Thioredoxin targets of the plant chloroplast lumen and their implications for plastid function

RESEARCH ARTICLE

Thioredoxin targets of the plant chloroplast lumen and

their implications for plastid function

Michael Hall1, Alejandro Mata-Cabana2, Hans-Erik Akerlund3, Francisco J. Florencio2,Wolfgang P. Schroder1, Marika Lindahl2 and Thomas Kieselbach1

1 Department of Chemistry, Umea University, Umea, Sweden2 Instituto de Bioquimica Vegetal y Fotosıntesis, Consejo Superior de Investigaciones Cientıficas – Universidad de

Sevilla, Spain3 Department of Biochemistry, Molecular Protein Science, Lund University, Lund, Sweden

Received: September 17, 2009

Revised: November 19, 2009

Accepted: November 22, 2009

The light-dependent regulation of stromal enzymes by thioredoxin (Trx)-catalysed disulphide/

dithiol exchange is known as a classical mechanism for control of chloroplast metabolism.

Recent proteome studies show that Trx targets are present not only in the stroma but in all

chloroplast compartments, from the envelope to the thylakoid lumen. Trx-mediated redox

control appears to be a common feature of important pathways, such as the Calvin cycle, starch

synthesis and tetrapyrrole biosynthesis. However, the extent of thiol-dependent redox regulation

in the thylakoid lumen has not been previously systematically explored. In this study, we

addressed Trx-linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using

complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more

than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of

thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II.

Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de-

epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indi-

cate that redox-controlled reactions in the chloroplast lumen play essential roles in the function

of photosystem II and the regulation of adaptation to light intensity.

Keywords:

D1-processing / Disulphide / Immunophilin / Pentapeptide protein / Plant

proteomics / Violaxanthin de-epoxidase

1 Introduction

The chloroplast lumen is the site of water-splitting and

concomitant formation of the thylakoid proton gradient that

drives ATP synthesis in oxygenic photosynthesis. Global

studies on chloroplast proteomics have highlighted several

remarkable features of the lumenal proteome. Notably,

enzymes of the primary metabolism have not been found,

although these enzymes account for large proportions of the

proteomes of other cellular compartments in plants, such as

the cytoplasm and chloroplast stroma. Instead, the chloroplast

lumen proteome appears to consist of a small number of

protein families, each represented by several members. The

largest families are the lumenal immunophilins and PsbP-

domain proteins. At least ten lumenal immunophilins appear

to be present in the Arabidopsis chloroplast lumen – more

than in any other subcellular compartment [1, 2].

The functions of most lumenal proteins are not known as

yet, but there is evidence that they play important roles in

the assembly and proper function of the photosynthetic

Abbreviations: DMA, N,N-dimethylacrylamide; IAM, iodoaceta-

mide; mBBr, monobromobimane; NEM, N-ethylmaleimide; NTR,

NADPH-dependent thioredoxin reductase; PPIase, peptidyl-

prolyl cis-trans-isomerase; PrxQ, peroxiredoxin Q; PSII, photo-

system II; TLCK, Tosyl-L-lysine chloromethyl ketone; TPCK,

Tosyl-L-phenylalanine chloromethyl ketone; Trx, thioredoxin;

VDE, violaxanthin de-epoxidase

Correspondence: Dr. Thomas Kieselbach, Department of Chem-

istry, Umea University, SE-90187 Umea, Sweden

E-mail: [email protected]

Fax: 146-90-786-7661

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2010, 10, 987–1001 987DOI 10.1002/pmic.200900654

apparatus. For instance, the lumenal immunophilins Cyp38

and FKBP20-2 are required for photosystem II (PSII)

assembly and Arabidopsis mutants containing T-DNA

insertions in the corresponding genes display distinct

phenotypes, with effects on photosynthesis and assembly of

PSII [3–5]. It has been suggested that the lumenal extrinsic

PSII protein PsbO2 could participate in the turnover of the

reaction centre D1-protein during repair of PSII [6] and a

role in the PSII repair cycle has also been proposed for the

lumenal protein TL18.3 [7]. Finally, evidence has been

obtained indicating that the lumenal immunophilin

AtCYP20-2 associates with both PSII [8] and, more recently,

the thylakoid NAD(P)H dehydrogenase [9].

A significant contribution to the discovery of unknown

functions of the lumenal proteome has been provided by

indications, presented by several groups, that some lumenal

proteins are able to react with thioredoxins (Trx). First, a

structural study of the lumenal immunophilin FKBP13 from

Arabidopsis showed that this protein has a pair of disulphide

bonds that are essential for its peptidyl-prolyl isomerase

activity, and that these bonds can be reduced by Escherichia coliTrx in vitro, causing inactivation of FKBP13 [10]. Further

evidence for lumenal Trx targets has been obtained from

screening studies of protein extracts from Arabidopsis leaves,

which have demonstrated in vitro interactions between cyto-

plasmic Trx h3 and both extrinsic lumenal PsbO proteins and

the pentapeptide protein TL17 [11, 12]. In addition, there is

evidence that the peroxiredoxin Q (PrxQ) is located in the

chloroplast lumen [13, 14]. However, association of PrxQ with

the stromal side of the thylakoid membrane has also been

suggested [15]. Peroxiredoxins are thiol-dependent perox-

idases, which commonly use Trxs as electron donors [16]. In

conclusion, three independent lines of research indicate the

existence of lumenal Trx targets, and thus that Trx-mediated

disulphide�dithiol exchange is characteristic not only of the

chloroplast stroma but of the entire organelle.

Currently, there is no evidence for a soluble lumenal Trx

[17]. However, the hcf164 gene product is a Trx-like protein,

which is anchored in the thylakoid membrane with its cata-

lytic moiety facing the lumen. HCF164 has been implicated

in maturation or assembly of the cytochrome b6f complex

[18]. The topology of HCF164 indicates the existence of a

disulphide-reduction pathway across the thylakoid membrane

similar to the DsbD system in the bacterial plasma

membrane, which transports electrons from the cytoplasm to

the periplasm through disulphide�dithiol exchange [19]. It

has been recently reported that the stromal Trx m could

facilitate transfer of reducing equivalents across the thylakoid

membrane to the catalytic domain of HCF164 on the lumenal

side of the thylakoids [20]. However, little is known about the

pathways involving lumenal Trx-regulated proteins or their

significance for photosynthesis and other chloroplast

processes. Although the other chloroplast compartments,

including the stroma, thylakoids and envelope, have been

systematically screened for Trx target proteins [21–23], no

corresponding studies have focused on the thylakoid lumen.

Trx target proteins are often identified by detecting

protein disulphides that are reduced by Trx. The active site

of Trx commonly displays the motif – WCGPC – and the

N-terminal cysteine residue of this motif forms a mixed

disulphide intermediate with the target protein, which is

subsequently broken by nucleophilic attack of the second

cysteine residue. The products of this reaction are an

oxidised Trx and a reduced target protein [24]. The newly

formed thiol groups exposed in target proteins are suscep-

tible to labelling using various types of thiol reagents, e.g.

the fluorescent dye monobromobimane (mBBr) [25, 26] or

radioactive iodoacetamide (IAM) [11, 12], both of which have

been used to visualize Trx target proteins in complex

mixtures. Another experimental approach for the same

purpose is based on the formation of stable mixed disul-

phides between target proteins and monocysteinic Trxs,

which lack the second cysteine of the active site. Immobi-

lised monocysteinic Trxs have been used in many studies to

trap and isolate Trx target proteins, and this technique is

often referred to as Trx affinity chromatography [12, 20,

27, 28].

We have undertaken a systematic study of interactions

between Trxs and the soluble lumenal chloroplast proteome,

thus completing the effort to discover new chloroplast Trx

targets. To achieve optimal coverage of lumenal Trx target

proteins we applied a complementary approach including

both Trx affinity chromatography and reduction of targets by

Trx followed by thiol labelling with a fluorescent probe or

differential mass tags [26, 12]. We found that the soluble

content of the chloroplast lumen was enriched in proteins

that were able to bind to monocysteinic Trx in vitro and that

more than 40% of the currently known lumenal chloroplast

proteins of Arabidopsis [13] can be reduced by Trx. Notably,

the lumenal Trx targets include the important xanthophyll

cycle enzyme violaxanthin de-epoxidase (VDE) and the two

D1-processing proteases, indicating that Trx interactions are

involved in non-photochemical quenching and assembly of

PSII.

2 Materials and methods

2.1 Isolation of lumenal chloroplast proteins and

treatment with N-ethylmaleimide

Soluble lumenal chloroplast proteins were isolated from

Arabidopsis leaves as described previously [2], and alkylated

using N-ethylmaleimide (NEM) under non-denaturing

conditions [29]. Briefly, 1 mL aliquots of 3–5 mL Arabidopsis

lumen preparations, containing 0.2�0.3 mg protein/mL,

were mixed with 50mL of fresh 40 mM NEM in 0.2 M

sodium phosphate (pH 7.8), 5 mM magnesium chloride,

50 mM sodium chloride and 100 mM sucrose. The resulting

mixtures were then incubated for 1 h at 371C and concen-

trated in 10K Microsep concentrators (Pall, Lund, Sweden)

at 41C to a volume of about 0.1 mL. Each portion was diluted

988 M. Hall et al. Proteomics 2010, 10, 987–1001


and concentrated three consecutive times in the same 10K

Microsep concentrator using 1 mL of 50 mM sodium phos-

phate (pH 7.8), 5 mM magnesium chloride, 50 mM sodium

chloride and 100 mM sucrose. Controls to test the comple-

tion of the alkylation reaction were prepared under identical

conditions except that addition of NEM was omitted. Protein

concentrations of alkylated lumenal fractions and the

corresponding controls were determined by the Bradford

assay using BSA as a standard [30]. Usually, these prepara-

tions contained between 1.5 and 3 mg protein/mL.

2.2 Fluorescence labelling and differential alkylation

NEM-treated lumenal chloroplast proteins were reduced

with NADPH and E. coli Trx/NTR reductase (NADPH-

dependent thioredoxin reductase) and then thiol-labelled

using mBBr as previously described [26, 31]. Lumenal Trx

targets were also alkylated using IAM and N,N-dimethyla-

crylamide (DMA) under identical conditions. To label the

thiols exposed after reduction fresh solutions of the corre-

sponding reagents in Milli-Q water were added to final

concentrations of 2–3% w/v of IAM and 4% w/v of DMA.

2.3 Trx affinity chromatography

Chloroplast lumenal proteins that bind to the monocysteinic

version of the Synechocystis sp. PCC 6803 TrxA (TrxA35)

were isolated using previously reported procedures [32, 33].

Briefly, in each experiment 1 mg of recombinant histidine-

tagged TrxA35 was bound to the Ni-affinity matrix and

10 mL of lumenal fraction containing about 2 mg of total

protein was applied. Target-TrxA35 mixed disulphides were

then eluted in 250-mL fractions with a buffer containing 1 M

imidazole.

2.4 Electrophoresis

Alkylated lumenal chloroplast proteins were separated by

IEF/SDS-PAGE as previously described [2], with minor

modifications. For the first dimension separation, samples

were applied to Immobilone DryStrips (pH 3–10 NL 24 cm),

and for the second dimension proteins were resolved in

20 cm long 12–20% gradient gels using the Ettan Daltsix

electrophoresis unit (GE Healthcare, Uppsala, Sweden). The

amounts of lumenal proteins applied for the first dimension

varied between 60 and 210mg, but the best results were

obtained with 60mg of protein. Proteins were stained using

hot 0.025% w/v Coomassie R350 in 10% v/v acetic acid [34].

TrxA35-target proteins were separated by nonreducing/

reducing electrophoresis as previously described [32, 35].

For native PAGE, samples containing 44mg of protein and

0.01% w/v of bromopheno lblue were separated on native

10% acrylamide gels at 41C, using the Laemmli buffer

system [36] without SDS. For the second dimension, excised

lanes were incubated for 15 min in 66 mM sodium carbo-

nate, 2% w/v SDS and 2% v/v b-mercaptoethanol; then

proteins were separated by SDS-PAGE using 14% poly-

acrylamide gels. Analysis of samples assayed for redox-

mediated proteolysis (see below) was performed using 14%

polyacrylamide gels containing 2 M urea. The poly-

acrylamide gels were prepared using Acrylogel 2.6 (40%)

solution (BDH Chemicals LTD) that was purchased from

VWR International AB (Stockholm, Sweden).

2.5 Immunoblot analysis

For immunoblot analyses proteins were transferred onto a

PVDF membrane in a semidry electroblotter using standard

electrode buffer [37]. Polyclonal peptide antibodies directed

against PsbO, TL29, TL17 and TL20.3 from Arabidopsis

were produced in collaboration with Agrisera (V.ann.as,

Sweden) and used at 4000-fold dilution (a-PsbO), 5000-fold

dilution (a-TL29) or 2000-fold dilution (a-TL17 and

a-TL20.3). Polyclonal antibodies against FKBP13 from

Arabidopsis were prepared as described previously [38] and

used at 400-fold dilution. The immunoreactants were then

assayed using goat-anti-rabbit IgG HRP-conjugate (BioRad,

Sundbyberg, Sweden) and either an ECL reagent kit or ECL

Plus reagent kit from GE Healthcare.

2.6 Image acquisition

UV images of gels were acquired using a Gel Doc XR CCD

imaging system (BioRad). Images of CBB-stained gels were

acquired using an image scanner and Labscan software (GE

Healthcare). ECL-images of immunoblots were acquired

using a LAS3000 CCD camera (Fujifilm, Stockholm,

Sweden) with chemiluminescence settings.

2.7 MS

Peptides were prepared for MS and analysed by MALDI-

TOF and ESI-MS/MS MS using published procedures

[39–41].

2.8 Enzymatic assays

VDE activity in lumenal chloroplast fractions [2] was assayed

in citrate-phosphate buffer, pH 5.2, following published

protocols [42, 43] in the presence and absence of reduced

E. coli Trx [26]. Proteolytic activities in the lumenal fraction

and the influence of the thiol redox state on these activities

were studied using the E. coli NTR/Trx system as a

reductant [26]. Prior to the assays, the lumenal chloroplast

fraction from Arabidopsis was concentrated using 10K

Proteomics 2010, 10, 987–1001 989


Microsep concentrators (Pall) to obtain a preparation with a

protein concentration between 1.5 and 2 mg/mL. The assay

mixtures contained final protein concentrations of 1 mg/

mL. Each experiment was performed using three samples,

one of which contained the complete system of NTR/Trx

and NADPH, while another contained NTR/Trx without

NADPH and the third contained NADPH without NTR/Trx.

Aliquots from all three samples were withdrawn at the times

indicated in Fig. 5 and frozen immediately at �801C until

further analysis. Each of these aliquots contained 25mg of

protein for analysis by SDS-PAGE followed by CBB-staining

and 15 mg of protein for analysis by immunoblotting.

Controls were carried out under identical conditions in the

presence of 100 mM concentrations of the protease inhibitors

Tosyl-L-phenylalanine chloromethyl ketone (TPCK) and

Tosyl-L-lysine chloromethyl ketone (TLCK) [44, 45].

3 Results

3.1 Identification of lumenal proteins susceptible to

Trx-catalysed reduction by labelling of thiols

with mBBr

To start screening for lumenal proteins that might be

reduced by Trx, preparations of the chloroplast lumen from

Arabidopsis were treated with the commercially available

E. coli NTR/Trx system [26, 31]. Subsequently, thiols

generated upon reduction of disulphides were detected by

2-D fluorescence electrophoresis using mBBr as a marker.

To improve the specificity of the labelling by mBBr, surface-

exposed thiols of the lumenal proteins were blocked with

NEM prior to the treatment with reduced Trx. This pre-

treatment led to almost complete suppression of back-

ground signals. In each of these analyses three replicate

samples were used, one of which was reduced for 20 min

using both NTR/Trx and NADPH. The other two samples

served as controls, to one of which NTR/Trx was added but

not NADPH, while NADPH was added to the other, but not

NTR/Trx.

The fluorescent image of the 1-DE gel displayed in

Fig. 1A shows the samples prepared in this experiment. The

lane containing NEM-treated lumenal proteins labelled with

mBBr after reduction using NADPH and NTR/Trx exhibits

ten fluorescent bands (Fig. 1A, lane 1). Control samples

treated with NTR/Trx without NADPH displayed some

background signals that were caused by contaminating

proteins in the NTR batch used (Fig. 1A, lane 2). However,

in controls treated with NADPH but not NTR/Trx essen-

tially no background signals from free thiols were observed,

demonstrating that NEM efficiently alkylated these thiols

(Fig. 1A, lane 3). The results also show that no endogenous

NADPH-dependent Trx-like activity is present in the soluble

lumen fraction. In contrast, a clear band of reduced Trx,

with an apparent mass of 10 kDa, was obtained from blanks

(Fig. 1A, lane 4). Resolution of the mBBr-labelled lumenal

chloroplast proteins in 2-DE gels provided more detailed

observations (Fig. 1B). Identification by PMF showed that

the most strongly labelled proteins from the Arabidopsis

chloroplast lumen were the extrinsic PSII subunits PsbO1

and PsbO2, the immunophilin FKBP13 and the pentapep-

tide protein TL17 (Fig. 1B). Other labelled proteins we

detected included the xanthophyll cycle enzyme VDE, the

pentapeptide protein TL15 (At2g44920.2) and the 20 kDa

Figure 1. Trx targets in the Arabidopsis chloroplast thylakoid lumen were labelled with mBBr and visualised by fluorescence following

electrophoresis. (A) Analysis of the efficiency of NEM alkylation. Lumenal proteins were alkylated with NEM and then incubated with the

complete reduction system including NTR, Trx and NADPH (lane 1), NTR/Trx without NADPH (lane 2) or NADPH without NTR/Trx (lane 3).

Lane 4 was loaded with a control consisting of E. coli NTR/Trx incubated with NADPH in the absence of lumenal proteins. Proteins were

subsequently labelled with mBBr and separated by 1-D SDS-PAGE. Thirty micrograms of lumenal protein were loaded per lane. (B)

Lumenal proteins were treated with 2 mM NEM to block exposed thiols and were then reduced for 20 min by E. coli Trx/NTR in the

presence of NADPH. Newly formed thiols were labelled with the fluorescent compound mBBr and 60 mg lumenal proteins were separated

by 2-DE IEF/SDS-PAGE. Fluorescent spots corresponding to mBBr-labelled proteins were visualised by excitation in UV light, excised and

identified by PMF.



Tab

le1.

Trx

targ

ets

dete

cted

inth

elu

men

al

chlo

rop

last

fract

ion

of

Ara

bid

op

sis

thali

an

a

Pro

tein

nam

eG

en

elo

cus

Cyst

ein

es

inm

atu

rep

rote

ina)

mB

Br

flu

ore

sc.

ele

ctro

ph

.

Dif

fere

nti

al

alk

yla

tio

nT

rxaffi

nit

ych

rom

ato

gra

ph

y

mB

Br

IAM

DM

A

Lu

men

al

pro

tein

s

Xan

tho

ph

yll

cycl

eV

DE

(NP

Q1)

AT

1G

08550.1

13

(Z12)

Dete

cted

C120,

C122,

C127,

C185,

C362

C127,

C163,

C178,

C185,

C362

C127,

C163,

C178,

C185,

C362

Dete

cted

Pro

tease

sC

-term

.D

1p

roce

ssin

gA

T4G

17740.1

,A

T4G

17740.2

5(Z

2)

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

Dete

cted

Pu

tati

ve

C-t

erm

.D

1p

roce

ssin

gA

T5G

46390.1

,A

T5G

46390.2

4(Z

1)

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

Dete

cted

Deg

1A

T3G

27925.1

1(1

)N

ot

dete

cted

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

Dete

cted

Deg

5A

T4G

18370.1

2(Z

1)

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

Dete

cted

Ph

oto

syte

mII

sub

un

its

Psb

O1

AT

5G

66570.1

2(2

)D

ete

cted

C114,

C137

C114,

C137

C114,

C137

Dete

cted

Psb

O2

AT

3G

50820.1

2(2

)D

ete

cted

C114,

C137

C114,

C137

C114,

C137

Dete

cted

Psb

P1

AT

1G

06680.1

1(1

)D

ete

cted

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

Dete

cted

Ph

oto

syte

mI

sub

un

its

Psa

NA

T5G

64040.1

4(Z

2)

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

Dete

cted

Psb

Pd

om

ain

pro

tein

s20

kDa

Psb

Pp

rote

inA

T3G

56650.1

2(2

)D

ete

cted

C128,

C132

C128,

C132

C128,

C132

Dete

cted

Imm

un

op

hilin

sC

yp

38

AT

3G

01480.1

1(1

)N

ot

dete

cted

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

Dete

cted

FK

BP

20-2

AT

3G

60370.1

2(2

)N

ot

dete

cted

No

td

ete

cted

C241

C241

No

td

ete

cted

FK

BP

13

AT

5G

45680.1

4(4

)D

ete

cted

No

td

ete

cted

C185

C185

No

td

ete

cted

Pen

tap

ep

tid

ere

peat

pro

tein

sT

L17

AT

5G

53490.1

4(4

)D

ete

cted

C92,

C117,

C235

C92,

C117,

C220,

C235

C92,C

117,C

220,

C235

No

td

ete

cted

TL15

AT

2G

44920.2

2(2

)D

ete

cted

No

td

ete

cted

C223

No

td

ete

cted

No

td

ete

cted

TL20.3

AT

1G

12250.1

4(Z

2)

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

Dete

cted

Pero

xir

ed

oxin

sP

rxQ

AT

3G

26060.1

2(2

)D

ete

cted

No

td

ete

cted

C111,

C116

No

td

ete

cted

Dete

cted

Un

kno

wn

fun

ctio

ns

TL29

(Ap

x4)

AT

4G

09010.1

2(1

)D

ete

cted

No

td

ete

cted

C325

No

td

ete

cted

Dete

cted

TL19

AT

3G

63535.1

1(1

)N

ot

dete

cted

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

Dete

cted

Str

om

al

pro

tein

s

Ru

Bis

Co

(LS

)A

TC

G00490.1

9(8

)N

ot

dete

cted

No

tan

aly

sed

C84,

C172,

C192,

C221,

C247,

C284,

C427,

C449,

C459

No

tan

aly

sed

Dete

cted

Proteomics 2010, 10, 987–1001 991


PsbP-domain protein At3g56650. In further experiments we

also observed reproducible mBBr labelling of the peroxi-

redoxin PrxQ (At3g26060), the extrinsic PSII subunit PsbP1

(At1g06680) and the TL29 protein (At4g09010) (Table 1).

3.2 Identification of lumenal Trx target proteins by

differential alkylation

The analysis of lumenal Trx targets using 2-D fluorescence

electrophoresis indicated that the chloroplast lumen is rich

in proteins that could be reduced by Trx. However, due to

potential co-migration on the 2-DE gels, some of the

proteins that were apparently associated with fluorescence

may not have reacted with Trx. For this reason, a comple-

mentary analysis was performed using differential alkyla-

tion. In this experiment the thiols exposed upon reduction

by NTR/Trx were alkylated using the reagents IAM and

DMA, which results in different mass shifts of cysteine-

containing peptides in subsequent MS analysis. This strat-

egy provides not only unambiguous identification of the

target proteins, but also information about which cysteine

residues react with Trx. To screen for Trx targets, the

labelled lumenal chloroplast proteins were separated by

2-DE and visualised by CBB staining for subsequent

screening by MALDI-TOF-MS (Fig. 2). Proteins were

considered as targets of Trx if their mass spectra consis-

tently showed cysteine-containing peptides that were label-

led both in experiments using IAM and DMA (Supporting

Information Table S1). As an example, a MALDI spectrum

of the 20 kDa PsbP domain protein (At3g56650) is shown in

the insert in Fig. 2, and the peaks of the IAM-labelled

peptides are marked with asterisks. The proteins reduced by

Trx in this series of experiments are indicated on the CBB-

stained gel shown in Fig. 2, and the cysteines carrying

differential labels are listed in Table 1. Comparison with the

results from the 2-D fluorescence electrophoresis analysis

(Fig. 1) showed that essentially the same targets were

detected (Table 1). The only differences were that the PSII

subunit PsbP1 was detected by 2-D fluorescence electro-

phoresis, but not by differential alkylation, and vice versa for

the immunophilin FKBP20-2. A summary of the identifi-

cations by PMF of the Trx targets found in these thiol

labelling-based approaches is presented in Supporting

Information Table S1.

3.3 Isolation of proteins in the Arabidopsis

thylakoid lumen that interacted with Trx using

Trx affinity chromatography

In one form of Trx affinity chromatography a histidine-

tagged monocysteinic Trx immobilised on a nickel affinity

matrix is employed [32, 33]. In this study, we used a histi-

dine-tagged monocysteinic version of the m-type Trx from

the cyanobacterium Synechocystis sp. PCC 6803 (TrxA35),Tab

le1.

Co

nti

nu

ed

Pro

tein

nam

eG

en

elo

cus

Cyst

ein

es

inm

atu

rep

rote

ina)

mB

Br

flu

ore

sc.

ele

ctro

ph

.

Dif

fere

nti

al

alk

yla

tio

nT

rxaffi

nit

ych

rom

ato

gra

ph

y

mB

Br

IAM

DM

A

FE

NR

2A

T1G

20020.1

6(p

recu

rso

r)(Z

4)

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

Dete

cted

FE

NR

1A

T5G

66190.2

4(p

recu

rso

r)(Z

4)

No

td

ete

cted

No

tan

aly

sed

C39,

C88,

C160,

C178,

C183,

C318

No

tan

aly

sed

Dete

cted

Un

kno

wn

pro

tein

AT

3G

15840.3

7(p

recu

rso

r)(Z

3)

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

Dete

cted

Un

decid

ed

locati

on

Pla

stid

lip

id-a

sso

ciate

dp

rote

inA

T3G

23400.1

1(0

)N

ot

dete

cted

No

td

ete

cted

No

td

ete

cted

No

td

ete

cted

Dete

cted

a)

Nu

mb

ers

inb

rack

ets

are

the

nu

mb

er

of

con

serv

ed

cyst

ein

ere

sid

ues

ing

reen

pla

nts

.



since this enzyme has been shown to be a highly efficient

bait for Trx target proteins [46]. Following binding of

lumenal proteins to the immobilised TrxA35 by mixed

disulphide formation, the matrix was washed with a low

concentration solution of imidazole to remove non-specifi-

cally interacting proteins. Finally, the TrxA35-target

complexes were eluted using a high concentration solution

of imidazole. The 1-DE profiles from this chromatographic

procedure are shown in Fig. 3A, including the lumenal

proteins before application to the column, the non-retained

proteins, the wash and the eluate. A comparison of the lanes

displaying the initial lumenal fraction and the non-retained

proteins shows that some proteins (e.g. VDE, Deg1, TL29,

TL19 and PsbO) were specifically retained on the matrix.

Furthermore, the eluate lane displayed more than ten

different protein bands apart from the highly abundant

TrxA35 used as bait (Fig. 3A). Ten of the eluted proteins

were identified by PMF and are indicated in Fig. 3A and

Supporting Information Table S2.

3.4 Separation and identification of lumenal

proteins isolated by Trx affinity chromatography

To resolve the lumenal proteins isolated by Trx affinity

chromatography more thoroughly, eluates were subjected to

2-D SDS-PAGE under non-reducing conditions in the first

dimension and reducing conditions in the second [32, 33].

For the first dimension, proteins were separated using SDS-

PAGE in the absence of reducing agents. Lanes excised from

the resulting gels were treated with DTT and applied to a

second SDS-gel. The target-TrxA35 mixed disulphides

remained intact in the first dimension, but the targets were

released and separated from TrxA35 in the second dimen-

sion. Hence, lumenal proteins that bound to the mono-

cysteinic Trx35A migrated below the diagonal indicated by

the pre-stained standard protein markers (Fig. 3B). A mass-

shift between the first and second dimensions of approxi-

mately 15 kDa corresponds to the additional mass of one

bound Trx molecule, thus confirming its interaction with

the target. This pattern was observed for several proteins,

including the cyclophilin Cyp38, the protease Deg1 and

TL19. Other target proteins, such as VDE, displayed patterns

consistent with the binding of at least two molecules of Trx

in the first dimension and some targets, such as the

peroxiredoxin PrxQ and PsaN, were distributed between two

or more spots (Fig. 3B). An advantage of this protocol is that

it can discriminate between true targets and possible

contaminants that would be equally retained in both

dimensions. Therefore, lumenal proteins that migrated

exclusively along the diagonal, such as HCF136, were not

considered as Trx targets.

The TL29-protein could not be unambiguously identified

by MS among the targets eluted from Trx affinity chroma-

tography column and resolved by 2-DE (Fig. 3B), although it

was detected by mBBr-labelling (Table 1). However,

comparison of the TL29 band in the lumenal fraction and

non-retained protein lanes of the 1-DE gels used to analyse

the results of the Trx affinity chromatography (Fig. 3A)

indicated that TL29 bound to TrxA35. Therefore, we

analysed the fraction of eluted proteins by immunoblotting

using specific peptide antibodies raised against TL29 of

Arabidopsis. When the eluate was reduced using 20 mM

DTT the antibodies detected a double band with apparent

masses of 26 and 24.5 kDa, which is typical of TL29.

However, when the eluted proteins were not reduced, TL29

was detected as a weak band at an apparent mass of

45.7 kDa, indicating that TrxA35 formed a mixed disulphide

with TL29 (Fig. 3C).

The two low molecular mass proteins FKBP13 and TL17,

which had been identified as putative Trx targets in the

alkylation experiments, were also not confirmed as targets

Figure 2. Lumenal Trx target proteins

were identified by differential alkyla-

tion. NEM-treated lumenal proteins

were incubated with E. coli NTR/Trx

and NADPH, and newly formed thiols

were then alkylated with either IAM or

DMA. Sixty micrograms of protein

from each sample were separated by

2-DE and differentially alkylated

peptides were detected by MALDI-

TOF-MS. The insert shows a mass

spectrum of the 20 kDa PsbP domain

protein from a sample alkylated with

IAM and the peaks indicated with

asterisks correspond to the IAM-

labelled peptides.

Proteomics 2010, 10, 987–1001 993


B

A C

250

98

kDa

64

50

36

26

16

6

Lum

en fr

actio

n

Unb

ound

prot

eins

Firs

t was

h

Elu

ted

prot

eins

Deg1

PsbO, PAPTL29

TL19

RBCL

PsaN

VDE

PsbQ

FNR1

RBCLVDE

HCF136Deg1

PsbO, PAPTrxA35TrxA35PsbP1, TrxA35

TL19

PrxQ

PsaN, TrxA35

TrxA35

kDa25015010075

50

37

25

20

15

10

− D

TT

+ D

TT

− D

TT

+ D

TT

TL29

TL29+TrxA35

TL20.3DegradedTL20.3

kDa98 −

64 −

50 −

36 −

30 −

16 −PsbQ1

TL26 (PsbP domain)

FKBP2TL1929.8 kDa (PsbP domain)

20 kDa (PsbP domain)PsbP1

21.5 kDa (PsbP domain)

38.5 kDa (PsbP domain)

PsaN PsaN

PrxQ

TL19

PsbP1PsbP1

PsbO1PsbO1PsbO2PsbO1PsbO1

20 kDa (PsbPdomain)

PsbO1PsbO2

At3g15840

PsbO1Deg5

FENR2Deg1

Cyp38 Put. fibrillin (PAP)

Hcf136

processing proteaseD1 C-terminal

peptidase S41D1 C-terminal

VDE

Figure 3. (A) Protein fractions from Trx affinity chromatography were separated by 1-D SDS-PAGE using a 12% polyacrylamide gel under

reducing conditions and stained with CBB. For each fraction 25 mL was applied per lane. Lane 1, total lumenal protein prior to binding to

immobilised histidine-tagged monocysteinic Trx, TrxA35. Lane 2, non-retained proteins after incubation with the TrxA35 affinity matrix.

Lane 3, the fraction obtained by washing with 60 mM imidazole. Lane 4, the eluate following addition of 1 M imidazole. Proteins identified

by PMF are indicated including the recombinant TrxA35 used as bait. The presence of TrxA35 at different apparent masses indicates

oligomerization. (B) Resolution of Trx affinity-derived target proteins from the thylakoid lumen by 2-D SDS-PAGE under non-reducing/

reducing conditions. Trx affinity chromatography was performed using TrxA35, a histidine-tagged monocysteinic Trx, as bait and TrxA35-

target mixed disulphides were eluted with 1 M imidazole. For the first dimension separation, 150 mL of eluate was mixed with 10mL

SeeBlue pre-stained protein standard markers and the intact TrxA35-target mixed disulphides were resolved under non-reducing

conditions. Lanes were excised, incubated with 100 mM DTT and the target proteins, released from TrxA35, were again separated by SDS-

PAGE and stained with CBB. Separations in both dimensions were performed using 11% polyacrylamide gels. The target proteins,

characterised by their migration below the diagonal, were identified by PMF. The spots defining the diagonal corresponding to the pre-

stained protein standard markers are encircled. (C) Western blot analysis of the Trx affinity chromatography eluate using polyclonal

antibodies raised against TL29 and TL20.3 of Arabidopsis. For each lane 100 mL eluate was used, and proteins were precipitated with

80% v/v acetone prior to solubilisation for SDS-PAGE. Samples were solubilised without reduction (�DTT), or after reduction using 20 mM

DTT (1DTT). The dilutions of the antisera were 1: 5000 for assaying TL29 and 1: 2000 for detecting TL20.3.



using Trx affinity chromatography, possibly due to the high

abundance of the bait in this molecular mass region. Since

the chloroplast lumen has three pentapeptide proteins, two

of which (TL17 and TL15) were detected as Trx targets by

thiol alkylation (Table 1), we tested the possibility that the

third pentapeptide protein (TL20.3) might also be a Trx

target and present in the fraction of TrxA35-binding

proteins. Using specific peptide antibodies raised against

TL20.3 of Arabidopsis, the eluted proteins were tested for

the presence of TL20.3 before and after reduction by 20 mM

DTT. As shown in Fig. 3C, TL20.3 was detected among the

reduced eluted proteins at apparent masses of 28.5 and

24.4 kDa. However, TL20.3 was not detected among the

unreduced eluted proteins, indicating that the epitope

recognized by the antibodies in TL20.3 is not accessible in

the mixed disulphide formed with TrxA35. The apparent

presence of two forms of TL20.3 with slightly differing

masses in the sample of reduced eluted proteins indicates

that reduced TL20.3 was degraded, since only a 28.5 kDa

band was detected in analyses of samples of purified

chloroplast lumen.

3.5 The effect of Trx-mediated reduction on the

oligomeric lumenal proteome

Eight lumenal proteins were detected along the diagonal of

the 2-DE gel displayed in Fig. 3, which were not Trx-targets

but co-eluted with true targets and perhaps represented

subunits of protein complexes. Therefore, to detect possible

complexes among the soluble lumen proteins, and examine

the effect of disulphide reduction on their oligomerization,

we resolved samples of lumenal proteins by native PAGE/

SDS-PAGE 2-DE before and after pre-treatment with the E.coli NTR/Trx reduction system in the presence and absence

of NADPH. The resulting CBB-stained gels showed that

most proteins migrated more slowly in the first than in the

second dimension (Fig. 4). Treatment of the lumenal frac-

tion with the complete reduction system prior to electro-

phoresis had two surprising consequences. First, the

amounts of PsbO1, PsbO2 and TL17 decreased after

reduction with E. coli NTR/Trx, indicating degradation of

these proteins. Second, a fraction of the PsbO proteins and

TL17 appeared to associate with higher mass oligomers that

were not found in the unreduced control (Fig. 4). The

molecular mechanism behind the formation of these

possible oligomers requires further investigation.

3.6 Reduction by Trx inactivates VDE in vitro

The identification of VDE as a Trx target suggested that

reduction by Trx might regulate its enzymatic activity. To

test this hypothesis, VDE activity of the lumenal chloroplast

fraction was assayed in the presence and absence of reduced

E. coli Trx. In accordance with the hypothesis, more than

80% of the specific activity of VDE in lumenal chloroplast

fractions was lost in the presence of reduced Trx (Table 2).

3.7 Proteolysis of lumenal chloroplast proteins and

the influence of the thiol redox state

The 2-D gel of lumenal proteins isolated by Trx affinity

chromatography (Fig. 3B) displayed a series of spots in the

20�30 kDa region, corresponding to degradation products

of the extrinsic PSII proteins PsbO1 and PsbO2. Since PsbO

proteins are usually highly stable, this observation suggested

that reduction of disulphides might destabilise these

proteins and make them accessible to an intrinsic lumenal

protease. To test this hypothesis, we incubated lumenal

chloroplast proteins in the presence of reduced E. coli Trx

and monitored changes in protein levels during 5 h of

incubation. Analysis by 1-D SDS-PAGE (Fig. 5A) showed

Reduced (Trx/NTR/+ NADPH) Non-reduced (Trx/NTR/-NADPH)

PsbO1

TL17

kDa755037

2520

15

10

kDa 440

232

140

67 kDa 440

232

140

67

PsbO2

TL17TL17

PsbO2 PsbO1

Figure 4. Analysis of the oligomeric lumenal proteome by native

PAGE/SDS-PAGE and the effect of Trx-dependent reduction.

Lumenal proteins were incubated for 1 h with E. coli NTR, Trx

and NADPH. Unreduced control samples were prepared under

identical conditions, except that NADPH was omitted. A portion

of each sample containing 44mg of lumenal proteins was

resolved using 10% polyacrylamide native gels (top panels), then

separated in the second dimension under denaturing conditions

using 14% polyacrylamide SDS-PAGE gels. Proteins were

stained with CBB and analysed by MALDI-TOF-MS. The strong

spots on the left and right sides at an apparent mass of

approximately 25 kDa do not represent proteins but are the

reference markers for the Ettan spot picking station.

Table 2. Inhibition of VDE activity by reduced E. coli Trx

Experiment VDE activity (nmol violaxanthin s�1

mg protein�1)

Lumenpreparation 1

Lumenpreparation 2

Lumenal fraction 6877103 655798Lumenal fraction and

Trx/NTR/NADPH132720 1773

Proteomics 2010, 10, 987–1001 995


that most lumenal proteins remained stable during the

experiment and some proteins, such as the cyclophilin

Cyp38 and the fibrillin PAP3, were degraded regardless of

whether or not reduced Trx was present. However, the

pentapeptide protein TL17 and the extrinsic PSII subunits

PsbO1 and PsbO2 showed pronounced degradation in the

presence of reduced Trx, but not in the presence of either

NTR/Trx without NADPH or NADPH without NTR/Trx

(Fig. 5A).

The redox-dependent degradation of PsbO1, PsbO2 and

TL17 was further analysed by immunoblotting (Fig. 5B).

Following 1 h of incubation in the presence of reduced Trx

the levels of PsbO1, PsbO2 and TL17 were dramatically

reduced and after 3 h they were virtually undetectable. In

addition, breakdown products of the PsbO proteins were

observed during the initial degradation process. The level of

the lumenal Trx target FKBP13 (Table 1) and [10] was

monitored for comparison and, as shown in Fig. 5B, it was

not degraded under any of the tested conditions. The redox-

dependent proteolysis of the PsbO proteins and of TL17 was

inhibited in the presence of a 100 mM concentration of

TPCK that inhibits trypsin-like serine-type proteases.

However, only a weak inhibition of this proteolysis was

observed, if the same experiment was performed in the

presence of a 100mM concentration of TLCK that inhibits

chymotrypsin-like serine proteases (data not shown).

4 Discussion

The chloroplast lumen proteome has unusual features,

including the presence of immunophilins, PsbP-domain

proteins and pentapeptide proteins [2, 13]. A further striking

feature, observed in this study, is that some members of all

the major protein families of the lumen, and all of the

lumenal pentapeptide proteins (TL17, TL15 and TL20.3),

interact with Trx. Indeed, more than 40% of the 45

empirically confirmed lumenal chloroplast proteins from

Arabidopsis (or 25% of the predicted lumenal proteins)

[2, 13] were found to be targets for Trx in vitro. These

numbers are surprisingly high, considering that there are

only 79 known Trx targets among the stromal chloroplast

proteins (less than 10% of the probable total number) [23].

These findings raise questions regarding the significance of

the lumenal Trx interactions observed in this study. Thirty

of the 45 confirmed lumenal chloroplast proteins of Arabi-

dopsis contain one or more cysteine residues in their

mature protein sequence. We identified 19 of these 30

proteins as potential Trx targets, while the other 11 did not

appear to interact with Trx in our experiments. Since the

concentrations of these 11 proteins are within the dynamic

range covered by our methods, they should also have been

detected if they were Trx targets, indicating that our meth-

ods were not unspecific. As detailed in Table 1, all 19

identified lumenal Trx targets contain cysteine residues that

are conserved in plants and thus are likely to play significant

functional roles. Eight lumenal Trx targets identified in this

study have also been detected in other studies that have

examined direct interactions of Trx: the extrinsic PSII

subunits PsbO1 and PsbO2 [11, 12, 47, 48], PsbP1 [12, 47],

the extrinsic PSI protein PsaN [20, 47], the immunophilins

FKBP13 and FKBP20-2 [5, 10], the pentapeptide protein

TL17 [11, 12] and the peroxiredoxin PrxQ [12, 27]. In

summary, there is no evidence that the lumenal Trx targets

found in this study represent false positives. To establish the

A

B

+ N

AD

PH

+ T

rx/N

TR

+ N

AD

PH

+ T

rx/N

TR

0 h 5 h

+ N

AD

PH

,T

rx/N

TR

37

2520

15

10

50

75100150200kDa

TL17

PsbO1PsbO2

Cyp38PAP3VDE

PsbP1

PsbQ1,PsbQ2

+ N

AD

PH

,T

rx/N

TR

15010075

50

37

25

20

15

kDa

10

FKBP13

TL17

PsbO1PsbO2

1 h0 h 3 h 5 h

+ N

AD

PH

+ Tr

x/N

TR

/N

AD

PH

+ Tr

x/N

TR

+ N

AD

PH

+ Tr

x/N

TR

/N

AD

PH

+ Tr

x/N

TR

+ N

AD

PH

+ Tr

x/N

TR

/N

AD

PH

+ Tr

x/N

TR

+ N

AD

PH

+ Tr

x/N

TR

/N

AD

PH

+ Tr

x/N

TR

Figure 5. Degradation of lumenal chloroplast proteins as a

function of Trx-dependent reduction. Lumenal proteins were

incubated at 371C for up to 5 h in the presence of E. coli NTR/Trx

and NADPH or, as controls, in the presence of NTR/Trx without

NADPH or NADPH without enzymes. (A) Lumenal proteins

separated by 1-DE, stained with CBB and analysed using MALDI-

TOF-MS. The extrinsic PSII proteins PsbO1 and PsbO2, PsbP1,

PsbQ1 and PsbQ2 were identified by PMF and are indicated, as

well as TL17, the immunophilin Cyp38 and the fibrillin PAP3. (B)

Immunoblot analysis of samples following 1, 3 and 5 h of incu-

bation using antibodies raised against PsbO1/PsbO2, TL17 and

FKBP13 at 1:4000, 1:2000 and 1:400 dilution, respectively.



physiological significance of these Trx interactions it would

be necessary to validate each target individually [49].

However, observations in this and previous relevant studies

provide indications of various intriguing possibilities, as

briefly outlined below.

Few lumenal proteins possess a known enzymatic

activity; thus the xanthophyll cycle enzyme VDE offers a

singular opportunity to corroborate the occurrence of

redox regulation in the chloroplast lumen. VDE catalyses

the de-epoxidation of violaxanthin to zeaxanthin during

non-photochemical quenching, which mediates thermal

dissipation of excess light energy [50]. The inhibitory effect

of DTT on violaxanthin de-epoxidation catalysed by VDE in

isolated thylakoids is well known [51, 52]. However, to our

knowledge, there are no previous reports of enzymatic

deactivation of VDE catalysed by Trx (Table 2), which may

provide a previously unrecorded mechanism of reversible

xanthophyll cycle regulation. Trx-mediated reduction of one

or more disulphide bridges in the cysteine-rich C-terminus

of VDE might provide a means to control the level of active

VDE within a short time frame. Reduced, inactive VDE

could be removed by proteolysis or regenerated by the ability

of the chloroplast lumen to form disulphide bridges [53, 54].

In this context, it is worth noting that one cysteine (C362)

close to the presumed substrate-binding pocket in the lipo-

calin domain was affected by Trx treatment (Table 1).

Immunophilins comprise the largest protein family of

the chloroplast lumen, with at least ten experimentally

confirmed members [1, 2]. FKBP13, one of the first lumenal

proteins to be linked to Trx-dependent redox regulation [10],

is responsible for nearly all peptidyl-prolyl cis-trans-isomer-

ase (PPIase) activity in the thylakoid lumen, and reduction

of the two disulphide bridges of FKBP13 by either DTT or

reduced Trx inactivates its PPIase activity [10, 38, 55]. The

function of FKBP13 appears to be related to the accumula-

tion of the cytochrome b6f Rieske protein [56], but the role of

its redox-dependent PPIase activity is not yet understood.

Another Arabidopsis immunophilin that has been shown to

interact with Trx is FKBP20-2, which has been proposed to

function in the assembly of PSII supercomplexes. This

protein has two conserved C-terminal cysteines, and

recombinant FKBP20-2 can receive electrons from E. coli Trx

in vitro [5], although reduction does not reportedly affect its

(already low) PPIase activity. Our Trx affinity chromato-

graphy analysis provides the first indications that a third

lumenal immunophilin of Arabidopsis, the cyclophilin

AtCYP38, might also be subject to redox control (Fig. 4 and

Table 1). This protein plays a critical role in the biogenesis

and assembly of PSII, particularly under high light inten-

sities [3, 4], and the spinach CYP38 homologue, TLP40, is a

negative regulator of the protein phosphatase that depho-

sphorylates thylakoid proteins, such as LHCII- and PSII

subunits [57]. The mature sequence of AtCYP38 contains a

single cysteine, C256, which is conserved among the plant

homologues and located at the interface between the acidic

domain and the PPIase-domain of the protein. However, the

molecular mechanisms whereby FKBP13, FKBP20-2 and

CYP38 and other immunophilins participate in thylakoid

protein assembly, and the roles that Trx-mediated signals

may play in their functions, remain unknown.

In this study, we also found that treatment of Arabidopsis

lumen preparations with reduced Trx results in significant

degradation of PsbO1, PsbO2 and TL17, but no significant

degradation of these proteins was observed if any compo-

nent of the NADPH/NTR/Trx system was missing. This is

interesting because previous studies have shown that the

intrinsic proteolytic activity in lumen preparations is low,

under standard non-reducing conditions, and most proteins

in them are not significantly degraded in vitro [58]. However,

proteases fulfil important functions in the regulation and

timing of biochemical pathways by processing and remov-

ing proteins that are no longer needed [59]. Our findings

indicate that in addition to reductive activation of proteases,

such as Deg1, there is a possibility that reduction of disul-

phides within the substrate proteins induces important

structural changes that stimulate their degradation. The

disulphide bridge of spinach PsbO has been previously

shown to be essential for its structural stability [60]. Hence,

reduction of the disulphide within the Arabidopsis PsbO

proteins could change their conformation and render them

accessible to proteolysis. By contrast, the Trx target FKBP13

did not display redox-dependent degradation, which is

consistent with a previous observation that reduction of the

disulphides of FKBP13 does not cause any major confor-

mational change of this protein [61]. Notably, enhanced

susceptibility to proteolysis after reduction by Trx was also

observed for the bovine milk allergen b-lactoglobulin [62]

and the Kunitz and Boman-birk soybean trypsin inhibitor

proteins [63]. Hence, redox-mediated proteolysis is not

restricted to plants and might be a more general mechanism

for the regulation of proteins.

The soluble lumenal chloroplast fraction contains no

other known proteases except the D1 processing proteases

and Deg1, Deg5 and Deg8. Cross-contamination by stromal

proteases in our lumen preparations has never been

observed and can thus be excluded [2], (Kieselbach, unpub-

lished). Therefore, the degradation of PsbO1, PsbO2 and

TL17 is probably due to the activity of the lumenal Deg

proteases. The Deg proteases of Arabidopsis share a trypsin-

like serine protease domain [64]. We observed that the redox-

dependent proteolysis of the PsbO proteins and TL17 was

inhibited by TPCK that inhibits trypsin-like serine proteases.

This observation would be consistent with the hypothesis

that these proteins were degraded by lumenal Deg protea-

ses. Deg5 has been suggested to function in a complex with

Deg8, which may catalyse the cleavage of photo-damaged D1

protein during the light-induced turnover of PSII [65]. Deg1

has also been implicated in the degradation of the D1

protein [66]. Redox dependence of casein degradation by

recombinant purified Deg1 has been recently reported [67],

and it is possible that the single conserved cysteine residue

of Deg1 could be involved in the formation of oligomers.

Proteomics 2010, 10, 987–1001 997


Previous evidence of the involvement of thiols in the

assembly and post-translational processing of the D1-

precursor indicates that proteases and possibly other lume-

nal proteins may be redox-regulated [68], but the roles of

disulphides in the biogenesis, assembly and turnover of the

D1 protein remain to be elucidated.

The central paradigm of Trx-mediated redox regulation in

the chloroplast stroma is that biosynthetic enzymes are inac-

tivated by disulphide formation in the dark and reactivated in

the light by Trx-catalysed reduction of the disulphides [21, 69].

Conversely, catabolic stromal enzymes are believed to be

activated in the dark by oxidation of cysteines and inactivated

in the light through disulphide reduction. The environmental

signals that may be transmitted to lumenal target proteins by

disulphide/dithiol exchange remain to be identified. Inti-

mately linked to this problem is the seemingly simple issue

regarding the kinds of light conditions that lead to reducing,

and the kinds that lead to oxidising states in the thylakoid

lumen. Buchanan and Luan (2005) [70] argue that the oxygen

evolved at the lumenal side of PSII in the light should be

sufficient to cause disulphide formation within newly

imported lumenal proteins. According to their model, the

precursor protein, of FKBP13 for example, is translocated

across the thylakoid membrane in an extended, reduced form

and thereafter acquires correct folding and is activated by

disulphide formation in the light. Moreover, it is possible that

disulphide formation in the lumen is controlled by catalysis

through a plant homologue of the cyanobacterial DsbA/DsbB-

like oxidoreductase [71]. It has also been proposed that cyto-

chrome c6A and plastocyanin can catalyse thiol oxidation of

lumenal proteins [72]. However, this would be a uni-direc-

tional process, which could not mediate reversible regulation

of any enzymatic activity unless further, unknown redox

proteins were also involved. For reduction of lumenal disul-

phides, a model has been presented [20] in which a stromal

FTR-dependent Trx functions as hydrogen donor for a trans-

thylakoid DsbD-like system that includes HCF164 and prob-

ably also CcdA [73]. The cited model is based on the obser-

vation that a lumenal Trx target protein, PsaN, could be

reduced in vitro following incubation of intact thylakoids with

reduced Trx m [20]. Taken together, these models suggest that

both oxidation and reduction occur in the light and it appears

difficult to predict which activity will predominate.

A crucial issue, which has been largely overlooked as yet, is

the extent to which a lumenal Trx, such as HCF164, would be

catalytically active in the light when the thylakoid lumen is

acidified. While the stromal pH in isolated chloroplasts rises

from about 7 in the dark to 7.5–8 in the light, the thylakoid

lumen is acidified, and under conditions of normal photo-

synthetic function lumenal pH is considered to be between

5.8 and 6.5 [74]. Recent measurements indicate that lumenal

pH in the light is between 5.4 and 6 [75]. Moderate acid-

ification of the chloroplast lumen under conditions of photo-

synthetic control is consistent with observations that support a

model of sequestered buffering domains in the thylakoid

membrane that link H1 release of the photosynthetic redox

reactions with the proton channel of the ATP synthase [76].

However, the acidic conditions in the thylakoid lumen during

photosynthetically active periods certainly would not favour

the formation of Trx active site thiolate anions, which are

essential for the first step of catalysis involving transient

mixed disulphides with target proteins. Hence, in contrast to

the chloroplast stroma, transfer of thiol equivalents to lumenal

target proteins might be restricted to dark periods when the

lumenal pH is neutral.

Another, so far unexplored, alternative is that reduction of

the DsbD-like trans-membrane system may be catalysed in the

dark by NTRC, a stromal Trx that is independent of light.

NTRC possesses both an NTR and a Trx domain in a single

polypeptide chain and constitutes a functional NADPH-disul-

phide reductase [77, 78]. This model would offer a reversible

regulatory mechanism, implying reduction in the dark and

oxidation in the light, in accordance with expectations that it

would be advantageous for enzymes such as VDE to be

reductively inactivated in the dark and oxidatively reactivated in

the light. Furthermore, it circumvents the problem of Trx

activity at low pH imposed by the model proposed in [20].

The strong representation of in vitro Trx targets among

the known lumenal proteins adds to the distinctive features

of the lumenal chloroplast proteome, and highlights the

impact of thiol-mediated signal transduction for the func-

tion of the thylakoid membrane. These signals appear to

regulate the turnover and function of lumenal proteins,

which participate in photosynthesis and adaptation to high

light intensities. Future experimental work is required to

clarify the nature of the thiol-based signals transmitted to

lumenal proteins and the transducers of these signals, in

order to gain understanding of the thylakoid lumen and its

interactions with other plastid compartments.

This study was supported by the Carl-Trygger Foundation(grants CTS 07:187, CTS 08:196) (T.K.), the Spanish Ministryof Science and Innovation (grant BFU 2007-60300) and theCOST action FA0603. We thank Prof. A. V. Vener for the gift ofthe FKPB13 antibody, and the Wallenberg and the KempeFoundations for funding the instruments and bioinformaticsinfrastructure at the Umea Protein Analysis Facility. Workcontributed: experimental work: M.H., A. M.-C., M.L., H.-E.A., and T.K.; laboratory resources: W.S., M.L., F.J.F., and T.K.;analytical material: A. M.-C., M.L., W.S., and T.K.; supervision:M.L., F.J.F., W.S., and T.K.; design of the study: T.K.; writingthe paper: M.L., and T.K.

The authors have declared no conflict of interest.

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