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

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

Thilo Kamphausen1, Jorg Fanghanel1, Dieter Neumann2, Burkhard Schulz3,y and Jens-U. Rahfeld1,�

1Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, Germany,2Institute of Plant Biochemistry, Weinberg 3, 06120 Halle, Germany, and3Universitat zu Koln, Botanisches Institut II, Max-Delbruck Labor in der MPG, Carl von Linne Weg 10, 50829 Koln,

Germany

Received 5 February 2002; revised 11 June 2002; accepted 23 June 2002.�For correspondence (fax þ49 34555 11972; e-mail [email protected]).yPresent address: ZMBP-Pflanzenphysiologie, Auf der Morgenstelle 5, 72076 Tubingen, Germany.

Summary

The twisted dwarf 1 (twd1) mutant from Arabidopsis thaliana was identified in a screen for plant architec-

ture 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 translo-

cates to the nucleus, AtFKBP42 was predicted to be membrane-localized, as shown by electron microscopy.

Keywords: PPIase, steroid hormone receptor, Hsp90, plasma membrane, Arabidopsis, signal transduction.

Introduction

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 leth-

ality 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 pub-

lished 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 5.2.1.8). Members of this

enzyme class specifically catalyse the peptidyl-bond iso-

merization preceding a proline (Fischer et al., 1984; Fischer

et al., 1989). PPIases are involved in de novo protein bio-

synthesis 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 tetratri-

copeptide 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

The Plant Journal (2002) 32, 263–276

� 2002 Blackwell Publishing Ltd 263

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) com-

plexes. 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 mam-

malian counterparts by one additional FKBP domain, gen-

erally 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 assem-

ble a functional SHR complex with immunopurified mam-

malian 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 mamma-

lian 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 brassi-

nolide (BL) is perceived extracellularly (Wang et al., 2001).

Brassinolide binding induces autophosphorylation of the

kinase domain (Oh et al., 2000). The following signal trans-

duction pathway is not well understood. There are still open

questions, such as phosphorylation targets or the oligo-

merization state of BRI1 (for review see Friedrichsen and

Chory, 2001; Mussig and Altmann, 2001). Two downstream

proteins of the BL-induced signalling cascade have been

analysed in more detail. The BIN2/UCU1 kinase of a glyco-

gen synthase–kinase 3/shaggy-like type is one phosphor-

ylation 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 pheno-

type 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 analy-

sis regards the protein–protein interactions of AtFKBP42

with calmodulin (CaM) and AtHsp90, employing cross-

linking experiments or isothermal titration calorimetry

approaches.

Results

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 align-

ment 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 degen-

erated 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 des-

cribed (Lam et al., 1995; Pedersen et al., 1999). A predicted

264 Thilo Kamphausen et al.

� Blackwell Publishing Ltd, The Plant Journal, (2002), 32, 263–276

Figure 1. Sequence alignment and domain composition of AtFKBp42. (a) Multiple sequence alignment of hFKBP12 (accession: P20071), AtFKBP42 (CAC00654),hFKBP38 (Q14318), DmFKBP45 (AAF57662) and hFKBP52 (Q02790), beginning with the second FKBP domain (aa 145). Within the FKBP domains, identical aminoacid residues (compared to hFKBP12) are shaded dark grey. The asterisks indicate essential residues, which are discussed in FK506 binding (Kay, 1996). Residueswhere each individual variation reduces hFKBP12 PPIase activity (<25%) are white reversed on black (DeCenzo et al., 1996; Timerman et al., 1995; Tradler et al.,1997), except the FKBP domain identical residues (compared to AtFKBP42) which are shaded light grey. Sequence motifs are indicated below the alignment. TheTPR motifs and calmodulin-binding sites are boxed, and the membrane anchor is underlined (predicted for hFKBP38 and DmFKBP45 by SMART). The sequenceswere aligned using the CLUSTALW program (www2.ebi.ac.uk/clustalw) (Thompson et al., 1994).(b) Domain composition of AtFKBP42. FKBP: FKBP12-like domain; TPR: tetratricopeptide repeat; CaM: putative calmodulin-binding region; MA membraneanchor.


Characterization of AtFKBP42 265

44.8 kDa protein from Drosophila melanogaster (Dm-

FKBP45; 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 seg-

ment – were predicted for all four proteins using the SMART

analysis tool (Figure 1). Putative CaM binding was pub-

lished for hFKBP38 and muFKBP38 (Lam et al., 1995; Ped-

ersen 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 pur-

ified 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 808C for (1–180)AtFKBP42, and

at 20 and 658C 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 tem-

perature from 20 to 808C for (1–180)AtFKBP42, and at

222 nm from 20 to 658C for (1–339)AtFKBP42 (Figure 2b).

The CD spectrum of the (1–180)AtFKBP42 at 208C shows

an absolute minimum at 208 nm, indicating a high content

of b-sheet, which is typical for FKBP domains. The structure

of hFKBP12 contains a b-sheet of five antiparallel strands, a

short helix and unstructured elements (Michnick et al.,

1991). The (1–339)AtFKBP42 CD spectrum, recorded at

208C, displays a double minimum at 222 and 209 nm, indi-

cating higher a-helical contents of the protein caused by the

additional TPR domain. TPR domains are mostly composed

of a-helical structures (Blatch and Lassle, 1999; Taylor et al.,

2001). The a-helical content of a protein can be calculated

from its CD spectra with the CDNN software (free down-

load: http://bioinformatic.biochemtech.uni-halle.de/cdnn).

CDNN analysis predicted 18% a-helical content for (1–

180)AtFKBP42 und 37% for (1–339)AtFKBP42, which con-

firms the higher a-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 �488C. The mid-point of the CD signal

change (Tm) was determined at �598C. The signal of

(1–339)AtFKBP42 changes little in the range from 25 to

408C. Above 408C, the signal changes more rapidly. Tm

was determined at �438C. 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 char-

acterized in vitro using short peptides and larger protein

fragments of Hsp90 (Pirkl and Buchner, 2001; Scheufler

et al., 2000). A competition experiment with high molecu-

lar mass wheat FKBP, Hsp90 and rat protein phosphatase5

Figure 2. CD spectra of (1–180)AtFKBP42 and (1–339)AtFKBP42.(a) Far-UV spectra of (1–180)AtFKBP42 (~) and (1–339)AtFKBP42 (*) from195 to 260 nm measured at 208C. The spectrum of (1–180)AtFKBP42 shows aminimum at 208 nm. Minima of the (1–339)AtFKBP42 spectrum are at 222and 209 nm.(b) Thermal stability of (1–180)AtFKBP42 and (1–339)AtFKBP42. The unfold-ing was followed at 205 nm for (1–180)AtFKBP42 (~) and at 222 nm for(1–339)AtFKBP42 (*).



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 Arabi-

dopsis 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 chro-

matography. 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 indi-

cates 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 mM 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 chaper-

one-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 mM (1–339)AtFKBP42 to CS (3 mM), 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 4mM (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 438C 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

Figure 3. Isothermal titration calorimetry measurement. Upper part: titra-tion curve of (559–700)AtHsp90.1 (28 mM) with (1–339)AtFKBP42 (283 mM) at208C. Lower part: integrated titration data, baseline corrected and fitted to a1 : 1 binding model. The determined constants were KD¼1.3 mM and astoichiometry of 0.48.

Figure 4. Influence of AtFKB42 proteins on the aggregation of citratesynthase (CS) at 408C. The increase of aggregation of 3 mM CS (*) wasfollowed for 1 h at 360 nm with various concentrations of (1–339)AtFKBP42:(!) 0.1 mM; (~) 2.7 mM; (~) 4 mM (1–339)AtFKBP42; and (1–180)AtFKBP42:(^) 45 mM (1–180)AtFKBP42; control: (*) 30 mM (1–339)AtFKBP42 withoutCS.



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

6mM with 0.5 mM 3, 30-dithiobis (sulfosuccinimidylpropio-

nate)(DTSSP). After SDS–PAGE separation and silver stain-

ing 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

Ca2þ-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

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-spectro-

scopy measurements indicate that (1–180)AtFKBP42 and

(1–339)AtFKBP42 are structured, neither showed any

PPIase activity with up to 6.35 mM 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, 200mg ml�1 in

buffer supplemented with 5% (NH4)2SO4 was the maxi-

mum 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 hydro-

phobic residues, as well as charged aa residues in the

substrate, a broad range of peptide substrates was exam-

ined. 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 mM 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 1mM

should have been detected.

Furthermore, we tested whether the missing PPIase activ-

ity 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 addi-

tional signal which could not be detected after staining with

Figure 5. Calmodulin binding of AtFKBP42.(a) Crosslinking experiment Lane 1, 6 mM (1–339)AtFKBP42; lane 2, 6 mM CaM(Sigma); lane 3, mix of 6 mM (1–339)AtFKBP42 with 6 mM CaM. For allreactions, 0.5 mM DTSSP was used. Lanes 1 and 2 show no correspondingband to protein dimers. Lane 3 shows an additional band, which has anapparent molecular weight of a (1–339)AtFKBP42–CaM complex.(b) CaM agarose binding. 10 mg (1–339)AtFKBP42, calcineurin and (1–180)AtFKBP42, respectively, were incubated with CaM agarose in CaCl2-supplemented buffer (þCaCl2) or EGTA-supplemented buffer (þEGTA).Supernatant (S), CaM agarose beads (B) and eluted (E) fractions wereanalysed by SDS–PAGE and Coomassie staining.



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-overex-

pressing 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 Arabidop-

sis 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).

Discussion

The overall structure of AtFKBP42 shares important fea-

tures 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

Figure 6. Electron microscopy pictures of (a) HA-TWD1 overexpressingplants; (b) wild-type Arabidopsis. The cellular compartments are indicatedas follows. C, cytosol; CH, chloroplast; CW, cell wall; PM, plasma membrane;T, tonoplast; V, vacuole. Scaling is indicated. Black dots represent gold-labelled proteinA-decorating anti-HA antibodies.

Figure 7. Detection of AtFKBP42 IN Arabidopsis plants.(a) Western blot analysis of plasma membrane preparation from HA-TWD1-overexpressing plants. Fractions (7.5 mg protein) were probed with anti-AtFKBP42 antibody. (1) 8000 g supernatant; (2) 48 000 g supernatant; (3)plasma membrane fraction resuspended after aqueous two-phase extrac-tion. The AtFKBP42 protein can be detected in the PM-enriched fraction at anapparent molecular weight of 55 kDa. The level of AtFKBP42 in whole-plantlysate (1) is below the detection limit.(b) Detection of glycosylated plasma membrane proteins and HA-TWD1 in7.5 mg PM protein. Left lane, signals after glycosylation detection, an�56 kDasingle signal is visible. Right lane, the same blot was reprobed with anti-AtFKBP42 antibody. In addition to the �56 kDa signal an �55 kDa protein(arrow) is detected only with anti-AtFKBP42 antibody, but not with theglycosylation detection module. The apparent molecular weight corre-sponds with that of HA-TWD1 in 10% SDS gels.



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 dimer-

ization that we identified for (559–700)AtHsp90.1 fragment

is consistent with the dimerization data published for mam-

malian 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 frag-

ment and monomeric (1–339)AtFKBP42 was measured with

1.3 mM. 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 med-

iate 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 stoichiome-

try 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 conser-

vation 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 com-

plex 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 plas-

mon resonance and isothermal titration calorimetry were

performed. With the design of the measurements a KD

value up to the low mM 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 deter-

mined.

The purified protein fragments of AtFKBP42 were ana-

lysed 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, respec-

tively (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 media-

ted 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). Exp-

eriments 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 immuno-

localization 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 tono-

plast and the plasma membrane. The preparation techni-

que 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, un-

structured areas are distinguishable. It is likely that high-

level overexpression of a membrane-localized protein modi-

fies 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 inter-

action 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 mam-

malian 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 trans-

mit 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 pro-

teins of the FKBP family were shown for the soluble SHR,

the membrane bound TGF-b receptor and ryanodyne recep-

tor (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.

Experimental procedures

All chemicals and column resins were purchased from MerckEurolab (Darmstadt, Germany) or Sigma (Munchen, Germany),unless indicated otherwise. Restriction enzymes were obtainedfrom 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 (50-gat cga cca tgg atg aat ctc tggagc atc-30) and TWD3a (30-tca gaa gct tag tct gct gca cca atc c). ThePCR product was restricted with NcoI, HindIII followed by ligationinto pET28a. This construct encodes (1–180)AtFKBP42, a proteinfragment from residues 1–180. The recombinant protein was pro-duced at 308C in BL21 codonþRIL cells (Stratagene, La Jolla, USA)for 5 h after induction with 1 mM isopropyl-b-D-thiogalactopyra-nosid (IPTG).

After disruption of harvested E. coli cells in a French Press (SLMAminco, Rochester, NY, USA), the supernatant of a 100 000 gcentrifugation was loaded on a Fractogel EMD-DEAE-650(M) col-umn equilibrated with 10 mM Hepes buffer (pH 7.5) and eluted witha linear KCl gradient (0–2 M). The (1–180)AtFKBP42-containing



fractions were pooled, dialysed in 10 mM Hepes (pH 7.5), andpassed through a Fractogel TSK-AF-Blue column. (1–180)At-FKBP42 did not bind. The (1–180)AtFKBP42-containing fractionswere concentrated using VivaSpin columns (Sartorius, Gottingen,Germany). Preparative size-exclusion chromatography wasperformed with a HiLoad Superdex 75 HR 16/60 column (Amer-sham Pharmacia, Freiburg, Germany) in 10 mM Hepes buffer(pH 7.5) containing 150 mM NaCl. The resulting homogeneouslypurified (1–180)AtFKBP42 was concentrated again and storedat �808C.

The construct (1–339)AtFKBP42 was amplified using primersTWD5a and TWD3b (50-tca cta agc tta aag gct ctt tga ctt agc acc-30). 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 byPCR amplification with primers HSP5a (50-gat gca cca tgg ttg tgg tctcag aca gga ttg-30) and HSP3a (50-gca gtc aag ctt agt cga ctt cct ccatc-30), NcoI, HindIII restriction and ligation into pET28a. Theencoded protein was expressed at 378C in BL21 codonþRIL cells(Stratagene) for 5 h after induction with 1 mM IPTG.

Cell lysate was obtained as described and loaded onto a Frac-togel 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 wasadded to a final concentration of 25% saturation and loaded onto aFractogel EMD-Propyl-650(M) column equilibrated with 10 mM

Hepes (pH 7.5) containing 25% (NH4)2SO4. The (558–700)At-Hsp90.1 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, wasco-purified.

The integrity of all recombinant proteins was confirmed withautomated gas-phase sequencing and mass spectrometry.Amounts of 500 mg (1–339)AtFKBP42 and (559–700)AtHsp90.1protein were rpHPLC purified and used for the generation ofrabbit polyclonal antibodies (Pab productions, Hebertshausen,Germany).

SDS–PAGE and Western blotting

SDS–PAGE was performed with 10% Tris/glycine gels and standardLaemmli buffer (Laemmli, 1970). The gels were either stained with amix of Coomassie G-250 and Coomassie R-250 (Serva, Heidelberg,Germany) or silver according to standard protocols, or blotted tonitrocellulose ina tank blotapparatus (Bio-Rad,Munchen,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.

Electron microscopy

Wild-type and HA-TWD1-overexpressing Arabidopsis plants (M.Geisler and co-workers, unpublished results) were grown on soilfor 14–18 days under long-day conditions at 228C. Plants were fixedand immunostained as described previously (Neumann et al.,1987). HA-TWD1 was detected with monoclonal anti-HA high-affinity antibody (Roche Diagnostics, Mannheim, Germany). Asthe anti-HA-antibody was generated in rat cell lines, antiserumagainst rat antibodies raised in rabbits was used. In order tovisualize the protein antibody complex, staining with gold-labelledproteinA 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 liquidMurashige & Skoog medium in 1 l Erlenmeyer flasks at 150 r.p.m.,like the plants grown for electron microscopy. For liquid cultivationthe 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–KOHpH 7.5, 0.5 M sucrose, 2 mM DTT, 0.1 mg ml�1 butylated hydroxy-toluene, 1% polyvinylpyrrolidone MG: 40 000). After filtrationthrough two layers of Miracloth (Calbiochem, La Jolla, CA, USA)the debris was removed by centrifugation (8000 g, 10 min). Themicrosomal 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 bufferB (50 mM Hepes–KOH pH 7.5, 0.33 M sucrose and one tablet ofcomplete EDTA-free (Roche Diagnostics) protease inhibitor mix,pelleted (48 000 g, 30 min), resolved in a small volume of buffer Band stored in aliquots at �808C. All centrifugation steps werecarried out at 48C.

Analysis of AtFKBP42 glycosylation

Proteins of enriched plasma membrane fractions from HA-TWD1-overexpressing Arabidopsis plants were tested for glycosylation ofAtFKBP42. Plasma membrane fractions were separated on SDS–PAGE (gels 10%) and transferred to nitrocellulose. This blot wasused to identify glycosylated proteins using the glycoproteindetection module (Amersham Pharmacia) following the manufac-turer’s protocol. The glycosylation was visualized by enhancedchemiluminescence (ECL) detection kit (Amersham Pharmacia).AtFKBP42 protein was visualized with antibody detection, and theECL reaction by reprobing the same blot.

Circular dichroism spectroscopy

Far-UV CD measurements were performed with a Jasco J-710 CDspectrometer (Gross-Umstadt, Germany). Temperature was con-trolled in a thermostated cuvette holder by a cryostat RTE111(Neslab Instruments, Portsmouth, NH, USA). The protein frag-ments (1–339)AtFKBP42 and (1–180)AtFKBP42 were dialysed in50 mM phosphate buffer (pH 7.5) overnight at 48C. The spectrawere recorded using a 1 mM protein solution in a 0.1 cm cuvettefrom 195 to 260 nm at constant temperatures (20, 65 and 808C).Buffer spectra were subtracted from the protein spectra, and themolar ellipticity spectra were calculated.

The thermal stability was observed from 20 to 658C at 222 nm for(1–339)AtFKBP42 and from 20 to 808C at 205 nm for (1–180)At-FKBP42. The temperature was increased by 18C min�1.

Citrate synthase assays

The effect of AtFKBP42 on temperature-induced aggregation of CSand the loss of CS activity was analysed as described by Buchneret al. (1998). Citrate synthase was obtained from Roche Diagnos-tics. The aggregation was measured at 408C with a diode arrayspectrophotometer (Hewlett Packard, Boblingen, Germany) at360 nm in 40 mM Hepes buffer (pH 7.5). The effects of variedconcentrations of (1–339)AtFKBP42 and (1–180)AtFKBP42 wereobserved.



Calmodulin binding

Recombinant (1–339)AtFKBP42 was tested for CaM binding. (1–339)AtFKBP42 was incubated with soluble bovine CaM (Sigma)and cross-linked by aminoreactive 3,30-dithiobis(sulfosuccinimi-dylpropionate) (DTSSP) reagent in 10 mM Hepes buffer (pH 7.8)supplemented with 3 mM CaCl2 and 150 mM NaCl. In order to stopthe reaction, excess of 1.8 M Tris buffer (pH 8.8) was added after30 min incubation at room temperature. The samples were sepa-rated by SDS–PAGE and silver-stained.

Binding to a CaM affinity matrix was analysed with CaM agaroseas previously described (Hueros et al., 1998). An amount of 10mg ofeither (1–339)AtFKBP42 or 10 mg (1–180)AtFKBP42 was incubatedwith 50 ml CaM agarose in a 40 mM Hepes (pH 7.5) incubation buffersupplemented with 3 mM CaCl2, 0.1 mM EDTA and 0.1 mM DTT for1 h at 48C with constant agitation. The supernatant was removed.After washing five times with incubation buffer, CaM agarose wasincubated for 15 min with elution buffer in which CaCl2 had beenreplaced by 3 mM EGTA. The test was controlled by using recom-binant calcineurin which was affinity-purified with CaM agarose asdescribed (Mondragon et al., 1997). In a second control, elutionbuffer was used for the incubation. Supernatant, CaM agarosebeads and eluted fractions were analysed by SDS–PAGE. AfterCoomassie staining, the bands were quantified using MULTIANA-

LYST software (Bio-Rad). The larger subunit of the heterodimericcalcineurin was used for quantification analysis.

Isothermal titration calorimetry

The interaction of (1–339)AtFKBP42 and (559–700)AtHsp90.1 wasanalysed by isothermal titration calorimetry (VP-ITC, MicroCal,Northampton, MA, USA) in order to determine the stoichiometryand dissociation constant of the complex (Pierce et al., 1999). AnAtFKBP42 fragment solution with the concentration of 283 mM wastitrated stepwise into a 28 mM (559–700)AtHsp90.1 solution. Beforetitration, both proteins were dialysed for 16 h in 50 mM phosphatebuffer (pH 7.5), to reduce the effects of buffer during titration. Theresulting titration curve was analysed using the manufacturer’ssoftware.

PPIase assays

PPIase activity of AtFKBP42 was examined with three differentassays.

Isomerspecific proteolytic assayPPIase activity to proline-containing peptide substrates was testedas described (Hani et al., 1999). In a competition assay, designedto detect putative FK506 binding, human FKBP12 was used in aconcentration that accelerated the non-enzymatic isomerizationof the peptide Suc-Ala-Phe-Pro-Phe-4NA threefold (three accelera-tion units). FK506 was kindly provided by Fujisawa GmbH(Munchen, Germany). By addition of FK506, this acceleration wasinhibited to one acceleration unit. If (1–339)AtFKBP42 boundto FK506, the inhibition efficiency of FK506 towards hFKBP12would have been reduced, indicated by an increase of accelerationunits.

Protease free assayTo overcome possible degradation of PPIase by proteases in theproteolytic assay, a protease-free test was performed as previously

described (Janowski et al., 1997). The disturbance of cis/transequilibrium is achieved by a solvent jump of the peptide fromLiCl/trifluorethanol to 35 mM Hepes buffer pH 7.8. Re-equilibration,which is accelerated by PPIases, is measured. AtFKBP42 was testedup to final concentrations of �1 mM protein.

Refolding of RCM-T1The refolding of RCM-T1 was measured as described (Schmidet 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 analysedby fluorescence spectroscopy. The refolding is limited to the cis/trans isomerization of the tyrosine38–proline39 peptide bond. Addi-tion of PPIases accelerates the refolding. AtFKBP42 was tested upto concentrations of 1.4 mM. The RCM-T1 concentration was173 mM.

Acknowledgements

We thank Dr Birte Hernandez Alvarez and Frank Edlich for readingthe manuscript critically, Dr Beate Saal for HA-TWD1 seeds, Joa-chim 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 Rucknageland Dr Angelika Schierhorn for NH2-terminal sequencing andmass spectrometry of the proteins. Part of this work were sup-ported by grants from the Deutsche Forschungsgemeinschaft, theEC (LATIN, BIOTEC 4), and the Ministerium fur Schule, Wis-senschaft und Forschung des Landes NRW to B.S.

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

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