Báo cáo Y học: The plant S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase is located in both envelope and thylakoid chloroplast membranes pot
The plant
S
-adenosyl-
L
-methionine:Mg-protoporphyrin IX
methyltransferase islocatedinbothenvelopeand thylakoid
chloroplast membranes
Maryse A. Block, Arun Kumar Tewari, Catherine Albrieux, Eric Mare
Â
chal and Jacques Joyard
Laboratoire de Physiologie Cellulaire Ve
Â
ge
Â
tale, CNRS/CEA/Universite
Â
Joseph Fourier, DBMS/PCV, Grenoble, France
Chlorophyll biosynthesis requires a metabolic dialog
between thechloroplastenvelopeand thylakoids where
biosynthetic activities are localized. Here, we report t he
®rst plant S-adenosyl-
L
-methionine:Mg-protoporphyrin
IX methyltransferase ( MgP
IX
MT) sequence identi®ed in
the Arabidopsis genome owing to its s imilarity with the
Synechocystis sp. MgP
IX
MT gene. After expr ession in
Escherichia coli, the recombinant Arabidopsis thaliana
cDNA was shown to encode a protein having MgP
IX
MT
activity. The full-length polypeptide exhibits a chloroplast
transit peptide that is processed d uring import into the
chloroplast. The m ature p rotein contains two functional
regions. T he C-terminal part aligns with the Synecho-
cystis full-length protein. The corresponding truncated
region binds to Ado-met, as assayed by UV crosslinking,
andisshowntoharbortheMgP
IX
MT activity. Down-
stream of the c leaved transit peptide, t he 40 N-terminal
amino acids of the mature protein are very hydrophobic
and enhance the association of the protein with the
membrane. I n A. thaliana and spinach, the MgP
IX
MT
protein has a dual localization i n chloroplast envelope
membranes as well as in thylakoids. The protein is active
in each membrane and has the s ame a pparent size cor-
responding to the processed mature protein. The protein
is very likely a monotopic m embrane p rotein embed ded
within one lea¯et of the membrane as indicated by ionic
and alkaline extraction of each membrane. The rationale
for a dual localization of the protein inthechloroplast is
discussed.
Keywords: protoporphyrin; methyltransferase; chloroplast;
membrane; chlorophyll.
The chlorophyll molecule is made up of two moieties of
distinct origin, c hlorophyllide and phytol. The initial steps
in c hlorophyllide synthesis, from the biosynthesis of
d-aminolevulinate to protoporphyrinogen IX, occur in
the soluble phase of plastids whereas the subsequent steps
are membrane-bound [1]. Envelopemembranes constitute
the main membrane system present in early development
of proplastids into chloroplasts. Although d evoid of
chlorophyll, they play a r ole in t he initial steps of
chlorophyll biosynthesis. Several results i ndicate that
chlorophyllide synthesis is at some point associated with
the envelope. Studies of ¯uorescence properties of isolated
envelope membranes f rom spinach chloroplasts demon-
strated the presence of small but signi®cant amounts of
protochlorophyllide and chlorophyllide [2,3]. Localization
of enzymatic activities has shown that several enzymes of
the biosynthetic pathway are linked to t he envelope; the
protoporphyrinogen oxidase is present inboththe thyla-
koids andtheenvelopemembranes [4]; the subunits of the
Mg chelatase are present in large amounts inthe stroma
but become associated with theenvelope at Mg
2+
concentration p resent in illuminated chloroplasts [5]; the
protochlorophyllide oxidoreductase (POR) that accumu-
lates in e tioplast p rolamellar bo dies [6] remains in low
amount in mature chlo roplasts where its activity is
detected intheenvelope [7]. POR w as immunologically
detected on the outer surface of thechloroplast envelope
[7]. Although not immunodetected inthe thylakoids [7],
POR could be also present inthe thylakoids considering
that in vitro import i nto chloroplasts of the POR precursor
from pea led to a main localization of the mature protein
in this membrane system [8,9].
So far, several e nzymatic steps of t he protoporphyri-
nogen to chlorophyllide pathway have not been localized.
In the present study, we f ocus on methylation of Mg-
protoporphyrin IX. Themethyltransferase has not yet
been identi®ed in plants but has bee n clearly recognized in
purple bacteria [10,11] andin cyanobacteria [12] by
functional complementation and b y expression of active
recombinant protein. In plants, the activity of the
S-adenosyl-
L
-methionine:Mg-protoporphyrin IX m ethyl-
transferase (MgP
IX
MT) was reported as membrane-bound
and r ather unstable [ 13±15]. The t etrapyrrole substrate
and product of this e nzyme never accumulate and t hey
may play a role in controlling t he expression of some light
dependent genes [16±20].
In this paper, we identify the Arabidopsis gene encoding
MgP
IX
MT, and characterize the encoded protein. We
further a nalyze the localization of theplant protein within
chloroplast membranesand discuss the results in relation to
chloroplast biogenesis.
Correspondence to M. Block, DBMS/PCV, CEA-Grenoble, F-38054,
Grenoble-cedex 9, France. Fax: + 33 4 38 78 50 91,
Tel.: + 33 4 38 7 8 49 85, E-mail: mblock@cea.fr
Abbreviations:Ado-met,S-adenosyl-
L
-methionine; MgP
IX
MT,
S-adenosyl-
L
-methionine:Mg-protoporphyrin IX methyltransferase;
MGDG, monogalactosyldiacylglycerol; MGD, monogalactosyldia-
cylglycerol synthase; IPTG, isopropyl thio-b-
D
-galactoside.
(Received 31 August 2 001, revised 25 October 2001, accepted 30
October 2001)
Eur. J. Biochem. 269, 240±248 (2002) Ó FEBS 2002
MATERIALS AND METHODS
Chemicals
Unlabeled Ado-met, p-toluenesulfonate salt was purchased
from Sigma and [methyl-
3
H]Ado-met
1
(3 TBqámmol
)1
)was
from NEN ( ref. NET-155H). Mg-protoporphyrin IX
disodium salt was purchased from Porphyrin products
Inc. and protoporphyrin IX disodium salt was from Sigma.
cDNA cloning
A 9 50-bp nucleic fragment was ampli®e d by PCR from
Arabidopsis t haliana cDNA prepared from 3-week-old leaf
poly(A) mRNA using the Clontech Advantage polymerase
and primers speci®c for the 5¢ and 3¢ ends of the coding
sequence of the putative MgP
IX
MT. The forward primer
was 5¢-CATATGCCGTTTGCTCCTTCC-3¢ and the
reverse primer 5¢-CATATGGCTTACATTGGAACAG
CTTC-3¢, each including a NdeIsiteattheendofthe
ampli®ed fragment. The ampli®ed cDNA was then blunt
endclonedintheSmaI restriction site of pBluescriptSK
+
and con®rmed by sequencing. The NdeI linearized insert
was cloned inthe modi®ed pET-Y3a p lasmid [21]. This
plasmid was used in order to overcome the problem raised
by the fact that the putative A. thaliana MgP
IX
MT protein
contains at the b eginning of its s equence numerous
arginines, encoded by AGG or AGA, which are poo rly
used in Escherichia coli. Orientation of the plasmid i nserts
was checked by BamHI digestion and PCR. Partial cDNA
fragments, corresponding to the protein shortened from the
®rst 79 amino a cids (D80-protein), or from the ®rst 1 60
amino acids (D161-protein), were prepared again by PCR
ampli®cation with forward primer 5¢-AGGCATAT
GTTGTTGCAGGCGGAGGAA-3¢ (80-NdeI) or forward
primer 5¢-CTCCATATGCCACTTGCTAAGGAAG-3¢
(161±NdeI) coupled to reverse primer 5¢-TTCAGGATCCT
CTACATTGGAACA-3¢ (sto p±BamHI). They were cloned
in NdeIandBa mHI restriction sites of pET-Y3a to express
truncated forms of the MgP
IX
MT.
Preparation of recombinant proteins
BL21(DE3) strain of E. coli was transformed with pET-Y3a
plasmid containing various inserts. Bacterial cultures w ere
grown at 37 °C under vigorous shaking until the
D
600
0.5. Expression of the recombinant proteins was
then induced by addition of 1 m
M
isopropyl thio-b-
D
-
galactoside (IPTG) inthe medium. The cultures were then
transfered to 28 °C for 2 h. Bacte ria w ere centrifuged and
the pellets stored at )70 °C.
Preparation of spinach chloroplast subfractions
All the procedures were carried out at 0±5 °C. Crude
chloroplasts were obtained from 3 to 4 kg o f spinach
(Spinacia oleracea L.) leaves andchloroplast subfractions
were puri®ed as described previously [7]. Puri®ed i ntact
chloroplasts were then lysed in buffer H (10 m
M
Mops
pH 7.8, 1 m
M
caproic acid, 1 m
M
phenylmethanesulfonyl
¯uoride with 4 m
M
MgCl
2
), andchloroplast subfractions
were separated by centrifugation on a linear s ucrose
gradient (0.6±4
M
sucrose in buffer H with 4 m
M
MgCl
2
).
To limit the sedimentation of soluble p roteins, such as
Rubisco, through the sucrose gradient, after 1 h centrifu-
gation at 70 000 g, the upper part of the tube content
corresponding to the loaded volume was replaced by the
same volume of buffer H with 4 m
M
MgCl
2.
The g radient
was further centrifuged at 70 000 g for 12 h . Chloroplast
envelope was collected as a yellow band inthe medium part
of the gradient whereas thylakoids were collected as a p ellet.
Each fraction was diluted three times in buffer H. Mem-
branes were then washed an d resuspended in 100 lLof
buffer H. monogalactosyldiacylglyce rol (MGDG)
2
synthase
activity was measured as described previously [21].
Puri®cation of envelope membranes
and thylakoids from
arabidopsis
chloroplasts
A. thaliana plants (e cotype ws) were gro wn for 6 weeks
under a 10-h p hotoperiod on compost
3
. The leaves (300 g)
were homogenized in a Waring Blender in 1.5 L ice-cold
buffer containing 0.45
M
sorbitol, 20 m
M
tricine/NaOH
pH 8.4, 10 m
M
EDTA, 1 0 m
M
NaHCO
3
,and0.1%BSA.
A crude chloroplast pellet was obtained by centrifugation at
1500 g for 3 min and further puri®ed in 0.33
M
sorbitol,
20 m
M
Mops pH 7.6, 5 m
M
MgCl
2
,2.5m
M
EDTA (buffer
P) on a Percoll gradient formerly prepared by centrifugation
of 45% (v/v) Percoll i n a SS90 vertical rot or at 10 000 g for
100 min. After centrifu gation at 5000 g for 10 min, intact
chloroplasts were recovered i n the Percoll gradient as a
heavy green layer. The c hloroplasts were washed twice with
buffer P and then broken in 10 m
M
Mops pH 7.6, 4 m
M
MgCl
2
,1m
M
phenylmethanesulfonyl ¯uoride and 1 m
M
caproic acid. Chloroplast subfractions were separated on a
step gradient of 0.93
M
to 0.6
M
sucrose in buffer R (10 m
M
Mops pH 7.6, 1 m
M
MgCl
2
) by centrifugation at 70 000 g
for 1 h. Theenvelope was co llected at the interface between
the layers of 0.6
M
and 0.93
M
sucrose and thylakoids as
a pellet. Each fraction was washed a nd resuspended in
buffer R.
In vitro
import of MgP
IX
MT into chloroplasts
The p BluescriptSK
+
plasmid c ontaining the f ull-length
cDNA of the MgP
IX
MT under T7 promoter was linearized
downstream of t he inserted DNA fragment using the
HindIII restriction site. Transcription a nd translation were
carried out in vitro with T7 polymerase and wheat germ
extract ( TNT C oupled Wheat Germ Extract System,
Promega) and [
35
S]methionine (37 TBqámmol
)1
,Amer-
sham). The import was performed at room temperature
for 15 m in on 10-day-old pea chloroplasts (15 lg chloro-
phyll) with 5 lL of t ranslated protein in 100 lL of i mport
buffer ( 330 m
M
sorbitol, 50 m
M
Hepes/KOH pH 7.6, 3 m
M
MgSO
4
,10m
M
methionine, 20 m
M
Kgluconate
4
,10m
M
NaHCO
3
,2%BSAand3m
M
ATP). Th i ntact chloroplasts
were then puri®ed by centrifugation through a 40% (v/v)
Percoll layer ( 300 lL) andthe pellet was washed twice by
centrifugation inchloroplast washing medium (330 m
M
sorbitol , 50 m
M
Hepes/KOH and 3 m
M
MgCl
2
). The pelle t
was either resuspended in SDS/PAGE loading buffer or in
100 lL washing medium containing 0.5 m
M
CaCl
2
and
0.03 mg ámL
)1
thermolysin (Boehringer). Inthe case o f
thermolysin treatment, chloroplasts were incu bated f or
20 min o n ice, then 10 m
M
EDTA was added and
Ó FEBS 2002 Theplant Mg-protoporphyrin IXmethyltransferase (Eur. J. Biochem. 269) 241
chloroplasts were immediately pelleted and resuspended in
SDS/PAGE sample buffer. Proteins were analyzed by
¯uorography on a 1 2% acrylamide gel under ®lms
(Amersham MP) at )70 °C for 1 week.
Assay of MgP
IX
MT activity
MgP
IX
MT activity was measured in an a ssay mixture
containing 20 m
M
Mops pH 7.8, 3 m
M
MgCl
2
,1m
M
dithiothreitol, 25 m
M
NaCl with the addition of 50 l
M
Mg-protoporphyrin IXand of 10 l
M
, S-[CH
3
-
3
H]Ado-met
(1 TBqámmol
)1
), prepared just before the use by adding
cold Ado-met and labeled Ado-met and neutralizing with
BaCO
3
, as described previously [22]. Incubation was
carried out with 5 ±20 lg protein in 24 lL of reaction
mixture at 30 °C for 1±10 min. The reaction was then
stopped by adding 1 mL of ice cold H
2
O. Extraction of
tetrapyrroles was performed a s described previously [10].
Proteins were precipitated by adding 3 m L of ice cold
acetone to the sample, mixing and centrifugation at 7700 g
for 5 min at 4 °C. The supernatant was retained and the
pellet w as resuspended in 500 lLof0.125
M
NH
4
OH and
1.5 mL o f a cetone. Centrifugation w as performed as
described above a nd boththe supernatants were com-
bined together and extracted with 7.5 mL of hexane, then
again with 2.5 mL of hexane and ®nally with 3 mL o f
2-methylbutane. Residual 2-methylbutane was removed by
a brief stream of argon. Saturated NaCl (1.7 mL) was then
added to the acetone fraction followed by suf®cient 0.25
M
maleic acid (pH 5.3) to neutralize the solution. The
tetrapyrroles were then extracted by two successive parti-
tions with 3 mL o f p eroxide f ree d iethyl ether. Ether
solution was then dried under argon and resuspended in
100 lL of solvent A (methanol/5 m
M
aqueous tetrabuty-
lammonium phosphate, 7 : 3, v/v) a nd stored at )20 °C
for further analysis with HPLC. The HPLC wa s per-
formed on a Lichrospher 100 RP-18 ( 5 l
M
) RT 125-4
Merck column p reequilibrated with s olvent A. Elution
(1 mLámin
)1
) was maintained with this solvent for ®rst
3 min then was carried out with solvent B (methanol/H
2
O,
7 : 3, v/v) for 45 min. Elution was followed by measuring
the absorbance a t 420 nm and radioactivity by scintillation
detection (Berthold H PLC radioactivity monitor) in pre-
sence of QuicksZint 302 (PerkinElmer). In these conditions
we veri®ed that Mg-protoporphyrin IX eluted at 10 min,
protoporphyrin IX at 12 min and m ethyl Mg-protoporph-
yrin IX at 15 min a fter injection. Further, each fraction
was t ested f or typical room temperature ¯uorescence
emission spectrum after excitation at 420 nm. A weak
radioactive peak was usually de tected at 1 min after
injection that corresponds to traces of Ado-met remaining
in the extract. The major peak for radioactivity was
detected at 15 min of elution time corresponding to the
labeled methyl Mg-protoporphyrin I X.
Photolabeling with Ado-met
Photolabeling was carried out as described previously [23].
S-[CH
3
-
3
H]Ado-met (3.5 l
M
, 3 TBqámmo l
)1
) was added t o
15 lL of ice cold protein fractions. The mixture was then
irradiated with UV for 15 m in before the addition of SDS/
PAGE sample buffer. After SDS/PAGE, labeled proteins
were analyzed by ¯uorography.
Western blot analyses
ArabidopsisMgP
IX
MT, containing a deletion of the ®rst 160
amino a cids (D161-protein) was expressed in E. coli,as
described above. The inclusion bodies, enriched i n the
expressed protein, were puri®ed and analyzed on an SDS/
PAGE gel. The D161-protein band was excised and used to
obtain rabbit polyclonal antibodies (Elevage Scienti®que
des Dombes, Chaà tillon-sur-Chalaronne, France). Western
blot analyses were performed using Arabidopsis, spinach or
transformed E. coli subcellular fractions, as described
previously [23]. Proteins (15±50 lg) were separated on an
SDS/12% polyacrylamide gel, as described previously [24].
Nonspeci®c binding sites on the blot were blocked using
Tris/HCl 10 m
M
,pH7.5,NaCl9gáL
)1
, and nonfat dried
milk (50 gáL
)1
). Arabidopsis MgP
IX
MT was detected with
antibodies at a 1 : 500 dilution using s econdary antibodies
coupled to alkaline phosphatase or horse radish peroxidase.
For spinach fractions, a 1 : 100 d ilution was u sed. Pre-
immune sera gave no signal.
Protein and chlorophyll determination
Protein c oncentration w as deter mined as described
previously [25], u sing BSA as a standard. C hlorophyll
concentration was also measured as described previously
[26].
RESULTS
Characterization of putative sequences for MgP
IX
MT
in plants
We analyzed nucleic databases starting from the sequ ence of
Synechocystis sp. MgP
IX
MT (BAA10812) and we identi®ed
several ORF sequences presenting a high alignment score
with the Synechocystis protein [27], in A. thaliana
(CAB36750), in Oryza sativa (BAA84812) a nd in Nicoti-
ana tabacum (AF213968) genomes. The resulting putative
plant proteins are very closely related
5
(69% identity over
250 amino acids between Arabidopsis and rice proteins). A
multiple alignment including the proteins from Arabidopsis
and rice a nd the MgP
IX
MT sequences from Synechocystis
and R. capsulatus (P26236) shows t hat theplant proteins are
82 and 96 amino acids longer than the Synechocystis
protein, respectively, containing a long N-terminal exten-
sion as compared to the p rokaryotic sequences (Fig. 1). In
the common C-terminal portion of the four sequences, we
detected a domain with 23% identity over 210 amino acids,
containing the three motifs for Ado-met methyltransferases
as identi®ed previously [28].
Identi®cation of the
arabidopsis
MgP
IX
MT
To id entify the activity associated with the protein encoded
by the A. thaliana s equence, th e 312 amino-acid protein
(33 795 Da) corresponding to the full-length ORF was
expressed in E. coli.MgP
IX
MT activity was assayed in the
bacterial pellet by measuring the formation of [
3
H]meth yl
Mg-protoporphyrin IX from Mg-protoporphyrin IX and,
S-[methyl-
3
H]Ado-met. After solvent extraction and HPLC
puri®cation, methyl Mg-protoporphyrin IX was further
identi®ed by r oom temperature ¯uorescen ce emission
242 M. A. Block et al. (Eur. J. Biochem. 269) Ó FEBS 2002
spectra
6
. Following IPTG induction, the MgP
IX
MT activity
was detected inthe transformed bacteria (Fig. 2A). Max-
imum MgP
IX
MT activity was observed after 2 h of
induction whereas no activity w as recorded in control
bacteria, i.e. bacteria transformed with either the plasmid
containing the i nverted insert o r containing soMGD1
cDNA, a control cDNA encoding a MGDG synthase from
spinach (Fig. 2A,B). These results demonstrate t hat the
characterized Arabidopsis cDNA sequence encodes a func-
tional M gP
IX
MT. Wh en the bacterial proteins w ere
analyzed by SDS/PAGE and C oomassie staining, no
recombinant protein could be visualized. Therefore,
although the recombinant protein was p resent in low
amounts in E. coli, it was in a highly active form. Expression
of truncated portions of the protein, [(a) D80-protein
missing the ®rst 79 amino acids and similar in size to the
prokaryotic MgP
IX
MT; and (b) D161-protein missing the
®rst 160 amino acids] led to high amounts of recombinant
proteins (Fig. 2B). The D161-protein was inactive, whereas
the D80-protein exhibited MgP
IX
MT activity. Therefore, the
methyltransferase domain of the MgP
IX
MT is clearly
contained after the 80 N-terminus amino acids of the f ull-
length protein.
MgP
IX
MT is a chloroplast protein and therefore should
contain a n N -terminal c hloroplast t argeting s equence.
CHLOROP
predicted that the Arabidopsis protein presents a
chloroplast t ransit peptide with a peptidase cleavage s ite
located between amino acids 39 and 40 leading to a putative
mature protein of 2 9 5 00 Da. Indeed, we analyzed whether
the in vitro rad iolabeled full-length precursor could be
imported into isolated pea chloroplasts (Fig. 3 ). We dem-
onstrated that the precursor protein was processed into a
mature protein with an apparent molecular mass of 31 kDa.
The processed p rotein was p rotected from thermolysin
treatment of chloroplasts, indicating that it was imported
inside the chloroplast. The apparent size of the processed
protein is close to the size of the predicted mature p rotein
suggesting that the
CHLOROP
predicted that the peptidase
cleavage site is probably correct. The mature Arabidopsis
protein is t herefore larger than the p rokaryotic proteins,
Fig. 1. Comparison of dierent MgP
IX
MT of
prokaryotic or eucaryotic origin. (A) Compar-
ison of the deduced amino acid sequences of
cDNA enco ding the MgP
IX
MT of Arabidops is
thaliana (At; AL035523.1), Oriza sativa (Os;
AP002542.2), Synechocystis (Scystis;
L47126.1), and Rhodobacter capsulatus
(Rhoca or Rc; P26236) using
CLUSTAL W
(1.8)
multiple alignment p rogram. Stars indicate
position of amino acids identical among t he
four sequences andthe position inthe align-
ment of the three classical motifs for Ado-met
dependent methyltransferaseis underlined
with a gray bar. The transit peptide cleavage
site for each plant pro tein as found by the
CHLOROP
program is indicated by an arrow.
Several putative transmembrane sequences
were determined by the
TMPRED
program.
They are i ndicated in white letters on black
background. The amino terminus end of the
recombinant truncated proteins (D80-protein
and D161-protein) is indicated by a box above
the Arabidopsis sequence. (B) Unrooted
phenogram drawn using an alignment of the
four sequences described above and of those
of: Nicotiana tabacum (Nt; AF213968.1),
Heliobacillus mobilis (Hm; AF080002),
Rhodospirullum rubrum (Rr, AF202319.1),
Rubrivivax gelatinosus (Rg; AB034704),
Rhodobacter spheroides (Rs; X81413.1).
The alignment was carried out over the 2 40
C-terminal amino acids of each sequence
according to [31]. Distance is indicated in
PAM (probability of accepted mutation).
The plant proteins appear ve ry close to each
other and as well as to the cyanobacterial
protein whereas all types are relatively far
from the proteins present in photosynthetic
bacteria (purple bacteria or heliobacteria).
Ó FEBS 2002 Theplant Mg-protoporphyrin IXmethyltransferase (Eur. J. Biochem. 269) 243
containing an additional domain of a bout 40 amino acids,
which are not involved inthemethyltransferase catalytic
activity.
Localization of the MgP
IX
MT in chloroplasts
In puri®ed spinach chloroplasts, we detected the M gP
IX
MT
activity in membrane pellets whereas no activity was found
in the s oluble fraction ( stroma). We then analyzed
MgP
IX
MT activity inchloroplast subfractions (envelope
membranes and thylakoids). MGDG synthase activity was
monitored as a reliable marker for the presence of envelope
[29] and chlorophyll as a marker for thylakoids. Whereas
protein pattern and distribution of both markers indicated
that en velope and t hylakoids were clearly separated, t he
MgP
IX
MT ac tivity was detected inboth t ype s of mem-
branes: theenvelopeandthe thylakoids (Fig. 4A). We
controlled
7
that MgP
IX
MT activity was linear with p rotein
amounts and time under our measurement conditions. The
MgP
IX
MT speci®c activity intheenvelope was 2.5-fold
higher than inthe thylakoids. In puri®ed Arabidopsis
chloroplast membranes, a similar distribution of the activity
was found between envelopeand t hylakoids (Fig. 4B).
Western blot analysis of Arabidopsis chloroplast membranes
using antibodies raised against the truncated recombinant
Arabidopsis protein (D161-protein) detected a p rotein with a
31-kDa apparent molecular mass intheenvelope fraction
and in t he thylakoids, w hereas the distribution of the E37
envelope marker [29] was clearly restricted to the envelope
(Fig. 4 B). I n spinach chloroplast membranes, envelope and
thylakoids, the D161-protein antibodies also detected a
31-kDa protein. T his size c orresponds to the size of the
processed protein after import. In addition, in a fraction
Fig. 2. Analysis of the MgP
IX
MT activity associated with the expression of recombinant proteins in E. coli BL21(DE3) . (A) MgP
IX
MT activity of
bacterial cultures a fter i nduction b y 1 m
M
isopropyl thio-b-
D
-galactoside (IPTG) . The black bars correspond to bacteria transformed w ith p ET Y3a
plasmid containing full-length Arabidopsis MgP
IX
MT ORF insert under T7 promoter andthe white bars correspond to the same construct but with
insert inthe opposite orientation. Enzyme activity was measured in b acterial pellet obtained from 200 lL of culture. (B) Co mparison of MgP
IX
MT
activity and protein composition o f bacteria 2 h after of induction. P corresponds to b acteria transformed with pE T Y3a plasmid containing full-
length Arabidopsis MgP
IX
MT ORF insert u nder T7 promoter, and R corresponds to the same construct b ut with insert inthe o pposite orientation.
P¢ corresponds to the same bacteria as in P; 80 and 161 are b acteria transformed with pET Y3a plasmid containing D80 and D161 truncated
Arabidopsis MgP
IX
MT ORF; M are bacter ia transformed with pET Y3a plasmid containing spinach MGDG synthase cDNA [21]. Positions of
clearly overexpressed recombinant proteins are indicated by triangles.
Fig. 3. Import of the [
35
S]methionine-labeled Arabidopsis MgP
IX
MT
precursor protein into isolated pea chloroplasts. Precursor protein or
chloroplast suspensions after in vitro import were analyzed by SDS/
PAGE, Coomassie staining and ¯ uoro graphy detection Lanes: P,
labeled precursor proteins (1 : 100 of what used for in vitro import); I,
proteins after in vitro import; It, proteins after in vitro import and
subsequent limited thermolysin treatment of chloroplasts.
244 M. A. Block et al. (Eur. J. Biochem. 269) Ó FEBS 2002
derived from spinach envelope membrane and devoid of
E37, a major methyltransferase of theenvelope [2 3]
removed by puri®cation on a DEAE column, a 31-k Da
protein was photolabeled with radioactive Ado-met under
UV light (Fig. 5) corresponding to the mature MgP
IX
MT.
From these results, we conclude that the m ature MgP
IX
MT
is locatedand active inbothchloroplast membrane systems,
i.e. envelopeand thylakoids, and that the protein has the
same size inboth fractions.
Association of MgP
IX
MT with the membranes
Analysis of the A rabidop sis MgP
IX
MT sequence by the
TMPRED
program predicted two transmembrane helices in
the mature Arabidopsis protein, one located i n the plant
speci®c N-terminal extension (amino acids 54±72), which is
very hydrophobic, and one inthemethyltransferase domain
(amino acids 142±162) (Figs 1A and 6A). We noticed that
this latter putative transmembrane helix is positioned on the
Ado-met methyltransferase motif and does not correspond
to a putative transmembrane domain inthe homologous
Rhodobacter MgP
IX
MT, suggesting t hat the transmem-
brane prediction was not reliable. Therefore, we analyzed
the association of the MgP
IX
MT with theenvelope and
with the t hylakoid membranes by ionic and alkaline
extractions. The same pattern of results was obtained for
both envelopeand thylakoids (Fig. 6B). The protein was
not released from the membrane by treatment with 1
M
NaCl, indicating that it was not peripherally associated with
the membrane. In contrast, the protein was solubilized by
treatment with 0.1
M
NaOH, indicating that the protein is
not a transmembrane protein. It is therefore possible that
the protein is associated with one lea¯et of the membrane via
lipid interaction. As theplant speci®c N-terminal e xtension
(amino acids 54±72) is very hydr ophobic, and a s t he
recombinant truncated protein ( D80-p rotein) deleted of this
zone was partially soluble i n E. coli (Fig. 6 C), we propose
that the N-terminal part of the mature plant protein may
favor the binding of the protein to the membranes.
DISCUSSION
Using enzymological studies in plants, several r eports had
proposed that a MgP
IX
MT might b e associated w ith plastid
membranes [14,15]. I n the present study, we show t hat an
Arabidopsis MgP
IX
MT is encoded by a gene located on
chromosome 4. One short intron (175 bp) is present in t he
Fig. 4. Localization of MgP
IX
MT inchloroplast membranes. (A) Analysis of s pinach c hloroplast envelopeand t hylakoids f ractions. The fractions
were analyzed by SDS/PAGE and C oomassie blue staining and MgP
IX
MT activity was compared to MGDG synthase activity a s a marker o f
envelope me mbranes and to chlo rophyll concentration as a marker o f thylakoids. (B) Analysis of A. thaliana chloroplastenvelopeand thylakoids
fractions. MgP
IX
MT activity was 3 nmoláh
)1
ámg protein
)1
in envelope fraction and 0.47 in thylakoids fraction. Western blot detection of
MgP
IX
MT was compared to detection of E 37, a marker of envelope m embran es. (E) chloroplast envelope; (T) thylakoids.
Fig. 5. Photolabeling of e nvelope subfractions with Ado-met. Spinach
chloroplast e nvelope was solubilized in buer S (6 m
M
CHAPS, 1 m
M
DTT, 50 m
M
Mops pH 7.8) at 1 mg p roteinámL
)1
. A fter centrifugation
at 100 000 g for 30 min, s olubilized fraction (1) was loaded on a DEAE
columnandwashedwithbuerStoremoveE37,anAdo-metmeth-
yltransferase present intheenvelope [23]. We veri®ed that eluted E37
hadnoMgP
IX
MT activity. DEAE bound protein fraction ( 2) was then
eluted in buer S containing 0.3
M
NaCl. MgP
IX
MT activity was
92 pmolámin
)1
ámL
)1
in fraction (1) and 50 pmolámin
)1
ámL
)1
in frac-
tion (2). Each fraction (18 lL aliquot) was assayed for binding of
3
H-Ado-met (3.5 l
M
) by crosslinking un der UV. Afte r SDS/PAGE a nd
Coomassie staining, labeled proteins were detected by ¯uorography. A
labeled 31 kDa protein ( ® ) is visible inthe puri®ed envelope fraction.
Ó FEBS 2002 Theplant Mg-protoporphyrin IXmethyltransferase (Eur. J. Biochem. 269) 245
middle of the ORF, between the lysine 185 and alanine 186
codons. W e i denti®ed three different regions inthe full-
length precu rsor. Firstly, an N-terminal domain of 40
amino acids includes a chloroplast transit peptide that is
cleaved after import into chloroplasts. Secondly, a long
C-terminal domain shows a strong homology to the full-
length prokaryotic MgP
IX
MT from Synechocystis and a
more distant homology to the MgP
IX
MT of photosynthetic
bacteria such as Rhodobacter capsulatus or Heliobacilus
mobilis (Fig. 1B). We demonstrated that this domain binds
Ado-met and contains the active MgP
IX
MT site. Thirdly,
between the two previous domains, inthe N-terminal end of
the m ature p lant protein, we found a very h ydrophobic
region possibly i nvolved inthe an choring of the protein to
the membranes. This part of the protein is absent in the
prokaryotic MgP
IX
MT.
From databank surveys, we detected highly similar
proteins in other plant species such as rice and tobacco.
Although these proteins have never been reported to
catalyze MgP
IX
MT activity, we can assume they do due
to their high similarity to A. thaliana MgP
IX
MT.
In spinach and A. thaliana chloroplasts, the MgP
IX
MT
protein is present in an active form inboth thylakoids and
envelope membranes. Its size is app arently identical in both
types of membranes although we cannot exclude some
limited modi®cations. The strong association of MgP
IX
MT
with either envelope or thylakoids membranes, and the
very low degree o f cross contamination of e nvelope and
thylakoids preparations, s upport that M gP
IX
MT is present
in both types of membrane. The speci®c activity is higher
in theenvelopemembranes than inthe thylakoids. Taking
into acco unt that the thylakoids network represents 50- to
100-fold more protein than the total envelope, the
MgP
IX
MT partition between envelopeand t hylakoids
can be considered close to 1 : 30 in mature chloroplasts.
On the other hand, during early development o f chloro-
plasts, MgP
IX
MT activity from theenvelope should be the
most important.
In Euglena,MgP
IX
MT was reported to be ® rmly attached
to chloroplastmembranes [30]. However 15± 25% of the
activity was also r ecovered i n a supernatant t hat was
probably enriched inchloroplastenvelope considering the
low density of envelope membranes.
To understand the role of the additional N-terminal
hydrophobic domain present intheplant MgP
IX
MT and
absent from the prokaryotic MgP
IX
MT, it would be
particularly interesting to know the localization of
MgP
IX
MT in cyanobacteria. We do not kn ow if it strictly
associates with membranesand with which membranes; the
cell envelope or the thylakoids?
Fig. 6. Analysis of the association of
MgP
IX
MT with membranes. (A) Hydropathy
pro®le of Arabidopsis MgP
IX
MT. The hydro-
pathy was analyzed as described previously
[32], using a 1 1 amino acid interval and
exponential weight variation model. The
highest hydrophobic region is found in the
vicinity of amino acid 63. (B) Analysis of the
association of the MgP
IX
MT with spin ach
chloroplast envelope or with Arabidopsis
thylakoids by ionic and alkaline extractions.
(B1) Each fraction, s oluble (S) and insoluble
(I), obtained after t reatment of 50 lgmem-
brane protein was analyzed by W estern-blot
with anti-MgP
IX
MT antibodies (dilution
1 : 100 for envelopeand 1 : 500 for t hylak-
oids). (B2) MgP
IX
MT was d etected by W est-
ern blot after two-dimensional analysis of
NaOH solubilized proteins from Arabidopsis
thylakoids (1 mg protein). T he NaOH solu-
bilized thylakoid MgP
IX
MT presents an
apparent molecular weight and an ap parent
isoelectric point close to those predicted b y
ChloroP for the mature protein (29.5 kDa and
pI 5.7 ). (C) Analysis of the solubility of
D80-protein ( ® )inE. coli.Expressionof
D80-protein was induced by IPTG for 2 h.
Frozen bacterial pellet was resuspended in
25 m
M
Mops pH 7.8, 1 m
M
MgCl
2
and soni-
cated on ice for 15 s. Soluble (S) and insoluble
(I) fractions were separated by centrifugation
(100 000 g, 30 min) and analyzed by SDS/
PAGE and Coomassie blue staining. (C2) The
identity and activity o f D80-p rotein were ver-
i®ed by UV photolabeling with Ado-met.
246 M. A. Block et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The dual localization of MgP
IX
MT in chloroplasts may
be related to different roles o f the protein i n each
membrane. For chlorophyll synthesis, the e nzyme must
be connected to other enzymes of the chlorophyll synthesis
pathway. Several enzymes were reported to be present in
or to associate with the envelope: the protoporphyrinogen
oxidase [4], the subunits of the Mg chelatase [5] and the
protochlorophyllide oxidoreductase (POR) [7]. The proto-
porphyrinogen oxidase is also present inthe thylakoids but
presumably involved in heme synthesis [4] and POR can
associate with the thylakoids [9]. Finally, the ®nal steps of
chlorophyll synthesis, such as addition and r eduction of
geranylgeranyl chain, are l ocated on the t hylakoids [1].
Therefore, we cannot exclude the possibility that there are
two locations
8
for chlorophyll synthesis inthe chloroplast
or a motion of intermediate compounds between the two
types of membrane. As the Mg chelatase does not seem to
associate w ith the thylak oids, a transfer of Mg-proto -
porphyrin IX from theenvelope to the thylakoids would
be required to supp ly thethylakoid MgP
IX
MT with
substrate.
Localization of MgP
IX
MT intheenvelope may play a
role in plastidic signaling. Mg-protoporphyrin IX or its
methyl derivative are thought to have a signaling function
outside of thechloroplastand it is therefore important to
regulate their abundance inthechloroplast envelope.
Kropat et al . [18] proposed that chlorophyll precursors
could act as plastidic signals to be transmitted to the
nucleus, thus affecting gene expression. Previous reports
have indicated that Mg-protoporphyrin IX or its methyl
derivative could modify transcription of cab or rbcs genes
[16,17]. In Chlamydomonas reinhardtii, Mg-protoporphyrin
IX or its methyl derivative could replace light in inducing
nuclear heat-shock protein genes (hsp70a and hsp70b)[18].
Furthermore, in this model, ac cessibility of Mg-proto-
porphyrin IX to the cytoplasm was reported to be crucial to
drive light signaling [19].
Finally, it is possible that the two pools of MgP
IX
MT
located intheenvelopeandinthe thylakoids play differ-
ential roles regarding chlorophyll biosynthesis and produc-
tion of plastidic signals r esponsible for c ontrolling nuclear
gene expression. The sorting of th e single MgP
IX
MT in two
different locations also presents a problem that will require
further exploration.
ACKNOWLEDGMENTS
We are grateful to Dr Ste
Â
phane Ravanel for providing us with the
Arabidopsis RACE library. This research work was supported in part
by a postdoctoral fellowship from t he French Ministe
Á
re de l'Education
Nationale, de la R echerche et de la Technolog ie (to A.K.T.).
SUPPLEMENTARY MATERIAL
The following material is available from http://www.ejbio
chem.com
Figure S1. Fluorescent spectra of tetrapyrroles c ompounds
issued from HPLC analysis.
REFERENCES
1. Ru
È
diger, W. & Schoch, S. (1988) Chlorophylls. InPlant Pigments
(Goodwin, T.W., ed.), pp. 1±59. Academic Press, London.
2. Pineau, B., Dubertret, G., Joyard, J. & Douce, R. (1986) Fluo-
rescence properties of the e nvelope m embranes fro m spinach
chloroplasts. Detection of protochlorophyllide. J. Biol. Chem. 261,
9210±9215.
3. Pineau, B., Ge
Â
rard-Hirne, C., Douce, R. & Joyard, J. (1993)
Identi®cation of the main species of tetrapyrrolic pigments in
envelope membranes from spinach chloroplasts. Plant Physiol.
102, 821±828.
4. Matringe, M., Camadro, J.M., Block, M.A., Joyard, J., Scalla, R.,
Labbe, P. & Douce, R. (1991) Localization within spinach chlo-
roplasts of protoporphyrinogen oxidase, the target enzyme for
diphenylether herbicides. J. Biol. Chem. 267, 4646±4651.
5. Nakayama, M., Masuda, T ., Bando, T., Yamagata, H., Ohta, H.
& T akamiya, K. (1998) Cloning and expression of the s oybean
chlH gene encoding a s ubunit of Mg-chelatase an d localization of
the M g
2+
concentration-depe ndent ChlH p rotein within the
chloroplast. Plant Cell Physiol. 39, 275±284.
6. Shaw, P., Henwood, J., Oliver, R. & Griths, T. (1985) Immu-
nogold localization of protochlorophyllide oxidoreductase in
barley etio plasts. Eur. J. Cell Biol. 39, 50±55.
7. Joyard,J.,Block,M.A.,Pineau,B.,Albrieux,C.&Douce,R.
(1990) Envelopemembranes from mature spinach chloroplasts
contain a NADPH: protochlorophyllide r eductase on the cytosolic
side of the outer membrane. J. Biol. Chem. 265 , 21820±)21827.
8. Dahlin, C., Sundqvist, C. & Timko, M.P. (1995) Thein vitro
assembly of the NADPH-protochlorophyllide oxidoreductase in
pea chloroplasts. Plant M ol. Biol. 29, 317±330.
9. Dahlin, C., Aronsson, H., Wilks, H.M., Lebedev, N., Sundqvist,
C. & Timko, M.P. (1999) The role of protein s urface charge in
catalytic activity andchloroplast membrane association of the
pea NADPH: protochlorophyllide o xidoreductase (POR) as
revealed by alanine scanning mutagenesis. Plant Mol. Biol. 39,
309±323.
10. Bollivar, D.W., Jiang, Z Y., Bauer, C.E. & Beale, S. (1994)
Heterologous expression of the bchM gene product from
Rhodobacter capsulatus and demonstration that it encodes
S-adenosyl-
L
-methionine: Mg-protoporphyrin IX methyltrans-
ferase. J. Bacteriol. 176, 5290±5296.
11. Gibson, L.C.D. & Hunter, C.N. (1994) The bacteriochorophyll
biosynthesis ge ne, bchM, of Rhodobacter sphaeroides en codes
S-adenosyl-
L
-methionine: Mg protoporphyrin IX me thyltrans-
ferase. FEBS Let. 352, 127±130.
12. Smith, C.A., Suzuki, J.Y. & Bauer, C.E. (1996) Cloning and
characterization of the chlorophyll bio synthesis gen e chlM from
Synechocystis PCC 6803 by complementation o f a bacteriochlo-
rophyll biosyn thesis m utant of Rhodobacter capsulatus. Plant Mol.
Biol. 30, 1307±1314.
13. Ellsworth, R.K. & Dullaghan, J.P. (1972) Activity and properties
of S-adeno syl-
L
-methionine: magnesium protoporphyrin IX
methyltransferase in crude homogenates from wheat seedlings.
Biochim. Biophys. Acta 268, 327±333.
14. Hinchigeri, S.B., Chan, J.C S. & Richards, W.R. (1981) Puri®-
cation of S-adenosyl-
L
-methionine: magnesium protoporphyrin
methyltransferase by a n ity chromatography. Photos ynthetica 15,
351±359.
15. Fuesler, T.P., Wong, Y.S. & Castelfranco, P.A. (1984) Localiza-
tion of Mg-c helatase and Mg-protoporphyrin IX mono methyl
ester (oxidative) cyclase activities within isolated developing
cucumber chloroplast s. Plant Physiol. 75, 662±664.
16. Johannin gmeier, U. & Howell, S.H. (1984) Re gulatio n of light-
harvesting chlorophyll-binding protein mRNA accumulation in
Chlamydomonas reinhardii. Possible involvement of chlorophyll
synthesis precursors. J. Biol. Chem. 259, 13541±13549.
17. Jasper, F., Qued nau, B., Korten jann, M. & Johannin gmeier, U.
(1991) Control o f cab g ene expre ssion in synchronized
Chlamydomonas reinhardtii cells. J. Photochem. Photobiol. 11,
139±150.
Ó FEBS 2002 Theplant Mg-protoporphyrin IXmethyltransferase (Eur. J. Biochem. 269) 247
18. K ropat, J., Oster, U., Ru
È
diger, W. & B eck, C.F. (1997) Chloro-
phyll precursors are s ignals of chloroplast origin involved i n light
induction of nuclear heat-shock genes. Proc. Natl Acad. Sci. USA
94, 14168±14172.
19.Kropat,J.,Oster,U.,Ru
È
diger, W. & Beck, C.F. (2000) Chlo-
roplast signalling inthe li ght induction of nuclear HSP70 gene s
requires the accumulation of chlorophyll precursors and their
accessibility to cytoplsm/nucleus. Plant J. 24, 523±531.
20. Mochizuki, N., Brusslan, J.A., Larkin, R., Nagatani, A. & Chory,
J. (2001) Arabidopsis geno mes uncoupled 5 (GUN5) mutant
reveals thein volvment of Mg-c helatase H s ubun it in plastid -to-
nucleus signal transduction. Proc. Natl Acad. Sci. USA 98 , 2053±
2058.
21. M ie
Á
ge, C., Ma re
Â
chal, E., Shimojima, M., Awai, K., B lock, M.A.,
Ohta, H., Takamiya, K., Douce, R. & Joyard, J. (1999) Bio-
chemical and topological properties of typ e A MGDG synthase, a
spinach c hloroplast envelope enzyme catalyzing the synthesis of
both prokaryotic and eukaryotic MGDG. Eur. J. Biochem. 265,
990±1001.
22. Som, S. & Friedman, S . (1990) Direct photolabeling of the EcorII
methyltransferase with S-adenosyl-
L
-methionine. J. Biol. Chem.
265, 4278±4283.
23. Teyssier, E., Block, M.A., Douce, R. & Joyard, J. (1996) Is E37, a
major p olypeptide of the inner membrane from plastid envelope,
an S-denosyl methionine-dependent methyltransferase? Plant J.
10, 903±912.
24. Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bactriophage T
4
. Nature 227, 680±685.
25. Lowry, O.H., Rosebr ough, N.J., Far r, A.L. & Randall, R.J.
(1951) Protein measurement with the Folin phenol reagent. J. Biol.
Chem. 193, 265±275.
26. Arnon, D .I. (1949) Copper enzymes in isolated chloroplasts.
Polyphenoloxydase i n Beta vulgaris. Plant Physiol. 24 ,1±5.
27. Altsch ul, S .F., M adde n, T.L., Scha
È
er, A.A., Zhang, J ., Miller, W.
& Lipman, D.J. (1997) Gapped BLAST and PSI-Blast: a new
generation of protein database search programs. Nucleic Acids
Res. 25, 3389±3402.
28. Kagan, R.M. & Clarke, S. (1994) Widespread occurence of three
sequence motifs in diverse S-adenosylmethionine d epende nt
methyltransferases suggests a common s tructure of these enzymes.
Arch. Biochem. Biophys. 310, 417±427.
29. Joyard,J.,Grossman,A.,Bartlett,S.G.,Douce,R.&Chua,N.H.
(1982) Characterization of envelope membrane po lypeptides from
spinach chloroplasts. J. Biol. Chem. 257, 1095±1101.
30. Ebbon, J.G. & Tait, G.H. (1969) Studies on S-aden osylmethio-
nine-magnesium protoporphyrin methyltransferasein Euglena
gracilis strain z. Biochem. J. 111, 573±582.
31. Co rpet, F. (1988) Multiple sequence align ment with hierarchical
clustering. Nucleic Acids Res. 16, 10881±10890.
32. Rao, M.J.K. & Argos, P. (1986) A conformational preference
parameter to predict helices in i ntegral membrane p roteins.
Biochem. Biophys. Acta 869, 197±214.
248 M. A. Block et al. (Eur. J. Biochem. 269) Ó FEBS 2002
. The plant
S
-adenosyl-
L
-methionine:Mg-protoporphyrin IX
methyltransferase is located in both envelope and thylakoid
chloroplast membranes
Maryse. hlorophyllide and phytol. The initial steps
in c hlorophyllide synthesis, from the biosynthesis of
d-aminolevulinate to protoporphyrinogen IX, occur in
the soluble