Báo cáo khoa học: Identification of the N-termini of NADPH : protochlorophyllide oxidoreductase A and B from barley etioplasts (Hordeum vulgare L.) ppt

Identification of the N-termini of
NADPH : protochlorophyllide oxidoreductase A and B
from barley etioplasts (Hordeum vulgare L.)
Matthias Plo
¨
scher
1
, Bernhard Granvogl
1
, Veronika Reisinger
1
and Lutz A. Eichacker
2
1 Department of Biology I, Ludwig-Maximilians-University Munich, Germany
2 Center for Organelle Research (CORE), Universitetet i Stavanger, Norway
The first step of plant greening is catalysed by
NADPH : protochlorophyllide oxidoreductase (POR),
which is one of the most abundant enzymes found in
etioplasts. The enzyme catalyses the light-activated
reduction of protochlorophyllide (Pchlide) to chloro-
phyllide [1]. In Arabidopsis thaliana, three isoforms of
the protein, PORA, PORB and PORC, are known
[2,3]. In barley (Hordeum vulgare L.), only PORA and
PORB are found, and only one isoform of POR is
present in pea (Pisum sativum L.) [4,5]. The isoforms
accumulate as membrane-associated extrinsic proteins
in the prolamellar body and to a lesser extent in
prothylakoids [6]. The photoactive POR comprises a
stable ternary NADPH–Pchlide–POR complex that
may assemble into higher-molecular-weight oligomers
in vivo [7].

Although the various POR proteins appear to be
structurally very similar [3], their expression has been
shown to be differentially regulated by light. Expres-
sion of PORA mRNA is dependent on darkness,
whereas PORB mRNA is continuously expressed after
illumination [2,8]. Upon illumination, the prolamellar
body and prothylakoids are transformed to thylakoids,
the concentration of PORA decreases, and only PORB
remains in the chloroplasts [4].
This differential accumulation of PORA and PORB
in the inner etioplast membrane system has attracted
considerable scientific attention. The nucleus-encoded
precursor proteins of POR (pPOR) are expressed in
the cytosol. Experiments have centred on study of the
regulation of protein transport into the plastid. Two
hypotheses have been published [9]. The first hypothe-
Keywords
etioplast; N-terminus; PORA;
protochlorophyllide oxidoreductase; transit
peptide
Correspondence
L. A. Eichacker, Center for Organelle
Research (CORE), Universitetet I Stavanger,
Kristine Bonnevis vei 22, N-4036 Stavanger,
Norway
Fax: +47 518 31860
Tel: +47 518 31896
E-mail:
(Received 11 October 2008, revised 7
December 2008, accepted 10 December

2008)
doi:10.1111/j.1742-4658.2008.06850.x
The N-termini of the NADPH : protochlorophyllide oxidoreductase (POR)
proteins A and B from barley and POR from pea were determined by acet-
ylation of the proteins and selective isolation of the N-terminal peptides
for mass spectrometry de novo sequence analysis. We show that the cleav-
age sites between the transit peptides and the three mature POR proteins
are homologous. The N-terminus in PORA is V48, that in PORB is A61,
and that in POR from pea is E64. For the PORB protein, two additional
N-termini were identified as A62 and A63, with decreased signal intensity
of the corresponding N-terminal peptides. The results show that the transit
peptide of PORA is considerably shorter than previously reported and
predicted by ChloroP. A pentapeptide motif that has been characterized as
responsible for binding of protochlorophyllide to the transit peptide of
PORA [Reinbothe C, Pollmann S, Phetsarath-Faure P, Quigley F, Weis-
beek P & Reinbothe S (2008) Plant Physiol 148, 694–703] is shown here to
be part of the mature PORA protein.
Abbreviations
Pchlide, protochlorophyllide; POR, NADPH : protochlorophyllide oxidoreductase; pPOR, precursor of NADPH : protochlorophyllide
oxidoreductase; SPP, stromal processing peptidase; TNBS, 2,4,6-trinitrobenzoesulfonic acid; UPLC, ultra performance liquid chromatography.
1074 FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS
sis states that translocation across the envelope mem-
brane is mediated by the general import pathway,
utilizing the translocons of the outer and inner chloro-
plast envelope membrane, TOC and TIC [10–13]. The
second hypothesis proposes that only pPORB is
imported by the general import pathway, whereas
pPORA requires an additional mechanism, as import
was described as being dependent on Pchlide binding
to the precursor peptide [14–17]. Various protochloro-

phyllide-dependent translocon proteins have been
described [18–21]. Recently, a pentapeptide motif was
described for binding of Pchlide to the transit peptide
of pPORA [22].
The N-terminus of mature PORA from Hordeum
vulgare L. was first determined by Edman degradation
[23], and resulted in identification of G75 as the first
amino acid of the mature protein. Shortly thereafter, a
tryptic peptide, with G68 as the N-terminus was identi-
fied [24]. The N-terminus of PORB is described in the
SwissProt database, and is classified as ‘potential’
(Q42850). The database entry refers to an older charac-
terization of the N-terminus of POR from pea [5], but
this N-terminus shows no homology to the N-terminus
of PORA described by Schulz et al. [23]. Despite dis-
agreement between the results of the scientific studies,
the highly tentative determinations of these N-termini,
especially of PORA, have not been challenged experi-
mentally using modern mass spectrometric techniques.
We therefore used modern methods for precise
determination of the N-terminal cleavage sites of
PORA and PORB from barley and POR from pea.
We modified a published method to enable selective
LC-MSMS based sequencing of N-terminal peptides at
low concentration or if blocked at the N-terminus [25].
We show that the N-termini of PORA and PORB of
barley and POR from pea are homologous, and that
Pchlide cannot bind to the PORA transit peptide of
pPORA as recently proposed [22].
Results

Acetylation of the POR protein and selective
isolation of the N-terminal peptide
Proteins extracted from plant or animal tissue are well
separated by polyacrylamide gel electrophoresis to
decrease the complexity of the sample. Proteins of
equal molecular weight are concentrated in a gel band
or spot where they are accessible for identification and
further investigations. Here, we used mass spectrome-
try-based protein identification of the N-terminal
peptides of POR separated by SDS–PAGE to compare
the precursor cleavage site of various POR proteins.
For experimental determination of the mature N-ter-
minus of the gel-separated POR proteins, we modified
an experimental procedure for proteome-wide analysis
of N-terminal peptides to be used after gel separation
of proteins [25].
We used acetic anhydride for in-gel acetylation of
primary amino groups of the proteins and OMX-S
Ò
reaction tubes for efficient in-gel digestion. The
a-amino group at the N-termini and the e-amino
group of lysines were found to be completely acety-
lated, whereas serines and threonines were only
partially acetylated. Partial acetylation of the hydroxyl
groups was avoided by incubation of gel-trapped pro-
teins in hydroxylamine. Acetylated proteins were then
in-gel-digested by a rapid protocol as described in the
OMX-S
Ò
instruction manual. Instead of Tris buffer, a

disodium tetraborate buffer was used to avoid side
reactions during the following reaction steps. After
in-gel digestion, exclusively peptide sequences with
arginine at the C-terminus were identified throughout.
After extraction of the peptide solution from the poly-
acrylamide gel, the sample volume was split into two
equal parts. One part was directly separated by liquid
chromatography (UPLC), and the second part was
modified using trinitrobenzoesulfonic acid (TNBS)
before UPLC separation. As TNBS selectively modifies
the N-terminal amino groups of internal peptides, the
hydrophobicity of the corresponding peptides is
increased, leading to a delay in the retention time
during chromatographic separation. In contrast, the
N-terminal peptides were not modified at the peptide
level and hence could be easily identified as no reten-
tion shift was observed for these peptides (Fig. 1).
Finally, the exact amino acid sequence of the N-termi-
nal peptides was determined from the MS ⁄ MS spectra
recorded from peptides with unchanged chromato-
graphic separation.
Neutral solvents decrease the appearance
of alkali metal adducts
Interestingly, acetylated peptides showed significantly
increased mass signals of sodium and potassium alkali
metal and various di- and tri-alkali metal adducts if
standard solvents with 0.1% formic acid were used for
the UPLC separation (Fig. 2A). In addition, the alkali
metal adducts showed very low quality MS ⁄ MS
spectra, leading to difficulties for de novo sequencing

analysis. In order to increase the signal intensity of the
protonated signals, we exchanged the acidified solvent
containing 0.1% formic acid for a neutral solvent con-
taining 10 mm ammonium formate (Fig. 2B). Ammo-
nium adducts were eliminated completely compared to
M. Plo
¨
scher et al. N-terminus of protochlorophyllide oxidoreductase
FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS 1075
the standard method by decreasing the cone voltage to
35 V and increasing the capillary voltage to 3500 V
(see Experimental procedures).
Identification of the N-terminal amino acids from
various POR proteins
First, the N-terminal peptide of PORA from barley was
determined. A peak at 6.55 min appeared, with no
hydrophobic shift, in both chromatograms (Fig. 1). MS
analysis revealed a peptide with m ⁄ z 871.11 [M +
3H]
3+
, and fragmentation analysis resulted in a corre-
sponding amino acid sequence of VATAPSPVTT
SPGSTASSPSGKKTLR. The N-terminal amino acid
valine and both lysines in the sequence were acetylated.
Sequence comparison to the corresponding annotated
barley sequence identified V48 as first amino acid of the
mature PORA protein. In contrast, determination of
the N-terminal amino acid of PORB from barley
resulted in identification of not one but three N-termi-
nal peptides. A61 was identified as the first amino acid

of a peptide with m ⁄ z 693.31 [M + 3H]
3+
. This pep-
tide showed the highest signal intensity and had the
sequence AAAVSAPTATPASPAGKKTVR (Fig. 3).
Interestingly, we also found a second N-terminal pep-
tide signal with m ⁄ z 669.69 [M + 3H]
3+
and amino
acid sequence AAVSAPTATPASPAGKKTVR, and a
third N-terminal peptide signal with m ⁄ z 646.02 [M +
3H]
3+
and a corresponding amino acid sequence
AVSAPTATPASPAGKKTVR. All three N-terminal
peptides were acetylated at the N-terminal amino acid,
and the signal intensity decreased with lower molecular
masses. We therefore concluded that all three proteins
were acetylated exclusively at the level of the mature
protein, indicating that the various proteins resulted
from three different cleavages by the processing prote-
ase. All experimental repeats revealed an equal ratio
among the three N-terminal PORB peptides.
The N-termini of various POR proteins
are homologous
During identification of the N-terminus from POR of
pea, we also found only one N-terminal peptide, with
TOF MS ES +
BPI
1.02e

3
TOF MS ES +
BPI
728
4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00
min
0
100
%
0
100
A
B
%
N-terminal
peptide
Fig. 1. Base peak-intensity chromatogram of N-terminal and
internal peptides of PORA. Peptides of PORA were isolated from
barley etioplasts and separated by UPLC using neutral solvents
containing 10 m
M ammonium formate. One half of each sample
was separated by UPLC after in-gel acetylation and digestion with-
out further modification (A). The second part of the sample was
separated after further modification of internal peptides using
TNBS, resulting in increased hydrophobicity of internal peptides (B).
In (B), only the N-terminal peptide of PORA remains unaltered and
elutes at the same retention time of 6.55 min as in (A).
0
100
A

B
%
TOF MS ES +
1.11e3
TOF MS ES +
7.77e3
[M + 3H]
3+
[M + 2H + Na]
3+
[M + 2H + K]
3+
[M + H + 2Na]
3+
[M + 3H]
3+
[M + 2H + Na]
3+
[M + 3Na]
3+
[M + H + K + Na]
3+
870 872 874 876 878 880 882 884 886 888 890 892 894
m/z
0
100
%
878.40
883.71
878.44

871.44
871.41
891.06
885.73
Fig. 2. Mass spectra of the N-terminal pep-
tide from barley PORA. Mass spectra of the
N-terminal peptide (871.11 [M + 3H]
3+
)
were recorded after peptide ionization in
standard solvents containing 0.1% formic
acid (A) and neutral solvents containing
10 m
M ammonium formate (B). In standard
solvents, distinct sodium ([M + 2H + Na]
3+
),
disodium ([M + H + 2Na]
3+
), trisodium
(M + 3Na]
3+
) and potassium adducts
([M + 2H + K]
3+
and [M + H + K + Na]
3+
)
appeared (A). In neutral solvents, the
signal intensity of 871.11 [M + 3H]

3+
increased and only the sodium adduct
[M + 2H + Na]
3+
was detectable, with a
significantly lower signal intensity (B).
N-terminus of protochlorophyllide oxidoreductase M. Plo
¨
scher et al.
1076 FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS
m ⁄ z 804.39 [M + 3H]
3+
and amino acid sequence
ETAAPATPAVNKSSSEGKKTLR (data not shown).
The first amino acid of the mature protein was deter-
mined to be E64. This finding appeared at first to be
in conflict with the SwissProt database entry, in which
T65 is denoted as the N-terminal amino acid of the
mature POR (SwissProt entry Q01289). However, in
the reference publication cited by SwissProt, E64 has
been determined by Edman degradation to be the first
amino acid of the mature protein, corroborating
our finding [5]. The sequences of PORA and PORB
from barley and POR from pea were aligned using
clustal w ( />mat.pl?page=npsa_clustalw.html) (Fig. 4). Sequence
homology in the N-terminal region was found, indicat-
ing that the N-terminus of POR from pea is positioned
one amino acid upstream in comparison to PORA and
the main PORB protein identified in barley.
When we performed a theoretical calculation of

the N-terminus of PORA protein using the program
chlorop ( />A91 was predicted to be the first amino acid of the
mature protein (Fig. 4). Hence, there is a difference of
43 amino acids from the N-terminus determined here.
Previous descriptions of the N-terminus of PORA also
differ significantly from our results. Schultz et al.
described G75 as the first amino acid of the mature pro-
tein. This processing site was determined by a method
based on Edman degradation [23] (Fig. 4). Later, the
same group described a tryptic peptide with G68 as the
first amino acid [24] (Fig. 4). For PORB of barley, Chlo-
roP predicts A59 as the first amino acid. This prediction
is close to A61, which was experimentally determined to
be the first amino acid of the most intense signal of the
three N-termini of PORB. In the case of mature POR of
pea, chlorop predicted A63 as the first amino acid. This
prediction differs by only one amino acid from E64,
which is the first amino acid of the mature POR protein
according to our experimental determination.
Discussion
Pchlide binding motifs are only found in the
mature PORA
In contrast to previous publications [26], we found that
the N-termini of the various POR proteins show
strong sequence homology (Fig. 4), and our results
also indicate a significantly shorter transit peptide for
PORA. This finding is of importance with respect to a
hypothesis proposed regarding Pchlide-dependent
import of pPORA [15,17]. In favour of this hypothesis,
TOF MS ES +

621
640 645 650 655 660 665 670 675 680 685 690 695 700 705 710
m/z
0
100
%
[M – A + 3H]
3+
[M – 2A + 3H]
3+
[M + 3H]
3+
[M + 2H + Na]
3+
693.36
700.70
669.69
646.02
Fig. 3. Mass spectrum of N-terminal peptides from PORB. Pep-
tides were ionized in neutral solvents with 10 m
M ammonium for-
mate. Three peptides were identified with m ⁄ z 693.36 [M + 3H]
3+
,
669.69 [M + 3H]
3+
and 646.02 [M + 3H]
3+
. The highest signal
intensity at 693.36 [M + 3H]

3+
was identified by de novo sequence
analysis as a peptide form containing three N-terminal alanines. The
minor peptide signals were identified as two alternative N-terminal
PORB peptides containing two alanines with m ⁄ z 669.69
[M ) A + 3H]
3+
and one alanine with m ⁄ z 646.02 [M ) 2A + 3H]
3+
.
Fig. 4. Sequence alignment of pPORA and pPORB from barley and pPOR from pea. Arrowheads indicate the cleavage sites between the
transit peptide and the mature PORA protein. The transit peptide as described here is shown in bold type. The bold arrow (
) indicates the
position of the experimentally verified N-terminus of PORA. Previous descriptions of the position of the N-terminus according to Benli et al.
[23] are marked by a narrower arrow (
), and that according to Schulz et al. [24] by a short arrow ( ). The arrowhead ( ) indicates the pre-
dicted cleavage site according to the program
CHLOROP ( The pentapeptide motif proposed to be
responsible for binding of Pchlide according to Reinbothe et al. [22] is shown in bold, italic letters and is only present in PORA. Identical resi-
dues are indicated by asterisks, strongly similar residues are indicated by colons, and weaker similarity is indicated by dots to illustrate the
homology of the cleavage sites between PORA and PORB from barley and POR from pea. The complete protein sequence alignment was
performed using
CLUSTAL W [37].
M. Plo
¨
scher et al. N-terminus of protochlorophyllide oxidoreductase
FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS 1077
it has been proposed that the transit peptide contains
a Pchlide binding site [22,26], and that binding of Pch-
lide to pPORA is essential for import into the etioplast

stroma. The transit peptide of PORA is the only exam-
ple of a precursor protein for which a substrate bind-
ing site has been proposed to regulate import in
addition to the general import pathway [19–21]. This
hypothesis was based on chimeric fusion proteins in
which the transit peptide of pPORA was functionally
exchanged with the transit peptide of pPORB and vice
versa, and the transit peptides were fused to a reporter
protein of mouse. It was found that only the isolated
transit peptide of PORA bound Pchlide, with a stoichi-
ometry of 1 : 1 [26]. Recently, amino acids T56–G60
have been defined as a pentapeptide motif that is
responsible for binding Pchlide and for the import of
PORA [22] (Fig. 4). However, according to our find-
ings, amino acids T56–G60 of PORA are located
exclusively in the mature part of the PORA protein
and not in the transit peptide. The position of the
motif in the N-terminal region of the mature protein is
in conflict with a function of the motif in a Pchlide-
responsive transit peptide as described previously [22].
Pchlide-dependent import would require a binding of
Pchlide to the mature part of PORA, which is C-termi-
nal of the processing site. It remains open whether
Pchlide binding to the proposed motif in mature
PORA is of importance for regulation of PORA
import [22]. In addition to a regulatory function in
protein import, binding of Pchlide at this binding site
could be important for transient stabilization of the
PORA protein in the plastid stroma after import and
before the protein is assembled into an enzymatically

active form. As a number of groups have found that
accumulation of PORA in the plastid stroma is a
substrate-independent process, close inspection of pub-
lished data and development of new experimental
set-ups is essential to clarify this interesting topic
[6,10–13,27].
Alternative N-termini of PORB
In contrast to the one unique processing site that we
describe here for the PORA protein, we found three
possible N-termini for the PORB protein. The N-ter-
minal amino acid of the corresponding peptide signal
with the most intense signal is homologous to the
N-terminal amino acid of PORA (Fig. 4). The two
additional N-terminal peptides of PORB both start
with the amino acid alanine. In parallel with the loss
of one and two amino groups, the signals of the N-ter-
minal peptides decrease in intensity. This could be
indicative of a correspondingly lower concentration of
these two alternative PORB proteins. The reason for
differential processing of PORB could be error-prone
positioning of the processing peptidase at the cleavage
site in the presence of three consecutive alanines, or
could indicate that a second processing peptidase scans
the N-termini after the first cleavage.
The cleavage site is characterized by a conserved
arginine within the transit peptide, which is located
two amino acids upstream of the N-terminal cleavage
site. Aliphatic and non-polar amino acids are found
N-terminal to the arginine. Alanine or threonine is
found C-terminal to the processing site. Similar amino

acids are present around the processing site in POR of
pea. Glutamine has been found to be the last amino
acid of the transit peptide in PORA and B of barley,
whereas POR from pea contains a glutamic acid at this
position, which is the first amino acid of the mature
protein. The sequence homology around the amino
acids of the processing site therefore leads to the con-
clusion that the processing peptidase or peptidases
might be the same for all POR proteins [28,29].
Although a general stromal processing peptidase (SPP)
has been characterized, and a preferred consensus
sequence for cleavage between a basic amino acid
(arginine or lysine) and a C-terminal alanine was
described [30,31], it is open where exactly SPP cleaves.
Two alternative N-termini have been described for the
stromal cysteine synthase [32], similar to the three
N-termini of PORB described here. SPP may therefore
cleave specifically after R-58, with a second less specific
processing peptidase cleaving C-terminal of A59, Q60
and A61 ⁄ 62. Then, V48 of PORA at the homologous
position to A61 of PORB would position the second
processing protease to yield one exactly defined
N-terminus. Alternatively, SPP may be the only pro-
cessing peptidase. In this case, the cleavage site down-
stream of the SPP consensus motif has a lower
specificity for PORB.
Experimental procedures
Chemicals
All organic solvents and water used in this work were of
HPLC gradient quality and purchased from Fisher Scien-

tific (Schwerte, Germany). Acetic anhydride, ammonium
formate and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were
obtained from Fluka (Buchs, Switzerland), Coomassie
Ò
brilliant blue R250 was obtained from Serva (Heidelberg,
Germany), and disodium tetraborate decahydrate was
obtained from Merck (Darmstadt, Germany). Sequencing
grade modified trypsin was purchased from Promega
(Mannheim, Germany).
N-terminus of protochlorophyllide oxidoreductase M. Plo
¨
scher et al.
1078 FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS
Protein isolation and gel electrophoresis
Etioplasts were isolated from 4.5-day-old dark-grown bar-
ley seedlings (Hordeum vulgare L. var. Steffi) as described
previously [33]. Membrane proteins were solubilized in SDS
buffer (3% w ⁄ v SDS, 15% w ⁄ v sucrose, 100 mm sodium
carbonate, 0.04% w ⁄ v bromophenol blue, 0.3% v ⁄ v b-mer-
captoethanol) by heating for 2 min at 72 °C, and separated
on 12.5% SDS–polyacrylamide gels containing 4 m urea in
a Protean II electrophoresis system (Bio-Rad, Hercules,
CA, USA) [34]. For each lane, proteins from 1 · 10
8
plast-
ids were loaded. Gels were stained with Coomassie brilliant
blue.
POR from etiolated pea leaves (Pisum sativum L. var.
Violetta) was isolated from 14-day-old seedlings grown in
the dark on vermiculite. Due to the small size of the leaves

(approximately 1 mm
2
), membrane-associated proteins were
prepared from the whole leaf material. In brief, the leaves
were ground in TMK buffer (10 mm Tris ⁄ HCl pH 6.8,
10 mm magnesium chloride, 20 mm potassium chloride) at
4 °C and centrifuged at 16 000 g for 5 min. The superna-
tant was discarded, and the pellet was resuspended in TMK
buffer and centrifuged twice at 16 000 g for 5 min to
remove soluble proteins. Then the pellet was resuspended in
SDS buffer and proteins were solubilized by heating for
5 min at 72 °C before separation on a 12.5% SDS–poly-
acrylamide gel containing 4 m urea. Proteins were stained
with Coomassie brilliant blue and fixed in the gel matrix by
incubation in acetic acid (10%). Detection of POR was
carried out by gel-blot analysis [35].
In-gel acetylation and in-gel digestion
For sample preparation, the OMX-SÒ tool for in-gel diges-
tion was used (OMX, Wessling, Germany) [36]. Briefly, the
protein spot of interest was excised from the SDS gel and
the gel was ruptured by centrifugation at 13 000 g for 2 min.
Proteins were destained in 50 mm ammonium bicarbonate
and 50% acetonitrile at 37 °C for 5 min. Then the orienta-
tion of the OMX-SÒ tool in the centrifuge was inverted, and
the solution was removed from the reaction chamber by cen-
trifugation at 2500 g. For in-gel acetylation of intact pro-
teins, 22.5 lL of 50 : 50 (v ⁄ v) acetonitrile ⁄ water and 2.5 lL
of acetic anhydride were added to the reaction chamber, and
the mixture was incubated at 37 °C for 30 min. Thereafter,
acetic anhydride was removed completely by washing three

times with 25 lL of 50 : 50 (v ⁄ v) acetonitrile ⁄ water for 5
min each. Partial acetylation of serines and threonines was
avoided by adding 12 lL of a solution containing 0.5 mm
hydroxylamine and 100 mm NaOH to the reaction chamber
and incubating at 37 °C for 15 min. Thereafter, 12 lL aceto-
nitrile was added to the sample, and the solution was
removed by reverse centrifugation.
After in-gel acetylation, in-gel digestion was carried out
utilizing 20 lLof50mm disodium tetraborate buffer,
pH 8.5, and 2 lL of trypsin at 50 °C for 45 min. The pep-
tide mixture was removed from the gel pieces and split
into two equal parts (A and B). The volume of part A
was increased to 30 lL using 50 mm disodium tetraborate
buffer, pH 8.5, and acidified with 5 lL of 40% formic
acid to stop trypsin digestion. The peptide mixture in
part B was modified with 100 mm TNBS solution in
water. For this modification, the sample volume was
increased to 28 lL with disodium tetraborate buffer,
pH 9.8, resulting in a final pH of the sample of 9.5. Then
2 lL of 100 mm TNBS solution were added, and the mix-
ture was incubated at 37 °C for 1 h. Finally, the sample
was acidified using 5 lL of 40% formic acid.
UPLC separation and mass spectrometry
For peptide separation, a Waters nanoAquityÔ 10 000 psi
UPLC system (Waters Corporation, Milford, MA, USA)
was used, equipped with a BEH130 C18 nanoflow column,
particle size 1.7 lm, with an inner diameter of 100 lm and
a length of 100 mm, and a Symmetry C18 trapping column,
particle size 5 lm, and dimensions 180 lm · 20 mm. Two
solvent systems – standard solvents acidified with formic

acid and neutral solvents with ammonium formate – were
used. In the standard approach, solvent A was composed
of 95 : 5 (v ⁄ v) water ⁄ acetonitrile and the solvent was acidi-
fied by addition of 0.1% formic acid, and solvent B was
composed of 99.9 : 0.1 (v ⁄ v) acetonitrile ⁄ formic acid. In the
neutral approach, solvent A comprised 95 : 5 (v ⁄ v)
water ⁄ acetonitrile and 10 mm ammonium formate was
added, and solvent B comprised 100% acetonitrile. The
same solvent gradient was used for both approaches. The
sample was trapped for 2 min at a flow rate of 15 lLÆ
min
)1
, followed by a linear gradient from 1% to 80%
solvent B applied over 16 min with a flow rate of
1.2 lLÆmin
)1
.
The UPLC equipment was connected to a Micromass
Q-TOF Premier mass spectrometer (Waters Corporation,
Milford, MA, USA). Mass spectra were obtained by auto-
mated LC-MS and LC-MS ⁄ MS analysis, and peptides were
identified using masslynx version 4.1 (Waters Corpora-
tion). With standard solvents, a capillary voltage of 3000 V
and a cone voltage of 45 V was used. With neutral solvents,
a capillary voltage of 3500 V and a cone voltage of 35 V
was used. The two MS chromatograms from parts A and B
of each sample were aligned using masslynx.
Acknowledgements
The work was funded by the Deutsche Forschungs-
gemeinschaft and the Sonderforschungsbereich Trans-

regio1 (SFB TR1). The antibody against the mature
part of POR was donated by Professor Sundquvist,
Sweden.
M. Plo
¨
scher et al. N-terminus of protochlorophyllide oxidoreductase
FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS 1079
References
1 Schoefs B & Franck F (2003) Protochlorophyllide
reduction: mechanisms and evolutions. Photochem
Photobiol 78, 543–557.
2 Armstrong GA, Runge S, Frick G, Sperling U & Apel
K (1995) Identification of NADPH:protochlorophyllide
oxidoreductases A and B: a branched pathway for
light-dependent chlorophyll biosynthesis in Arabidopsis
thaliana. Plant Physiol 108, 1505–1517.
3 Oosawa N, Masuda T, Awai K, Fusada N, Shimada H,
Ohta H & Takamiya K (2000) Identification and light-
induced expression of a novel gene of NADPH-proto-
chlorophyllide oxidoreductase isoform in Arabidopsis
thaliana. FEBS Lett 474, 133–136.
4 Reinbothe S, Reinbothe C, Holtorf H & Apel K (1995)
Two NADPH:protochlorophyllide oxidoreductases in
barley: evidence for the selective disappearance of
PORA during the light-induced greening of etiolated
seedlings. Plant Cell 7, 1933–1940.
5 Spano AJ, He Z, Michel H, Hunt DF & Timko MP
(1992) Molecular cloning, nuclear gene structure, and
developmental expression of NADPH: protochlorophyl-
lide oxidoreductase in pea (Pisum sativum L.). Plant

Mol Biol 18, 967–972.
6 Dahlin C, Sundqvist C & Timko MP (1995) The
in vitro assembly of the NADPH-protochlorophyllide
oxidoreductase in pea chloroplasts. Plant Mol Biol 29,
317–330.
7 Boeddi B, Lindsten A, Ryberg M & Sundqvist C (1989)
On the aggregational states of protochlorophyllide and
its protein complexes in wheat etioplasts. Physiol Plant
76, 135–143.
8 Holtorf H, Reinbothe S, Reinbothe C, Bereza B &
Apel K (1995) Two routes of chlorophyllide synthesis
that are differentially regulated by light in barley
(Hordeum vulgare L.). Proc Natl Acad Sci USA 92,
3254–3258.
9 Aronsson H, Sundqvist C & Dahlin C (2003) POR –
import and membrane association of a key element in
chloroplast development. Physiol Plant 118, 1–9.
10 Philippar K, Geis T, Ilkavets I, Oster U, Schwenkert S,
Meurer J & Soll J (2007) Chloroplast biogenesis: the
use of mutants to study the etioplast–chloroplast transi-
tion. Proc Natl Acad Sci USA 104, 678–683.
11 Aronsson H, Sundqvist C & Dahlin C (2003) POR hits
the road: import and assembly of a plastid protein.
Plant Mol Biol 51, 1–7.
12 Aronsson H, Sohrt K & Soll J (2000) NADPH:proto-
chlorophyllide oxidoreductase uses the general
import route into chloroplasts. Biol Chem 381, 1263–
1267.
13 Dahlin C, Aronsson H, Almkvist J & Sundqvist C
(2000) Protochlorophyllide-independent import of two

NADPH:Pchlide oxidoreductase proteins (PORA and
PORB) from barley into isolated plastids. Physiol Plant
109, 298–303.
14 Kim C & Apel K (2004) Substrate-dependent and
organ-specific chloroplast protein import in planta.
Plant Cell 16, 88–98.
15 Reinbothe S, Mache R & Reinbothe C (2000) A second,
substrate-dependent site of protein import into chloro-
plasts. Proc Natl Acad Sci USA 97, 9795–9800.
16 Reinbothe S, Reinbothe C, Neumann D & Apel K
(1996) A plastid enzyme arrested in the step of precur-
sor translocation in vivo. Proc Natl Acad Sci USA
93,
12026–12030.
17 Reinbothe S, Runge S, Reinbothe C, van Cleve B &
Apel K (1995) Substrate-dependent transport of the
NADPH:protochlorophyllide oxidoreductase into iso-
lated plastids. Plant Cell 7, 161–172.
18 Kim C, Ham H & Apel K (2005) Multiplicity of different
cell- and organ-specific import routes for the NADPH-
protochlorophyllide oxidoreductases A and B in plastids
of Arabidopsis seedlings. Plant J 42, 329–340.
19 Reinbothe S, Pollmann S, Springer A, James RJ, Tich-
tinsky G & Reinbothe C (2005) A role of Toc33 in the
protochlorophyllide-dependent plastid import pathway
of NADPH:protochlorophyllide oxidoreductase (POR)
A. Plant J 42, 1–12.
20 Reinbothe S, Quigley F, Gray J, Schemenewitz A &
Reinbothe C (2004) Identification of plastid envelope
proteins required for import of protochlorophyllide

oxidoreductase A into the chloroplast of barley. Proc
Natl Acad Sci USA 101, 2197–2202.
21 Reinbothe S, Quigley F, Springer A, Schemenewitz A &
Reinbothe C (2004) The outer plastid envelope protein
Oep16: role as precursor translocase in import of proto-
chlorophyllide oxidoreductase A. Proc Natl Acad Sci
USA 101, 2203–2208.
22 Reinbothe C, Pollmann S, Phetsarath-Faure P, Quigley
F, Weisbeek P & Reinbothe S (2008) A pentapeptide
motif related to a pigment binding site in the major
light-harvesting protein of photosystem II, LHCII,
governs substrate-dependent plastid import of
NADPH:proto-chlorophyllide oxidoreductase (POR)
A. Plant Physiol 148, 694–703.
23 Schulz R, Steinmuller K, Klaas M, Forreiter C,
Rasmussen S, Hiller C & Apel K (1989) Nucleotide
sequence of a cDNA coding for the NADPH-protochlo-
rophyllide oxidoreductase (PCR) of barley (Hordeum
vulgare L.) and its expression in Escherichia coli. Mol
Gen Genet 217, 355–361.
24 Benli M, Schulz R & Apel K (1991) Effect of light
on the NADPH-protochlorophyllide oxidoreductase
of Arabidopsis thaliana. Plant Mol Biol 16, 615–
625.
25 Gevaert K, Goethals M, Martens L, Van Damme J,
Staes A, Thomas GR & Vandekerckhove J (2003)
Exploring proteomes and analyzing protein processing
N-terminus of protochlorophyllide oxidoreductase M. Plo
¨
scher et al.

1080 FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS
by mass spectrometric identification of sorted N-termi-
nal peptides. Nat Biotechnol 21, 566–569.
26 Reinbothe C, Lebedev N, Apel K & Reinbothe S (1997)
Regulation of chloroplast protein import through a pro-
tochlorophyllide-responsive transit peptide. Proc Natl
Acad Sci USA 94, 8890–8894.
27 Aronsson H, Sundqvist C, Timko M & Dahlin C (2001)
Characterization of the assembly pathway of the pea
NADPH : protochlorophyllide (Pchlide) oxidoreductase
(POR), with emphasis on the role of its substrate, Pch-
lide. Physiol Plant 111, 239–244.
28 Bruce BD (2001) The paradox of plastid transit
peptides: conservation of function despite diver-
gence in primary structure. Biochim Biophys Acta 1541,
2–21.
29 Emanuelsson O, Nielsen H & von Heijne G (1999)
ChloroP, a neural network-based method for predicting
chloroplast transit peptides and their cleavage sites.
Protein Sci 8, 978–984.
30 Richter S & Lamppa GK (2003) Structural properties
of the chloroplast stromal processing peptidase required
for its function in transit peptide removal. J Biol Chem
278, 39497–39502.
31 Richter S & Lamppa GK (1998) A chloroplast process-
ing enzyme functions as the general stromal processing
peptidase. Proc Natl Acad Sci USA 95, 7463–7468.
32 Zybailov B, Rutschow H, Friso G, Rudella A, Emanu-
elsson O, Sun Q & van Wijk KJ (2008) Sorting signals,
N-terminal modifications and abundance of the chloro-

plast proteome. PLoS ONE 3, e1994.
33 Eichacker LA, Muller B & Helfrich M (1996) Stabiliza-
tion of the chlorophyll binding apoproteins, P700,
CP47, CP43, D2, and D1, by synthesis of Zn-pheophy-
tin a in intact etioplasts from barley. FEBS Lett 395,
251–256.
34 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
35 Towbin H, Staehelin T & Gordon J (1979) Electro-
phoretic transfer of proteins from polyacrylamide gels
to nitrocellulose sheets: procedure and some
applications. Proc Natl Acad Sci USA 76, 4350–
4354.
36 Granvogl B, Gruber P & Eichacker LA (2007) Stan-
dardisation of rapid in-gel digestion by mass spectro-
metry. Proteomics 7, 642–654.
37 Thompson JD, Higgins DG & Gibson TJ (1994)
CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight
matrix choice. Nucleic Acids Res 22, 4673–
4680.
M. Plo
¨
scher et al. N-terminus of protochlorophyllide oxidoreductase
FEBS Journal 276 (2009) 1074–1081 ª 2009 The Authors Journal compilation ª 2009 FEBS 1081