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
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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.
990 M. Hall et al. Proteomics 2010, 10, 987–1001
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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
.
992 M. Hall et al. Proteomics 2010, 10, 987–1001
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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.
994 M. Hall et al. Proteomics 2010, 10, 987–1001
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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.
996 M. Hall et al. Proteomics 2010, 10, 987–1001
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com
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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