- Email: [email protected]
Profound redox sensitivity of peptidyl-prolyl isomerase activity in Arabidopsis thylakoid lumen
FEBS Letters 580 (2006) 3671–3676
Profound redox sensitivity of peptidyl-prolyl isomerase activity in Arabidopsis thylakoid lumen Alexey Shapiguzov1, Anna Edvardsson, Alexander V. Vener* Division of Cell Biology, Linko¨ping University, SE-581 85 Linko¨ping, Sweden Received 5 April 2006; revised 23 May 2006; accepted 25 May 2006 Available online 5 June 2006 Edited by Richard Cogdell
Abstract Proteomic, enzymatic, and mutant analyses revealed that peptidyl-prolyl isomerase (PPIase) activity in the chloroplast thylakoid lumen of Arabidopsis is determined by two immunophilins: AtCYP20-2 and AtFKBP13. These two enzymes are responsible for PPIase activity in both soluble and membraneassociated fractions of thylakoid lumen suggesting that other lumenal immunophilins are not active towards the peptide substrates. In thiol-reducing conditions PPIase activity of the isolated AtFKBP13 and of the total thylakoid lumen is suppressed several fold. Profound redox-dependence of PPIase activity implies oxidative activation of protein folding catalysis under oxidative stress and photosynthetic oxygen production in the thylakoid lumen of plant chloroplasts. Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Immunophilin; FKBP; Cyclophilin; Peptidyl-prolyl isomerase; Thylakoid lumen; Arabidopsis thaliana
1. Introduction Immunophilins are ubiquitous receptors for immunosuppressive drugs cyclosporine A and FK506. Receptors for cyclosporine A are defined as cyclophilins, while FK506 binding proteins are called FKBPs. Despite of little sequence homology, immunophilins of the two families share similar peptidyl-prolyl cis/trans isomerase (PPIase) enzymatic activity that might be involved in the catalysis of protein folding [1–3]. The assays of PPIase activity with prolyl-containing synthetic peptides have demonstrated little dependence of cyclophilincatalyzed isomerization on peptide sequence, while FKBPs were much more efficient in isomerization of peptides with a prolyl preceded by a bulky hydrophobic residue like leucine [3,4]. The cellular functions of cyclophilins and FKBPs extend far beyond their PPIase activity: these proteins are involved in cell signaling, protein biogenesis and trafficking, cell cycle control, heat shock responses and regulation of membrane recep-
* Corresponding author. Fax: +46 13 224314. E-mail address: [email protected] (A.V. Vener). 1 Permanent address: Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, 127276 Moscow, Russia.
Abbreviations: FKBP, FK506 binding protein; PPIase, peptidyl-prolyl cis/trans isomerase
tors, channels and pores, as reviewed in [1,2,5,6]. Nevertheless, the physiological roles of numerous immunophilins in different organisms are still unknown. The genomic analysis of Arabidopsis thaliana has revealed 52 genes encoding putative immunophilins in this model plant [7]. A significant fraction of Arabidopsis cyclophilins and FKBPs has been localized in the chloroplast, particularly in the small compartment of chloroplast thylakoid lumen. Proteomic analyses of Arabidopsis thylakoid lumen [8–10] identified 3 cyclophilins and 7 FKBPs, while none of these immunophilins has been analyzed for PPIase activity. The recombinant lumenal AtFKBP13 possessed this activity and was inactivated upon reduction of two redox-active disulfide bonds specific for this FKBP [11]. Two cyclophilins TLP40 [12] and TLP20 [13] possessing PPIase activity have been isolated from thylakoid lumen of spinach. In this study, we set out to map the PPIase activities of the immunophilins from the thylakoid lumen of A. thaliana by chromatographic separations and PPIase assays with two peptide substrates that should accommodate the specificities of both cyclophilins and FKBPs. Unexpectedly, we revealed that the PPIase activity of Arabidopsis thylakoid lumen is restricted to only AtFKBP13 and AtCYP20-2. The dominant activity belonged to AtFKBP13 and was strongly inhibited by the thiol-reducing agent DTT. These findings reveal dramatic redox sensitivity of the total PPIase activity in the chloroplast thylakoid lumen. 2. Materials and methods 2.1. Plant material, isolation and chromatographic fractionation of thylakoid lumen Arabidopsis thaliana ecotype Columbia plants were grown hydroponically [14] at 20–22 °C, 8 h light/16 h dark photoperiod and light intensity 120 lmol photons/m2/s. Arabidopsis insertion line SALK_009552 was obtained from the Salk Institute collection [15]. Homozygot plants with the T-DNA insertion were identified by PCR analysis with the AtCYP20-2 gene specific primers: 5 0 -TTTGCACAGGTAAATATGCTTCATAGGAT-3 0 (forward), 5 0 -AGATCGGACTTTAAATACAAATTGCTCGT-3 0 (reverse) and the T-DNA left border specific primer 5 0 -TGGTTCACGTAGTGGGCCATCG3 0 . Plant leaves were harvested after 16 h of dark period. Soluble lumenal proteins were isolated from 100 g of wild type leaves or from 25 g of mutant leaves according to the protocol described in [8,16] with minor modifications. For Yeda press treatment the following buffer was used: 20 mM Tricine, pH 7.8, 5 mM MgCl2, and 100 mM sucrose. For isolation of membrane-associated lumenal fraction 1 M NaCl was added to the same buffer and the membranes were subjected to additional Yeda press treatment. The high-salt protein extract was desalted using Amicon Ultra-15 centrifugal filter units with 5 kDa cut-off (Millipore). The lumenal proteins were fractionated by sequential FPLC on
0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.05.054
3672
A. Shapiguzov et al. / FEBS Letters 580 (2006) 3671–3676
Resource Q and Resource S columns (Amersham Biosciences) equilibrated with 20 mM Tricine, pH 7.8 and eluted with 0–1 M NaCl linear gradient. 2.2. Measurements of PPIase activity The PPIase activity was analyzed by chymotrypsin-coupled assay as described in [17]. Peptide substrates N-succinyl-Ala-Ala-Pro-Phe-pnitroanilide (A-peptide) and N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (L-peptide) (Bachem) were dissolved in trifluoroethanol with LiCl to increase the proportion of cis isomer [4]. The reactions were started by adding peptide substrate to a mixture of protein sample and chymotrypsin (final concentration 60 lM) in 0.1 M Tricine, pH 7.8 at 10 °C. The change in absorbance at 390 nm due to release of p-nitroanilide was monitored on Lambda 25 Perkin–Elmer spectrophotometer. Reaction rate coefficients were derived by non-linear curve fitting to a firstorder rate equation using Origin 7.5 software. The enzymatic activity was calculated as a difference in the rates of catalyzed and spontaneous reactions.
established protocol [8,16] and subjected them to chromatographic fractionation by FPLC. The PPIase activity in all fractions was measured using two substrate peptides, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (A-peptide) and N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (L-peptide), to match the specificities of both cyclophilins and FKBPs [3,4]. The anionexchange FPLC revealed two chromatographic peaks with PPIase activity, as was detected with both substrate peptides (Fig. 1A). The activity towards the A-peptide was significantly higher than towards the L-peptide (Fig. 1A) indicating the possible presence of cyclophilins in the active fractions. The
A
12 A280 A-peptide L-peptide
2.4. Identification of proteins by mass spectrometry The individual protein bands were excised from the gels and treated with trypsin (sequencing grade modified, Promega). MALDI-TOF analyses of the peptides were performed on Voyager-DE Pro (Applied Biosystems, Framingham, MA, USA). Electrospray ionization tandem mass spectra were acquired on a hybrid mass spectrometer API QSTAR Pulsar i (Applied Biosystems, Foster City, CA, USA) equipped with a nanoelectrospray ion source (MDS Protana, Odense, Denmark). The nanoelectrospray capillaries were loaded with 2 ll peptide solution in water/acetonitrile (50/50) with 1% formic acid. The collision-induced fragmentation of selected peptide ions was done using the instrument settings recommended by Applied Biosystems and manual control of collision energy.
8 6 4 2 0 AtCYP20-2
1
B
5 6 7 8 910
AtCYP38
15 20 Fraction
23 25 27
30
12 A280 A-peptide
10 PPIase activity (%)
2.3. SDS–PAGE and immunoblotting analyses Chromatographic fractions were subjected to SDS–polyacrylamide gel electrophoresis in 15% acrylamide gels that were either stained with silver or used for immunoblotting. In the latter case, the proteins were transferred to a polyvinylidene difluoride membrane (Immobilone-P, Millipore, MA, USA). The membrane was blocked with milk proteins and incubated with indicated specific antibodies, which was followed by ECL detection according to the protocol (Amersham Biosciences, Sweden). Polyclonal antiserum to AtCYP38 was raised against a synthetic peptide GPAEGFIDPSTEKTR corresponding to amino acids 211–225 of the mature protein. Polyclonal antibodies against AtCYP20-2, PsbO and plastocyanin were kindly provided by Drs. P.G.N. Romano, C. Spetea and H.V. Scheller.
PPIase activity (%)
10
W.t. Mutant AtCYP38
8
AtCYP20-2
6 4 2
3. Results
0
To map the PPIase activities in the thylakoid lumen of Arabidopsis we isolated lumenal proteins according to the
1
5
10
15 20 Fraction
c C 100
30
644.92+ y2 11 10 9 8 7
y102+
80 Intensity (%)
Fig. 1. Mapping of PPIase activity and cyclophilins separated by anion-exchange FPLC. (A) Fractionation of PPIase activity from Arabidopsis thylakoid lumen on Resource Q column (representative of three experiments). PPIase activity was measured with two different synthetic peptides as depicted by bold gray lines and indicated. The protein absorbance at 280 nm is shown in dashed black line. Insets: distribution of AtCYP20-2 and AtCYP38 cyclophilins according to immunoblotting. (B) Fractionation of PPIase activity from the thylakoid lumen of Arabidopsis mutant lacking AtCYP20-2. The inset shows the absence of AtCYP20-2 in the mutant and presence of AtCYP38 in both mutant and wild type plants, as determined by immunoblotting. The protein absorbance at 280 nm is shown in dashed black line; the PPIase activity measured with A-peptide is depicted by bold gray line. (C) Collision-induced fragmentation spectrum and sequencing of a tryptic peptide from AtCYP20-3 (fraction 9). The signal of the doubly charged peptide ion is indicated at m/z 644.9. The sequence of the peptide (corresponding to amino acids 111–122 in the initial translation product of AtCYP20-3) is shown and the major detected b (N-terminal) and y (C-terminal) fragment ions are indicated.
25
4 3 2
IVMGLFGEVVPK bn 2 3 4 5
9 10
60 b2 40
y10
b5 a2 20
b4 y3 b3 y4a5
y7
y 9 b9
b10 y11
y8
0 200
400
600
800 m/z
1000
1200
yn
A. Shapiguzov et al. / FEBS Letters 580 (2006) 3671–3676
3673
c Fig. 2. Identification of AtFKBP13 as a single active PPIase in cationexchange FPLC fractionation and inhibition of its activity by DTT. (A) Separation of proteins from the Resource Q void on the Resource S column (representative of three experiments). The protein absorbance at 280 nm is shown in dashed black line. PPIase activity was measured with two different synthetic peptides before and after incubation of the fractions with 2 mM DTT, as indicated by the corresponding lines. Inset: silver stained gel with the fraction 6 that corresponds to the peak of PPIase activity. (B,C) Collision-induced fragmentation spectra and sequencing of two tryptic peptides from AtFKBP13 (fraction 6). The sequences of the peptides are shown and the major detected fragment b-ions and y-ions are indicated. (B) The fragmentation spectrum of the doubly charged ion at m/z 494.2, as indicated. The determined peptide sequence corresponds to the positions 125–132 in the initial translation product of AtFKBP13. (C) The fragmentation spectrum of the doubly charged ion at m/z 409.7, as indicated. The peptide sequence corresponds to the positions 133–139 in the initial translation product of AtFKBP13.
The largest portion of the total lumenal PPIase activity was recovered in the void from the anion-exchange column and was subsequently applied on and completely bound to a cation-exchange column. Analysis of the fractions eluted by salt gradient revealed a single peak of PPIase activity in the fractions 5–7 (Fig. 2A). The activity in this peak was variable in three different preparations performed in the study (Table 1). The corresponding fractions contained a major or single protein band of about 14 kDa (Fig. 2A, inset) correlating with the activity profile. MALDI-TOF analysis revealed a peptide mass fingerprint that matched AtFKBP13 (locus At5g45680). The identity of AtFKBP13 was confirmed by sequencing of A
35
Fraction 6
PPIase activity (%)
30
kDa 45 -
A280 A-peptide A-peptide, 2 mM DTT L-peptide L-peptide, 2 mM DTT
25 20
30 20 -
15 14.3 10
6.5 -
5 0 1
B
5
15 20 Fraction
10
25
30
100 494.22+ 80
Intensity (%)
7 6 5 4 3 2 1 yn
60
VFDSSYNR
a1
bn
2 3
a2 40 b2 20
y1
y5 y2
b3
y6
y4
y3
y7
0 100
C
300
500 m/z
700
900
100 5 4 3 2 1 yn
GKPLTFR
80 Intensity (%)
remaining PPIase activity was recovered in the void containing unbound proteins and was further separated by cationexchange FPLC (see below). Immunoblotting analysis revealed cyclophilin AtCYP20-2 (locus At5g13120) in the fractions 6–8 containing the major PPIase peak from the first separation (Fig. 1A, inset). The fractions in this peak contained many proteins (data not shown), which implied a possible presence of several PPIases. To check this possibility we isolated thylakoid lumen from Arabidopsis mutant lacking AtCYP20-2 (see inset in Fig. 1B) and subjected it to an identical chromatographic separation. Fig. 1B shows that the first peak of PPIase activity was specifically absent in the fractions 6–8 from the mutant thylakoid lumen, as compared with the fractions containing AtCYP20-2 from the wild type plants (Fig. 1A). Thus, we concluded that PPIase activity in the fractions 6–8 (Fig. 1A) was determined by AtCYP20-2. To identify the PPIase responsible for the second, minor peak of activity (Fig. 1A) we separated the proteins from fraction 9 by SDS–PAGE and analyzed seven individual protein bands found in this fraction by peptide mass fingerprinting. The MALDI-TOF analyses revealed that two most abundant proteins were ‘‘20-kDa protein with a PsbP domain’’ (At3g56650) [8] and ‘‘11.6-kDa pentapeptide protein’’ (At2g44920) [8] (data not shown). The 20 kDa cyclophilin AtCYP20-3 (locus At3g62030) was the only immunophilin found in this fraction. This cyclophilin has been earlier described as an active PPIase localized in the stroma of chloroplast [18]. The identity of AtCYP20-3 was confirmed by sequencing of two peptides using electrospray ionization mass spectrometry: the fragmentation spectrum and corresponding sequence of one of these peptides are shown in Fig. 1C. Accordingly, the peak of PPIase activity detected in fractions 9 and 10 was assigned to the stromal cyclophilin AtCYP20-3 that appeared in thylakoid lumen preparations as a contamination [8–10]. As the result, the anionexchange separation of thylakoid lumenal proteins revealed only one active PPIase from the lumen: AtCYP20-2. Notably, three other lumenal immunophilins with low isoelectric points, AtFKBP16-3 (locus At2g43560), AtCYP37 (locus At3g15520) and AtCYP38 (locus At3g01480) [8,10], were also expected to bind to this column. Cyclophilin AtCYP38 that has the lowest isoelectric point of all lumenal immunophilins, was indeed tightly bound to the column and eluted only with 250– 400 mM NaCl (fractions 24–27), as was revealed by immunoblotting analysis (Fig. 1A, inset). Surprisingly, no PPIase activity was detected in the corresponding fractions.
bn
60
2
409.72+
40
y5
b2
20 y1
y52+ y2
y3
y4
0 150
300
450 m/z
600
3674
A. Shapiguzov et al. / FEBS Letters 580 (2006) 3671–3676
Table 1 Distribution of PPIase activity from thylakoid lumen of Arabidopsis between the individual immunophilins Synthetic substrate peptide
A-peptide L-peptide
% of the total lumenal PPIase activity AtFKBP13 (locus At5g45680, fractions 3–8 from the cation-exchange column)
AtCYP20-2 (locus At5g13120, fractions 6–8 from the anion-exchange column)
AtCYP20-3 (locus At3g62030, fractions 9–10 from the anion-exchange column)
42.3 ± 6.2 61.3 ± 14.5
17.2 ± 2.1 3.0 ± 0.7
8.3 ± 3.6 0.9 ± 0.5
The activity is assayed with two different synthetic substrate peptides. The results presented are the mean values from chromatographic separations of proteins from three independent preparations of the thylakoid lumen ± S.D. soluble membranelumen associated lumen
A
AtCYP38 AtCYP20-2 PsbO Plastocyanin
B PPIase activity (µmol/min)
five peptides from this protein using electrospray ionization mass spectrometry. The fragmentation spectra for two of these peptides are shown in Fig. 2B and C. These findings demonstrated that the major PPIase activity in thylakoid lumen of Arabidopsis belonged to an FKBP-type immunophilin AtFKBP13, as summarized in Table 1. The sum of the PPIase activities recovered in the chromatographic fractions constituted about 70% of the activity in the corresponding preparations of Arabidopsis lumen (Table 1). The major loss of activity was observed during cation-exchange chromatography. This separation yielded virtually pure AtFKBP13 (insert in Fig. 2A), however, PPIase activity of this FKBP was not very stable. PPIase activity of recombinant AtFKBP13 was previously shown to be redox regulated and diminishing upon treatment with reducing agents [11]. To check if this is true for the isolated plant-derived enzyme, we incubated the fractions containing AtFKBP13 with 2 mM DTT for 30 min at room temperature, which resulted in 3- to 4-fold decrease in PPIase activity with both peptide substrates (Fig. 2A). The redox sensitivity of AtFKBP13 might explain variability in PPIase activity that was observed in the corresponding fractions. The inhibition of the enzyme from the plant differed from that of the recombinant AtFKBP13, which was most susceptible to reduction by a combination of thioredoxin and DTT, while DTT alone had a minimal effect [11]. Importantly, activity analyses with both substrate peptides revealed that addition of 2 mM DTT to the isolated thylakoid lumen of Arabidopsis also resulted in 2- to 3-fold inhibition of the total PPIase activity. We also attempted to increase/restore the PPIase activity of AtFKBP13 by oxidizing agents. However, oxidized DTT (20 mM) or Cu2+ (100 lM) were found inefficient. Two cyclophilins from thylakoid lumen, spinach TLP40 [12,19] and Arabidopsis AtCYP20-2 [20], have been shown to bind to thylakoid membranes. To check if the established protocol of thylakoid lumen isolation [8,16] enables efficient release of membrane-associated PPIases, we made an additional protein extraction from thylakoid membranes with a buffer containing 1 M NaCl. The immunoblotting analysis demonstrated that the high-salt treatment removed both AtCYP20-2 and AtCYP38 (Arabidopsis homolog of TLP40) from the membranes (Fig. 3A). The amount of AtCYP38 in the membraneassociated lumen (high-salt fraction) was higher than in the ‘‘conventional’’ soluble lumen fraction, while AtCYP20-2 was about equally distributed between the two fractions (Fig. 3A). The high-salt treatment of the membranes also released substantial PPIase activity, comparable to that in the soluble lumen preparation (Fig. 3B). To characterize this activity, the proteins of the membrane-associated lumen were desalted and subjected to sequential anion- and cation-exchange chromatography, as
20
A-peptide L-peptide
16 12 8 4 0
soluble lumen
membraneassociated lumen
Fig. 3. Distribution of immunophilins AtCYP38 and AtCYP20-2, and PPIase activity between the soluble lumen and the membraneassociated fraction of Arabidopsis thylakoids. (A) Immunoblotting analysis of AtCYP38 and AtCYP20-2 and control proteins PsbO and plastocyanin. The controls show that PsbO (the extrinsic component of PS II) was distributed between the lumen and the thylakoid membranes, while plastocyanin was almost completely removed from the membranes within the soluble lumen. (B) Distribution of PPIase activity between the soluble and the membrane-associated protein fractions obtained from the thylakoids containing 30 mg of chlorophyll. Activity was measured with two different substrate peptides, as indicated, and expressed in the enzymatic units.
described above. The pattern of PPIase activity peaks appeared to be almost identical to that of the soluble lumen shown in Figs. 1A and 2A. The only difference was the absence of the minor peak corresponding to the stromal cyclophilin AtCYP20-3. Thus, AtCYP20-3 was effectively removed from the membranes during the preparation of the soluble lumenal proteins.
4. Discussion In this study we mapped the distribution of PPIase activity among immunophilins of chloroplast thylakoid lumen from the model plant A. thaliana. It was expected that the combination of enzymatic and proteomic analyses would unravel
A. Shapiguzov et al. / FEBS Letters 580 (2006) 3671–3676
numerous PPIases corresponding to a complex repertoire of immunophilins previously defined in this cellular compartment by proteomics and bioinformatics [8,10,21]. Surprisingly, we found that all PPIase activity measured in thylakoid lumen with two synthetic substrate peptides was determined by only two immunophilins, AtFKBP13 and AtCYP20-2. The lack of detectable PPIase activity towards these peptides in case of the other lumenal immunophilins suggests that they have evolved high substrate specificities for particular protein partners. Indeed, some immunophilins are known to perform site-specific docking to distinct target proteins and to trigger structural rearrangements of protein complexes [1,2,5,6]. It has also been suggested [22] that lumenal immunophilins have specific receptors in the thylakoid membrane. In agreement with this, we revealed that significant amounts of active PPIases, AtFKBP13 and AtCYP20-2, as well as of the cyclophilin AtCYP38 were bound to the thylakoid membranes. We detected no PPIase activity of AtCYP38, while its spinach homolog TLP40 (82% of sequence identity to AtCYP38 in the mature form) was an active PPIase [12]. The molecular basis for this difference is unknown. TLP40 is a multi-domain cyclophilin that interacts with a thylakoid membrane protein phosphatase regulating thylakoid protein dephosphorylation [12,19,23]. Notably, Arabidopsis mutants lacking AtCYP38 demonstrate drastically retarded growth and abnormal properties of thylakoid membranes (Khrouchtchova, Edvardsson, Shapiguzov, Paakkarinen, Hansson, Haldrup, Aro, Scheller and Vener, unpublished data), which proves the importance of this cyclophilin for assembly and regulation of the photosynthetic apparatus despite of the lack of PPIase activity towards the peptide substrates. Significant PPIase activity measured in thylakoid lumen from spinach using the A-peptide substrate was ascribed to cyclophilin TLP20 [13]. Here we show that AtCYP20-2, an Arabidopsis homolog of TLP20, is also the active PPIase in the assay with the A-peptide, while its activity towards the L-peptide is relatively low. Another active PPIase in the thylakoid lumen of Arabidopsis is AtFKBP13. Characterization of recombinant AtFKBP13 demonstrated that its PPIase activity was redox regulated [11]. Our finding that this enzyme is the most active lumenal PPIase supports the novel paradigm postulating oxidative control of enzymatic activities in the thylakoid lumen [24]. The activity of the isolated natural AtFKBP13 was inhibited 3- to 4-fold by the addition of the thiol-reducing agent DTT. On the contrary, this enzyme was not activated by simple chemical oxidation with either oxidized DTT or Cu2+. These data imply that oxidative activation of AtFKBP13 in the lumen is enzymatically controlled and the protein catalysts involved in this process are yet to be found. Being the site of photosynthetic oxygen production, the chloroplast thylakoid lumen is highly susceptible to oxidative stress. Our work suggests that molecular mechanisms protecting photosynthetic machinery against oxidative stress may involve redox regulated catalysis of protein folding in the thylakoid lumen and at the surface of thylakoid membrane, as it is judged on the basis of the PPIase activity. Acknowledgements: We thank Dr. Gennady Bronnikov for advices on kinetic analyses of PPIase reactions and Dr. Inger Carlberg for providing purified oxidized DTT. This work was supported by grants from the Swedish Research Council, the Swedish Research Council for Environment, Agriculture and Space Planning, and Nordiskt Kontaktorgan fo¨r Jordbruksforskning.
3675
References [1] Fischer, G. and Aumuller, T. (2003) Regulation of peptide bond cis/trans isomerization by enzyme catalysis and its implication in physiological processes. Rev. Physiol. Biochem. Pharmacol. 148, 105–150. [2] Fischer, G., Tradler, T. and Zarnt, T. (1998) The mode of action of peptidyl prolyl cis/trans isomerases in vivo: binding vs. catalysis. FEBS Lett. 426, 17–20. [3] Harrison, R.K. and Stein, R.L. (1990) Substrate specificities of the peptidyl prolyl cis–trans isomerase activities of cyclophilin and FK-506 binding protein: evidence for the existence of a family of distinct enzymes. Biochemistry 29, 3813–3816. [4] Kofron, J.L., Kuzmic, P., Kishore, V., Colon-Bonilla, E. and Rich, D.H. (1991) Determination of kinetic constants for peptidyl prolyl cis–trans isomerases by an improved spectrophotometric assay. Biochemistry 30, 6127–6134. [5] Schiene-Fischer, C. and Yu, C. (2001) Receptor accessory folding helper enzymes: the functional role of peptidyl prolyl cis/trans isomerases. FEBS Lett. 495, 1–6. [6] Vener, A.V. (2001) Peptidyl-prolyl isomerases and regulation of photosynthetic functions in: Regulation of Photosynthesis (Aro, E.M. and Andersson, B., Eds.), pp. 177–193, Kluwer Academic Publishers, Dordrecht. [7] He, Z., Li, L. and Luan, S. (2004) Immunophilins and parvulins. Superfamily of peptidyl prolyl isomerases in Arabidopsis. Plant Physiol. 134, 1248–1267. [8] Schubert, M., Petersson, U.A., Haas, B.J., Funk, C., Schro¨der, W.P. and Kieselbach, T. (2002) Proteome map of the chloroplast lumen of Arabidopsis thaliana. J. Biol. Chem. 277, 8354– 8365. [9] Peltier, J.B., Friso, G., Kalume, D.E., Roepstorff, P., Nilsson, F., Adamska, I. and van Wijk, K.J. (2000) Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins. Plant Cell 12, 319– 341. [10] Peltier, J.B., Emanuelsson, O., Kalume, D.E., Ytterberg, J., Friso, G., Rudella, A., Liberles, D.A., Soderberg, L., Roepstorff, P., von Heijne, G. and van Wijk, K.J. (2002) Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction. Plant Cell 14, 211–236. [11] Gopalan, G., He, Z., Balmer, Y., Romano, P., Gupta, R., Heroux, A., Buchanan, B.B., Swaminathan, K. and Luan, S. (2004) Structural analysis uncovers a role for redox in regulating FKBP13, an immunophilin of the chloroplast thylakoid lumen. Proc. Natl. Acad. Sci. USA 101, 13945–13950. [12] Fulgosi, H., Vener, A.V., Altschmied, L., Herrmann, R.G. and Andersson, B. (1998) A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen. EMBO J. 17, 1577–1587. [13] Edvardsson, A., Eshaghi, S., Vener, A.V. and Andersson, B. (2003) The major peptidyl-prolyl isomerase activity in thylakoid lumen of plant chloroplasts belongs to a novel cyclophilin TLP20. FEBS Lett. 542, 137–141. [14] Nore´n, H., Svensson, P. and Andersson, B. (2004) A convenient and versatile hydroponic cultivation system for Arabidopsis thaliana. Physiol. Plant. 121, 343–348. [15] Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H., Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C., Jeske, A., Koesema, E., Meyers, C.C., Parker, H., Prednis, L., Ansari, Y., Choy, N., Deen, H., Geralt, M., Hazari, N., Hom, E., Karnes, M., Mulholland, C., Ndubaku, R., Schmidt, I., Guzman, P., Aguilar-Henonin, L., Schmid, M., Weigel, D., Carter, D.E., Marchand, T., Risseeuw, E., Brogden, D., Zeko, A., Crosby, W.L., Berry, C.C. and Ecker, J.R. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657. ˚ ., Andersson, B. and Schro¨der, W.P. [16] Kieselbach, T., Hagman, A (1998) The thylakoid lumen of chloroplasts. Isolation and characterization. J. Biol. Chem. 273, 6710–6716. [17] Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T. and Schmid, F.X. (1989) Cyclophilin and peptidyl-prolyl cis– trans isomerase are probably identical proteins. Nature 337, 476–478.
3676 [18] Lippuner, V., Chou, I.T., Scott, S.V., Ettinger, W.F., Theg, S.M. and Gasser, C.S. (1994) Cloning and characterization of chloroplast and cytosolic forms of cyclophilin from Arabidopsis thaliana. J. Biol. Chem. 269, 7863–7868. [19] Vener, A.V., Rokka, A., Fulgosi, H., Andersson, B. and Herrmann, R.G. (1999) A cyclophilin-regulated PP2A-like protein phosphatase in thylakoid membranes of plant chloroplasts. Biochemistry 38, 14955–14965. [20] Romano, P.G.N., Edvardsson, A., Ruban, A.V., Andersson, B., Vener, A.V., Gray, J.E. and Horton, P. (2004) Arabidopsis AtCYP20-2 is a light-regulated cyclophilin-type peptidyl-prolyl cis –trans isomerase associated with the photosynthetic membranes. Plant Physiol. 134, 1244–1247.
A. Shapiguzov et al. / FEBS Letters 580 (2006) 3671–3676 [21] Romano, P.G.N., Gray, J., Horton, P. and Luan, S. (2005) Plant immunophilins: functional versatility beyond protein maturation. New Phytol. 166, 753–769. [22] Kieselbach, T. and Schro¨der, W.P. (2003) The proteome of the chloroplast lumen of higher plants. Photosynth. Res. 78, 249–264. [23] Rokka, A., Aro, E.-M., Herrmann, R.G., Andersson, B. and Vener, A.V. (2000) Dephosphorylation of photosystem II reaction center proteins in plant photosynthetic membranes as an immediate response to abrupt elevation of temperature. Plant Physiol. 123, 1525–1536. [24] Buchanan, B.B. and Luan, S. (2005) Redox regulation in the chloroplast thylakoid lumen: a new frontier in photosynthesis research. J. Exp. Bot. 56, 1439–1447.
Profound redox sensitivity of peptidyl-prolyl isomerase activity in Arabidopsis thylakoid lumen Alexey Shapiguzov1, Anna Edvardsson, Alexander V. Vener* Division of Cell Biology, Linko¨ping University, SE-581 85 Linko¨ping, Sweden Received 5 April 2006; revised 23 May 2006; accepted 25 May 2006 Available online 5 June 2006 Edited by Richard Cogdell
Abstract Proteomic, enzymatic, and mutant analyses revealed that peptidyl-prolyl isomerase (PPIase) activity in the chloroplast thylakoid lumen of Arabidopsis is determined by two immunophilins: AtCYP20-2 and AtFKBP13. These two enzymes are responsible for PPIase activity in both soluble and membraneassociated fractions of thylakoid lumen suggesting that other lumenal immunophilins are not active towards the peptide substrates. In thiol-reducing conditions PPIase activity of the isolated AtFKBP13 and of the total thylakoid lumen is suppressed several fold. Profound redox-dependence of PPIase activity implies oxidative activation of protein folding catalysis under oxidative stress and photosynthetic oxygen production in the thylakoid lumen of plant chloroplasts. Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Immunophilin; FKBP; Cyclophilin; Peptidyl-prolyl isomerase; Thylakoid lumen; Arabidopsis thaliana
1. Introduction Immunophilins are ubiquitous receptors for immunosuppressive drugs cyclosporine A and FK506. Receptors for cyclosporine A are defined as cyclophilins, while FK506 binding proteins are called FKBPs. Despite of little sequence homology, immunophilins of the two families share similar peptidyl-prolyl cis/trans isomerase (PPIase) enzymatic activity that might be involved in the catalysis of protein folding [1–3]. The assays of PPIase activity with prolyl-containing synthetic peptides have demonstrated little dependence of cyclophilincatalyzed isomerization on peptide sequence, while FKBPs were much more efficient in isomerization of peptides with a prolyl preceded by a bulky hydrophobic residue like leucine [3,4]. The cellular functions of cyclophilins and FKBPs extend far beyond their PPIase activity: these proteins are involved in cell signaling, protein biogenesis and trafficking, cell cycle control, heat shock responses and regulation of membrane recep-
* Corresponding author. Fax: +46 13 224314. E-mail address: [email protected] (A.V. Vener). 1 Permanent address: Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, 127276 Moscow, Russia.
Abbreviations: FKBP, FK506 binding protein; PPIase, peptidyl-prolyl cis/trans isomerase
tors, channels and pores, as reviewed in [1,2,5,6]. Nevertheless, the physiological roles of numerous immunophilins in different organisms are still unknown. The genomic analysis of Arabidopsis thaliana has revealed 52 genes encoding putative immunophilins in this model plant [7]. A significant fraction of Arabidopsis cyclophilins and FKBPs has been localized in the chloroplast, particularly in the small compartment of chloroplast thylakoid lumen. Proteomic analyses of Arabidopsis thylakoid lumen [8–10] identified 3 cyclophilins and 7 FKBPs, while none of these immunophilins has been analyzed for PPIase activity. The recombinant lumenal AtFKBP13 possessed this activity and was inactivated upon reduction of two redox-active disulfide bonds specific for this FKBP [11]. Two cyclophilins TLP40 [12] and TLP20 [13] possessing PPIase activity have been isolated from thylakoid lumen of spinach. In this study, we set out to map the PPIase activities of the immunophilins from the thylakoid lumen of A. thaliana by chromatographic separations and PPIase assays with two peptide substrates that should accommodate the specificities of both cyclophilins and FKBPs. Unexpectedly, we revealed that the PPIase activity of Arabidopsis thylakoid lumen is restricted to only AtFKBP13 and AtCYP20-2. The dominant activity belonged to AtFKBP13 and was strongly inhibited by the thiol-reducing agent DTT. These findings reveal dramatic redox sensitivity of the total PPIase activity in the chloroplast thylakoid lumen. 2. Materials and methods 2.1. Plant material, isolation and chromatographic fractionation of thylakoid lumen Arabidopsis thaliana ecotype Columbia plants were grown hydroponically [14] at 20–22 °C, 8 h light/16 h dark photoperiod and light intensity 120 lmol photons/m2/s. Arabidopsis insertion line SALK_009552 was obtained from the Salk Institute collection [15]. Homozygot plants with the T-DNA insertion were identified by PCR analysis with the AtCYP20-2 gene specific primers: 5 0 -TTTGCACAGGTAAATATGCTTCATAGGAT-3 0 (forward), 5 0 -AGATCGGACTTTAAATACAAATTGCTCGT-3 0 (reverse) and the T-DNA left border specific primer 5 0 -TGGTTCACGTAGTGGGCCATCG3 0 . Plant leaves were harvested after 16 h of dark period. Soluble lumenal proteins were isolated from 100 g of wild type leaves or from 25 g of mutant leaves according to the protocol described in [8,16] with minor modifications. For Yeda press treatment the following buffer was used: 20 mM Tricine, pH 7.8, 5 mM MgCl2, and 100 mM sucrose. For isolation of membrane-associated lumenal fraction 1 M NaCl was added to the same buffer and the membranes were subjected to additional Yeda press treatment. The high-salt protein extract was desalted using Amicon Ultra-15 centrifugal filter units with 5 kDa cut-off (Millipore). The lumenal proteins were fractionated by sequential FPLC on
0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.05.054
3672
A. Shapiguzov et al. / FEBS Letters 580 (2006) 3671–3676
Resource Q and Resource S columns (Amersham Biosciences) equilibrated with 20 mM Tricine, pH 7.8 and eluted with 0–1 M NaCl linear gradient. 2.2. Measurements of PPIase activity The PPIase activity was analyzed by chymotrypsin-coupled assay as described in [17]. Peptide substrates N-succinyl-Ala-Ala-Pro-Phe-pnitroanilide (A-peptide) and N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (L-peptide) (Bachem) were dissolved in trifluoroethanol with LiCl to increase the proportion of cis isomer [4]. The reactions were started by adding peptide substrate to a mixture of protein sample and chymotrypsin (final concentration 60 lM) in 0.1 M Tricine, pH 7.8 at 10 °C. The change in absorbance at 390 nm due to release of p-nitroanilide was monitored on Lambda 25 Perkin–Elmer spectrophotometer. Reaction rate coefficients were derived by non-linear curve fitting to a firstorder rate equation using Origin 7.5 software. The enzymatic activity was calculated as a difference in the rates of catalyzed and spontaneous reactions.
established protocol [8,16] and subjected them to chromatographic fractionation by FPLC. The PPIase activity in all fractions was measured using two substrate peptides, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (A-peptide) and N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide (L-peptide), to match the specificities of both cyclophilins and FKBPs [3,4]. The anionexchange FPLC revealed two chromatographic peaks with PPIase activity, as was detected with both substrate peptides (Fig. 1A). The activity towards the A-peptide was significantly higher than towards the L-peptide (Fig. 1A) indicating the possible presence of cyclophilins in the active fractions. The
A
12 A280 A-peptide L-peptide
2.4. Identification of proteins by mass spectrometry The individual protein bands were excised from the gels and treated with trypsin (sequencing grade modified, Promega). MALDI-TOF analyses of the peptides were performed on Voyager-DE Pro (Applied Biosystems, Framingham, MA, USA). Electrospray ionization tandem mass spectra were acquired on a hybrid mass spectrometer API QSTAR Pulsar i (Applied Biosystems, Foster City, CA, USA) equipped with a nanoelectrospray ion source (MDS Protana, Odense, Denmark). The nanoelectrospray capillaries were loaded with 2 ll peptide solution in water/acetonitrile (50/50) with 1% formic acid. The collision-induced fragmentation of selected peptide ions was done using the instrument settings recommended by Applied Biosystems and manual control of collision energy.
8 6 4 2 0 AtCYP20-2
1
B
5 6 7 8 910
AtCYP38
15 20 Fraction
23 25 27
30
12 A280 A-peptide
10 PPIase activity (%)
2.3. SDS–PAGE and immunoblotting analyses Chromatographic fractions were subjected to SDS–polyacrylamide gel electrophoresis in 15% acrylamide gels that were either stained with silver or used for immunoblotting. In the latter case, the proteins were transferred to a polyvinylidene difluoride membrane (Immobilone-P, Millipore, MA, USA). The membrane was blocked with milk proteins and incubated with indicated specific antibodies, which was followed by ECL detection according to the protocol (Amersham Biosciences, Sweden). Polyclonal antiserum to AtCYP38 was raised against a synthetic peptide GPAEGFIDPSTEKTR corresponding to amino acids 211–225 of the mature protein. Polyclonal antibodies against AtCYP20-2, PsbO and plastocyanin were kindly provided by Drs. P.G.N. Romano, C. Spetea and H.V. Scheller.
PPIase activity (%)
10
W.t. Mutant AtCYP38
8
AtCYP20-2
6 4 2
3. Results
0
To map the PPIase activities in the thylakoid lumen of Arabidopsis we isolated lumenal proteins according to the
1
5
10
15 20 Fraction
c C 100
30
644.92+ y2 11 10 9 8 7
y102+
80 Intensity (%)
Fig. 1. Mapping of PPIase activity and cyclophilins separated by anion-exchange FPLC. (A) Fractionation of PPIase activity from Arabidopsis thylakoid lumen on Resource Q column (representative of three experiments). PPIase activity was measured with two different synthetic peptides as depicted by bold gray lines and indicated. The protein absorbance at 280 nm is shown in dashed black line. Insets: distribution of AtCYP20-2 and AtCYP38 cyclophilins according to immunoblotting. (B) Fractionation of PPIase activity from the thylakoid lumen of Arabidopsis mutant lacking AtCYP20-2. The inset shows the absence of AtCYP20-2 in the mutant and presence of AtCYP38 in both mutant and wild type plants, as determined by immunoblotting. The protein absorbance at 280 nm is shown in dashed black line; the PPIase activity measured with A-peptide is depicted by bold gray line. (C) Collision-induced fragmentation spectrum and sequencing of a tryptic peptide from AtCYP20-3 (fraction 9). The signal of the doubly charged peptide ion is indicated at m/z 644.9. The sequence of the peptide (corresponding to amino acids 111–122 in the initial translation product of AtCYP20-3) is shown and the major detected b (N-terminal) and y (C-terminal) fragment ions are indicated.
25
4 3 2
IVMGLFGEVVPK bn 2 3 4 5
9 10
60 b2 40
y10
b5 a2 20
b4 y3 b3 y4a5
y7
y 9 b9
b10 y11
y8
0 200
400
600
800 m/z
1000
1200
yn
A. Shapiguzov et al. / FEBS Letters 580 (2006) 3671–3676
3673
c Fig. 2. Identification of AtFKBP13 as a single active PPIase in cationexchange FPLC fractionation and inhibition of its activity by DTT. (A) Separation of proteins from the Resource Q void on the Resource S column (representative of three experiments). The protein absorbance at 280 nm is shown in dashed black line. PPIase activity was measured with two different synthetic peptides before and after incubation of the fractions with 2 mM DTT, as indicated by the corresponding lines. Inset: silver stained gel with the fraction 6 that corresponds to the peak of PPIase activity. (B,C) Collision-induced fragmentation spectra and sequencing of two tryptic peptides from AtFKBP13 (fraction 6). The sequences of the peptides are shown and the major detected fragment b-ions and y-ions are indicated. (B) The fragmentation spectrum of the doubly charged ion at m/z 494.2, as indicated. The determined peptide sequence corresponds to the positions 125–132 in the initial translation product of AtFKBP13. (C) The fragmentation spectrum of the doubly charged ion at m/z 409.7, as indicated. The peptide sequence corresponds to the positions 133–139 in the initial translation product of AtFKBP13.
The largest portion of the total lumenal PPIase activity was recovered in the void from the anion-exchange column and was subsequently applied on and completely bound to a cation-exchange column. Analysis of the fractions eluted by salt gradient revealed a single peak of PPIase activity in the fractions 5–7 (Fig. 2A). The activity in this peak was variable in three different preparations performed in the study (Table 1). The corresponding fractions contained a major or single protein band of about 14 kDa (Fig. 2A, inset) correlating with the activity profile. MALDI-TOF analysis revealed a peptide mass fingerprint that matched AtFKBP13 (locus At5g45680). The identity of AtFKBP13 was confirmed by sequencing of A
35
Fraction 6
PPIase activity (%)
30
kDa 45 -
A280 A-peptide A-peptide, 2 mM DTT L-peptide L-peptide, 2 mM DTT
25 20
30 20 -
15 14.3 10
6.5 -
5 0 1
B
5
15 20 Fraction
10
25
30
100 494.22+ 80
Intensity (%)
7 6 5 4 3 2 1 yn
60
VFDSSYNR
a1
bn
2 3
a2 40 b2 20
y1
y5 y2
b3
y6
y4
y3
y7
0 100
C
300
500 m/z
700
900
100 5 4 3 2 1 yn
GKPLTFR
80 Intensity (%)
remaining PPIase activity was recovered in the void containing unbound proteins and was further separated by cationexchange FPLC (see below). Immunoblotting analysis revealed cyclophilin AtCYP20-2 (locus At5g13120) in the fractions 6–8 containing the major PPIase peak from the first separation (Fig. 1A, inset). The fractions in this peak contained many proteins (data not shown), which implied a possible presence of several PPIases. To check this possibility we isolated thylakoid lumen from Arabidopsis mutant lacking AtCYP20-2 (see inset in Fig. 1B) and subjected it to an identical chromatographic separation. Fig. 1B shows that the first peak of PPIase activity was specifically absent in the fractions 6–8 from the mutant thylakoid lumen, as compared with the fractions containing AtCYP20-2 from the wild type plants (Fig. 1A). Thus, we concluded that PPIase activity in the fractions 6–8 (Fig. 1A) was determined by AtCYP20-2. To identify the PPIase responsible for the second, minor peak of activity (Fig. 1A) we separated the proteins from fraction 9 by SDS–PAGE and analyzed seven individual protein bands found in this fraction by peptide mass fingerprinting. The MALDI-TOF analyses revealed that two most abundant proteins were ‘‘20-kDa protein with a PsbP domain’’ (At3g56650) [8] and ‘‘11.6-kDa pentapeptide protein’’ (At2g44920) [8] (data not shown). The 20 kDa cyclophilin AtCYP20-3 (locus At3g62030) was the only immunophilin found in this fraction. This cyclophilin has been earlier described as an active PPIase localized in the stroma of chloroplast [18]. The identity of AtCYP20-3 was confirmed by sequencing of two peptides using electrospray ionization mass spectrometry: the fragmentation spectrum and corresponding sequence of one of these peptides are shown in Fig. 1C. Accordingly, the peak of PPIase activity detected in fractions 9 and 10 was assigned to the stromal cyclophilin AtCYP20-3 that appeared in thylakoid lumen preparations as a contamination [8–10]. As the result, the anionexchange separation of thylakoid lumenal proteins revealed only one active PPIase from the lumen: AtCYP20-2. Notably, three other lumenal immunophilins with low isoelectric points, AtFKBP16-3 (locus At2g43560), AtCYP37 (locus At3g15520) and AtCYP38 (locus At3g01480) [8,10], were also expected to bind to this column. Cyclophilin AtCYP38 that has the lowest isoelectric point of all lumenal immunophilins, was indeed tightly bound to the column and eluted only with 250– 400 mM NaCl (fractions 24–27), as was revealed by immunoblotting analysis (Fig. 1A, inset). Surprisingly, no PPIase activity was detected in the corresponding fractions.
bn
60
2
409.72+
40
y5
b2
20 y1
y52+ y2
y3
y4
0 150
300
450 m/z
600
3674
A. Shapiguzov et al. / FEBS Letters 580 (2006) 3671–3676
Table 1 Distribution of PPIase activity from thylakoid lumen of Arabidopsis between the individual immunophilins Synthetic substrate peptide
A-peptide L-peptide
% of the total lumenal PPIase activity AtFKBP13 (locus At5g45680, fractions 3–8 from the cation-exchange column)
AtCYP20-2 (locus At5g13120, fractions 6–8 from the anion-exchange column)
AtCYP20-3 (locus At3g62030, fractions 9–10 from the anion-exchange column)
42.3 ± 6.2 61.3 ± 14.5
17.2 ± 2.1 3.0 ± 0.7
8.3 ± 3.6 0.9 ± 0.5
The activity is assayed with two different synthetic substrate peptides. The results presented are the mean values from chromatographic separations of proteins from three independent preparations of the thylakoid lumen ± S.D. soluble membranelumen associated lumen
A
AtCYP38 AtCYP20-2 PsbO Plastocyanin
B PPIase activity (µmol/min)
five peptides from this protein using electrospray ionization mass spectrometry. The fragmentation spectra for two of these peptides are shown in Fig. 2B and C. These findings demonstrated that the major PPIase activity in thylakoid lumen of Arabidopsis belonged to an FKBP-type immunophilin AtFKBP13, as summarized in Table 1. The sum of the PPIase activities recovered in the chromatographic fractions constituted about 70% of the activity in the corresponding preparations of Arabidopsis lumen (Table 1). The major loss of activity was observed during cation-exchange chromatography. This separation yielded virtually pure AtFKBP13 (insert in Fig. 2A), however, PPIase activity of this FKBP was not very stable. PPIase activity of recombinant AtFKBP13 was previously shown to be redox regulated and diminishing upon treatment with reducing agents [11]. To check if this is true for the isolated plant-derived enzyme, we incubated the fractions containing AtFKBP13 with 2 mM DTT for 30 min at room temperature, which resulted in 3- to 4-fold decrease in PPIase activity with both peptide substrates (Fig. 2A). The redox sensitivity of AtFKBP13 might explain variability in PPIase activity that was observed in the corresponding fractions. The inhibition of the enzyme from the plant differed from that of the recombinant AtFKBP13, which was most susceptible to reduction by a combination of thioredoxin and DTT, while DTT alone had a minimal effect [11]. Importantly, activity analyses with both substrate peptides revealed that addition of 2 mM DTT to the isolated thylakoid lumen of Arabidopsis also resulted in 2- to 3-fold inhibition of the total PPIase activity. We also attempted to increase/restore the PPIase activity of AtFKBP13 by oxidizing agents. However, oxidized DTT (20 mM) or Cu2+ (100 lM) were found inefficient. Two cyclophilins from thylakoid lumen, spinach TLP40 [12,19] and Arabidopsis AtCYP20-2 [20], have been shown to bind to thylakoid membranes. To check if the established protocol of thylakoid lumen isolation [8,16] enables efficient release of membrane-associated PPIases, we made an additional protein extraction from thylakoid membranes with a buffer containing 1 M NaCl. The immunoblotting analysis demonstrated that the high-salt treatment removed both AtCYP20-2 and AtCYP38 (Arabidopsis homolog of TLP40) from the membranes (Fig. 3A). The amount of AtCYP38 in the membraneassociated lumen (high-salt fraction) was higher than in the ‘‘conventional’’ soluble lumen fraction, while AtCYP20-2 was about equally distributed between the two fractions (Fig. 3A). The high-salt treatment of the membranes also released substantial PPIase activity, comparable to that in the soluble lumen preparation (Fig. 3B). To characterize this activity, the proteins of the membrane-associated lumen were desalted and subjected to sequential anion- and cation-exchange chromatography, as
20
A-peptide L-peptide
16 12 8 4 0
soluble lumen
membraneassociated lumen
Fig. 3. Distribution of immunophilins AtCYP38 and AtCYP20-2, and PPIase activity between the soluble lumen and the membraneassociated fraction of Arabidopsis thylakoids. (A) Immunoblotting analysis of AtCYP38 and AtCYP20-2 and control proteins PsbO and plastocyanin. The controls show that PsbO (the extrinsic component of PS II) was distributed between the lumen and the thylakoid membranes, while plastocyanin was almost completely removed from the membranes within the soluble lumen. (B) Distribution of PPIase activity between the soluble and the membrane-associated protein fractions obtained from the thylakoids containing 30 mg of chlorophyll. Activity was measured with two different substrate peptides, as indicated, and expressed in the enzymatic units.
described above. The pattern of PPIase activity peaks appeared to be almost identical to that of the soluble lumen shown in Figs. 1A and 2A. The only difference was the absence of the minor peak corresponding to the stromal cyclophilin AtCYP20-3. Thus, AtCYP20-3 was effectively removed from the membranes during the preparation of the soluble lumenal proteins.
4. Discussion In this study we mapped the distribution of PPIase activity among immunophilins of chloroplast thylakoid lumen from the model plant A. thaliana. It was expected that the combination of enzymatic and proteomic analyses would unravel
A. Shapiguzov et al. / FEBS Letters 580 (2006) 3671–3676
numerous PPIases corresponding to a complex repertoire of immunophilins previously defined in this cellular compartment by proteomics and bioinformatics [8,10,21]. Surprisingly, we found that all PPIase activity measured in thylakoid lumen with two synthetic substrate peptides was determined by only two immunophilins, AtFKBP13 and AtCYP20-2. The lack of detectable PPIase activity towards these peptides in case of the other lumenal immunophilins suggests that they have evolved high substrate specificities for particular protein partners. Indeed, some immunophilins are known to perform site-specific docking to distinct target proteins and to trigger structural rearrangements of protein complexes [1,2,5,6]. It has also been suggested [22] that lumenal immunophilins have specific receptors in the thylakoid membrane. In agreement with this, we revealed that significant amounts of active PPIases, AtFKBP13 and AtCYP20-2, as well as of the cyclophilin AtCYP38 were bound to the thylakoid membranes. We detected no PPIase activity of AtCYP38, while its spinach homolog TLP40 (82% of sequence identity to AtCYP38 in the mature form) was an active PPIase [12]. The molecular basis for this difference is unknown. TLP40 is a multi-domain cyclophilin that interacts with a thylakoid membrane protein phosphatase regulating thylakoid protein dephosphorylation [12,19,23]. Notably, Arabidopsis mutants lacking AtCYP38 demonstrate drastically retarded growth and abnormal properties of thylakoid membranes (Khrouchtchova, Edvardsson, Shapiguzov, Paakkarinen, Hansson, Haldrup, Aro, Scheller and Vener, unpublished data), which proves the importance of this cyclophilin for assembly and regulation of the photosynthetic apparatus despite of the lack of PPIase activity towards the peptide substrates. Significant PPIase activity measured in thylakoid lumen from spinach using the A-peptide substrate was ascribed to cyclophilin TLP20 [13]. Here we show that AtCYP20-2, an Arabidopsis homolog of TLP20, is also the active PPIase in the assay with the A-peptide, while its activity towards the L-peptide is relatively low. Another active PPIase in the thylakoid lumen of Arabidopsis is AtFKBP13. Characterization of recombinant AtFKBP13 demonstrated that its PPIase activity was redox regulated [11]. Our finding that this enzyme is the most active lumenal PPIase supports the novel paradigm postulating oxidative control of enzymatic activities in the thylakoid lumen [24]. The activity of the isolated natural AtFKBP13 was inhibited 3- to 4-fold by the addition of the thiol-reducing agent DTT. On the contrary, this enzyme was not activated by simple chemical oxidation with either oxidized DTT or Cu2+. These data imply that oxidative activation of AtFKBP13 in the lumen is enzymatically controlled and the protein catalysts involved in this process are yet to be found. Being the site of photosynthetic oxygen production, the chloroplast thylakoid lumen is highly susceptible to oxidative stress. Our work suggests that molecular mechanisms protecting photosynthetic machinery against oxidative stress may involve redox regulated catalysis of protein folding in the thylakoid lumen and at the surface of thylakoid membrane, as it is judged on the basis of the PPIase activity. Acknowledgements: We thank Dr. Gennady Bronnikov for advices on kinetic analyses of PPIase reactions and Dr. Inger Carlberg for providing purified oxidized DTT. This work was supported by grants from the Swedish Research Council, the Swedish Research Council for Environment, Agriculture and Space Planning, and Nordiskt Kontaktorgan fo¨r Jordbruksforskning.
3675
References [1] Fischer, G. and Aumuller, T. (2003) Regulation of peptide bond cis/trans isomerization by enzyme catalysis and its implication in physiological processes. Rev. Physiol. Biochem. Pharmacol. 148, 105–150. [2] Fischer, G., Tradler, T. and Zarnt, T. (1998) The mode of action of peptidyl prolyl cis/trans isomerases in vivo: binding vs. catalysis. FEBS Lett. 426, 17–20. [3] Harrison, R.K. and Stein, R.L. (1990) Substrate specificities of the peptidyl prolyl cis–trans isomerase activities of cyclophilin and FK-506 binding protein: evidence for the existence of a family of distinct enzymes. Biochemistry 29, 3813–3816. [4] Kofron, J.L., Kuzmic, P., Kishore, V., Colon-Bonilla, E. and Rich, D.H. (1991) Determination of kinetic constants for peptidyl prolyl cis–trans isomerases by an improved spectrophotometric assay. Biochemistry 30, 6127–6134. [5] Schiene-Fischer, C. and Yu, C. (2001) Receptor accessory folding helper enzymes: the functional role of peptidyl prolyl cis/trans isomerases. FEBS Lett. 495, 1–6. [6] Vener, A.V. (2001) Peptidyl-prolyl isomerases and regulation of photosynthetic functions in: Regulation of Photosynthesis (Aro, E.M. and Andersson, B., Eds.), pp. 177–193, Kluwer Academic Publishers, Dordrecht. [7] He, Z., Li, L. and Luan, S. (2004) Immunophilins and parvulins. Superfamily of peptidyl prolyl isomerases in Arabidopsis. Plant Physiol. 134, 1248–1267. [8] Schubert, M., Petersson, U.A., Haas, B.J., Funk, C., Schro¨der, W.P. and Kieselbach, T. (2002) Proteome map of the chloroplast lumen of Arabidopsis thaliana. J. Biol. Chem. 277, 8354– 8365. [9] Peltier, J.B., Friso, G., Kalume, D.E., Roepstorff, P., Nilsson, F., Adamska, I. and van Wijk, K.J. (2000) Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins. Plant Cell 12, 319– 341. [10] Peltier, J.B., Emanuelsson, O., Kalume, D.E., Ytterberg, J., Friso, G., Rudella, A., Liberles, D.A., Soderberg, L., Roepstorff, P., von Heijne, G. and van Wijk, K.J. (2002) Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction. Plant Cell 14, 211–236. [11] Gopalan, G., He, Z., Balmer, Y., Romano, P., Gupta, R., Heroux, A., Buchanan, B.B., Swaminathan, K. and Luan, S. (2004) Structural analysis uncovers a role for redox in regulating FKBP13, an immunophilin of the chloroplast thylakoid lumen. Proc. Natl. Acad. Sci. USA 101, 13945–13950. [12] Fulgosi, H., Vener, A.V., Altschmied, L., Herrmann, R.G. and Andersson, B. (1998) A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen. EMBO J. 17, 1577–1587. [13] Edvardsson, A., Eshaghi, S., Vener, A.V. and Andersson, B. (2003) The major peptidyl-prolyl isomerase activity in thylakoid lumen of plant chloroplasts belongs to a novel cyclophilin TLP20. FEBS Lett. 542, 137–141. [14] Nore´n, H., Svensson, P. and Andersson, B. (2004) A convenient and versatile hydroponic cultivation system for Arabidopsis thaliana. Physiol. Plant. 121, 343–348. [15] Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H., Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C., Jeske, A., Koesema, E., Meyers, C.C., Parker, H., Prednis, L., Ansari, Y., Choy, N., Deen, H., Geralt, M., Hazari, N., Hom, E., Karnes, M., Mulholland, C., Ndubaku, R., Schmidt, I., Guzman, P., Aguilar-Henonin, L., Schmid, M., Weigel, D., Carter, D.E., Marchand, T., Risseeuw, E., Brogden, D., Zeko, A., Crosby, W.L., Berry, C.C. and Ecker, J.R. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657. ˚ ., Andersson, B. and Schro¨der, W.P. [16] Kieselbach, T., Hagman, A (1998) The thylakoid lumen of chloroplasts. Isolation and characterization. J. Biol. Chem. 273, 6710–6716. [17] Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T. and Schmid, F.X. (1989) Cyclophilin and peptidyl-prolyl cis– trans isomerase are probably identical proteins. Nature 337, 476–478.
3676 [18] Lippuner, V., Chou, I.T., Scott, S.V., Ettinger, W.F., Theg, S.M. and Gasser, C.S. (1994) Cloning and characterization of chloroplast and cytosolic forms of cyclophilin from Arabidopsis thaliana. J. Biol. Chem. 269, 7863–7868. [19] Vener, A.V., Rokka, A., Fulgosi, H., Andersson, B. and Herrmann, R.G. (1999) A cyclophilin-regulated PP2A-like protein phosphatase in thylakoid membranes of plant chloroplasts. Biochemistry 38, 14955–14965. [20] Romano, P.G.N., Edvardsson, A., Ruban, A.V., Andersson, B., Vener, A.V., Gray, J.E. and Horton, P. (2004) Arabidopsis AtCYP20-2 is a light-regulated cyclophilin-type peptidyl-prolyl cis –trans isomerase associated with the photosynthetic membranes. Plant Physiol. 134, 1244–1247.
A. Shapiguzov et al. / FEBS Letters 580 (2006) 3671–3676 [21] Romano, P.G.N., Gray, J., Horton, P. and Luan, S. (2005) Plant immunophilins: functional versatility beyond protein maturation. New Phytol. 166, 753–769. [22] Kieselbach, T. and Schro¨der, W.P. (2003) The proteome of the chloroplast lumen of higher plants. Photosynth. Res. 78, 249–264. [23] Rokka, A., Aro, E.-M., Herrmann, R.G., Andersson, B. and Vener, A.V. (2000) Dephosphorylation of photosystem II reaction center proteins in plant photosynthetic membranes as an immediate response to abrupt elevation of temperature. Plant Physiol. 123, 1525–1536. [24] Buchanan, B.B. and Luan, S. (2005) Redox regulation in the chloroplast thylakoid lumen: a new frontier in photosynthesis research. J. Exp. Bot. 56, 1439–1447.