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].

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-

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

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

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

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]

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 +

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

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

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

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

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
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