A peptide methionine sulfoxide reductase highly expressedin photosynthetic tissue in Arabidopsis thaliana can protectthe chaperone-like activity of a chloroplast-localized smallheat shock protein

Niklas Gustavsson1, Bas PA. Kokke2, Ulrika HaÈ rndahl1, Maria Silow1, Ulrike Bechtold3, Zaruhi Poghosyan4,

Denis Murphy3,², Wilbert C. Boelens2 and Cecilia Sundby1,*

1Department of Biochemistry, Lund University, Sweden, 2Department of Biochemistry, University of Nijmegen,

PO Box 9101, 6500 HB Nijmegen, The Netherlands,3School of Applied Sciences, University of Glamorgan, Cardiff CF37 1DL, UK, and4School of Biological Sciences, University of East Anglia, Norwich, UK

Received 16 July 2001; revised 9 November 2001; accepted 23 November 2001.*For correspondence (fax +46 46 222 4872; e-mail Cecilia@biochem.lu.se).²Present address: Lipoprotein Research Centre, 81 Christchurch Road, Norwich, NR2 3NG, UK.

Summary

The oxidation of methionine residues in proteins to methionine sulfoxides occurs frequently and protein

repair by reduction of the methionine sulfoxides is mediated by an enzyme, peptide methionine

sulfoxide reductase (PMSR, EC 1.8.4.6), universally present in the genomes of all so far sequenced

organisms. Recently, ®ve PMSR-like genes were identi®ed in Arabidopsis thaliana, including one

plastidic isoform, chloroplast localised plastidial peptide methionine sulfoxide reductase (pPMSR) that

was chloroplast-localized and highly expressed in actively photosynthesizing tissue (Sadanandom A

et al., 2000). However, no endogenous substrate to the pPMSR was identi®ed. Here we report that a set

of highly conserved methionine residues in Hsp21, a chloroplast-localized small heat shock protein, can

become sulfoxidized and thereafter reduced back to methionines by this pPMSR. The pPMSR activity

was evaluated using recombinantly expressed pPMSR and Hsp21 from Arabidopsis thaliana and a direct

detection of methionine sulfoxides in Hsp21 by mass spectrometry. The pPMSR-catalyzed reduction of

Hsp21 methionine sulfoxides occurred on a minute time-scale, was ultimately DTT-dependent and led to

recovery of Hsp21 conformation and chaperone-like activity, both of which are lost upon methionine

sulfoxidation (HaÈ rndahl et al., 2001). These data indicate that one important function of pPMSR may be

to prevent inactivation of Hsp21 by methionine sulfoxidation, since small heat shock proteins are crucial

for cellular resistance to oxidative stress.

Keywords: peptide methionine sulfoxide reductase, small heat shock protein, methionine sulfoxidation,

oxidative stress, chaperone-like activity, redox regulation.

Introduction

Oxidation of proteins occurs frequently in cells and can

have deleterious effects on protein structure and func-

tion. Apart from general oxidative damage leading to

fragmentation and carbonylation of the peptide back-

bone, speci®c modi®cations of certain amino acid side

chains are common during oxidative stress. Cysteine

and methionine both contain a sulfur atom in their side

chains and are among the most easily oxidized amino

acids (Vogt, 1995).

Loss of protein function due to methionine sulfoxidation

has been reported in numerous cases (Vogt, 1995). For

example, the accumulation of methionine sulfoxides in

calmodulin from brains of aging rats is accompanied by a

decrease in the ability of calmodulin to activate the plasma

membrane Ca-ATPase (Gao et al., 1998). Another interest-

ing example is the oxidation-induced inactivation of a

protease in two different forms of the human infectious

virus (HIV), HIV-1 and HIV-2 (Davis et al., 2000). In the HIV-1

The Plant Journal (2002) 29(5), 545±553

ã 2002 Blackwell Science Ltd 545

protease, inactivation is mediated by glutathionylation of

cysteine residue 95, an oxidative cross-linking reaction

between glutathione and the cysteine residue, forming a

disul®de bridge. This reaction is reversible by thiol-

transferase, a glutathione-removing enzyme. In the other

viral form, HIV-2, the protease differs from the protease in

HIV-1 in that the cysteine at position 95 is replaced with a

methionine. Upon methionine sulfoxidation with hydro-

gen peroxide, the protease activity is inhibited. Thus,

although substitution of methionine for a cysteine residue

in a protein is usually considered a non-conservative

amino acid change, they both share the property of a

susceptibility to oxidative modi®cation which is reversible.

Methionine sulfoxides can be reduced back to the

methionines by a thioredoxin-dependent enzyme, peptide

methionine sulfoxide reductase, PMSR (Abrams et al.,

1981; Brot et al., 1981; Moskovitz et al., 1995;

Sadanandom et al., 1996). Enzymatic reduction of methio-

nine sulfoxides by PMSR provides the cell with a way to

repair proteins damaged by reactive oxygen species

instead of having them degraded followed by de novo

synthesis. It has also been suggested that methionines can

act as endogenous antioxidants by shielding other oxida-

tion-sensitive amino acids in the reactive center of

enzymes, such as the glutamine synthetase, from being

oxidized (Levine et al., 1996). This would keep the active

site intact in spite of the oxidative stress, after which the

methionine sulfoxides so formed can be reduced by PMSR

back to methionines, once again ready to defend the

enzyme against an oxidative attack.

Indication on methionine sulfoxide reductase activity in

the chloroplast was ®rst shown using extracts from

chloroplasts and an arti®cial substrate (SaÂnchez et al.,

1983). Recently, ®ve PMSR-like genes were identi®ed in

Arabidopsis thaliana, including one plastidic isoform

(pPMSR) that was highly expressed in actively photo-

synthesizing tissue (Sadanandom et al., 2000). The gene

for the pPMSR was cloned and an enzymatic activity of

recombinantly expressed pPMSR was determined by

measuring repair of a model substrate protein, oxidized

bovine a-1-proteinase inhibitor (Sadanandom et al., 2000).

Since the pPMSR was differently regulated than the

cytoplasmic form of PMSR, a novel function was sug-

gested for the pPMSR compared with the cytosolic PMSR,

but the actual function of the pPMSR, and the identity of its

endogenous substrate proteins, remained to be deter-

mined.

This prompted us to investigate whether methionine

sulfoxidation of the many conserved methionines in a

chloroplast-localized protein, Hsp21, could be reversed by

pPMSR. Since both proteins are localized to the chlor-

oplast, there is the possibility for them to act in a co-

operating mode. The Hsp21 protein is a small heat shock

protein (sHsp) (Caspers et al., 1995) and has a unique

conserved region towards the N-terminus, which is extra-

ordinary rich in methionine residues and predicted to form

an amphipathic a-helix with all the methionines exposed

on one side (Chen and Vierling, 1991). The methionines in

the amphipathic helix of Hsp21 can presumably recognize

various hydrophobic stretches in partially unfolded

proteins. Hsp21 like other sHsps shows a chaperone-like

activity such that partially unfolded proteins can be

protected and prevented from aggregation by binding to

sHsps (Ehrnsperger et al., 1997; Horwitz, 1992; Jakob et al.,

1993) and then be refolded by other chaperones and ATP

(Lee and Vierling, 2000). Following methionine sulfoxida-

tion, the chaperone-like activity of Hsp21 is lost concomi-

tant with a conformational change (HaÈ rndahl et al., 2001).

We have used a reconstituted system with puri®ed

recombinant Arabidopsis thaliana proteins and evaluated

pPMSR activity by direct determination of methionine

sulfoxides by mass spectrometry. We found that methio-

nine sulfoxides in the amphipathic a-helix of Hsp21 could

indeed be reduced by the pPMSR, that the pPMSR activity

was DTT-dependent and that oxidized Hsp21 was func-

tionally restored, both in terms of conformation and

chaperone activity.

Figure 1. PMSR activity on a synthetic oxidized peptide measured byMALDI/TOF mass spectrometry.MALDI/TOF-MS spectra of a synthetic peptide corresponding to aminoacid 40±67 in the Arabidopsis thaliana Hsp21 sequence. (a) and (b)represent control and oxidized sample, respectively, and (c) shows anoxidized sample after reduction with pPMSR and DTT.

546 Niklas Gustavsson et al.

ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 545±553

Results

Methionine sulfoxides in Hsp21 are reduced by pPMSR

in a DTT-dependent manner

We have previously shown that matrix assisted lasr

desporption/ionization/time-of-¯ight-mass spectrometry

(MALDI/TOF-MS) is a very useful tool for evaluating

the methionine sulfoxidation state in Hsp21 (Gustavsson

et al., 1999). Here we applied the same procedure to

judge if pPMSR can convert the methionine sulfoxide

residues in Hsp21 back to methionines. First, an

oxidized synthetic peptide corresponding to the amino

acids 40±67 in the Arabidopsis thaliana Hsp21 sequence

was used as a substrate for pPMSR. The peptide

contains six methionines (Met-49, Met-52, Met-55, Met-

59, Met-62, Met-67) and by circular dichroism spectros-

copy the peptide showed no secondary structure either

in the control or in the oxidized form (data not shown).

In Figure 1, panel a shows the MALDI/TOF spectra of

the non-oxidized control peptide at its molecular mass

of 3381 Da (detected mass: 3381.30 Da, predicted mass:

3381.04 Da). In panel b, representing the peptide after

exposure to 5 mM hydrogen peroxide, its molecular

mass is increased to 3477 Da (detected mass:

3477.20 Da, predicted mass: 3477.04 Da), an increase of

96 Da. This mass difference corresponds to a mass

increase of 16 Da for each of the six methionines being

oxidized. After incubation of the oxidized peptide with

pPMSR in the presence of DTT (c) the degree of

oxidation in the peptide is much less than in (b), now

appearing with mainly three or four methionine sulf-

oxides. The spectrum represents a mixture of peptides,

some with two, but the majority with three or four

methionine sulfoxides. Such a statistical behaviour is

also seen in the oxidized sample in (b), where the

majority of the peptide population is in its fully oxidized

state and a smaller fraction of peptides contains only

®ve of the six methionines in sulfoxide form.

Next we used oxidized Hsp21 protein as a substrate for

pPMSR. After oxidation and pPMSR treatment, Hsp21 was

enzymatically digested with Staphylococcus aureus V8

endoproteinase Glu-C (V8 protease) and the peptide

mixture was analyzed. Figure 2 shows MALDI/TOF spectra

for the peptide representing the amino acid residues 27±64

in Hsp21, containing the six methionine residues Met-35,

Met-49, Met-52, Met-55, Met-59, Met-62. The ®rst spectrum

(a) shows how in a control sample, the peptide has a

molecular mass of 4571 Da (detected mass: 4571.79 Da,

predicted mass: 4571.46 Da) and is virtually non-oxidized,

while after oxidation with 5 mM hydrogen peroxide (b), the

peptide mass is increased to 4667 Da (detected mass:

4667.87 Da, predicted mass: 4667.46 Da) with all six

methionines in sulfoxide form. After incubation of the

oxidized Hsp21 with pPMSR in presence of 15 mM DTT (c),

the methionine sulfoxide content is reduced just as in the

synthetic peptide in Figure 1. This effect is clearly a result

of enzymatic reduction by a DTT-dependent pPMSR, since

the oxidized methionines were not reduced by DTT alone

(d). PMSR is dependent on a reducing agent such as DTT

for its activity, and no reduction of methionine sulfoxides

is detected in the absence of DTT (e). To ensure that the

pPMSR effect is indeed an enzymatic activity, pPMSR was

digested with trypsin prior to the reduction assay, and this

trypsinated pPMSR (f) showed no reducing activity as

expected. Incubation with higher concentrations of pPMSR

(pPMSR:Hsp21 molar ratio 1 : 5) or longer incubation

times (up to 6 h) did not increase the reducing effect of

pPMSR (data not shown).

Figure 2. PMSR activity on oxidized Hsp21 protein measured by MALDI/TOF mass spectrometry.MALDI/TOF-MS spectra of the peptide covering amino acid 27±64,obtained after digestion of Hsp21 with V8-protease. (a), control Hsp21with the peak representing the peptide with no methionine sulfoxidesmarked at m/z 4571. (b), oxidized Hsp21, the peak corresponding to thefully oxidized peptide with 6 methionine sulfoxides at m/z 4667 ismarked. (c), oxidized Hsp21 incubated with pPMSR and DTT (d) onlypPMSR (e) only DTT and (f) DTT and pPMSR that had been digested withtrypsin.

PMSR protects chaperone-like activity in Hsp21 547

ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 545±553

Reduction of methionine sulfoxides restores oligomeric

conformation of Hsp21

Oxidation of the methionines in the N-terminal part of

Hsp21 correlates with a change in oligomeric conform-

ation for Hsp21 as detected by non-denaturing PAGE

(Gustavsson et al., 1999). Since pPMSR was capable of at

least partly reducing methionine sulfoxides back to

methionines (Figures 1 and 2), we then wanted to see

whether this also could affect the conformation of the

Hsp21 oligomer. Apart from the methionines there is also

one cysteine residue, Cys-151, per Hsp21 monomer. To

exclude the possibility that cysteine oxidation, commonly

known to induce conformational changes, is responsible

for the conformational changes in Hsp21, we also created a

mutant in which Cys-151 was replaced by alanine (C151A).

The non-denaturing PAGE in Figure 3 shows the effect of

methionine sulfoxide reduction by pPMSR in wild-type

and C151A mutant Hsp21. Both wild-type and mutant

Hsp21 appear as 400 kDa bands. Upon oxidation, the

conformational change in the Hsp21 oligomer is seen as a

shift to 450 kDa with some smearing in the lanes below the

band, which probably re¯ects heterogeniety in the oligo-

meric conformation of oxidized Hsp21. After incubation of

the oxidized samples with pPMSR the Hsp21 oligomer is

again found as a distinct band close to its original

molecular weight of 400 kDa. These data strongly corrob-

orate that the conformational change is not caused by any

other oxidative modi®cation, since it is reversed by

pPMSR, which is speci®c to methionine sulfoxidation.

Samples incubated with pPMSR for 4 h did not differ from

the 30 min samples. As the change in mobility of the

oligomer upon oxidation and reduction by pPMSR was

identical for both wild-type and C151A mutant Hsp21,

cysteine oxidation and disul®de bridging of Hsp21 mono-

mers are not involved in these changes in oligomeric

conformation.

Reduction of methionine sulfoxides restores the

chaperone activity of Hsp21

Several sHsps show a chaperone-like activity in vitro,

preventing the aggregation of various substrate proteins,

keeping them in a refolding competent state during

transient heat stress. We have used light scattering

based assays with citrate synthase (CS) or insulin as the

substrate and previously found that the chaperone-like

activity of Hsp21 is abolished upon methionine sulfoxida-

tion (HaÈ rndahl et al., 2001). We therefore wanted to see if

the pPMSR incubation that restored methionines to their

reduced form as seen in the mass spectra (Figure 2) also

could restore the chaperone-like activity of Hsp21. In

Figure 4, showing the thermal aggregation of CS, the

presence of Hsp21 decreased the CS aggregation to 60%. If

Hsp21 was oxidized prior to the chaperone assay it lost this

ability to prevent CS aggregation. However, after a 30-min

pPMSR incubation of the oxidized Hsp21, its chaperone-

like activity was restored to that of untreated control

Hsp21. During the initial phase of denaturation and

aggregation the pPMSR treated Hsp21 showed even better

Figure 3. The oxidation induced conformational change is reversed bypPMSR in wild-type Hsp21 and a cysteine-less mutant of Hsp21.Non-denaturing PAGE of wild-type Hsp21 and the C151A mutantshowing how the oxidation-induced conformational change in the Hsp21oligomer is reversed by 30 or 240 min incubation with pPMSR.

Figure 4. Restoration by pPMSR of chaperone activity of Hsp21measured by light scattering.Thermal aggregation of citrate synthase (37.5 nM) was induced at 43°C.The aggregation curves show CS only, CS with a 30x molar excess(monomer to monomer basis) of oxidized (1.5 mM hydrogen peroxide)Hsp21 (Hsp21 ox), CS with a 30x molar excess of oxidized and PMSRtreated Hsp21 (Hsp21 PMSR), CS incubated with a 30x molar excess ofcontrol Hsp21 (Hsp21 control). The insert shows the partial recovery ofchaperone activity after PMSR-treatment when 5.0 mM, instead of 1.5 mM

hydrogen peroxide was used for Hsp21 oxidation. Absolute lightscattering (A.U) is shown instead of percentage (as error bars wouldotherwise be non-informative). Graphs are the average of fourexperiments.

548 Niklas Gustavsson et al.

ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 545±553

activity compared with the control Hsp211. Compared with

Figures 1, 2 and 3, the inactivation of the chaperone-like

activity of Hsp21 in Figure 4 was obtained after oxidation at

a lower hydrogen peroxide concentration (1.5 mM). Under

these conditions only some of the six methionines are in

sulfoxide form (Gustavsson et al., 1999), which may be a

better starting point for repair and recovery. However, the

insert in Figure 4 shows that also after oxidation with 5 mM

hydrogen peroxide, as in Figures 1,2 and 3, the pPMSR

incubation recovered the chaperone-like activity of Hsp21,

although to a smaller but still signi®cant extent.

Discussion

Reducing methionine sulfoxidation of Hsp21 may be one

important function of the pPMSR which is highly

expressed is photosynthetically active tissues

PMSR is a universal enzyme present in all organisms and

belonging to the minimal gene set for cellular life

(Mushegian and Koonin, 1996). Methionine sulfoxidation,

and a de®cient PMSR function, is implicated in biological

ageing as well as in a number of diseases. Microbial and

animal, including human, genomes described to date only

contain a single PMSR encoding gene. However, by

screening a leaf cDNA library and an EST database, ®ve

PMSR-like genes were recently identi®ed in the

Arabidopsis thaliana genome, and also found to be

expressed at high levels (Sadanandom et al., 2000). Of

these ®ve, three were corresponding to cytoplasmic

isoforms (cPMSR), and two of them for plastidic, chlor-

oplast-localized isoforms (pPMSR). These two isoforms

were differently expressed in various tissues and in

response to both development and stress indicating that

PMSR may play additional and complex roles in plants.

Interestingly, the cPMSR but not the pPMSR responded to

an exposure to the cauli¯ower mosaic virus. The expres-

sion of the pPMSR was restricted to actively photosynthe-

sizing tissues, but no endogenous substrate for pPMSR

was identi®ed.

In this paper we provide evidence that one such

chloroplast-localized endogenous substrate for pPMSR

is Hsp21. Methionine sulfoxidation destroys the chaper-

one-like activity of Hsp21 (HaÈ rndahl et al., 2001) and the

methionines in Hsp21 must therefore continuously be

kept reduced to maintain good Hsp21 function.

Probably, the hydrophobic methionines are involved in

binding of the partially unfolded substrate proteins.

Following methionine sulfoxidation, the rotational ¯exi-

bility of the methionine side chain is greatly decreased

as is the hydrophobicity (Gellman, 1991). Interestingly,

the methionines in Hsp21 can be substituted by non-

oxidizable leucines with largely retained chaperone-like

activity (Gustavsson et al., 2001). The conserved methio-

nines in Hsp21, which evolved during land-plant evolu-

tion (Waters and Vierling, 1999), possibly could do so

only if the chloroplast could keep them reduced by the

action of pPMSR. Since PMSR is part of the minimal

gene set for cellular life (Mushegian and Koonin, 1996)

it was likely present in the endosymbiont that gave rise

to plastids and thus preceded the evolution of methio-

nine-containing sHsps. Thus one can speculate that

evolution of the methionine-containing Hsp21 could

take place because pPMSR was already present in the

chloroplast.

In this paper, pPMSR activity was measured by a direct

determination of the amount of methionine sulfoxides in

Hsp21. No reduction of methionine sulfoxides in Hsp21

took place if just DTT was added without pPMSR (Figure

2e). Furthermore, the addition of 15 mM DTT was an

absolute requirement for the pPMSR-activity since no

reduction at all was obtained in absence of DTT (Figure

2d). Previously described forms of PMSR also depend on a

reductant for activity, with thioredoxin most likely to be the

endogenous reductant (Brot et al., 1981; Moskovitz et al.,

1996). A functional restoration of activity, as seen for the

chaperone-like activity of Hsp21 in Figure 4, has previously

been shown for a-1-proteinase inhibitor (Abrams et al.,

1981), calmodulin (Sun et al., 1999) and the HIV-2 protease

(Davis et al., 2000).

Reversibility of methionine sulfoxidation and

stereospeci®city of PMSR

Only partial reduction was achieved in the pPMSR assay,

leaving about half of the six methionine sulfoxides in

oxidized form. Steric hindrance is probably not the reason

as the same result was obtained with the synthetic peptide

(Figure 1) as with intact Hsp21 (Figure 2). Also, since

methionine sulfoxidation of Hsp21 leads to a loss of a-

helical secondary structure (HaÈ rndahl et al., 2001), the

methionine sulfoxides in Hsp21 may be in random coil

rather than the original a-helical conformation. There are

at least two possibilities as to why the recovery of reduced

methionines after the pPMSR treatment is not 100%. One

possibility could be that some of the methionines in Hsp21

are unaffected by oxidation while others at the same time

could be oxidized not only to methionine sulfoxides but in

two steps to methionine sulfones, which are not reducable

by pPMSR. However, this seems unlikely as we have seen

that, using the same system, small amounts of sulfones

1Treatment with pPMSR was in fact frequently observed to increase Hsp21activity above that of `control Hsp21', presumably because `control Hsp21'even without added hydrogen peroxide, to varying extent shows abackground methionine sulfoxidation, as revealed by mass spectrometry(data not shown). To avoid this, Hsp21 protein should be kept and storedunder reducing conditions in, for example 10 mM DTT.

PMSR protects chaperone-like activity in Hsp21 549

ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 545±553

could be detected only when radically higher concentra-

tions (> 30 mM) of hydrogen peroxide were used (N.

Gustavsson et al., unpublished results).

Another possibility, more likely to explain the only

partial reduction of methionine sulfoxides by pPMSR

could be the PMSR stereospeci®city for only one of the

two diastereomers of methionine sulfoxide (Sharov et al.,

1999). Since in vitro oxidation of methionine leads theor-

etically to an equal distribution of the d- and l-isomers of

methionine sulfoxides, our results that about half of the

methionine sulfoxides were available for pPMSR to reduce

are reasonable and expected. A similar partial rather than

complete recovery has been reported for other PMSRs,

e.g. from yeast and from bovine liver (Moskovitz et al.,

2000; Sharov et al., 1999). Although all methionines in

proteins are in l-form, equal amounts of d-and l-isomers of

the methionine sulfoxide occurs upon chemical oxidation

with hydrogen peroxide (Sharov et al., 1999).

Stereoselective oxidation of methionine may occur

in vivo. A ¯avin-containing monooxygenase in rat liver

and kidney microsomes can oxidize methionine, yielding

mainly the d-isomer (Krause et al., 1996), and different

biologically relevant radical species for the oxidation of

methionines give different stereoselectivity (Miller et al.,

1998). However, very interestingly, a new methionine

sulfoxide reductase, MsrB, was recently identi®ed

(Grimaud et al., 2001) and suggested to be diastereoselec-

tive for the l-isomer of methionine sulfoxide. This MsrB

had no sequence similarity to the established form MsrA

form of PMSR, but was detected in almost every genome.

The chaperone-like activity of oxidized Hsp21 was fully

restored upon pPMSR treatment (Figure 4) and the

oligomeric conformation was almost completely restored

to that of control Hsp21 (Figure 3), although only 50% of

the methionine sulfoxides were reduced by pPMSR (Figure

2). This can simply be due to the amount of Hsp21 being in

vast excess of the amount of CS (30-fold on a Hsp21

monomer to CS monomer basis), but perhaps also some

degree of sulfoxidation may be tolerated so that conform-

ation as well as activity appear normal even when some of

the methionines still remain in sulfoxide form after the

pPMSR-treatment. Indeed, sulfoxidation of only the two

methionines (Met-62 and Met-67) in a Hsp21 mutant with

the four most conserved methionines (Met-49, Met-52,

Met-55, Met-59) exchanged for leucines gave no confor-

mational change in response to oxidation showing that full

sulfoxidation of these two, out of the six, methionines can

be tolerated without affecting the Hsp21 conformation

(Gustavsson et al., 2001).

Role of PMSR in stress protection in chloroplasts

One possible function for PMSR is to act as a general

line of defense against oxidative damage to methionine

residues by repairing proteins that have been moder-

ately damaged by reactive oxygen species rather than

having the cell to pay the large cellular costs of protein

degradation and de novo synthesis. Being unusually

rich in methionine residues, Hsp21 could be one

important endogenous substrate for a chloroplast-loca-

lized PMSR, which would repair Hsp21 and return its

oxidized methionines back to a reduced state during or

after a heat and oxidative stress period. Of course there

may also be several other endogenous substrate

proteins in the chloroplast, but keeping Hsp21 in good

shape and active may be especially important since the

sHsps are so crucial for the resistance against oxidative

stress (Arrigo, 1998).

In the chloroplast, heat stress is often coupled with

oxidative stress with the formation of a number of different

reactive oxygen species (ROS). However, the chloroplast

also contains several NADPH-dependent ROS scavenging

enzymes such as the glutathione and thioredoxin systems.

Thioredoxin is generally believed to supply the reducing

equivalent to PMSR in vivo. Given all these scavenging

systems in the chloroplast, is Hsp21 ever found with its

methionines sulfoxidized in vivo? To address this question

we have developed procedures for immunoprecipitation of

Hsp21 from plant extracts. These experiments are now

underway and by comparing immunoprecipitated Hsp21

from control and heat stressed plants, detection of differ-

ences in the degree of methionine sulfoxidation will be

feasible.

We show here that several or all of the oxidized

methionines in the amphipathic a-helix of Hsp21 were

accessible for reduction by pPMSR. This is by no means

self-evident. Not all methionine sulfoxides are necessar-

ily substrates for PMSR, for example in HIV-2, two

methionines were oxidized (Met-95, Met-76) but only

one was restored by the PMSR-treatment (Davis et al.,

2000). The ability of PMSR to reduce protein bound

methionines will depend on their accessibility to this

enzyme and may require a random coil conformation or

surface location of the methionine sulfoxides. Of course

not all methionines in proteins are necessarily prone to

oxidation. For example, Rubisco is another chloroplast-

localized protein, which hypothetically could be an

endogenous substrate for the pPMSR since it is

inactivated by oxidative stress (Desimone et al., 1998).

However, we also investigated methionine sulfoxidation

in Rubisco at the same protein and hydrogen peroxide

concentrations as used for Hsp21 in Figure 2, but

Rubisco exhibited hardly any methionine sulfoxidation

when analyzed by MALDI/TOF-MS (data not shown). For

Rubisco, the inactivating effect of oxidation may instead

be due to cystein oxidation. The only methionine in

Rubisco that we could detect in methionine sulfoxide

form, even after treatment with 20 mM hydrogen per-

550 Niklas Gustavsson et al.

ã Blackwell Science Ltd, The Plant Journal, (2002), 29, 545±553

oxide, was M212 in the large subunit. The oxidized

sidechain of M212 was also reduced back to methionine

upon incubation with the pPMSR (data not shown). This

shows that Rubisco may be one out of several

chloroplast proteins that to some extent need to be

repaired by pPMSR. However, Rubisco seems only very

slightly sensitive to methionine sulfoxidation compared

with Hsp21, whose conserved methionines in Hsp21 are

indeed very prone to methionine sulfoxidation, and in

addition accessible for reduction by pPMSR.

Reactive oxygen species may affect the cellular

response to oxidative stress by alterations in the gene

expression. One example involves disul®de bridging of

cysteine residues in, for example transcription factors as

a mechanism to sense changes in the redox state of the

cell (Arrigo, 1999; AÊ slund and Beckwith, 1999). For

expression of the antioxidant defense, plants have

developed a systemic signaling system by hydrogen

peroxide signaling from parts of the plant irradiated

with photoinhibitory light intensities to parts of the

plant not yet experiencing these conditions (Karpinski

et al., 1999). The sHsps are known to be involved not

only in protection against oxidative stress but also in

regulation of cellular events involving changes in the

redox potential such as apoptosis and differentiation

(Arrigo, 1998). A cyclic system including oxidation of

methionine residues and enzymatic reduction of methio-

nine sulfoxides by PMSR could function as a redox-

dependent regulatory system. With its unusually high

content of readily oxidized methionines in the amphi-

pathic helix, Hsp21 would provide an excellent target for

such regulatory events involving a chloroplast-localized

PMSR.

Experimental procedures

Recombinant expression and puri®cation of Hsp21 and

pPMSR

Pure, recombinant Hsp21 was obtained using size exclusionchromatography as described earlier (HaÈ rndahl et al., 2001;HaÈ rndahl et al., 1998). The E. coli strain BL21(DE3) was trans-formed with the plasmid, pAZ376 (obtained from Dr E. Vierling,Department of Biochemistry, University of Arizona, Tucson, AZ,USA), encoding Hsp21 from Arabidopsis thaliana without thechloroplast signal sequence. Isolation, expression and puri®ca-tion of recombinant pPMSR was done as described in(Sadanandom et al., 2000).

Site-directed mutagenesis

Mutagenesis of the expression vector pAZ376, encoding themature form of Arabidopsis thaliana Hsp21, was done using theQuickChange Site-Directed Mutagenesis Kit (Stratagene, CA,USA) to replace cysteine 151 with an alanine as described in(Gustavsson et al., 2001).

Oxidation of Hsp21

Puri®ed Hsp21 (0.4 mg ml±1) was oxidized in 0.1 M ammoniumbicarbonate buffer pH 7.8, with 5 mM H2O2 as described before(Gustavsson et al., 1999). After incubation at 37°C for 2 h thesamples were precipitated with acetone and lyophilized in aSpeed Vac (Savant New York, NY, USA). For the non-denaturingPAGE in Figure 3 oxidation was performed in 10 mM potassiumphosphate buffer pH 7.0 and the oxidized protein subsequentlyfrozen at ±20°C to minimize further oxidation and after storagethawed and precipitated as above. A synthetic peptide (5 mM)corresponding to amino acids 40±67 in Arabidopsis thalianaHsp21 was oxidized with 20 mM H2O2.

Enzymatic reduction assay by pPMSR

Lyophilized oxidized Hsp21 or peptide was resuspended in 0.1 M

ammonium bicarbonate buffer pH 7.8 and added MgCl2±12 mM,KCl to 34 mM and pPMSR giving a pPMSR/Hsp21 molar ratio of1 : 50. DTT was added to 15 mM together with ammonium bicar-bonate buffer to give an Hsp21 concentration of 0.2 mg ml±1. Thesamples were incubated at 25°C for 30 min followed by acetoneprecipitation and lyophilization. After incubation with pPMSR at25°C for 30 min, fractions were taken out for non-denaturing PAGEor precipitated with acetone for enzymatic digestion and MALDI/TOF-MS analysis.

Light-scattering assays for chaperone activity with citrate

synthase as substrate

Thermal aggregation/denaturation of citrate synthase (37.5 nM, in40 mM HEPES pH 7.0) was induced by incubation at 43°C asdescribed in (Ehrnsperger et al., 1997), in the presence or absenceof Hsp21, and the light-scattering was recorded in a RF-5301PCShimadzu spectro¯uorimeter. To analyze the chaperone activitiesof oxidized and pPMSR treated Hsp21, pre-incubation of Hsp21(0.4 mg ml±1) was carried out with 1.5 mM or 5 mM H2O2 at 37°Cfor 2 h and with pPMSR and 15 mM DTT at 25°C for 30 min

Enzymatic digestion

Lyophilized Hsp21 samples were resuspended in ammoniumbicarbonate buffer, pH 7.8 and digested with V8 protease(Staphylococcus aureus V8 endoproteinase Glu-C, Sigma,Stockholm, Sweden) in a 1 : 40 ratio as described before(Gustavsson et al., 1999).

MALDI/TOF-MS analysis

The Hsp21 V8-digests and the synthetic peptide were analysed ona Bruker Bi¯ex workstation (Leipzig, Germany), equipped withdelayed ion extraction, in positive ion mode. The samples wereprepared as in (Gustavsson et al., 1999) with a-cyano-4-hydroxycinnamic acid (Sigma) as the matrix. Spectra were obtained byaccumulating 150±300 single shot spectra and calibrated intern-ally using known Hsp21 peptide masses.

Acknowledgements

We thank Ulf Nilsson at the department of Organic Chemistry 2,Lund University, Sweden, for letting us use the MALDI/TOF

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equipment. This work was supported by a grant from the SwedishNatural Sciences Reseach Council and the Crafoord Foundation.B.P.A.K was supported by the Netherlands Organisation forScienti®c Research (NWO). U.B. was funded by a John InnesFoundation studentship and Z.P. by Vavilov-Frankel and BBSRCfellowships.

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