hsp90 was characterized in vitro using short peptides and larger protein fragments of Hsp90 (Pirkl and Buchner, 2001; Scheufler et al., 2000). A competition experiment with high molecular mass wheat FKBP, Hsp90 and rat protein phosphatase5 suggests a similar mode of action in the plant system (Reddy et al., 1998). Nonetheless, no dissociation constants have been published for these plant proteins.
Here we report the first KD data measured for the Arabidopsis proteinAtFKBP42, and a COOH-terminal fragment (aa 559–700) of AtHsp90.1. Evidence of an interaction of (1–339)AtFKBP42 and (557–700)AtHsp90.1 is provided by a shift of (1–339)AtFKBP42 in analytical size-exclusion chromatography. The retention time of (1–339)AtFKBP42 was reduced in the presence of (559–799)AtHsp90.1. The AtHsp90.1 fragment itself had a retention time that indicates a molecular mass higher than the theoretical value (data not shown). These data emphasize that the putative interaction of AtFKBP42 and AtHsp90.1 is similar to that of mammalian Hsp90 and TPR domains of mammalian FKBPs. To compare binding affinities, the dissociation constant and stoichiometry of the in vitro complex were determined by isothermal titration calorimetry (ITC). The data for the titration curve of (1–339)AtFKBP42 and (559–700)AtHsp90.1 were fitted to a 1 : 1 binding model (Figure 3). A dissociation constant of 1.3 µm was calculated with a stoichiometry of two molecules of (559–700)AtHsp90 to one molecule of (1–339)AtFKBP42.
Citrate synthase assay
The PPIases Cyp40, FKBP51 and FKBP52 display (in addition to their PPIase activity and the binding to Hsp90) a chaperone-like activity which was found to be associated with the TPR domain (Pirkl and Buchner, 2001). The observed domain similarity suggests that this activity is also present in AtFKBP42. Thus we analysed the effect of AtFKBP42 using the citrate synthase (CS) aggregation assay. (1–339)AtFKBP42 reduced the aggregation of CS efficiently. Aggregation was reduced more than 15% by addition of 0.1 µm (1–339)AtFKBP42 to CS (3 µm), and largely prevented by less than equimolar (1–339)AtFKBP42 concentrations. Total prevention was achieved with excess of (1–339)AtFKBP42 at a concentration of 4 µm (Figure 4). This means a ratio of one molecule of CS to 1.3 molecules of (1–339)AtFKBP42. (1–339)AtFKBP42 did not aggregate in the absence of CS. (1–180)AtFKBP42 was examined to identify the region responsible for this effect. The FKBP domain fragment was not sufficient to prevent aggregation using up to 10-fold higher concentrations compared to CS. Despite the prevention of aggregation, the inactivation of CS enzyme activity at 43°C was not slowed down by (1–339)AtFKBP42.
CaM binding of AtFKBP42
After the first publication of a CaM binding site in FKBP52 (Callebaut et al., 1992), the binding of CaM was shown for several large immunophilins, including maize FKBP66 (Hueros et al., 1998). CaM binding is predicted for AtFKBP42 and DmFKBP45, whereas others have postulated CaM binding for human and murine FKBP38 (Lam et al., 1995; Pedersen et al., 1999). We showed CaM binding for (1–339)AtFKBP42 in a cross-linking experiment. Both proteins were cross-linked separately or mixed at a concentration of 6 µm with 0.5 mm 3, 3′-dithiobis (sulfosuccinimidylpropionate)(DTSSP). After SDS–PAGE separation and silver staining of the gel, a band with the additive molecular weight of both proteins appeared in the lane corresponding to the protein mix reaction. This band is absent in single protein cross-link reactions (Figure 5a).
The binding was further analysed by (1–339)AtFKBP42 pulldown using agarose-immobilized CaM. Quantification of the bands in a Coomassie-stained gel showed that 70% (eluted and non-eluted fractions) of (1–339)AtFKBP42 bound to CaM agarose. From these amounts, 5% were eluted with EGTA. Total amounts of 95% calcineurin were bound, 31% eluted under the given conditions, whereas little binding (19%) was detected for (1–180)AtFKBP42 without any elution by EGTA (Figure 5b). Even under Ca2+-free conditions, ≈21% of the (1–339)AtFKBP42 bound to CaM agarose. Similar values were found for calcineurin (26%), and a less intense binding for (1–180)AtFKBP42, with ≈4% (Figure 5b).
PPIase activity can be measured with several methods. (i) The most conventional test is the protease-coupled assay (Fischer et al., 1984; Hani et al., 1999). (ii) A re-equilibration of the cis/trans equilibrium can be followed directly after a solvent jump of the peptide substrate without protease (Janowski et al., 1997). (iii) The third test utilizes a protein substrate: the increase of fluorescence during the refolding of a denaturated, reduced S-carboxy-methylated S54G/P55N-variant of RNaseT1 (RCM-T1) was measured (Schmid et al., 1996). Although the data obtained by the CD-spectroscopy measurements indicate that (1–180)AtFKBP42 and (1–339)AtFKBP42 are structured, neither showed any PPIase activity with up to 6.35 µm final concentration in the proteolytic assay. This concentration is clearly higher than the upper range of protein concentrations used for measuring weak PPIase activities, as performed for hPar14 (Uchida et al., 1999). Full-length AtFKBP42 was also tested in the proteolytic test. Due to aggregation, 200 µg ml−1 in buffer supplemented with 5% (NH4)2SO4 was the maximum protein concentration used. Similarly to (1–339)AtFKBP42, no PPIase activity could be detected.
PPIases exhibit preferences concerning different residues preceding proline. To test effects of large and small hydrophobic residues, as well as charged aa residues in the substrate, a broad range of peptide substrates was examined. Additionally, the protease-free assay and the RCM-T1 assay were performed to detect PPIase activity of the FKBP domain and (1–339)AtFKBP42. A PPIase activity of AtFKBP42 could not be detected by one of these assays with any kind of substrate. We examined the binding of FK506 to (1–339)AtFKBP42 by isothermal titration calorimetry and in a competition assay with hFKBP12. The addition of (1–339)AtFKBP42 with a final concentration of 6.35 µm in the competition assay affected neither the PPIase activity of hFKBP12 without FK506, nor the inhibition of hFKBP12 by FK506 (data not shown). An excess of (1–339)AtFKBP42 of >2000-fold was used. The constant for FK506 inhibition of hFKBP12 is 0.4 nm (Siekierka et al., 1989). Thus binding of FK506 to (1–339)AtFKBP42 with a hypothetical KD up to 1 µm should have been detected.
Furthermore, we tested whether the missing PPIase activity of AtFKBP42 is caused by a lack of post-translational glycosylation in vitro. Plasma membrane preparations were analysed for glycosylated proteins after SDS–PAGE and Western blotting. The same blot was reprobed with anti-AtFKBP42 antibody, and both signals were compared. The detection using anti-AtFKBP42 antibody gave an additional signal which could not be detected after staining with the glycosylation detection module (see Figure 7b). These results indicate that AtFKBP42 is not glycosylated.
Membrane localization of the AtFKBP42 protein
The last 27 COOH-terminal aa of AtFKBP42 were predicted to anchor AtFKBP42 in membranes of Arabidopsis cells. The cellular localization of hemagglutinin epitope (HA)-tagged AtFKBP42 (HA-TWD1) overexpressing plants was analysed by electron microscopy. Using immunogold detection, HA-TWD1 was found to be embedded in the plasma membrane and the tonoplast that separates the vacuole from the cytosol. The gold-labelled proteinA appears as black dots (Figure 6). Signals for HA-TWD1 were mostly identified in plasma membrane and tonoplast. The white areas appear only in the HA-TWD1-overexpressing plants. Wild-type Arabidopsis plants were used as controls.
The plasma membrane of HA-TWD1-overexpressing plants was prepared with aqueous two-phase extraction to confirm these observations. HA-TWD1 was visualized in samples taken at different preparation stages of Arabidopsis plasma membrane with anti-AtFKBP42 antibody after Western blotting. In contrast to whole lysate and soluble fraction, a signal can be detected only in the enriched plasma membrane fraction (Figure 7a).
The overall structure of AtFKBP42 shares important features with the mammalian SHR-interacting PPIases FKBP51 and FKBP52, which consist of FKBP domains followed by the large immunophilin typical tripartite TPR domain. The interaction of TPR domains and mammalian Hsp90 is essential for chaperone cycle of the SHR activation (Richter and Buchner, 2001). We showed an in vitro interaction of (1–339)AtFKBP42 and (559–700)AtHsp90.1, and found a stoichiometry of 1 : 2. The same stoichiometry is known for a mammalian complex of Hsp90 and FKBP52, gained by cross-linking experiments (Silverstein et al., 1999). Recent ITC experiments reveal complexes of two PPIase molecules with a Hsp90 dimer (Pirkl and Buchner, 2001). The dimerization that we identified for (559–700)AtHsp90.1 fragment is consistent with the dimerization data published for mammalian Hsp90 (Carrello et al., 1999), whereas no indication was found for a dimerization of (1–339)AtFKBP42. Those would have been detectable in the cross-linking reactions and the size exclusion chromatography.
The dissociation constant of the dimeric AtHsp90.1 fragment and monomeric (1–339)AtFKBP42 was measured with 1.3 µm. Binding affinities for human hFKBP51, hFKBP52 and hCyp40 with hHsp90 were determined with of 174, 55 and 226 nm, respectively (Pirkl and Buchner, 2001). The very COOH-terminal aa residues ‘EEVD’ of Hsp90 generally mediate binding to TPR domains. A partial loss of this motif due to C-terminal degradation during protein purification would influence the interaction with the TPR domain of AtFKBP42; however, the probability of determining a 1 : 2 stoichiometry caused by degradation effects is low. This would require the loss of the EEVD motif for 50% of the molecules.
The specific binding of the C-terminal domain of Hsp90 to TPR domains is achieved by hydrophobic interactions upstream of the EEVD motif (Scheufler et al., 2000). In Arabidopsis four cytoplasmic isoforms of AtHsp90 are described, all showing the COOH-terminal EEVD motif (aa 697–700 for AtHsp90.1, aa 696–699 for AtHsp90.2–90.4) (Krishna and Gloor, 2001; Milioni and Hatzopoulos, 1997). Although Hsp90 is a highly conserved protein, the conservation between plant Hsp90s and mammalian Hsp90s is much lower than between mammalian Hsp90s (Krishna and Gloor, 2001).
The reported interaction of AtHsp90.1 fragment with (1–339)AtFKBP42 is based on the same principles as the mammalian Hsp90–TPR binding. As AtFKBP42 is localized to the Arabidopsis plasma membrane and tonoplast, and there are no indications that it is dimeric, the KD and stoichiometry are adapted to the specific localization of this complex. No data exist about the corresponding complex of hHsp90 and hFKBP38.
A chaperone-like activity localized to the TPR domain of the human SHR complex-associated PPIases has been found (Pirkl and Buchner, 2001). (1–339)AtFKBP42 prevents aggregation of CS 14-fold better than hFKBP52, and even threefold better than hCyp40 and hFKBP51. The single FKBP domain (1–180)AtFKBP42 did not prevent any aggregation. Therefore the effect is localized to the TPR domain and the following residues. Thus the properties of the AtFKBP42 TPR domain are similar to the human PPIase TPR domains.
Another aspect was the CaM binding of AtFKBP42. The published binding experiments of multidomain PPIases showed an affinity of FKBP52, maize FKBP66 and AtFKBP72 to CaM agarose (Carol et al., 2001; Hueros et al., 1998; Massol et al., 1992). We used cross-linking and CaM pull-down to analyse this interaction. CaM binding to AtFKBP42 was not found to be quantitative in either experiment. To analyse the AtFKBP42–CaM binding, constant surface plasmon resonance and isothermal titration calorimetry were performed. With the design of the measurements a KD value up to the low µm range should have been detected, but no binding constants were obtained. Therefore the KD of AtFKBP42 and CaM should be greater than this range. Both methods were used to determine binding constants in combination of CaM and a protein ligand (Fischer et al., 2001; Liang et al., 2000; Moorthy et al., 1999). The KD for the well characterized CaM–calcineurin interaction was found to be in the range of 1 nm (Hubbard and Klee, 1987). Thus the CaM binding predicted for AtFKB42 appears to be caused by a CaM-like binding motif, which implies the possibility of a different function, that remains to be determined.
The purified protein fragments of AtFKBP42 were analysed by CD spectroscopy. The CD spectra of the FKBP domain and (1–339)AtFKBP42 have a similar shape to the published CD spectra of hFKBP12 and hFKBP52, respectively (Pirkl and Buchner, 2001; Tradler et al., 1997). The rapid change of the ellipticity signal during heating is due to a temperature-induced unfolding process. Compared to the transition curve of (1–180)AtFKBP42, the thermal stability is reduced by the TPR domain. Similar effects were observed by Pirkl and Buchner (2001) for hCyp40, hFKBP51 and hFKBP52. These data show that the purified AtFKBP42 protein fragments are structured. The question of structural identity will finally be answered by solving the crystal or solution structures of (1–339)AtFKBP42 and (1–180)AtFKBP42.
In contrast to the activities of FKBP51 and FKBP52 (Callebaut et al., 1992; Nair et al., 1997; Pirkl and Buchner, 2001), no PPIase activity was detected for AtFKBP42. In the case of FKBP52, the PPIase activity was exclusively mediated by the first of two FKBP domains (Pirkl et al., 2001). The characteristic aa residues, important for FK506 binding and PPIase activity of hFKBP12, are conserved for the first FKBP domain of hFKBP52, but not for the second. Sequence analysis of AtFKBP42 showed direct parallels between the FKBP domain and the inactive FKBP domain of hFKBP52 concerning the conserved residues. The same was found for the identified homologous proteins hFKBP38, muFKBP38 and DmFKBP45. In agreement, hFKBP38 purified from insect cells was described to be inactive (Lam et al., 1995). Experiments to restore an artificial PPIase activity by exchanging the residues A76G, E86D, E105V, L106I, L109W, N142I and Y151F did not lead to detectable PPIase activity.
In addition to PPIase activity, the first domain of hFKBP52 is also involved in binding to cytoplasmic dynein, which mediates the nuclear transport of glucocorticoid receptor (Galigniana et al., 2001). Binding to rabbit dynein was also found for wheat FKBP77 (Pratt et al., 2001). If this represents the in vivo function of the PPIase active domain of FKBP52, this active domain would not be important for AtFKBP42, as this protein is membrane-anchored.
HA-TWD1-overexpressing plants were used for immunolocalization of AtFKBP42. Signals of gold-labelled proteinA were clearly detected in the tonoplast and the plasma membrane. Small background reactivity was discovered in wild-type plants. Thus HA-TWD1 is localized to the tonoplast and the plasma membrane. The preparation technique used for immunogold detection of proteins in electron microscopy is known to reduce the structural stability of cell compartments, compared to standard techniques. Loss of structure is less in the wild-type plant preparation. In the HA-TWD1 overexpressing plant preparation, white, unstructured areas are distinguishable. It is likely that high-level overexpression of a membrane-localized protein modifies the structure of the membranes, which may result in white, unstructured areas that are determined in sections.
Growth defects of two Arabidopsis mutant lines were identified in two different genes encoding multidomain AtFKBP with TPR motifs. As the mutant phenotypes show, both play an important role in Arabidopsis development. Both phenotypes were discussed to be caused by defects in the brassinosteroid signalling pathway: (i) pasticcino1 (AtFKBP72), a soluble, nuclear localized PPIase with three FKBP domains and a TPR domain that shows a low PPIase activity (Carol et al., 2001); and (ii) AtFKBP42 (Harrar et al., 2001). The localization of AtFKBP72 for a direct interaction with BRI1 is questionable, as it is described as a nuclear protein. The interaction of BRI1 and AtFKBP42 is more likely. Not only the determined localization and the interaction with AtHsp90, but also genetic studies, strongly support this interaction thesis. Twd1, like bri1, is insensitive to exogenous application of BL. The insensitivity against exogenous application of BL of twd1 mutants and the occurrence of BL-insensitive double mutants of twd1 with BR biosynthesis mutants indicates that AtFKBP42 is involved in perception or signal transduction of BL (B.S. and co-workers, unpublished results). Data for the mammalian SHR lead to the conclusion that AtFKBP42, together with AtHsp90, may also be part of an analogous SHR complex in Arabidopsis. In addition, AtFKBP42 and BRI1 are located to the same membrane (Friedrichsen et al., 2000; M. Geisler and co-workers, unpublished results). Further phosphorylation and cross-linking experiments with AtFKBP42 and BRI1 might provide more evidence for this assumption.
The soluble SHR complex mediates gene regulation by transcription activation. In addition to the soluble SHR, there is a second type of steroid hormone receptor in mammals that is localized to the plasma membrane. This type of receptor is responsible for ‘non-genomic steroid action’, as it does not directly influence the transcription of genes like the soluble SHR complex (Borski, 2000; Schmidt et al., 2000). The involvement of PPIases in the functioning of this type of receptor remains to be investigated.
Although the receptor complex assembly of mammalian SHR is possibly with wheat FKBP (Owens-Grillo et al., 1996; Reddy et al., 1998), a similar signal transduction process, as found with mammalian soluble SHRs, seems unlikely. The membrane-bound BRI1 SHR affects the gene expression of different proteins (Bishop and Yokota, 2001; Friedrichsen and Chory, 2001). The recently described proteins BIN2/UCU1 and BES1 indicate a signal transduction and resulting gene regulation via altered phosphorylation levels (Li and Nam, 2002; Perez-Perez et al., 2002; Yin et al., 2002). Thus BRI1 can be seen as more closely related to the function of non-genomic mammalian SHR, which also seem to transmit signals by the change of phosphorylation levels (Flores-Delgado et al., 2001). There is growing evidence that, in most investigated receptor complexes, PPIases play a role in receptor activation or regulation. Interactions with proteins of the FKBP family were shown for the soluble SHR, the membrane bound TGF-β receptor and ryanodyne receptor (Schiene-Fischer and Yu, 2001), the insect's ecdysone SHR (Arbeitman and Hogness, 2000; Song et al., 1997), and have also been suggested for the BRRI1 receptor complex. Further experiments will show if human and murine FKBP38 and DmFKBP45 play a role in the non-genomic steroid action of these organisms.
All chemicals and column resins were purchased from Merck Eurolab (Darmstadt, Germany) or Sigma (München, Germany), unless indicated otherwise. Restriction enzymes were obtained from New England Biolabs (Beverly, MA, USA).
Cloning and purification of AtFKBP42 protein fragments
The TWD1 template was amplified by polymerase chain reaction (PCR) using the primers TWD5a (5′-gat cga cca tgg atg aat ctc tgg agc atc-3′) and TWD3a (3′-tca gaa gct tag tct gct gca cca atc c). The PCR product was restricted with NcoI, HindIII followed by ligation into pET28a. This construct encodes (1–180)AtFKBP42, a protein fragment from residues 1–180. The recombinant protein was produced at 30°C in BL21 codon + RIL cells (Stratagene, La Jolla, USA) for 5 h after induction with 1 mm isopropyl-β-d-thiogalactopyranosid (IPTG).
After disruption of harvested E. coli cells in a French Press (SLM Aminco, Rochester, NY, USA), the supernatant of a 100 000 g centrifugation was loaded on a Fractogel EMD-DEAE-650(M) column equilibrated with 10 mm Hepes buffer (pH 7.5) and eluted with a linear KCl gradient (0–2 m). The (1–180)AtFKBP42-containing fractions were pooled, dialysed in 10 mm Hepes (pH 7.5), and passed through a Fractogel TSK-AF-Blue column. (1–180)AtFKBP42 did not bind. The (1–180)AtFKBP42-containing fractions were concentrated using VivaSpin columns (Sartorius, Göttingen, Germany). Preparative size-exclusion chromatography was performed with a HiLoad Superdex 75 HR 16/60 column (Amersham Pharmacia, Freiburg, Germany) in 10 mm Hepes buffer (pH 7.5) containing 150 mm NaCl. The resulting homogeneously purified (1–180)AtFKBP42 was concentrated again and stored at −80°C.
The construct (1–339)AtFKBP42 was amplified using primers TWD5a and TWD3b (5′-tca cta agc tta aag gct ctt tga ctt agc acc-3′). It was cloned, expressed and purified as described above.
Cloning and purification of (559–700)AtHsp90.1
We constructed a plasmid expressing the COOH-terminal region (residues 559–700) of AtHsp90.1. A cDNA template was cloned by PCR amplification with primers HSP5a (5′-gat gca cca tgg ttg tgg tct cag aca gga ttg-3′) and HSP3a (5′-gca gtc aag ctt agt cga ctt cct cca tc-3′), NcoI, HindIII restriction and ligation into pET28a. The encoded protein was expressed at 37°C in BL21 codon + RIL cells (Stratagene) for 5 h after induction with 1 mm IPTG.
Cell lysate was obtained as described and loaded onto a Fractogel EMD-DEAE-650(M) column, equilibrated with 10 mm MES (pH 6.0), and eluted with a linear KCl gradient (0–2 m). The (559–700)AtHsp90.1-containing fractions were pooled. (NH4)2SO4 was added to a final concentration of 25% saturation and loaded onto a Fractogel EMD-Propyl-650(M) column equilibrated with 10 mm Hepes (pH 7.5) containing 25% (NH4)2SO4. The (558–700)AtHsp90.1 was eluted with a gradient of 0–5% (w/v) glycerol, 0–0.5% (w/v) Chaps and 25–0% (NH4)2SO4. During preparation of (559–700)AtHsp90.1 a smaller fragment, (587–700)AtHsp90.1, was co-purified.
The integrity of all recombinant proteins was confirmed with automated gas-phase sequencing and mass spectrometry. Amounts of 500 µg (1–339)AtFKBP42 and (559–700)AtHsp90.1 protein were rpHPLC purified and used for the generation of rabbit polyclonal antibodies (Pab productions, Hebertshausen, Germany).
SDS–PAGE and Western blotting
SDS–PAGE was performed with 10% Tris/glycine gels and standard Laemmli buffer (Laemmli, 1970). The gels were either stained with a mix of Coomassie G-250 and Coomassie R-250 (Serva, Heidelberg, Germany) or silver according to standard protocols, or blotted to nitrocellulose in a tank blot apparatus (Bio-Rad, München, Germany) with 400 mA for 2 h and transfer buffer containing 25 mm Tris, 150 mm glycine, 0.1% SDS and 10% MeOH at pH 8.3.
Wild-type and HA-TWD1-overexpressing Arabidopsis plants (M. Geisler and co-workers, unpublished results) were grown on soil for 14–18 days under long-day conditions at 22°C. Plants were fixed and immunostained as described previously (Neumann et al., 1987). HA-TWD1 was detected with monoclonal anti-HA high-affinity antibody (Roche Diagnostics, Mannheim, Germany). As the anti-HA-antibody was generated in rat cell lines, antiserum against rat antibodies raised in rabbits was used. In order to visualize the protein antibody complex, staining with gold-labelled proteinA was performed using standard protocols.
Plasma membrane preparation of A. thaliana by aqueous two-phase extraction
Arabidopsis plants were cultivated on soil or in 400 ml of liquid Murashige & Skoog medium in 1 l Erlenmeyer flasks at 150 r.p.m., like the plants grown for electron microscopy. For liquid cultivation the seeds were surface-sterilized by treatment with ethanol (70%, 1 min) and hypochloride (6%, 10 min) and washed with sterile water.
Plants were harvested and homogenized with an Ultra-Turrax (six × 30 sec) in 10 vol prechilled buffer A (50 mm Hepes–KOH pH 7.5, 0.5 m sucrose, 2 mm DTT, 0.1 mg ml−1 butylated hydroxytoluene, 1% polyvinylpyrrolidone MG: 40 000). After filtration through two layers of Miracloth (Calbiochem, La Jolla, CA, USA) the debris was removed by centrifugation (8000 g, 10 min). The microsomal fraction was pelleted from the supernatant (48 000 g, 30 min). The resulting pellet was purified with an aqueous two-phase system as described (Kammerloher et al., 1994).
The enriched plasma membranes were diluted 10-fold in buffer B (50 mm Hepes–KOH pH 7.5, 0.33 m sucrose and one tablet of complete EDTA-free (Roche Diagnostics) protease inhibitor mix, pelleted (48 000 g, 30 min), resolved in a small volume of buffer B and stored in aliquots at −80°C. All centrifugation steps were carried out at 4°C.
Analysis of AtFKBP42 glycosylation
Proteins of enriched plasma membrane fractions from HA-TWD1-overexpressing Arabidopsis plants were tested for glycosylation of AtFKBP42. Plasma membrane fractions were separated on SDS–PAGE (gels 10%) and transferred to nitrocellulose. This blot was used to identify glycosylated proteins using the glycoprotein detection module (Amersham Pharmacia) following the manufacturer's protocol. The glycosylation was visualized by enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia). AtFKBP42 protein was visualized with antibody detection, and the ECL reaction by reprobing the same blot.
Circular dichroism spectroscopy
Far-UV CD measurements were performed with a Jasco J-710 CD spectrometer (Gross-Umstadt, Germany). Temperature was controlled in a thermostated cuvette holder by a cryostat RTE111 (Neslab Instruments, Portsmouth, NH, USA). The protein fragments (1–339)AtFKBP42 and (1–180)AtFKBP42 were dialysed in 50 mm phosphate buffer (pH 7.5) overnight at 4°C. The spectra were recorded using a 1 µm protein solution in a 0.1 cm cuvette from 195 to 260 nm at constant temperatures (20, 65 and 80°C). Buffer spectra were subtracted from the protein spectra, and the molar ellipticity spectra were calculated.
The thermal stability was observed from 20 to 65°C at 222 nm for (1–339)AtFKBP42 and from 20 to 80°C at 205 nm for (1–180)AtFKBP42. The temperature was increased by 1°C min−1.
Citrate synthase assays
The effect of AtFKBP42 on temperature-induced aggregation of CS and the loss of CS activity was analysed as described by Buchner et al. (1998). Citrate synthase was obtained from Roche Diagnostics. The aggregation was measured at 40°C with a diode array spectrophotometer (Hewlett Packard, Böblingen, Germany) at 360 nm in 40 mm Hepes buffer (pH 7.5). The effects of varied concentrations of (1–339)AtFKBP42 and (1–180)AtFKBP42 were observed.
Recombinant (1–339)AtFKBP42 was tested for CaM binding. (1–339)AtFKBP42 was incubated with soluble bovine CaM (Sigma) and cross-linked by aminoreactive 3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP) reagent in 10 mm Hepes buffer (pH 7.8) supplemented with 3 mm CaCl2 and 150 mm NaCl. In order to stop the reaction, excess of 1.8 m Tris buffer (pH 8.8) was added after 30 min incubation at room temperature. The samples were separated by SDS–PAGE and silver-stained.
Binding to a CaM affinity matrix was analysed with CaM agarose as previously described (Hueros et al., 1998). An amount of 10 µg of either (1–339)AtFKBP42 or 10 µg (1–180)AtFKBP42 was incubated with 50 µl CaM agarose in a 40 mm Hepes (pH 7.5) incubation buffer supplemented with 3 mm CaCl2, 0.1 mm EDTA and 0.1 mm DTT for 1 h at 4°C with constant agitation. The supernatant was removed. After washing five times with incubation buffer, CaM agarose was incubated for 15 min with elution buffer in which CaCl2 had been replaced by 3 mm EGTA. The test was controlled by using recombinant calcineurin which was affinity-purified with CaM agarose as described (Mondragon et al., 1997). In a second control, elution buffer was used for the incubation. Supernatant, CaM agarose beads and eluted fractions were analysed by SDS–PAGE. After Coomassie staining, the bands were quantified using MultiAnalyst software (Bio-Rad). The larger subunit of the heterodimeric calcineurin was used for quantification analysis.
Isothermal titration calorimetry
The interaction of (1–339)AtFKBP42 and (559–700)AtHsp90.1 was analysed by isothermal titration calorimetry (VP-ITC, MicroCal, Northampton, MA, USA) in order to determine the stoichiometry and dissociation constant of the complex (Pierce et al., 1999). An AtFKBP42 fragment solution with the concentration of 283 µm was titrated stepwise into a 28 µm (559–700)AtHsp90.1 solution. Before titration, both proteins were dialysed for 16 h in 50 mm phosphate buffer (pH 7.5), to reduce the effects of buffer during titration. The resulting titration curve was analysed using the manufacturer's software.
PPIase activity of AtFKBP42 was examined with three different assays.
Isomerspecific proteolytic assay
PPIase activity to proline-containing peptide substrates was tested as described (Hani et al., 1999). In a competition assay, designed to detect putative FK506 binding, human FKBP12 was used in a concentration that accelerated the non-enzymatic isomerization of the peptide Suc-Ala-Phe-Pro-Phe-4NA threefold (three acceleration units). FK506 was kindly provided by Fujisawa GmbH (München, Germany). By addition of FK506, this acceleration was inhibited to one acceleration unit. If (1–339)AtFKBP42 bound to FK506, the inhibition efficiency of FK506 towards hFKBP12 would have been reduced, indicated by an increase of acceleration units.
Protease free assay
To overcome possible degradation of PPIase by proteases in the proteolytic assay, a protease-free test was performed as previously described (Janowski et al., 1997). The disturbance of cis/trans equilibrium is achieved by a solvent jump of the peptide from LiCl/trifluorethanol to 35 mm Hepes buffer pH 7.8. Re-equilibration, which is accelerated by PPIases, is measured. AtFKBP42 was tested up to final concentrations of ≈1 µm protein.
Refolding of RCM-T1
The refolding of RCM-T1 was measured as described (Schmid et al., 1996). RCM-T1 is denaturated in low-salt, 100 mm Tris buffer (pH 8.0). Spontaneous refolding can be induced by diluting RCM-T1 into the same buffer supplemented with 2 m NaCl and analysed by fluorescence spectroscopy. The refolding is limited to the cis/trans isomerization of the tyrosine38–proline39 peptide bond. Addition of PPIases accelerates the refolding. AtFKBP42 was tested up to concentrations of 1.4 µm. The RCM-T1 concentration was 173 µm.
We thank Dr Birte Hernandez Alvarez and Frank Edlich for reading the manuscript critically, Dr Beate Saal for HA-TWD1 seeds, Joachim Berger for providing cDNA clones of AtHsp90, and Dr Jun O. Liu for the calcineurin clone. We are grateful to Matthias Weiwad, who supplied the recombinant calcineurin, and Dr Peter Rücknagel and Dr Angelika Schierhorn for NH2-terminal sequencing and mass spectrometry of the proteins. Part of this work were supported by grants from the Deutsche Forschungsgemeinschaft, the EC (LATIN, BIOTEC 4), and the Ministerium für Schule, Wissenschaft und Forschung des Landes NRW to B.S.
- 2000) The genome sequence of Drosophila melanogaster. Science, 287, 2185–2195. , , , , et al. (
- 2001) FKBP12, the 12-kDa FK506-binding protein, is a physiologic regulator of the cell cycle. Proc. Natl Acad. Sci. USA, 98, 2425–2430. , , , , , , and (
- 1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410. , , , and (
- 2000) Molecular chaperones activate the Drosophila ecdysone receptor, an RxR heterodimer. Cell, 101, 67–77. and (
- 1998) Studies on the expression of the wheat prolyl isomerase FKBP73 during plant development. Plant Sci. 139, 149–158. , , , , and (
- 2001) Plant steroids recognized at the cell surface. Trends Genet. 17, 60–62. (
- 2001) Regulation of vegetative phase change in Arabidopsis thaliana by cyclophilin 40. Science, 291, 2405–2407. , , and (
- 2001) Plant steroid hormones, brassinosteroids: current highlights of molecular aspects on their synthesis/metabolism, transport, perception and response. Plant Cell Physiol. 42, 114–120. and (
- 1999) The tetratricopeptide repeat: a structural motif mediating protein–protein interactions. Bioessays, 21, 932–939. and (
- 2000) Nongenomic membrane actions of glucocorticoids in vertebrates. Trends Endocrin. Met. 11, 427–436. (
- 1998) Analysis of chaperone function using citrate synthase as nonnative substrate protein. Meth. Enzymol. 290, 323–338. , and (
- 1992) An immunophilin that binds Mr 90 000 heat shock protein: main structural features of a mammalian p59 protein. Proc. Natl Acad. Sci. USA, 89, 6270–6274. , , , , , and (
- 2001) Pasticcino1 (AtFKBP70) is a nuclear-localised immunophilin required during Arabidopsis thaliana embryogenesis. Plant Sci. 161, 527–535. , , , and (
- 1999) The common tetratricopeptide repeat acceptor site for steroid receptor-associated immunophilins and hop is located in the dimerization domain of Hsp90. J. Biol. Chem. 274, 2682–2689. , , , and (
- 1996) FK506-binding protein mutational analysis – defining the active-site residue contributions to catalysis and the stability of ligand complexes. Protein Eng. 9, 173–180. , , , , , and (
- 1998a) An Arabidopsis immunophilin, AtFKBP12, binds to AtFIP37 (FKBP interacting protein) in an interaction that is disrupted by FK506. Plant J. 15, 783–789. , and (
- 1998b) The PASTICCINO genes of Arabidopsis thaliana are involved in the control of cell division and differentiation. Development, 125, 909–918. , , , , , , , and (
- 1994) Peptidyl-prolyl cis/trans isomerases and their effectors. Angew. Chem. Int. Ed. Engl. 33, 1415–1436. (
- 1984) Nachweis einer Enzymkatalyse fur die cis–trans-Isomerisierung der Peptidbindung in prolinhaltigen Peptiden. Biomed. Biochim. Acta, 43, 1101–1111. , and (
- 1989) Cyclophilin and peptidyl-prolyl cis–trans isomerase are probably identical proteins. Nature, 337, 476–478. , , , and (
- 2001) Application of different surface plasmon resonance biosensor chips to monitor the interaction of the Cam-binding site of nitric oxide synthase I and calmodulin. Biochem. Biophys. Res. Commun. 285, 463–469. , and (
- 2001) Nongenomic estrogen action in human lung myofibroblasts. Biochem. Biophys. Res. Commun. 283, 661–667. , , , and (
- 2001) Steroid signaling in plants: from the cell surface to the nucleus. Bioessays, 23, 1028–1036. and (
- 2000) Brassinosteroid-insensitive-1 is a ubiquitously expressed leucine-rich repeat receptor serine/threonine kinase. Plant Physiol. 123, 1247–1255. , , , and (
- 2001) Evidence that the peptidylprolyl isomerase domain of the Hsp90-binding immunophilin FKBP52 is involved in both dynein interaction and glucocorticoid receptor movement to the nucleus. J. Biol. Chem. 276, 14884–14889. , , , and (
- 1996) Methods to estimate the conformation of proteins and polypeptides from circular dichroism data. Anal. Biochem. 235, 1–10. (
- 1999) Mutations in a peptidylprolyl-cis/trans-isomerase gene lead to a defect in 3′-end formation of a pre-mRNA in Saccharomyces cerevisiae. J. Biol. Chem. 274, 108–116. , , , , , and (
- 2001) FKBPs: At the crossroads of folding and transduction. Trends Plant Sci. 6, 426–431. , and (
- 1987) Calmodulin binding by calcineurin. Ligand-induced renaturation of protein immobilized on nitrocellulose. J. Biol. Chem. 262, 15062–15070. and (
- 1998) A maize FK506-sensitive immunophilin, mzFKBP-66, is a peptidylproline cis–trans-isomerase that interacts with calmodulin and a 36-kDa cytoplasmic protein. Planta, 205, 121–131. , , and (
- 2000) Immunophilins: switched on protein binding domains? Med. Res. Rev. 20, 452–484. (
- 1997) A protease-free assay for peptidyl prolyl cis/trans isomerases using standard peptide substrates. Anal. Biochem. 252, 299–307. , , and (
- 1994) Water channels in the plant plasma-membrane cloned by immunoselection from a mammalian expression system. Plant J. 6, 187–199. , , and (
- 1996) Structure–function relationships in the FK506-binding protein (FKBP) family of peptidylprolyl cis–trans isomerases. Biochem. J. 314, 361–385. (
- 2001) The Hsp90 family of proteins in Arabidopsis thaliana. Cell Stress Chaperones, 6, 238–246. and (
- 1999) The wheat peptidyl prolyl cis–trans-isomerase FKBP77 is heat induced and developmentally regulated. Plant Physiol. 119, 693–703. , , , and (
- 1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature, 227, 680. (
- 1995) Isolation of a cDNA encoding a novel human FK506-binding protein homolog containing leucine zipper and tetratricopeptide repeat motifs. Gene, 160, 297–302. , and (
- 1995) Tetratrico peptide repeat interactions – to TPR or not to TPR. Trends Biochem. Sci. 20, 257–259. , and (
- 2000) The Arabidopsis thaliana Pin1At gene encodes a single-domain phosphorylation-dependent peptidyl prolyl cis/trans isomerase. J. Biol. Chem. 275, 10577–10581. , , , , , , , and (
- 2002) Regulation of brassinosteroid signaling by a GSK3/SHAGGY-like kinase. Science, 295, 1299–1301. and (
- 2001) Bin2, a new brassinosteroid-insensitive locus in Arabidopsis. Plant Physiol. 127, 14–22. , , and (
- 2000) Calmodulin binds to and inhibits the activity of the membrane distal catalytic domain of receptor protein-tyrosine phosphatase alpha. J. Biol. Chem. 275, 30075–30081. , , , and (
- 1999) Function of WW domains as phosphoserine- or phosphothreonine-binding modules. Science, 283, 1325–1328. , , and (
- 1992) Rabbit FKBP59-heat shock protein binding immunophillin (HBI) is a calmodulin binding protein. Biochem. Biophys. Res. Commun. 187, 1330–1335. , , , and (
- 2001) Functional replacement of the essential Ess1 in yeast by the plant parvulin DlPar13. J. Biol. Chem. 276, 13524–13529. , , , , , and (
- 1991) Solution structure of FKBP, a rotamase enzyme and receptor for FK506 and rapamycin. Science, 252, 836–839. , , , and (
- 1997) Genomic organization of Hsp90 gene family in Arabidopsis. Plant Mol. Biol. 35, 955–961. and (
- 1997) Overexpression and purification of human calcineurin alpha from Escherichia coli and assessment of catalytic functions of residues surrounding the binuclear metal center. Biochemistry, 36, 4934–4942. , , , , and (
- 1999) Thermodynamics of target peptide recognition by calmodulin and a calmodulin analogue: implications for the role of the central linker. FEBS Lett. 461, 19–24. , , , , , and (
- 2001) Brassinosteroid signaling in plants. Trends Endocrin. Met. 12, 398–402. and (
- 1997) Molecular cloning of human FKBP51 and comparisons of immunophilin interactions with Hsp90 and progesterone receptor. Mol. Cell. Biol. 17, 594–603. , , , , , and (
- 1987) Intracellular localization of heat-shock proteins in tomato cell cultures. Eur. J. Cell Biol. 43, 71–81. , , , , and (
- 2000) Recombinant brassinosteroid insensitive 1 receptor-like kinase autophosphorylates on serine and threonine residues and phosphorylates a conserved peptide motif in vitro. Plant Physiol. 124, 751–765. , , , , and (
- 1996) Binding of immunophilins to the 90 kDa heat shock protein (Hsp90) via a tetratricopeptide repeat domain is a conserved protein interaction in plants. Biochemistry, 35, 15249–15255. , , , and (
- 1999) muFKBP38: a novel murine immunophilin homolog differentially expressed in Schwannoma cells and central nervous system neurons in vivo. Electrophoresis, 20, 249–255. , , and (
- 2001a) Mutations in the ULTRACURVATA2 gene of Arabidopsis thaliana, which encodes a FKBP-like protein, cause dwarfism, leaf epinasty and helical rotation of several organs. Int. J. Dev. Biol. 45, S49–S50. , and (
- 2001b) ULTRACURVATA1, a SHAGGY-like Arabidopsis gene required for cell elongation. Int. J. Dev. Biol. 45, S51–S52. , and (
- 2002) The UCU1 Arabidopsis gene encodes a SHAGGY/GSK3-like kinase required for cell expansion along the proximodistal axis. Dev. Biol. 242, 161–173. , and (
- 1999) Isothermal titration calorimetry of protein–protein interactions. Methods — a companion to Methods in Enzymology, 19, 213–221. , and (
- 2001) Functional analysis of the Hsp90-associated human peptidyl prolyl cis/trans isomerases FKBP51, FKBP52 and cyp40. J. Mol. Biol. 308, 795–806. and (
- 2001) Localization of the chaperone domain of FKBP52. J. Biol. Chem. 276, 37034–37041. , , and (
- 1998) The Hsp90-based chaperone system – involvement in signal transduction from a variety of hormone and growth factor receptors. Proc. Soc. Exp. Biol. Med. 217, 420–434. (
- 1997) Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocrin. Rev. 18, 306–360. and (
- 2001) Hsp90-binding immunophilins in plants: the protein movers. Trends Plant Sci. 6, 54–58. , and (
- 1998) High-molecular-weight FK506-binding proteins are components of heat-shock protein 90 heterocomplexes in wheat germ lysate. Plant Physiol. 118, 1395–1401. , , , , and (
- 2001) Hsp90: chaperoning signal transduction. J. Cell. Physiol. 188, 281–290. and (
- 2000) Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70–Hsp90 multichaperone machine. Cell, 101, 199–210. , , , , , , and (
- 2000) Enzymes that catalyse the restructuring of proteins. Curr. Opin. Struct. Biol. 10, 40–45. and (
- 2001) Receptor accessory folding helper enzymes: the functional role of peptidyl prolyl cis/trans isomerases. FEBS Lett. 495, 1–6. and (
- 1996) Catalyzed and assisted protein folding of ribonuclease T1. Biol. Chem. 377, 417–424. , , and (
- 2000) Rapid, nongenomic steroid actions: a new age?. Front. Neuroendocrinol. 21, 57–94. , , , , and (
- 1998) SMART, a simple modular architecture research tool: identification of signaling domains. Proc. Natl Acad. Sci. USA, 95, 5857–5864. , , and (
- 2000) SMART: a web-based tool for the study of genetically mobile domains. Nucl. Acids Res. 28, 231–234. , , , and (
- 1998) Cardiac defects and altered ryanodine receptor function in mice lacking FKBP12. Nature, 391, 489–492. , , et al. (
- 1989) A cytosolic binding protein for the immunosuppressant FK506 has peptidyl-prolyl isomerase activity but is distinct from cyclophilin. Nature, 341, 755–757. , , , and (
- 1999) Different regions of the immunophilin FKBP52 determine its association with the glucocorticoid receptor, hsp90, and cytoplasmic dynein. J. Biol. Chem. 274, 36980–36986. , , , , and (
- 1997) An immunophilin is a component of the insect ecdysone receptor (EcR) complex. Insect Biochem. Mol. Biol. 27, 973–982. , , and (
- 1996) Animal and plant cell lysates share a conserved chaperone system that assembles the glucocorticoid receptor into a functional heterocomplex with Hsp90. Biochemistry, 35, 554–561. , , and (
- 2001) Two structures of cyclophilin 40: folding and fidelity in the TPR domains. Structure, 9, 431–438. , , , , and (
- 1994) ClustalW – improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673–4680. , and (
- 1995) Characterization of an exchange reaction between soluble FKBP-12 and the FKBP–ryanodine receptor complex. Modulation by FKBP mutants deficient in peptidyl-prolyl isomerase activity. J. Biol. Chem. 270, 2451–2459. , , and (
- 1997) Comparative mutational analysis of peptidyl prolyl cis/trans isomerases: active sites of Escherichia coli trigger factor and human FKBP12. FEBS Lett. 407, 184–190. , , , , and (
- 1999) Identification and characterization of a 14 kDa human protein as a novel parvulin-like peptidyl prolyl cis/trans isomerase. FEBS Lett. 446, 278–282. , , , and (
- 2001) Peptidyl-prolyl isomerases and regulation of photosynthetic functions. Regul. Photosynth. 11, 177–193. (
- 1998) Mutation in the Arabidopsis Pasticcino1 gene, which encodes a new FK506-binding protein-like protein, has a dramatic effect on plant development. Mol. Cell. Biol. 18, 3034–3043. , , , and (
- 1996) Novel structure of a high molecular weight FK506 binding protein from Arabidopsis thaliana. Mol. Gen. Genet. 252, 510–517. and (
- 2001) BRI1 is a critical component of a plasma-membrane receptor for plant steroids. Nature, 410, 380–383. , , , and (
- 1995) Circular dichroism. Meth. Enzymol. 246, 34–71. (
- 2002) BES1 accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell, 109, 181–191. , , , , , and (
- 1998) Specific binding of tetratricopeptide repeat proteins to the C-Terminal 12-kDa domain of Hsp90. J. Biol. Chem. 273, 18007–18010. , and (