Miriam Laxa

Janine König

Karl-Josef Dietz
email: karl-josef.dietz@uni-bielefeld.de

Andrea Kandlbinder

Role of the cysteinyl

Keywords: Arabidopsis thaliana, cyclophilin, chloroplast, immunophilin, prolyl-peptidyl cistrans isomerase, peroxiredoxin, redox regulation AMS: 4-acetoamido-4'-maleimidyl-stilbene-2, 2

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Introduction

Cyclophilins are members of the immunophilin protein superfamily. The Arabidopsis genome encodes at least 52 genes for immunophilins, immunophilin-like proteins and multidomain proteins with immunophilin domains [START_REF] He | Immunophilins and parvulins. Superfamily of peptidyl prolyl isomerases in Arabidopsis[END_REF]. Thus, plants possess the largest number of immunophilin family members as compared to other organisms with sequenced genomes at present [START_REF] Buchanan | Redox regulation in the chloroplast thylakoid lumen: a new frontier in photosynthesis research[END_REF]. Among these, 29 gene products belong to the cyclophilin (CYP) subfamily [START_REF] Romano | The Arabidopsis cyclophilin gene family[END_REF].

The functions of the plant CYPs mostly are unknown and unexplored. In mammals they were first identified as targets of the immunosuppressive drug cyclosporin A [START_REF] Handschumacher | Cyclophilin: A specific cytosolic binding protein for cyclosporin A[END_REF]. They constitute a family of phylogenetically old proteins occurring ubiquitously in bacteria, animals and plants [1; 3; 5] where they are known for their function in the isomerisation of Xaa-Pro-peptide bonds during the folding and assembly of proteins. Furthermore they act as chaperones [6; 7; 8; 9; 10; 11] and are involved in regulatory signal transduction pathways [2; 12; 13] during stress responses [14; 15; 16], plant development [17] and affect RNA splicing reactions [5; 18; 19]. Lee et al. observed the binding of human CypA to human peroxiredoxins Prdx I-VI by MALDI-TOF analysis and confirmed it via protein overlay assays and subsequent Western immunoblot analysis [20]. Recently it was shown in a DNA protection assay that a cyclophilin from Pisum sativum is able to reduce oxidised 2-Cys Prx and regenerates the peroxide detoxifying activity [21]. The reaction mechanism is still unclear.

Plant peroxiredoxins are non-heme containing peroxidases [22; 23]. They detoxify hydrogen peroxide, alkyl hydroperoxides and peroxynitrite [24; 25; 26]. The active centre contains a conserved cysteinyl residue, which reduces different peroxides. Its regeneration is coupled to electron donors such as thioredoxins, glutaredoxins and cyclophilins [21; 26]. Prxs are grouped into four clans, namely 1-Cys Prx, 2-Cys Prx, type II-Prx und PrxQ, according to sequence similarities, presence of an additional resolving cysteinyl group at variable positions, as well as the mechanisms of catalysis and regeneration [23; 25]. The Arabidopsis thaliana genome encodes 10 different Prxs with distinct subcellular localisation, tissue distribution, transcriptional regulation and biochemical properties [22; 27]. Prxs play a role in the antioxidative protection system of photosynthesis, respiration and stress response. analyses, a role in protein unfolding and degradation has been proposed under stress conditions such as heat shock [START_REF] Romano | The Arabidopsis cyclophilin gene family[END_REF]. Additionally it was identified as a target protein for chloroplast 31]. CYP20-3 contains four cysteinyl residues, which form two disulfide bonds under oxidising conditions, namely Cys 54 -Cys 171 and Cys 129 -Cys 176 , indicating redox-dependent conformational changes shown by 5,5'-dithio-bis(2-nitrobenzoic acid) binding and MALDI-TOF mass spectrometry [31].

This study aims at improving our understanding of the recently found redox properties of CYP20-3 and its interaction with the different chloroplast located peroxiredoxins. To this end, each Cys-residue of CYP20-3 was mutagenised and the variants were heterologously expressed in E. coli. His-tagged variants as well as the wild type protein were analysed for peptidyl-prolyl cis-trans isomerase (PPI) activity, redox-dependent electrophoretic mobility using band shift-experiments as well as for their redox-dependent conformational changes by intrinsic Trp fluorescence analysis. The midpoint redox potential of wild type and variant proteins was determined to place CYP20-3 into the redox system of the plastids. Furthermore, the disulfide reducing property of CYP20-3 was analysed in DNA-protection assays with all four chloroplast located peroxiredoxins.

Material and Methods

RNA Isolation and cDNA Synthesis. Total RNA was isolated from rosette leaves of

Arabidopsis thaliana (ecotype Col-0) plants grown in soil culture (1:1:1 mixture of Frühsdorfer Erde Klocke P, perlite and vermiculite) in a controlled environment (10 h of light, 100 µmol quanta m -2 s -1 , 23°C and 14 h darkness at 18°C; 50% relative humidity) for 28-32 days. The plant material was frozen in liquid nitrogen and ground to a fine powder. Total RNA was extracted using an RNA-isolation kit (Promega, Mannheim, D) according to the manufacturer's instructions. Following DNase I-treatment, cDNA was synthesised from 5 µg of total RNA with M-MLV RT II [H-] (Promega, Mannheim, D) and oligo-dT-priming in 30 µl reactions.

Heterologous Expression and Site-Directed Mutagenesis of CYP20-3. CYP20-3 was

amplified from Arabidopsis cDNA without the N-terminal sequence encoding the signal peptide, using Pfu (Pyrococcus furiosus) polymerase (Stratagene, La Jolla, CA, USA). Sitedirected mutations were introduced with two subsequent PCRs according to Montemartini et al. [32]. The primers used for mutagenesis are listed in the supplementary table S1. CYP20-3 cDNA was cloned into the pCR ® T7/NT-Topo ® vector and transformed into BL21(DE3)pLysS cells (Invitrogen, La Jolla, CA, U) for heterologous expression of wild type and variants of CYP20-3 as His-tagged fusion proteins. The mutations were verified by sequencing.

Culture Growth and Purification of His-tagged Proteins. 2 l LB medium containing 100 µg/ml ampicillin was inoculated with 150 ml of a non-induced overnight bacteria culture and incubated at 37 °C until an optical density of 0.6 -0.8 was reached at 600 nm using a spectrophotometer (Uvikon 930, Kontron-Instruments). Expression of recombinant protein was induced by addition of isopropyl-1-thio-β-D-galactopyranoside to a final concentration of 0.4 mM. After 4 h, the cells were harvested by centrifugation (4000 x g, for 30 min at 4°C) and the cell pellet was stored overnight at -20 °C. Heterologously expressed proteins [CYP20-3 WT , CYP20-3 C54S , CYP20-3 C129S , CYP20-3 C171S and CYP20-3 C176 and CYP20-3 ∆PPI ] were purified as described previously [33] either under reducing (10 mM β-mercaptoethanol in the lysis buffer) or non-reducing (no reducing reagent) conditions as indicated. The protein concentrations were determined spectrophotometrically using the Bio-Rad protein assay (BioRad, München D) using bovine serum albumine as a tentative reference. The protein was stored at -80° C. E. coli Trx was overexpressed and purified as described by Yamamoto et al.

[34], 2-Cys PrxA, 2-Cys PrxB, PrxII E and PrxQ according to Horling et al. [22] Excitation and emission spectra were obtained using a spectrofluorimeter (SFM 25, Kontron-Instruments). Trp fluorescence was excited at a wavelength of 290 nm and the emission spectrum was recorded as relative fluorescence over a range from 300 -400 nm. At least 9 independent measurements were carried out for each sample. Data were fitted with the software program FindGraph using an assymetric Gauss equation.

Peptidyl-Prolyl cis-trans Isomerisation Assay.

The PPI assay was performed as described previously [36] with minor modifications. A 1-ml assay consisted of 35 mM Hepes buffer (pH 8.0), 250 µg α-chymotrypsin (Sigma), 75 nM Cyp, 100 µM N-succinyl-Ala-Ala-Pro-Phep-nitroanilide (Sigma, Deissenhofen D) as chromogenic substrate (10 mM stock solution was dissolved in 100% methanol) and either 10 mM DTT (AppliChem, Darmstadt D) or 2.5 mM H 2 O 2 (Roth). All components of the assay mixture except the α-chymotrypsin were combined and preincubated at 10°C for 10 min. α-Chymotrypsin was added and quickly mixed to initiate the reaction. The absorbance was read every 6 s over a period of 5 min at 390 nm using a spectrophotometer (Uvikon 930, Kontron-Instruments). Data were fitted to a firstorder rate equation (A 360 = A 1 + A 0 e -kt , with k as rate constant) and rate constants (k obs ) derived as described by Motohashi et al. [31]. The kcat/Km values were calculated according to the

equation kcat/Km = (k obs -k 0 )/[PPIase],
where k 0 is the first-order rate constant for spontaneous cis-trans isomerisation [37]. Nicking and fragmentation of supercoiled plasmid DNA were monitored by gel electrophoresis in a 1% agarose gel.

Oxidation-Reduction Midpoint

Results

Generation of CYP20-3 WT and Variants. Site-directed mutations were introduced in the primary aa sequence to investigate the functional significance of the Cys residues in CYP20-3. Overexpressed N-terminally His-tagged CYP20-3 WT and variants were isolated and purified by Ni-NTA chromatography with yields of 0.25 to 2 mg/l E. coli culture. In nonreducing SDS-PAGE separations, CYP20-3 WT and all variants with the exception of CYP20-3 C54S were detected as reduced monomers after purification under reducing conditions (Figure 1A-E, 1 st lane). CYP20-3 C176S revealed a doublet band with a predominant form of lower electrophoretic mobility indicating a partially abnormal conformation, while in wild type protein, the faster migrating form was abundant. Following purification under non-reducing conditions, CYP20-3 C176S was detected as a dimer (Figure 1F).

Redox-Dependent Electrophoretic Mobility of the SDS-Solubilized Proteins under Oxidising

and Reducing Conditions. As reported by Motohashi et al. [31] two disulfide bonds (Cys 54 -Cys 171 and Cys 129 -Cys 176 ) can be formed in CYP20-3 WT that are sensitive to the redox milieu as shown by AMS-labelled protein mobility on SDS-PAGE. Thiol/disulfide transitions, e.g. by generating a more compact structure through an intramolecular disulfide bridge formation, often alter apparent electrophoretic mobilities of proteins. Therefore, the CYP20-3 WT and the diverse variants were checked under oxidising and reducing conditions. In the presence of H 2 O 2 CYP20-3 WT showed a higher mobility than after treatment with DTT (Figure 1A). Interestingly, the CYP20-3 C176S protein showed aberrant mobility compared to the wild type and all other Cys→Ser variants. The observed difference in electrophoretic mobility between both monomeric forms was considerably larger than that between the reduced and oxidised forms of the wild type protein. CYP20-3 C176S was partially oxidised after purification, separated as doublet band and showed no further conformational change upon addition of hydrogen peroxide (Figure 1E). The purification under non-reducing conditions of CYP20-3 C176S showed a dimeric form of this variant that converted to the doublet monomeric form upon DTT treatment (Figure 1F). CYP20-3 C54S existed in an oxidised and reduced form, but the presence of the oxidised form after purification under reducing conditions indicates that the reduced form is not stable and highly sensitive to oxidation (Figure 1B). The disulfide bond between Cys 129 and Cys 176 forms spontaneously.

The deletion of either Cys129 or Cys176 that form a disulfide bond in the oxidised protein [31] caused no conformational changes if purified under reducing conditions. Compared to the wild type protein CYP20-3 C171S is also less sensitive towards hydrogen peroxide, suggesting that no stable disulfide bridge is formed as Cys 54 sterically inhibits the spontaneous formation of the disulfide bond between Cys 129 and 176 (see below, Figure 6A). Formation of a disulfide bond between the adjoining Cys 54 and Cys 176 in the tertiary structure can be excluded. On the contrary, CYP20-3 C129S and CYP20-3 C171S were detected mostly in the reduced form and their mobility was not altered under oxidising conditions (Figure 1C,D). This observation indicates that major intramolecular changes associated with the transition from the reduced to the oxidised molecule were absent and probably no disulfide bonds were formed in these variants (Figure 1C,D). Apparently, both disulfide bonds are necessary to stabilise the conformation of the oxidised CYP20-3 molecule. These results support the hypothesis that the transition from the reduced to the oxidised form of CYP20-3 as well as the stability of the oxidised form depend on the formation of both disulfide bonds. Furthermore, the two disulfide bonds appear to be formed in a coordinated and ordered way.

Intrinsic Fluorescence Reveals Conformational Changes during the Transition from the

Reduced to the Oxidised State. Analysis of the intrinsic fluorescence offers a powerful approach for studying conformational changes in proteins. The aromatic amino acid tryptophan is rather specifically excited at a wavelength of 290 nm [39]. [START_REF] Burstein | Fluorescence and the location of tryptophan residues in protein molecules[END_REF] [ Emission spectra of CYP20-3 WT and its Cys-variants were compared under oxidising and reducing conditions. For CYP20-3 WT a blue shift of λ em-max of 1.6 nm occurred during transition from reducing to oxidising conditions indicating that Trp 135 is more shielded from the solvent in the presence of H 2 O 2 (Figure 2A, Table 1). In the presence of DTT the maximum Trp 135 -fluorescence was blue-shifted in all Cys→Ser variants compared to the wild type with exception of the CYP20-3 C129S , whereas in the presence of H 2 O 2 the fluorescence maximum was either blue-shifted in CYP20-3 C54S and CYP20-3 C176S or red-shifted in CYP20-3 C129S and CYP20-3 C171S (Figure 2, Table 1). Interestingly, the fluorescence emission spectra of the variants mutated in the interacting disulfide bond partners were similar (Figure 2). While λ em-max of CYP20-3 C54S and CYP20-3 C171S was red-shifted by 2.7 and 2.8 nm, respectively, upon oxidation only a slight shift in maximum fluorescence λ em-max was observed for CYP20-3 C129S as well as CYP20-3 C176S (Figure 2, Table 1). This indicates either an The Significance of the Cysteinyl Residues for the Isomerisation Activity of CYP20-3. CYP20-3 promotes the isomerisation of peptidyl prolyl residues from the cisto the trans-form. To investigate the requirement of the conserved cysteinyl residues for the isomerisation activity, wild type CYP20-3 and the different Cys variants were analysed for PPI activity. The catalytic efficiency as expressed by kcat/Km-ratio is derived from the slope of the concentration-dependent k obs values of the corresponding protein. They were determined under reducing and oxidising conditions, respectively (Figure 3, Table 2). The kcat/Km-ratio was 4.23 x 10 5 M -1 s -1 for the reduced form and 1.12 x 10 5 M -1 s -1 for the oxidised form of wild 2) confirming previous reports of oxidation-induced inactivation [31]. Elimination of three out of the four cysteinyl residues decreased the maximum catalytic PPI activity of the reduced CYP20-3. Activity remained unaltered in the case of CYP20-3 C176S (CYP20-3 C176S : kcat/Km = 3.97 x 10 5 M -1 s -1 ) (Figure 3, Table 2). Under reducing conditions the mutation of Cys 54 , Cys 129 and Cys 171 into serine decreased the PPI activity by 60 %, 51 % and 75 % compared to the wild type, respectively. The isomerisation activities of the oxidised forms of CYP20-3 C54S , CYP20-3 C129S and CYP20-3 C171S showed no significant differences compared to CYP20-3 WT , whereas a significantly higher activity was observed for CYP20-3 C171S (Figure 3, Table 2). Moreover, simultaneous mutation of Arg 69 and Phe 74 , which are essential amino acids for PPI activity and part of the so called RWFHmotif [START_REF] Romano | The Arabidopsis cyclophilin gene family[END_REF], into Ala 69 and Leu 74 respectively, abolished the PPI activity (data not shown).

The Midpoint Redox Potential of CYP20-3 WT is -319 mV. The midpoint redox potential of CYP20-3 WT and the variants was determined using a fluorimetric test. The proteins were incubated in redox buffers adjusted with defined ratios of oxidised to reduced DTT, followed by labelling with excess monobromobimane (Figure 4). The wild type CYP20-3 midpoint redox potential was determined as -319 ± 2mV. The midpoint redox potentials of the Cys mutants were more negative compared to the wild type, but only CYP20-3 C176S showed a significant decrease (CYP20-3 C54S : -320 ± 4 mV, CYP20-3 C129S : -326 ± 6 mV, CYP20-3 C171S :

-322 ± 4 mV and CYP20-3 C176S : -327 ± 3 mV; Figure 4).

The Cysteinyl Residues 129 and 171 of CYP20-3 are Required for the Regeneration of 2-Cys

PrxA and 2-Cys PrxB. Recently, Bernier-Villamor et al. [21] showed that the CYP20-3 homologue from Pisum sativum is able to enhance the protective activity of 2-Cys Prx in a DNA protection assay. The chloroplasts of Arabidopsis thaliana contain four peroxiredoxins, namely 2-Cys PrxA and B, PrxQ and PrxII E [41]. To address the question whether Arabidopsis CYP20-3 is able to specifically reduce Prx the DNA protection assay in the presence of DTT/Fe 3+ was employed with different combinations of CYP20-3 and Prx. In a first set of experiments CYP20-3 WT and the different CYP20-3 variants were tested for their ability to protect the plasmid-DNA either directly or by interaction with Prx. Neither reduced nor oxidized CYP20-3 WT was able to protect the plasmid DNA (Figure 5A). Likewise, the CYP20-3 variants also showed no protection of DNA (Figure 5B). In a converse manner, CYP20-3 WT increased the protective activity in the presence of 2-Cys PrxA (Figure 5C) and 2-Cys PrxB (Figure 5D). However, no reducing interaction was observed between CYP20-3 WT and PrxQ (Figure 5E) or PrxII E (Figure 5F). To prove the functionality of the assay, the DNA protection was tested with known regenerators of PrxQ and 2-Cys PrxB. The results confirmed that 2-Cys Prx is regenerated by E. coli Trx [33], and PrxQ by the A. thaliana Trx y 2 [35], respectively, whereas no regenerator for PrxII E was found yet (Figure 5). On a comparative basis, the regeneration of 2-Cys PrxB was more efficient with Trx than with CYP20-3 WT (Figure 5D). Until now, the mechanism of the CYP20-3/2-Cys Prx interaction is still not clarified. To identify the cysteines responsible for the reduction of 2-Cys PrxA and 2-Cys PrxB, the different CYP20-3 variants were tested. The addition of the variants CYP20-3 C54S and CYP20-3 C176S enhanced the fraction of supercoiled DNA and thus showed a better protective activity than the wild type. The other two cysteinyl variants had a similar protective pattern compared to the wild type (Figure 5C,5D). In conclusion the cysteinyl residues Cys 129 and Cys 171 are most important for the interaction with 2-Cys PrxA and B. Interestingly, the CYP20-3 ∆PPI variant showed a greater reducing activity than the wild type protein indicated by the lowest level of plasmid-DNA cleavage (Figure 5C,D).

Discussion

CYP20-3 Undergoes Major Conformational Changes in Dependence on Redox State. CYP20-

3 WT shows altered electrophoretic mobility during the transition from the reduced to the oxidised form when separated under non-reducing conditions in the SDS-PAGE (Figure 1A). Motohashi et al. [31] have shown by 5,5'-dithio-bis(2-nitrobenzoic acid) labelling and MALDI-TOF mass spectrometry that oxidised CYP20-3 contains two redox-sensitive disulfide bonds, namely Cys 54 -Cys 171 and Cys 129 -Cys 176 , respectively. The formation of the disulfide bonds Cys 54 -Cys 171 and Cys 129 -Cys 176 [31] and the two observed redox-states of the CYP20-3 WT protein (Figure 1A) indicate redox-dependent conformational changes as a basic mechanism for regulating CYP20-3 function. The electrophoretic mobility assays and the intrinsic fluorescence determinations (Figure 2, Table 1) revealed that the transition from the reduced to the oxidised state of CYP20-3 as well as the stability of the oxidised form depend on the formation of both disulfide bonds. In addition, the results from intrinsic fluorescence analysis (Figure 2 [31] since the corresponding variants revealed identical alterations.

Cysteinyl Residues Affect CYP20-3 Isomerisation Activity. CYP20-3 promotes the isomerisation of prolyl bonds from the cisto the trans-form. This study provides answers to two redox-related questions, namely: (i) does the isomerisation activity depend on the thiol redox state, and (ii) are all cysteinyl residues equally important in respect to the PPI activity.

In accordance with previous reports [31] the isomerisation activity of CYP20-3 WT was modulated by the redox milieu being less active under oxidising conditions when disulfide bonds between Cys 54 and Cys 171 as well as Cys 129 and Cys 176 are formed (Figure 3, Table 2).

The kcat/Km-value for reduced CYP20-3 WT (4.23 x 10 5 M -1 s -1 ) was 6-to 40-fold lower than previously reported for CYP20-3 WT of A. thaliana (8.2 x 10 6 M -1 s -1 ; [31]), CeCyp3 (kcat/Km: 2.4 x 10 6 M -1 s -1 ; [42]) or human HsCypA (1.4 x 10 7 M -1 s -1 ; [37]. All mutations caused changes regarding the isomerisation activity under reducing and oxidising conditions compared to CYP20-3 WT . This implies alterations in the chemical vicinity of the RWFH-motif in the variants. The low catalytic efficiency observed in the apparently reduced CYP20-3 C171S variant (Figures 1D and3D, Table 2) supports the conclusion that Cys 171 is most important to maintain maximum PPI activity. The wild type-like catalytic activity of reduced CYP20-3 C176S on the other hand indicates a minor role in PPI activity, but a major role in oxidative inactivation since CYP20-3 C176S maintained a very high catalytic efficiency. The thioldependency of CYP20-3 PPI activity contrasts the insensitivity of human CypA (HsCypA) PPI to mutation of the cysteinyl residues 52, 62, 115 and 161 [37] although two of the four Cys-residues of HsCypA are located within the PPI-motif. Interestingly only Cys 129 and Cys 176 are conserved among plant and human Cyp, whereas Cys 54 and Cys 171 in the plant protein is absent from the human protein (Fig. 6B). Accordingly, the disulfide bridge between Cys 54 and Cys 171 is essential for the redox control of the PPI activity.

Importance of the Disufide Bonds. The midpoint redox potential of CYP20-3 WT was determined to be -319 ± 2 mV (Figure 4, Table 3). The midpoint redox potentials of the variants were similar to that of the wild type. It may be postulated that the oxidation of one disulfide bond alters the redox potential of the second disulfide bond causing a easier/faster oxidation. However, it was impossible to determine the midpoint redox potentials for each disulfide bond independently. The variants CYP20-3 C54S (320 mV) and CYP20-3 C171S (322 mV) have a slightly less negative redox potential than the other two variants (CYP20-3 C129S :

Copyright 2006 Biochemical Society

Arabidopsis thaliana cyclophilin CYP20-3 13 13 326 mV and CYP20-3 C176S : 326 mV). In the variants CYP20-3 C54S and CYP20-3 C171S the midpoint redox potential of the disulfide bridge between Cys 129 and Cys 176 is less negative indicating an easier reduction. In a converse manner, the disulfide bridge between Cys 54 and Cys 171 is more easily formed than the disulfide bridge between Cys 129 and Cys 176 . This interpretation is still speculative in the light of the similarity and the standard deviation of the determined redox midpoint potentials. But this hypothesis fits into the emerging picture that the disulfide bond between Cys 54 and Cys 171 is the more important one for the redox properties of functional CYP20-3. These two cysteinyl residues are conserved in other Arabidopsis cyclophilins. It will have to be tested whether these proteins also are regulated by thiol/disulfide transition. On the other hand, amino acids corresponding to Cys 176 lack in all other members of the Arabidopsis cyclophilin protein family [START_REF] Romano | The Arabidopsis cyclophilin gene family[END_REF]. CYP20-3 as Regenerator of Chloroplast Peroxiredoxins. With -319 was the midpoint redox potential of CYP20-3 WT more negative than that of 2-Cys Prx A with an Em of -307 mV [33].

Reductive activation of CYP20-3 is likely to occur under conditions of excess electron pressure in photosynthesis. In accordance to other publications, in vitro experiments revealed Trx-m as an interaction partner of CYP20-3: Trx-m with an Em of -300 mV is able to reduce CYP20-3 and to activate its PPI activity [30; 31; 43]. Recently, Lee et al. [20] reported that HsCypA activates human peroxiredoxins and Bernier-Villamor et al. [21] showed that a CYP20-3-homologue from Pisum sativum supports the peroxide-detoxifying activity of plant 2-Cys Prx. The midpoint redox potentials of chloroplast Prxs range between -290 mV for PrxII E, -307 mV for 2-Cys Prx A, -322 mV for 2-Cys Prx B and -325 mV for PrxQ [22; 44]. Thus based on redox potentials, all chloroplast Prxs are potential targets for CYP20-3, considering that an efficient reduction of the regulatory disulfides still can occur in the presence of a ∆Em of up to 20 mV [44]. The DNA-protection assay manifested the ability of CYP20-3 to reduce 2-Cys PrxA and 2-Cys PrxB (Figure 5) similar to E. coli Trx [33] and Arabidopsis . In a converse manner the combination of PrxII E and PrxQ, respectively, in combination with CYP20-3 gave no protection of plasmid-DNA in the DNA-cleavage assay (Figure 5E,F). It is concluded that CYP20-3 is unable to efficiently reduce PrxQ and PrxII E. This suggests that 2-Cys PrxA and 2-Cys PrxB are specific interaction partners of CYP20-3 at least in vitro.

The redox-dependent properties of cyclophilins may also be relevant in other organisms.

Caenorhabditis elegans Cyp3 (CeCyp3) that shows a similarity at the protein primary Copyright 2006 Biochemical Society sequence level of 63% to CYP20-3 possesses four cysteine residues at conserved positions homologous to the four in CYP20-3 suggesting similar function (Figure 6B) [42]. The authors proposed that CeCyp3 might play a role in a signalling response to oxidative stress.

The DNA-cleavage assay proved Prx-reducing activity of all four variants, but the particular importance of Cys 129 and Cys 171 in the reduction of 2-Cys PrxA and 2-Cys PrxB (Figure 5C, D). Lee et al. [20] found Cys 115 and Cys 161 of human CypA corresponding to Cys 129 and Cys 176 of CYP20-3 (Figure 6B) to be important for the reduction of human Prx II using the glutamine synthetase protection assay. Taking together the results of the PPI activity measurements (Figure 3) and the DNA-protection assay (Figure 5) suggest that Cys 171 is involved in both redox-related functions. The CYP20-3 ∆PPI variant showed an increased reducing activity towards 2-Cys PrxA and 2-Cys PrxB (Figure 5C, D) implicating that Cys 129 or Cys 171 is better accessible for oxidised 2-Cys PrxA and 2-Cys PrxB after changing the thiols in close vicinity of the active centre. As the CYP20-3 ∆PPI variant showed no isomerisation activity (data not shown), but greater reducing activity, it can be concluded that both functions of CYP20-3, i.e. the catalytic activity as PPIase and the function as reductant, are independent from each other. Nevertheless both functions are regulated in dependence on the redox status of the active centre of CYP20-3. The best-known mechanism to modify cyclophilin activity is the binding of cyclosporin A (CsA), an immunosuppressive drug that specifically inhibits the cyclophilin PPIase activity [START_REF] Handschumacher | Cyclophilin: A specific cytosolic binding protein for cyclosporin A[END_REF]. Subsequently, the CsA-Cyp complex binds to subunit B of the Ca 2+ /calmodulin-dependent Ser-/Thr-phosphatase calcineurin leading to its inactivation and, finally, resulting in altered gene transcription in the nucleus [45; 46]. Thus, the electron donating property of CYP20-3 to 2-Cys Prx as observed in the DNA-protection assay not necessarily indicates a major role of CYP20-3 in the antioxidant defence of the chloroplast but a role in the redox signalling network adjusting the redox state and thereby the activity of CYP20-3. In this scenario, the 2-Cys Prx serves as peroxide sensor transmitting redox information to CYP20-3 that in turn modulates target protein functions [START_REF] Dietz | Redox regulation, redox signalling and redox homeostasis in plant cells[END_REF]. It will be necessary to identify other interacting partners and thus regulatory targets of CYP20-3. Furthermore, the analysis of Arabidopsis lines lacking or overexpressing CYP20-3 protein will possibly allow clarifying its physiological role within the plant redox network in the adaptation process to stress. nm (n ≥ 9 for each protein). Data were fitted with the software program FindGraph. The wavelengths of the emission maxima (λ em-max ) are given in Table 1. Table 1: Emission maximum wavelengths (λ em-max ) for CYP20-3 WT and the Cys→ Ser variants under reducing and oxidising conditions. Data were fitted by an asymmetric Gaussian equation using the software program FindGraph and analysed for the wavelength of maximum emission. Also given are the calculated differences between the maximum wavelengths of variants minus wildtype under reducing (column 3) and oxidising conditions (column 5) and the difference between oxidising and reducing conditions (column 6).

Reduced

Oxidised Difference

λ

  Additional functions are proposed in signal transduction during plant development and adaptation [25; 24; 27]. The 28.2 kDa cyclophilin CYP20-3 (at3g62030; Roc4) has been characterised previously. It is located in the stroma of chloroplasts [28; 29; 3] and the four conserved amino acids Arg 69 , Phe 74 , Trp 135 , His 140 (R-F-W-H-motive) are involved in peptidyl-prolyl-isomerase activity and Biochemical Journal Immediate Publication. Published on 24 Aug 2006 as manuscript BJ20061092 Copyright 2006 Biochemical Society 4 cyclosporin A-binding (Trp) implicating a function in protein folding. From transcript

  Potential. Recombinant CYP20-3 was titrated at pH 7.0. After 3 h incubation in MOPS buffer (100 mM) containing 2 mM total DTT (in different ratios of reduced and oxidised DTT) at ambient temperature, the reduced fraction of Cyp was labelled with monobromobimane (10 mM) and analysed for fluorescence [38]. Data were fitted with the software program GraFit5 using the Nernst equation with n = 4 (in case of Biochemical Journal Immediate Publication. Published on 24 Aug 2006 as manuscript BJ20061092 Copyright 2006 Biochemical Society 7 CYP20-3 WT ) and n = 3 (for each mutant protein as one cysteine was missing) based on a value of -330 mV for the Em of DTT at pH 7.0. DNA-Protection Assay. CYP20-3-dependent reduction of recombinant chloroplastidic Prx was tested in a DNA-cleavage assay as described previously [21] with minor modifications. ROS production was induced by incubation of 3 µM FeCl 3 in the presence of 10 mM DTT for 3 h at 37 °C. Supercoiled plasmid DNA (pCR ® T7/NT-Topo ® vector; 2 µg) was added and incubated for 5 h at 37 °C. The reaction contained 6 µg Prx protein and 3 µg CYP20-3 or Trx protein as indicated. The Arabidopsis peroxiredoxin proteins, 2-Cys PrxA and 2-Cys PrxB, PrxII E and PrxQ [22] were preincubated with 5 mM DTT for 5 min followed by incubation with 20 mM H 2 O 2 for 1 h, CYP20-3 WT with either 10 mM DTT or 10 mM H 2 O 2 for 1 h. The proteins were dialysed overnight against 40 mM potassium-phosphate buffer (pH 7.0).

  40] introduced two different classes of emission spectra equivalent to different Trp locations within the protein structure. The class I-Trp is characterised by λ em-max =330-332 nm Biochemical Journal Immediate Publication. Published on 24 Aug 2006 as manuscript BJ20061092 Copyright 2006 Biochemical Society Arabidopsis thaliana cyclophilin CYP20-3 9 9 corresponding to a location in the interior of the protein, being shielded from the solvent, whereas class II-Trp residues are exposed to the solvent at the protein surface with λ em- max =340-342 nm. CYP20-3 contains one Trp residue (Trp 135 ) which is part of the R-W-F-Hmotif and located close to the surface of the protein in the vicinity of Cys 129 (Figure 6A, B).

  increased exposure of Trp 135 to the solvent or a stimulated quenching effect of Cys 129 in the oxidised form. In case of CYP20-3 C129S only a slight shift in the fluorescence maximum by λ em-max = 344.1 nm (red) and λ em-max = 344.9 nm (ox) was observed indicating a solventexposure of the Trp[40]. The evidence that Cys 129 is located in the vicinity of Trp 135 (see below: Figure6A, B) together with the observation of the pronounced red shift of λ em-max compared to the wild type under reducing and oxidising conditions suggests a strong quenching effect of Cys 129 on Trp 135 -fluorescence. In conclusion a major conformational change takes place during the transition from the reduced to the oxidised state, whereby the formation of the disulfide bond between Cys54 and Cys171 is likely to play a significant role.

Figure 1 :

 1 Figure 1: Protein mobility shift of the recombinant CYP20-3 WT and Cys→ Ser variants. 0.5 µg Cyp was incubated with various concentrations of either H 2 O 2 or DTT at RT for 15 min. All samples were analysed by SDS-16 % PAGE separation and subsequent silver staining (A-F).

Figure 2 :

 2 Figure 2: Intrinsic Trp fluorescence emission spectra of CYP20-3 WT and the Cys → Ser variants as a function of thiol redox state. Trp fluorescence of the proteins was measured under reducing (black line) and oxidising (grey line) conditions after excitation at λ ex = 290

Figure 3 :

 3 Figure 3: Observed velocity, k obs , with different concentrations of wild type CYP20-3 and the Cys → Ser variants under reducing or oxidising conditions. The PPI activity was assayed using N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Variation of the amount of enzyme led to a family of curves and generated the linear correlation of k obs versus protein concentrations. As described in ´Material and Methods`, the slope of each line equals kcat/Km for the corresponding protein either under reducing or oxidising conditions as listed in Table 2. Assay (A) wild type CYP20-3, (B) CYP20-3 C54S , (C) CYP20-3 C129S , (D) CYP20-3 C171S and (E) CYP20-3 C176S (n ≥ 3 ± SD). Symbols, regression lines and error bars represent reducing and oxidising conditions as follows: ο, ____ and black error bars: 10 mM DTT; ■, --and grey error bars: 10 mM H 2 O 2 (n ≥ 3 ± SD).

Figure 4 :

 4 Figure 4: Titration of the midpoint redox potential of CYP20-3 WT and the Cys → Ser variants. The redox potential of the samples was adjusted by varying the ratio of DTT oxidised /DTT reduced . Reduced thiol groups were labelled with monobromobimane and the samples analysed for bound fluorophore. Experimental data were fitted to the Nernst equation with n = 4 (wild type) or n = 3 (variants) using GraFit5. Midpoint redox potentials (Em; means ± SD of two or more experiments for each protein) are indicated in the figure.

Figure 5 :

 5 Figure 5: DNA protection assay as indicator of CYP20-3-dependent reduction of the chloroplastic peroxiredoxins. In each experiment (1) represents the control without 3 µM FeCl 3 /10 mM DTT and (2) the maximum cleavage of the plasmid DNA achieved in that

Figure 6 : 3 (

 63 Figure 6: (A) Localisation of the cysteinyl residues 54, 129, 171 and 176 as well as Trp 135 in the tertiary structure and in comparison to the active centre represented by the R-F-W-H-motif of the CYP20-3 molecule. The Cys54 and 176 are arranged very close together in the structure of CYP20-3 and are part of the surface of the molecule, therefore, possibly being accessible to reactive oxygen species (ROS). The Cys129 and 171 are located on the opposite site of the molecule compared to Cys54 and 176. The model was calculated based on known structures of human CypB and Cyp3 from C. elegans, respectively, using Deep View/Swiss-Pdb Viewer 3.7 (www.expasy.org/spdbw/) and further processed by using Rasmol. (B) Amino acid sequence alignment of the A. thaliana cyclophilin CYP20-3 (B53422, without the signal peptide of 77 amino acids), CypA from H. sapiens (P62937) and Cyp3 from C. elegans (NP_506751). The conserved cysteinyl residues are shaded and the catalytic motif of a typical PPI is underlined.

Figure 1 :

 1 Figure 1:

Biochemical Journal Immediate Publication. Published on 24 Aug 2006 as manuscript BJ20061092 Copyright 2006 Biochemical Society

Maslow Biochemical Journal Immediate Publication. Published on 24 Aug 2006 as manuscript BJ20061092 Copyright 2006 Biochemical Society

  . Trx y1 was kindly provided by Dr. Emmanuelle Issakidis-Bourguet and Dr. Myroslawa Miginiac-

	Arabidopsis thaliana cyclophilin CYP20-3	6
	[35].
	Electrophoretic mobility. A typical assay (50 µl) consisted of 40 mM potassium-phosphate
	buffer (pH 7.0), 1.25 µg Cyp protein, various concentrations of H 2 O 2 and DTT (1-10 mM).
	Following incubation for 15 min at room temperature, the samples were supplemented with
	equal volumes of 3x-concentrated non-reducing loading buffer (375 mM Tris-HCl, pH 6.8, 30
	% glycerin, 12 % SDS, 0.03 % bromophenol blue), and 20 µl aliquots each separated on a
	SDS-16% PAGE. Proteins were detected by silver staining.
		6

Excitation and Emission Spectrum of Trp Fluorescence. 2

	µM Cyp protein dissolved in
	phosphate-buffered saline pH 7.0 (137.0 mM NaCl, 18.8 mM Na 2 HPO 4 ; 2.7 mM KCl; 2.0
	mM KH 2 PO 4 ) was incubated either with 10 mM DTT or 20 mM H 2 O 2 for 5 min at RT.

Biochemical Journal Immediate Publication. Published on 24 Aug 2006 as manuscript BJ20061092 Copyright 2006 Biochemical Society

	Arabidopsis thaliana cyclophilin CYP20-3	8
		8

Biochemical Journal Immediate Publication. Published on 24 Aug 2006 as manuscript BJ20061092 Copyright 2006 Biochemical Society 12

  , Table1) assign a predominant role in conformational dynamics to the disulfide bridge formation between Cys 54 and Cys 171 . The intrinsic fluorescence analysis also confirms that Cys 54 and Cys 171 , and Cys 129 and Cys 176 cooperate in disulfide bond formation

Biochemical Journal Immediate Publication. Published on 24 Aug 2006 as manuscript BJ20061092

Biochemical Journal Immediate Publication. Published on 24 Aug 2006 as manuscript BJ20061092

Biochemical Journal Immediate Publication. Published on 24 Aug 2006 as manuscript BJ20061092 Copyright 2006 Biochemical Society

			Arabidopsis thaliana cyclophilin CYP20-3	23
			Laxa et al., 2006
		em-max	shift	λ em-max	shift	oxidized-reduced
		[nm]	(variant-wt)	[nm]	(variant-wt)	[nm]
			[nm]		[nm]
	CYP20-3 WT	339.2	± 0.0	337.6	± 0.0	-1.6
	CYP20-3 C54S	333.7	-5.5	336.4	-1.2	+ 2.7
	CYP20-3 C129S	344.1	+ 4.9	344.9	+ 7.3	+ 0.8
	CYP20-3 C171S	335.2	-4.0	338.0	+ 0.4	+ 2.8
	CYP20-3 C176S	334.4	-4.8	334.1	-3.5	-0.3
						23

Table 2 : Kcat/Km-values [s -1 M -1 ] for the PPIs of CYP20-3 WT and the Cys→ Ser variants.

 2 Various concentrations of Cyp were incubated with either 10 mM DTT or 10 mM H 2 O 2 . Kcat/Km-values were determined as described in ´Materials and Methods´.

		Reduced		Oxidised
		Kcat/Km-values		Kcat/Km-values
		[s -1 M -1 ]	[%]	[s -1 M -1 ]	[%]
	CYP20-3 WT	4.23 x 10 5	100	1.12 x 10 5	100
	CYP20-3 C54S	1.71 x 10 5	40.4	1.38 x 10 5	123.2
	CYP20-3 C129S	2.06 x 10 5	48.7	0.94 x 10 5	83.9
	CYP20-3 C171S	1.06 x 10 5	25.1	0.97 x 10 5	86.6
	CYP20-3 C176S	3.97 x 10 5	93.9	2.59 x 10 5	231.3

Biochemical Journal Immediate Publication. Published on 24 Aug 2006 as manuscript BJ20061092 Copyright 2006 Biochemical Society Laxa et al., 2006

Acknowledgements:

Expert technical assistance by Heike Bogunovic is gratefully acknowledged. Trx y1 was kindly provided by Dr. Emmanuelle Issakidis-Bourguet and Dr. Myroslawa Miginiac-Maslow. We thank Dr. Andreas Brockhinke for helpful discussion. Research was supported by Bielefeld University within the FIF-initiative and by the Deutsche Forschungsgemeinschaft within the SFB 613 (D9), but also FOR 387 (TP3).

Laxa et al., 2006


Richard Thorogate

Dr Katalin Török

Dr N N Kasri

Thorogate and Török CaM nuclear binding

Keywords: pdfauthor=Richard Thorogate, Katalin Török, pdfkeywords=Life Sciences, pdfcreator=HAL, pdfproducer=P CaM, nucleus, nucleolus, chromatin, calcium

published or not. The documents may come

,

Introduction

Calmodulin (CaM), a ubiquitous Ca 2+ -binding protein belonging to the EF-hand homolog family, regulates a whole host of processes within the cytoplasm and nucleus of all eukaryotic cells [START_REF] Bachs | Calmodulin and calmodulin-binding proteins in the nucleus[END_REF][START_REF] Persechini | The EF-hand family of calcium modulated proteins[END_REF]. Within the nucleus, CaM has been found to be involved in RNA synthesis [START_REF] Pardo | Effect of calcium and calmodulin on RNA synthesis in isolated nuclei from rat liver cells[END_REF], DNA synthesis [START_REF] Wang | Targeted neutralization of calmodulin in the nucleus blocks DNA synthesis and cell cycle progression[END_REF][START_REF] King | Temporal inhibition of calmodulin in the nucleus[END_REF][START_REF] Subramanyam | Nuclear localization of 68 kDa calmodulin-binding protein is associated with the onset of DNA replication[END_REF][START_REF] Reddy | Growth factor modulated calmodulin-binding protein stimulates nuclear DNA synthesis in hemopoietic progenitor cells[END_REF] and the function of a number of nuclear enzymes and receptors including the estrogen receptor-α [START_REF] Bouhoute | Modulation of estradiol and DNA binding to estrogen receptor upon association with calmodulin[END_REF][START_REF] Li | The transcriptional activity of estrogen receptor-alpha is dependent on Ca 2+ /calmodulin[END_REF]. Ca 2+ /CaM dependent phosphorylation of three nuclear proteins of 50-60 kDa [START_REF] Maizels | Ca 2+ -calmodulin-dependent phosphorylation of soluble and nuclear proteins in the rat ovary[END_REF], CREB [START_REF] Sheng | CREB: a Ca 2+ -regulated transcription factor phosphorylated by calmodulin-dependent kinases[END_REF] and of rat liver heterogenous nuclear ribonucleoproteins hnRNP A2 and C has previously been reported [START_REF] Bosser | Phosphorylation of rat liver heterogeneous nuclear ribonucleoproteins A2 and C can be modulated by calmodulin[END_REF]. A structural role for CaM is demonstrated by the finding that elimination of the nuclear function of Ca 2+ /CaM causes disruption of nuclear structure [START_REF] Wang | Functional elimination of calmodulin within the nucleus by targeted expression of an inhibitor peptide[END_REF] and a nuclear scaffold protease activity is Ca 2+ /CaM modulated [START_REF] Madsen | Regulation of nuclear scaffold protease activity by calcium[END_REF]. Ca 2+ /CaM also regulates nuclear entry of numerous proteins involved in cell division and proliferation and has been shown to localize to the mitotic apparatus and be essential for mitotic transitions of early sea urchin embryos [START_REF] Török | Imaging the spatial dynamics of calmodulin activation during mitosis[END_REF].

A number of previous studies have shown that upon cell stimulation, CaM translocates from the cytoplasm into the nucleus. Indeed, nuclear translocation of CaM has been proposed to support CREB phosphorylation [START_REF] Deisseroth | Signaling from synapse to nucleus: postsynaptic CREB phosphorylation during multiple forms of hippocampal synaptic plasticity[END_REF][START_REF] Mermelstein | Calmodulin priming: nuclear translocation of a calmodulin complex and the memory of prior neuronal activity[END_REF] and morphine stimulation induced rapid CaM nuclear translocation that was associated with increased CREB phosphorylation [START_REF] Wang | Nuclear Ca 2+ /calmodulin translocation activated by mu-opioid (OP3) receptor[END_REF]. CaM nuclear accumulation was reported in response to corticotrophin stimulation of adrenal cortex cells suggesting a role for CaM in the regulation of hormone effects on nuclear processes [START_REF] Harper | Localization of calmodulin in rat tissues[END_REF]. Ca 2+ /CaM nuclear translocation was shown to require high frequency Ca 2+ signals, whereas, brief or low frequency signals did not change free Ca 2+ /CaM concentration in the nucleus [START_REF] Teruel | Differential codes for free Ca(2+)-calmodulin signals in nucleus and cytosol[END_REF]. It has, however, been shown that passive diffusion of CaM into the nucleus in low Ca 2+ is too slow to serve as a means of signal transduction and that upon Ca 2+ stimulation, CaM is transported into the nucleus by Ca 2+ -dependent facilitated transport [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF]. Further experiments showed an effect of CaM to increase the permeability of the nuclear pore complex [START_REF] Thorogate | Nuclear pore gating by calmodulin[END_REF]. lobe CaM fragment CaM 76-148 , pT7/7.CaM 1-80 , pT7/7.CaM 76-148 vectors were kindly provided by Professor M. A. Shea and Dr. B. Sorenson, University of Iowa, USA.

Protein expression was carried out in BL21-Gold cells (Stratagene)

Proteins and peptides

Protein purification was carried out as previously described for wild type CaM [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF]. CaM 1-80 and CaM 76-148 were expressed and purified as described for wild type CaM.

The concentration of wild type CaM and CaM1234 was measured at 278 nm (A 278 0.18 = 1 mg/mL CaM). CaM 1-80 and CaM 76-148 were measured by weight. mTrp peptide and its characterization was carried out as previously described [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF].

Fluorescent CaM, CaM 1-80 and CaM 76-148 fragments

CaM and CaM1234 were labeled with 6ROX as previously described [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF]. CaM 1- 80 and CaM 76-148 were labeled in the same conditions and purified by HPLC to homogeneity as previously described [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF]. Labeled protein was digested by trypsin for mass spectrometric analysis as previously described for wild type CaM labeled with 6ROX and DTAF from Molecular probes and TA-Cl [27]. Maldi-tof mass spectrometry was carried out as previously described [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF]. Fluorescently labeled tryptic peptides were sequenced by post-source decay analysis to identify the labeled residue. The CaM 1-80 fragment was determined to be labeled at Lys 75 and the CaM 76-148 fragment was modified by the α-amino group of Met 76 (data not shown). The fluorescence of FL-CaM 1-80 , 6-ROX-CaM 1-80 , TA-CaM 76-148 , FL-CaM 76-148 and 6ROX-CaM 76-148 did not significantly change upon Ca 2+ and mTrp peptide binding (data not shown). TA-CaM 1-80 fluorescence was, however, sensitive to both Ca 2+ and mTrp peptide binding as shown in Figure 7.

Cell culture and stimulation

Cell culture and electroporation were carried out as previously described [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF].

HeLa cells were electroporated with fluorescent CaM or CaM mutant in 2 mM EGTA.

2.1 mM Ca 2+ was added to the extracellular medium and cells were imaged for up to 30 minutes. Fluo-4 was administered as AM ester at 4 µM concentration and the cells were incubated at room temperature for 15 minutes before stimulation. mTrp peptide was added to the external medium and the cells were incubated for 15 minutes prior to stimulation. Fluorescein-labeled wheat germ agglutinin (FL-WGA, Sigma) was coelectroporated to an estimated final concentration of 20 µg/mL with fluorescent CaM derivatives to test for facilitated nuclear transport processes.

Confocal imaging and image analysis

Cells were imaged on a Leica SP system with a 63 X 0.9 water immersion lens, using a 568 nm Krypton laser to excite 6ROX-CaM, a 488 nm Argon laser to excite fluo-

Statistics

Results are expressed as mean ± s.e.m. (standard error of the mean) and n = number of experiments. All statistical analysis was performed using the parametric test ANOVA-one way. Significant difference was accepted at P<0.01.

Results

6ROX-CaM binding to nuclear targets

We have previously reported two properties of CaM regarding its nuclear entry; firstly, we showed Ca 2+ -dependent and independent entry to the nucleus upon cell stimulation [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF] and secondly, we showed that upon robust Ca 2+ stimulation of the cell, CaM within minutes of stimulation, induces an increase in the permeability of the nuclear pore complex [START_REF] Thorogate | Nuclear pore gating by calmodulin[END_REF]. Here we investigate which structures within the nucleus of HeLa cells, CaM becomes associated with upon its Ca 2+ -stimulated entry into the nucleus. This is important as these sites may represent the sites of downstream activities of nuclear CaM.

6ROX-CaM nuclear entry was stimulated by the addition of 2.1 mM Ca 2+ to HeLa cells [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF]. As seen in Figure 1, an immediate rise in [Ca 2+ ] was followed by CaM translocation into the nucleus and by association of 6ROX-CaM with the nucleoli.

Nucleolic association of CaM occurred with a t 1/2 of 73 ± 6 s (n = 12). The nucleolic to cytoplasmic fluorescence ratio of 6ROX-CaM significantly increased from 0.92 ± 0.04 to 2.8 ± 0.2. Over the course of the experiment, typically, after 486 ± 43 s the fluorescence intensity of 6ROX-CaM in the nucleoplasm became lower than that associated with other nuclear targets and the nuclear membrane. The nucleoplasmic-cytoplasmic ratio of 6ROX-CaM fluorescence was 1.63 ± 0.05 at its peak, after 317 ± 25 s. However, by 1200 s this had decreased to 1.38 ± 0.12 reflecting the binding of nucleoplasmic 6ROX-CaM to other nuclear targets. An example of the time course of binding to nuclear targets and to the nuclear membrane is shown in Figure 1 suggesting that the structures CaM becomes associated with in the nucleus following Ca 2+ stimulation, in addition to nucleoli and the nuclear membrane, may be chromatin.

6ROX-CaM co-localization with Hoechst dye

The association of CaM with chromatin has been previously reported [START_REF] Iwasa | Interaction of calmodulin with chromatin associated proteins and myelin basic protein[END_REF]. Using the Hoechst dye, we checked whether nuclear CaM and DNA co-localize in HeLa cells.

In 2 mM EGTA, before 2.1 mM Ca 2+ stimulation (see panels at t 0 in Figure 2), 6ROX-CaM showed no co-localization with the Hoechst dye, indicating that CaM did not bind to nucleic acid containing structures in the absence the Ca 2+ . However, over several minutes after stimulation by extracellular Ca 2+ addition (513 ± 31 s (n = 3)), 6ROX-CaM fluorescence intensity in the nucleoplasm became lower than that in areas stained by the Hoechst dye, indicating that it was binding to chromatin. However, the Hoechst dye did not appear to co-localize with 6ROX-CaM at the nucleoli (Figure 2).

6ROX-CaM1234 binding to nuclear targets

To determine the role of Ca 2+ stimulation and if Ca 2+ /CaM was required for the binding to nuclear targets we electroporated HeLa cells with the Ca 2+ -non-binding probe 6ROX-CaM1234. The localization of 6ROX-CaM1234 to nuclear targets was analyzed to determine if the binding to nucleoli and chromatin required Ca 2+ Upon stimulation with 2.1 mM Ca 2+ no initial translocation into the nucleus was seen with the 6ROX-CaM1234 consistent with the fact that Ca 2+ -dependent facilitated CaM translocation could not take place if Ca 2+ binding to CaM was inhibited [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF].

However, 6ROX-CaM1234 induced an increase in the permeability of the nuclear pores and entered the nucleus with a few minutes delay, as previously described [START_REF] Thorogate | Nuclear pore gating by calmodulin[END_REF]. Typically after 425 ± 65 s (n = 8) there was a slow association with a t 1/2 of 268 ± 27 s of the 6ROX-CaM1234 in the nucleus with chromatin and the nuclear membrane. Although the fluorescence intensity of 6ROX-CaM1234 in the nucleoli and chromatin did not appear as a large increase, in the nucleoli it was a 2.9 ± 0.5-fold higher compared to the cytoplasm, and 2.3 ± 0.3-fold greater in the nuclear membrane than in the nucleoplasm.

This indicated an association of CaM1234 with the subnuclear structures, similarl to that observed with wild type CaM. An example of Ca 2+ -induced 6ROX-CaM1234 nuclear localization is presented in Figure 3. This indicated that Ca 2+ was causing a conformational change in these nuclear targets allowing CaM to bind rather than Ca 2+ causing a conformational change in CaM allowing it to bind.

To totally confirm that the late nuclear binding was due to Ca 2+ induced changes of these nuclear targets rather than to Ca 2+ binding to CaM and then allowing it to bind to targets, 10 µM of mTrp peptide was added to the cells after they were electroporated with 6ROX-CaM1234. As shown in Figure 4, it was found that the nuclear entry of 6ROX-CaM1234 in the presence of the mTrp peptide occurred more slowly. The presence of the mTrp peptide did, however, not prevent the binding of 6ROX-CaM1234 to the nuclear targets, firstly confirming that the mTrp peptide can only bind to Ca 2+ -CaM and that the binding of CaM to chromatin and the nuclear membrane and to some extent to nucleoli, was driven by Ca 2+ induced changes allowing CaM to bind independently of Ca 2+ . Upon analysis it was found that for 6ROX-CaM 76-148 in 2 mM EGTA, the nucleoplasmic to cytoplasmic ratio was 1.95 ± 0.49, the nucleolic to cytoplasmic ratio was 2.41 ± 0.97 and the nuclear membrane to cytoplasmic ratio was 1.61 ± 0.42 (n = 7). For FL-CaM 76- 148 , the nucleoplasmic to cytoplasmic ratio of was found to be 1.9 ± 0.43, the nucleolic to cytoplasmic ratio was 2.52 ± 0.93 and the nuclear membrane to cytoplasmic ratio was 1.86 ± 0.45 (n = 7) (Figure 8 illustrates such an example). The values for the CaM 76-148 fragment thus were significantly different from the values found for 6ROX-CaM in 2 mM EGTA which had a nucleoplasmic to cytoplasmic ratio of 1.18 ± 0.03 and a nucleolic to cytoplasmic ratio = 0.92 ± 0.04 (n = 19). Thus, both FL-CaM 76-148 and 6ROX-CaM 76-148 were bound to nucleoli, chromatin and the nuclear membrane before Ca 2+ stimulation unlike full-length fluorescently labeled CaM. A possible explanation for this phenomenon may be that due to its small size, the CaM 76-148 fragment unlike full length CaM does not require Ca 2+ -dependent facilitated transport but enters the nucleus by diffusion and it becomes trapped there. This may occur due to high affinity binding to nuclear components and/or by impairment of a nuclear export system which requires full length CaM for functioning.

Nuclear localization and activation of fluorescently labeled C-and N-lobe

Nuclear target binding of CaM 76-148 in high intracellular [Ca 2+ ]

Upon cell stimulation with 2.1 mM Ca 2+ no significant nuclear translocation of either 6ROX-CaM 76-148 (Figure 5) or FL-CaM 76-148 (Figure 8B) was observed. However, the C-lobe CaM from the nucleoplasm demonstrated further rapid binding to the nucleoli, nuclear membrane and chromatin with a t 1/2 of 25 ± 4.1 for 6ROX-CaM 76-148 (n = 4) and 35 ± 5.4 for FL-CaM 76-148 (n = 4). The nucleolic to nucleoplasm ratio significantly increased from 1.2 ± 0.06 to 3.3 ± 0.17 (6ROX-CaM 76-148 ) and from 1.3 ± 0.08 to 3.87 ± 0.33 (FL-CaM 76-148 ). The nuclear membrane to nucleoplasm ratio also significantly increased from 0.82 ± 0.02 to 1.92 ± 0.19 (FL-CaM 76-148 ) and from 0.98 ± 0.03 to 1.97 ± 0.1 (6ROX-CaM 76-148 ). Thus, C-lobe CaM demonstrated a similar response to full length CaM upon Ca 2+ stimulation.

Nuclear target binding of CaM 76-148 inhibited by mTrp peptide

We checked the specificity of the C-lobe CaM nuclear binding by using the CaM inhibitor mTrp peptide. The application of 10 µM mTrp peptide to HeLa cells loaded with 6ROX-CaM 76-148 in 2 mM EGTA and then stimulated with 2.1 mM Ca 2+ largely prevented binding to nuclear targets and 6ROX-CaM 76-148 remained distributed in the nucleoplasm (n = 3) (Figure 6). These data show that the association of 6ROX-CaM 76-148 with nuclear targets seen above was specific to CaM rather than representing trapping in subnuclear structures.

Lack of nuclear target binding of CaM 1-80 in low [Ca 2+ ]

HeLa cells were electroporated with 6ROX-CaM 1-80 in 2 mM EGTA to analyse N-lobe nuclear binding in low Ca 2+ conditions. Upon analysis it was found that 6ROX-CaM 1-80 , unlike 6ROX-CaM 76-148 , was not bound to nucleoli, chromatin or the nuclear membrane before Ca 2+ stimulation. The nucleoplasmic to cytoplasmic ratio of 6ROX-CaM 1-80 was found to be 1.39 ± 0.04 (n = 9) and the nucleolic to cytoplasmic ratio was 0.99 ± 0.04 (Figure 7). This was significantly different from the nucleoplasmic to cytoplasmic ratio of 1.95 ± 0.49 and the nucleolic to cytoplasmic ratio of 2.41 ± 0.97 found with 6ROX-CaM 76-148 in the same conditions. This indicated that the C-lobe of CaM was able to bind to nuclear targets with a higher affinity than the N-lobe. However the nucleoplasmic to cytoplasmic ratio of 6ROX-CaM 1-80 was still significantly higher than 6ROX-CaM (1.39 ± 0.04 compared to 1.18 ± 0.03, p < 0.001). It is noteworthy that in contrast with C-lobe, neither the N-lobe nor full length CaM was able to bind to nucleoli, chromatin or the nuclear membrane in the absence of Ca 2+ .

Lack of nuclear target binding of CaM 1-80 in high [Ca 2+ ]

Upon cell stimulation with 2.1 mM Ca 2+ no further significant nuclear translocation of fluorescently-labeled CaM 1-80 was observed. In comparison to the Clobe, no significant binding of the N-lobe to nucleoli, chromatin or the nuclear membrane was found and 6ROX-CaM 1-80 remained in the nucleoplasm. After 500 s, the nucleoplasmic to cytoplasm ratio was 1.4 ± 0.05 showing no significant increase and the nucleolic to nucleoplasmic ratio slightly increased, but not significantly, from 0.72 ± 0.03 to 0.79 ± 0.06 (Figure 7). This suggested that either the N-lobe CaM was unable to bind to nucleoli, chromatin and the nuclear membrane or it had a lower affinity for either Ca 2+ or target binding than endogenous full length CaM.

Ca 2+ activation of TA-CaM 1-80 activation upon stimulation

To check that CaM 1-80 was binding to Ca 2+ , HeLa cells were electroporated with TA-CaM 1-80 in 2 mM EGTA and stimulated with 2.1 mM Ca 2+ . Upon cell stimulation there was a 1.88 ± 0.12 (n = 13) fold increase in the nucleoplasmic fluorescence indicating that TA-CaM 1-80 was binding to Ca 2+ and possibly to unidentified nucleoplasmic proteins. There was also a 2.89 ± 0.12 fold increase in the nucleolar fluorescence indicating Ca 2+ binding and some nucleolar accumulation of TA-CaM 1-80 (Figure 8A).

Simultaneous imaging of C-and N-CaM domains in the same cell

Mechanism of nuclear accumulation of C-and N-CaM domains in resting HeLa cells

In order to determine if the inhibition of facilitated transport would prevent nuclear entry of the C and N-domains as was seen in cells electroporated with full length 6ROX-CaM [START_REF] Sorensen | Paramecium calmodulin mutants defective in ion channel regulation associate with melittin in the absence of calcium but require it for tertiary collapse[END_REF], FL-WGA (estimated final concentration 20 µg/mL) was coelectroporated into HeLa cells with either 6ROX-CaM 76-148 or 6ROX-CaM 1-80 in 2 mM EGTA. FL-WGA was found to permeate both the plasma and the nuclear membrane in a subsection of electroporated cells (Figure 9A andC), but in a different subsection of cells, FL-WGA presented only in the plasma membrane but not in the nuclear membrane (Figure 9B andD). Surprisingly, upon visualization of C-domain CaM, it was found that cells containing the FL-WGA inhibitor associated with the nuclear membrane showed 6ROX-CaM 76-148 concentrated in the nucleus with none in the cytoplasm (Figure 9A). In cells that also contained FL-WGA but only in the plasma membrane and not in the nuclear membrane, 6ROX-CaM 76-148 was distributed in the normal way (Figure 9B). A similar pattern was seen with 6ROX-CaM 1-80 , again in the presence of FL-WGA inhibiting facilitated transport, the N-lobe was concentrated in the nucleus with none present in the cytoplasm (Figure 9C). Cells that did not have the association of FL-WGA with the nuclear membrane showed normal distribution of 6ROX-CaM 1-80 (Figure 9D). This was the complete opposite of what was found in cells containing 6ROX-CaM and FL-WGA [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF]. In those cells 6ROX-CaM nuclear entry was blocked whereas here 6ROX-CaM 76-148 and 6ROXCaM 1-80 were both prevented from exiting the nucleus.

Discussion

CaM has been reported to be localized to the nucleoli, non-peripheral chromatin and peripheral chromatin associated with the nuclear membrane [START_REF] Wong | Association of calmodulin with isolated nuclei from rat hepatocytes[END_REF]. Here 6ROX-CaM in low Ca conditions was found to be 1.2-fold higher in the nucleoplasm of HeLa cells compared to the cytoplasm. However, no binding to nucleoli, chromatin or the nuclear membrane was detected in these conditions. Upon Ca stimulation 6ROX-CaM immediately bound to nucleoli and translocation from the cytoplasm to the nucleus was observed. Typically 7 -9 minutes later, 6ROX-CaM began to associate with other structures and cells loaded with the nucleic acid stain Hoechst dye and 6ROX-CaM suggested that these structures were chromatin. 2+ 2+ 6ROX-CaM1234 similarly showed no association with nucleoli, chromatin and the nuclear membrane before addition with Ca 2+ and as expected no immediate binding to the nucleoli was seen upon cell stimulation. However, 6ROX-CaM1234 also showed late binding to chromatin and the nuclear membrane minutes after stimulation. This suggested that Ca 2+ was inducing a conformational change in these nuclear targets allowing CaM to bind rather than Ca 2+ inducing a change on CaM allowing it to bind. This is indeed possible; an interaction of Ca 2+ with chromatin material has been reported in sympathetic neuronal cells [START_REF] Przywara | Stimulated rise in neuronal calcium is faster and greater in the nucleus than the cytosol[END_REF]. It was also shown in vitro that Ca 2+ and ATP induced structural changes on the histone H1, a major constituent of chromatin that contributes to chromatin condensation [START_REF] Tarkka | Nucleotide and calcium-induced conformational changes in histone H1[END_REF]. It is possible therefore that Ca 2+ -induced chromatin changes were allowing CaM or a CaM-binding protein to bind to chromatin. Indeed, it has been found that a 62 kDa CaM-binding protein, p62, which has a low-affinity for CaM, was localized over condensed chromatin and the nuclear matrix in quiescent astrocytes, hepatocytes and cortical astroglia cells [START_REF] Portoles | Nuclear calmodulin/62 kDa calmodulinbinding protein complexes in interphasic and mitotic cells[END_REF]. Here, the results suggest that 6ROX-CaM or 6ROX-CaM1234 may not have bound to chromatin directly but was bound to a CaM-binding protein independently of Ca 2+ . The conformational change on chromatin induced by Ca 2+ then allowed CaM to associate via a CaM-binding protein.

The binding of the N and C-lobe CaM fragments to nucleoli, chromatin and the nuclear membrane were also investigated in HeLa cells. The C-lobe is in a semi-open conformation in apo conditions, whereas the N-lobe is in a closed conformation and in the presence of Ca 2+ it opens allowing it to bind to targets [START_REF] Swindells | Pre-formation of the semi-open conformation by the apo-calmodulin C-terminal domain and implications binding IQ-motifs[END_REF]. It has been found that Ca 2+ binding sites III and IV in the C-lobe have a 10-fold higher affinity for Ca 2+ than sites I and II in the N-lobe [START_REF] Sorensen | Interactions between domains of apo calmodulin alter calcium binding and stability[END_REF][START_REF] Vanscyoc | Calcium binding to calmodulin mutants monitored by domainspecific intrinsic phenylalanine and tyrosine fluorescence[END_REF]. However, the isolated N-lobe has a higher Ca 2+ affinity than when part of the full length CaM indicating that Ca 2+ binding to sites I and II is of a lower affinity in the presence of the C-lobe [START_REF] Sorensen | Interactions between domains of apo calmodulin alter calcium binding and stability[END_REF]. Previous studies have also shown that C-domain was able to bind to NR1 C0 [START_REF] Akyol | Apo-calmodulin binds with its C-terminal domain to the Nmethyl-D-aspartate receptor NR1 C0 region[END_REF] and melittin [START_REF] Sorensen | Paramecium calmodulin mutants defective in ion channel regulation associate with melittin in the absence of calcium but require it for tertiary collapse[END_REF] in apo conditions, whereas the N-domain only became associated in Ca 2+ conditions.

The ability of apo-CaM 76-148 to bind to nuclear targets was seen here with both 6ROX-CaM 76-148 and FL-CaM 76-148 demonstrating binding to nucleoli, chromatin and the nuclear membrane in low Ca 2+ conditions. This contrasted with the finding that full length CaM did not bind to nucleoli, chromatin or the nuclear membrane in the absence of Ca 2+ . It was also observed that the C-lobe was 1.9-fold higher in the nucleoplasm compared to the cytoplasm indicating that the C-lobe bound unidentified nucleoplasmic proteins in apo conditions. In contrast with the similarly sized 9.5 kDa FL-dextran, which only showed a ratio of 0.92 as it remained unbound in the nucleus [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF], CaM 76-148 was able to bind to nucleoplasmic targets, nucleoli, chromatin and the nuclear membrane therefore keeping it at a higher concentration the nucleus. Upon the addition of Ca 2+ both 6ROX-CaM 76-148 and FL-CaM 76-148 from the nucleoplasm were seen to further bind to nucleoli, chromatin and the nuclear membrane with t 1/2 values of 25 s and 35 s, respectively. This was significantly faster than the binding of full-length 6ROX-CaM to nucleoli which occurred with a t 1/2 of 72 s consistent with the C-lobe having a higher affinity for nucleolar CaM binding proteins and a smaller size than full-length CaM.

Another interesting difference was the lack of delay of C-lobe CaM to bind to chromatin and the nuclear membrane whereas full length CaM typically did not bind to these targets until 8 minutes post stimulation. If the theory presented here that chromatin undergoes a conformational change before CaM can bind is correct then the same restrictions might not apply to the C-lobe CaM. A pool of FL-CaM 76-148 and 6ROX-CaM 76-148 must have been bound to unidentified targets in the nucleoplasm in low free Ca 2+ otherwise apo-CaM 76-148 would have all bound to nucleoli, chromatin and the nuclear membrane. Upon Ca 2+ stimulation it was possible that this pool of bound CaM 76-148 in the nucleoplasm was released and became free to bind with nucleoli, chromatin and the nuclear membrane.

Indeed it has been reported that only 5 % of CaM was free in Ca 2+ -independent conditions and upon Ca 2+ stimulation a large pool of this CaM was released [START_REF] Luby-Phelps | Ca(2+)-regulated dynamic compartmentalization of calmodulin in living smooth muscle cells[END_REF].

6ROX-CaM 1-80 showed a nucleoplasmic to cytoplasmic ratio of 1.4 in low Ca 2+ conditions, which was still significantly higher than the ratio of 0.92 for 6ROX-CaM. This indicated that the N-lobe was able to bind to nucleoplasmic targets in the absence of Ca 2+ which was surprising due to the closed conformation of the N-domain in apo conditions preventing it from binding to targets. However, certain targets appear to bind to the N-domain in the absence of Ca 2+ even though it is in a closed conformation, for example, the ryanodine receptor RYR1 [START_REF] Luby-Phelps | Ca(2+)-regulated dynamic compartmentalization of calmodulin in living smooth muscle cells[END_REF] and voltage-gated Ca 2+ channels [START_REF] Zhang | A noncontiguous, intersubunit binding site for calmodulin on the skeletal muscle Ca 2+ release channel[END_REF]. The nucleoplasmic to cytoplasmic ratio was still found to be lower compared to the C-lobe (1.4 compared to 1.9) indicating that the C-lobe had a higher affinity for nucleoplasmic targets.

Upon Ca 2+ stimulation, there was no significant increase in the binding of 6ROX-CaM 1-80 to nucleoli, chromatin or the nuclear membrane, indicating two possibilities.

Firstly, it was possible that the N-domain could not bind to the nucleolar and chromatin CaM-binding proteins in the absence of a C-domain. A second possibility was that these targets had a lower affinity for Ca 2+ -CaM 1-80 than for endogenous full length CaM. As seen in Figure 7F, Trp peptide had a 6 x 10 4 -fold lower affinity for Ca 2+ /TA-CaM 1-80 than for Ca 2+ /CaM [27]. The 2-fold increase in fluorescence of TA-CaM 1-80 indicated that it was binding to Ca 2+ causing the N-lobe to enter an open conformation. Therefore it is more than likely that endogenous Ca 2+ -CaM competed out the binding of the N-lobe.

Similarly in cells electroporated with both FL-CaM 76-148 and TA-CaM 1-80 only the C-lobe was seen to bind to nucleoli and chromatin with very limited binding of the N-lobe.

Again this demonstrated that the C-lobe had a higher affinity for these targets than the Nlobe.

Upon the co-electroporation of 6ROX-CaM 76-148 or 6ROX-CaM 1-80 with WGA, both lobes were unexpectedly trapped in the nucleus and over a period of 30 minutes no diffusion back into the cytoplasm of either CaM lobe was seen. This was the complete opposite of what was seen with 6ROX-CaM or 6ROX-CaM1234 in apo conditions, where in the presence of WGA, they were significantly more cytoplasmic [START_REF] Thorogate | Ca 2+ -dependent and -independent mechanisms of calmodulin nuclear translocation[END_REF]. It is known that WGA only prevents facilitated transport, import as well as export, but not passive diffusion [START_REF] Middeler | The tumor suppressor p53 is subject to both nuclear import and export, and both are fast, energy-dependent and lectininhibited[END_REF], therefore, the observations suggest that both C and N-lobes of CaM are in a bound state in the nucleus otherwise both would diffuse back into the cytoplasm. One obvious question is why neither lobe is distributed similarly in the absence of WGA i.e. all in the nucleus. The possible answer to this is that upon electroporation both lobes of CaM are able to diffuse into the nucleus attracted to nucleoplasmic targets and nucleoli and chromatin in the case of CaM 76-148 . Once there they bind to these nuclear targets in the absence of Ca 2+ . These nucleoplasmic targets that either lobe binds to in the absence of Ca 2+ could be shuttling proteins that shuttle continuously between the nucleus and cytoplasm and contain import and export sequences. Therefore in the presence of WGA they might become trapped in the nucleus due to the inhibition of facilitated transport. One such protein family could be the heterogeneous nuclear RNA-binding proteins (hnRNP proteins) [START_REF] Vautier | Transcription-dependent nucleocytoplasmic distribution of hnRNP A1 protein in early mouse embryos[END_REF]. Full-length CaM, however, does not show the same properties as the two separate lobes. Upon electroporation CaM is too large to pass through the nuclear pores in the absence of Ca 2+ and import into the nucleus requires it to associate with a CaM-binding protein with an NLS sequence. In the presence of WGA this complex cannot be taken up into the nucleus due the inhibition of facilitated transport.

Therefore, in conclusion it is shown that CaM and CaM1234 can bind to chromatin upon Ca 2+ stimulation due to a conformational change induced by Ca 2+ on chromatin rather than a conformational change on CaM. Both the C and N-lobes of CaM appear to be able to associate with nucleoplasmic targets in apo conditions, as indicated by higher nucleoplasmic to cytoplasmic ratios than full-length CaM and by the observation that both domains are trapped in the nucleus in the presence of WGA. The C terminal lobe of CaM, CaM 76-148 is also able to bind to chromatin and nucleoli in apo conditions but the N terminal lobe, CaM 1-80 does not show any association and is competed out upon Ca 2+ stimulation due to its lower affinity for targets than full-length endogeous CaM.

Acknowledgement

4

  and a 351/365 nm Argon ion UV laser to excite TA-CaM fluorescence. Images were taken at 6 s intervals for 1200 s at 22 o C in electroporation buffer and had a box size of 512 X 512 pixels. Image analysis is presented using intensities in representative areas as shown in the figures. The presented single values are within 10% of the mean values obtained by comparing 3-5 areas of interest (not shown).

  CaM mutants The role of the individual C-and N-terminal lobes of CaM in subnuclear targeting was tested by using fluorescently labeled CaM fragments corresponding to residues 76-8 80 were labeled with fluorophores to report localization and and/or activation by Ca 2+ and target binding and were introduced into HeLa cells by electroporation. The associations with unidentified nuclear targets, nucleoli, chromatin and the nuclear membrane of 6ROX-CaM 76-148 or FL-CaM 76-148 , representing the C-lobe, and 6ROX-CaM 1-80 or TA-CaM 1-80 , representing the N-lobe, were analyzed in low Ca 2+ and in a Ca 2+ stimulated state and compared to the binding of full length CaM in similar conditions. The mTrp peptide was added to the cells to determine if the inhibitor would prevent CaM 76-148 or CaM 1-80 binding. Nuclear target binding of CaM 76-148 in low [Ca 2+ ] HeLa cells were electroporated with either FL-CaM 76-148 or 6ROX-CaM 76-148 in 2 mM EGTA to analyze C-lobe binding in low Ca 2+ conditions. As seen in the example shown in Figure 5A, 6ROX-CaM 76-148 accumulated in the nucleus of unstimulated cells.

FL-

  CaM 76-148 and TA-CaM 1-80 were electroporated into the same cells to directly compare the binding differences between the N and C-domains. Upon Ca 2+ stimulation, FL-CaM 76-148 immediately bound to nucleoli, chromatin and the nuclear membrane as seen for 6ROX-CaM 76-148 (Figure4), whereas TA-CaM 1-80 showed very little binding to these targets but demonstrated typical nucleoplasmic and nucleolar increases in 11 Biochemical Journal Immediate Publication. Published on 16 Oct 2006 as manuscript BJ20061111 Copyright 2006 Biochemical Society Thorogate and Török CaM nuclear binding fluorescence (n = 4) (Figure 8A-D). In Figure 8D, due to a large reduction in fluorescence of FL-CaM 76-148 that occurred upon Ca 2+ stimulation likely as a result of a decrease in pH, fluorescence intensities measured in nuclear regions were related to that in cytoplasm in each frame. Solution fluorescence measurements showed that the fluorescence of TA-CaM 1-80 increased 1.9-fold upon Ca 2+ binding giving an apparent dissociation constant K d for Ca 2+ of 3.85 ± 1.2 µM (Figure 8E) and the addition of Trp peptide up to 600 nM increased the fluorescence a further 2.7-fold in these Ca 2+ saturated conditions (Figure 8F). A K d value of 208 ± 26 nM was measured for mTrp peptide binding to TA-CaM 1-80 . However, in the absence of Ca 2+ the fluorescence of TA-CaM 1-80 only increased 1.2-fold upon additions of mTrp peptide up to 500 nM. This suggested that CaM 1-80 needed to be in an open conformation induced by Ca 2+ binding to be able to bind to the mTrp peptide.

:

  This work was supported by the Medical Research Council, UK.

Figure 1 :

 1 Figure 1: 6ROX-CaM binding to nuclear targets after stimulation. A) HeLa cell electroporated with 6ROX-CaM in 2 mM EGTA (t 0 ) and stimulated with 2.1 mM Ca 2+ (t 43 ). In the example shown, 6ROX-CaM bound to nucleoli upon stimulation with a t 1/2 of 92 s and the fluorescence intensity in the nucleolus relative to the cytoplasm increased 2.91-fold. As seen in the image taken at t 601 and onwards, redistribution of 6ROX-CaM from the nucleoplasm to other nuclear targets and the nuclear membrane became pronounced. Intensity measurements were made in single windows selected in representative areas. Time course of (B) 6ROX-CaM nuclear translocation and (C) the Ca 2+ transient indicated by fluo-4 over 1200 s are shown in the (♦, orange) nucleolus, (▲, blue) nucleoplasm, (■, mauve) nuclear membrane and the (*, green) cytoplasm. It is noteworthy that the time courses of Ca 2+ decay in this experiment and those shown in Figure 4C, 5C were slower to decay with t 1/2 -s of > 400 s than those presented in Figures 6D and 7D which had a t 1/2 of decay of ~ 200 s. We have not identified any significant difference between the CaM cellular redistribution patterns and time courses as a result of difference in the Ca 2+ sequestration time courses. Arrow indicates 2.1 mM Ca 2+ addition.

Figure 2 :

 2 Figure 2: 6ROX-CaM and Hoechst nuclear localization upon stimulation. HeLa cell loaded with A) 6ROX-CaM, B) Hoechst dye and C) overlay of 6ROX-CaM and Hoechstimages. Cells were stimulated with 2.1 mM Ca 2+ . Upon Ca 2+ stimulation, 6ROX-CaM bound immediately to the nucleoli with a t 1/2 of 75 s and then showed a second slower binding step at t 500 where it became localized with the Hoechst dye, indicating that it was possibly binding to chromatin (t 900 ).

Figure 3 : 22 Biochemical

 322 Figure 3: 6ROX-CaM1234 binding to nuclear targets after Ca 2+ stimulation. A) HeLa cell electroporated with 6ROX-CaM1234 and fluo-4 in 2 mM EGTA and stimulated with 2.1 mM Ca 2+ (t 72 ). No translocation of 6ROX-CaM1234 was seen unlike 6ROX-CaM upon Ca 2+ stimulation, however binding to nucleoli, chromatin and the nuclear membrane began at t 510 and showed a slow association with a t 1/2 of 314 s. Time course of (B) 6ROX-CaM1234 nuclear binding and (C) the Ca 2+ transient measured by fluo-4 over 1800 s in the (♦, orange) nucleolus, (▲, blue) nucleoplasm, (■, mauve)

Figure 4 :

 4 Figure 4: 6ROX-CaM1234 binding to nuclear targets after Ca 2+ stimulation in the presence of mTrp peptide. A) HeLa cells electroporated with 6ROX-CaM1234 and fluo-4 in 2 mM EGTA, loaded with 10 µM mTrp peptide and stimulated with 2.1 mM Ca 2+ (t 130 ). Time course of B) 6ROX-CaM1234 nuclear binding and C) the Ca 2+ transient measured by fluo-4 in the (♦, orange) nucleolus, (▲, blue) nucleoplasm, (■, mauve) nuclear membrane and the (*, green) cytoplasm. Arrow indicates 2.1 mM Ca 2+ addition.

Figure 5 :

 5 Figure 5: 6ROX-CaM 76-148 nuclear binding upon stimulation. A) HeLa cell was electroporated with 6ROX-CaM 76-148 in 2 mM EGTA and stimulated with 2.1 mM Ca 2+ . Most of 6ROX-CaM 76-148 , which was already present in the nucleus, rapidly accumulated in the nucleoli with a t 1/2 of 20 s and also bound to chromatin and the nuclear membrane. Time course of (B) 6ROX-CaM 76-148 nuclear binding and (C) the Ca 2+ transient measured by fluo-4 over 400 s in the (♦, orange) nucleolus, (▲, blue) nucleoplasm, (■, mauve) nuclear membrane and the (*, green)cytoplasm. Arrows indicate 2.1 mM Ca 2+ addition.

Figure 6 :

 6 Figure 6: 6ROX-CaM 76-148 nuclear binding inhibited by mTrp peptide. HeLa cell electroporated with (A) 6ROX-CaM 76-148 and (B) fluo-4 and loaded with 10 µM mTrp peptide. Cell was stimulated with 2.1 mM Ca 2+ , however, no additional binding was seen to nuclear targets. Time course of (C) 6ROX-CaM 76-148 nuclear binding and (D) the Ca 2+ transient measured by fluo-4 over 1200 s in the (♦, orange) nucleolus, (▲, blue) nucleoplasm and the (*, green)cytoplasm. Arrow indicates 2.1 mM Ca 2+ addition.

Figure 7 : 23 Biochemical

 723 Figure 7: 6ROX-CaM 1-80 nuclear binding upon stimulation. HeLa cell was electroporated with (A) 6ROX-CaM 1-80 and (B) fluo-4 in 2 mM EGTA and stimulated with 2.1 mM Ca 2+ . No nuclear translocation was observed and no significant binding to nucleoli, chromatin or the nuclear membrane was seen. Time course of (C) 6ROX-CaM 1- 80 nuclear binding and (D) the Ca 2+ transient measured by fluo-4 over 1200 s in the (♦,

Figure 8 :

 8 Figure 8: TA-CaM 1-80 and FL-CaM 76-148 nuclear binding upon stimulation. HeLa cell electroporated with (A) TA-CaM 1-80 and (B) FL-CaM 76-148 in 2 mM EGTA and stimulated with 2.1 mM Ca 2+ . FL-CaM 76-148 bound to nucleoli with a t 1/2 of 15 s and TA-CaM 1-80 showed a 2.1 and 2.5-fold rise in fluorescence in the nucleoplasm and nucleoli respectively at F max . Time course of (C) TA-CaM 1-80 nuclear binding and (D) the fluorescence increase in FL-CaM 76-148 upon Ca 2+ and target binding over 400 s in the (♦, orange) nucleolus, (▲, blue) nucleoplasm, (■, mauve) nuclear membrane and the (*, green)cytoplasm. Arrow indicates 2.1 mM Ca 2+ addition. (E) Ca 2+ titration of TA-CaM 1- 80 . Buffer contained 2 mM EGTA, 100 mM KCl, 2 mM MgCl 2 and 50 mM PIPES-K + , pH 7.0 at 21 o C and 30 nM TA-CaM 1-80 . Free Ca 2+ concentrations were calculated using a K d of 4.35 x 10 -7 M for EGTA determined in similar buffer and ionic strength conditions and calculated [Ca 2+] were checked by titration of fluo-3 fluorescence as previously described[START_REF] Tzortzopoulos | (C) & (D) HeLa cells were electroporated with 6ROX-CaM 1-80 (left-hand panels) and FL-WGA (right-hand panels) in 2 mM EGTA. (C) When FL-WGA was incorporated into the nuclear membrane, 6ROX-CaM 1-80[END_REF]. Upon Ca 2+ additions up to 1 mM free Ca 2+ there was a 1.92-fold fluorescence increase (excitation = 365 nm; emission = 420 nm). Best fit K d value of 2.75 ± 0.57 µM (Hill coefficient n = 2) was obtained to the Hill equation using Grafit4 (Erithacus Software Ltd.). (F) mTrp peptide titration of TA-CaM 1-80 . Conditions as in (E) except for 1 mM Ca 2+ in the solution from the start. Upon mTrp additions of up to 600 nM to 30 nM TA-CaM 1-80 there was a 2.8-fold fluorescence increase. Best fit K d value of 360 ± 174 nM was obtained to the equation for the analysis of the binding curve representing bound versus total ligand concentrations using Grafit4 (Erithacus Software Ltd.) (excitation = 365 nm; emission = 420 nm).

Figure 9 :

 9 Figure 9: 6ROX-CaM 76-148 and 6ROX-CaM 1-80 distributions in the presence of FL-WGA blocked nuclei and in the presence of FL-WGA not inhibiting nuclear transport. (A) & (B) HeLa cells were electroporated with 6ROX-CaM 76-148 (left-hand panels) and FL-WGA (right-hand panels) in 2 mM EGTA. (A) When FL-WGA was incorporated into the nuclear membrane, 6ROX-CaM 76-148 was found to be concentrated in the nucleus but none was present in the cytoplasm. Note the typical binding of 6ROX-

Figure 4 29

 4 Figure 4

-bound CaM or required 7 Biochemical Journal Immediate Publication. Published on 16 Oct 2006 as manuscript BJ20061111 Copyright 2006 Biochemical Society

	Thorogate and Török	CaM nuclear binding
	Ca 2+ to alter the conformation of nuclear targets allowing CaM to bind in a Ca 2+ -
	independent form.

and 1-80 representing the C-and N-lobes of CaM, respectively. CaM 76-148 and CaM 1-

Biochemical Journal Immediate Publication. Published on 16 Oct 2006 as manuscript BJ20061111