Báo cáo Y học: The plant S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase is located in both envelope and thylakoid chloroplast membranes pot

thaliana and spinach, the MgP;xMT protein has a dual localization in chloroplast envelope membranes as well as in thylakoids.. Received 31 August 2001, revised 25 October 2001, accepted

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Eur J Biochem 269, 240-248 (2002) © FEBS 2002

The plant S-adenosyl-.-methionine:Mg-protoporphyrin IX

methyltransferase is located in both envelope and thylakoid

chloroplast membranes

Maryse A Block, Arun Kumar Tewari, Catherine Albrieux, Eric Maréchal and Jacques Joyard

Laboratoire de Physiologie Cellulaire Végétale, CNRS/CEA/Université Joseph Fourier, DBMS/PCV, Grenoble, France

Chlorophyll biosynthesis requires a metabolic dialog

between the chloroplast envelope and thylakoids where

biosynthetic activities are localized Here, we report the

first plant S-adenosyl-L-methionine:M g-protoporphyrin

IX methyltransferase (MgPxMT) sequence identified in

the Arabidopsis genome owing to its similarity with the

Synechocystis sp MgP;yMT gene After expression in

Escherichia coli, the recombinant Arabidopsis thaliana

cDNA was shown to encode a protein having MgP;xMT

activity The full-length polypeptide exhibits a chloroplast

transit peptide that is processed during import into the

chloroplast The mature protein contains two functional

regions The C-terminal part aligns with the Synecho-

cystis full-length protein The corresponding truncated

region binds to Ado-met, as assayed by UV crosslinking,

and is shown to harbor the MgP;xMT activity Down-

stream of the cleaved transit peptide, the 40 N-terminal amino acids of the mature protein are very hydrophobic and enhance the association of the protein with the membrane In A thaliana and spinach, the MgP;xMT protein has a dual localization in chloroplast envelope membranes as well as in thylakoids The protein is active

in each membrane and has the same apparent size corresponding to the processed mature protein The protein

is very likely a monotopic membrane protein embedded within one leaflet of the membrane as indicated by ionic and alkaline extraction of each membrane The rationale for a dual localization of the protein in the chloroplast is discussed

Keywords: protoporphyrin; methyltransferase; chloroplast; membrane; chlorophyll

The chlorophyll molecule is made up of two moieties of

distinct origin, chlorophyllide and phytol The initial steps

in chlorophyllide synthesis, from the biosynthesis of

é-aminolevulinate to protoporphyrinogen IX, occur in

the soluble phase of plastids whereas the subsequent steps

are membrane-bound [1] Envelope membranes constitute

the main membrane system present in early development

of proplastids into chloroplasts Although devoid of

chlorophyll, they play a role in the initial steps of

chlorophyll biosynthesis Several results indicate that

chlorophyllide synthesis is at some point associated with

the envelope Studies of fluorescence properties of isolated

envelope membranes from spinach chloroplasts demon-

strated the presence of small but significant amounts of

protochlorophyllide and chlorophyllide [2,3] Localization

of enzymatic activities has shown that several enzymes of

the biosynthetic pathway are linked to the envelope; the

protoporphyrinogen oxidase is present in both the thyla-

koids and the envelope membranes [4]; the subunits of the

Mg chelatase are present in large amounts in the stroma

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 78 49 85, E-mail: mblock@cea.fr

Abbreviations: Ado-met, S-adenosyl-L-methionine; MgP;xMT,

S-adenosyl-L-methionine:Mg-protoporphyrin [X methyltransferase;

MGDG, monogalactosyldiacylglycerol; MGD, monogalactosyldia-

cylglycerol synthase; IPTG, isopropyl thio-B-p-galactoside

(Received 31 August 2001, revised 25 October 2001, accepted 30

October 2001)

but become associated with the envelope at Mg" concentration present in illuminated chloroplasts [5]; the protochlorophyllide oxidoreductase (POR) that accumu- lates in etioplast prolamellar bodies [6] remains in low amount in mature chloroplasts where its activity is detected in the envelope [7] POR was immunologically detected on the outer surface of the chloroplast envelope [7] Although not immunodetected in the thylakoids [7], POR could be also present in the thylakoids considering that in vitro import into 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 enzymatic steps of the protoporphyrinogen to chlorophyllide pathway have not been localized

In the present study, we focus on methylation of Mg- protoporphyrin IX The methyltransferase has not yet been identified in plants but has been clearly recognized in purple bacteria [10,11] and in cyanobacteria [12] by functional complementation and by expression of active recombinant protein In plants, the activity of the S-adenosyl-L-methionine:Mg-protoporphyrin [IX methyltransferase (MgP;xMT) was reported as membrane-bound and rather unstable [13-15] The tetrapyrrole substrate and product of this enzyme never accumulate and they may play a role in controlling the expression of some light dependent genes [16-20]

In this paper, we identify the Arabidopsis gene encoding MegPixMT, and characterize the encoded protein We further analyze the localization of the plant protein within chloroplast membranes and discuss the results in relation to chloroplast biogenesis

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MATERIALS AND METHODS

Chemicals

Unlabeled Ado-met, p-toluenesulfonate salt was purchased

from Sigma and [methyl H]Ado-met (3 TBg:‘mmol') was

from NEN (ref NET-155H) Mg-protoporphymnn IX

disodium salt was purchased from Porphyrin products

Inc and protoporphyrin [X disodium salt was from Sigma

cDNA cloning

A 950-bp nucleic fragment was amplified by PCR from

Arabidopsis thaliana cDNA prepared from 3-week-old leaf

poly(A) mRNA using the Clontech Advantage polymerase

and primers specific for the 5’ and 3’ ends of the coding

sequence of the putative MgP;xMT The forward primer

was 5-CATATGCCGTTTGCTCCTTCC-3’ and the

reverse primer 5’ CATATGGCTTACATTGGAACAG

CTTC-3’, each including a NdeI site at the end of the

amplified fragment The amplified cDNA was then blunt

end cloned in the Smal restriction site of pBluescriptSK *

and confirmed by sequencing The NdeI linearized insert

was cloned in the modified pET-Y3a plasmid [21] This

plasmid was used in order to overcome the problem raised

by the fact that the putative A thaliana MgP;{xMT protein

contains at the beginning of its sequence numerous

arginines, encoded by AGG or AGA, which are poorly

used in Escherichia coli Orientation of the plasmid inserts

was checked by BamHI digestion and PCR Partial cDNA

fragments, corresponding to the protein shortened from the

first 79 amino acids (A80-protein), or from the first 160

amino acids (Al61-protein), were prepared again by PCR

amplification with forward primer 5-AGGCATAT

GTTGTTGCAGGCGGAGGAA-3’ (80-Ndel) or forward

primer 5’ CTCCATATGCCACTTGCTAAGGAAG-3’

(161—Ndel) coupled to reverse primer 5-TTCAGGATCCT

CTACATTGGAACA-3’ (stop—BamH]) They were cloned

in Ndel and BamHI restriction sites of pET-Y3a to express

truncated forms of the MgP;xMT

Preparation of recombinant proteins

BL21(DE3) strain of E coli was transformed with pET-Y3a

plasmid containing various inserts Bacterial cultures were

grown at 37°C under vigorous shaking until the

Deo = 0.5 Expression of the recombinant proteins was

then induced by addition of 1 mm isopropyl thio-B-p-

galactoside (IPTG) in the medium The cultures were then

transfered to 28 °C for 2 h Bacteria were 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 4kg of spinach

(Spinacia oleracea L.) leaves and chloroplast subfractions

were purified as described previously [7] Purified intact

chloroplasts were then lysed in buffer H (10 mm Mops

pH 7.8, 1 mm caproic acid, 1 mm phenylmethanesulfonyl

fluoride with 4mm MgCl), and chloroplast subfractions

were separated by centrifugation on a linear sucrose

gradient (0.6—-4 m sucrose in buffer H with 4 mm MgCl)

To limit the sedimentation of soluble proteins, such as Rubisco, through the sucrose gradient, after 1 h centrifugation 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 4mm MgCl, The gradient was further centrifuged at 70 000 g for 12 h Chloroplast envelope was collected as a yellow band in the medium part

of the gradient whereas thylakoids were collected as a pellet Each fraction was diluted three times in buffer H Mem- branes were then washed and resuspended in 100 pL of buffer H monogalactosyldiacylglycerol (MGDG) synthase activity was measured as described previously [21]

Purification of envelope membranes and thylakoids from arabidopsis chloroplasts

A thaliana plants (ecotype ws) were grown for 6 weeks under a 10-h photoperiod on compost The leaves (300 g) were homogenized in a Waring Blender in 1.5 L ice-cold buffer containing 0.45 m sorbitol, 20 mm tricine/NaOH

pH 8.4, 10 mm EDTA, 10 mm NaHCO;, and 0.1% BSA

A crude chloroplast pellet was obtained by centrifugation at

1500 g for 3 min and further purified in 0.33 mM sorbitol,

20 mm Mops pH 7.6, 5 mm MgCh, 2.5 mm EDTA (buffer P) on a Percoll gradient formerly prepared by centrifugation

of 45% (v/v) Percoll in a SS90 vertical rotor at 10 000 g for

100 min After centrifugation at 5000 g for 10 min, intact chloroplasts were recovered in the Percoll gradient as a heavy green layer The chloroplasts were washed twice with buffer P and then broken in 10 mm Mops pH 7.6, 4 mm MgCl, 1 mm phenylmethanesulfonyl fluoride and 1 mm caproic acid Chloroplast subfractions were separated on a step gradient of 0.93 m to 0.6 m sucrose in buffer R (10 mm Mops pH 7.6, 1 mm MgCl) by centrifugation at 70 000 g for | h The envelope was collected at the interface between the layers of 0.6 M and 0.93 m sucrose and thylakoids as

a pellet Each fraction was washed and resuspended in buffer R

In vitro import of MgP,xMT into chloroplasts The pBluescriptSK* plasmid containing the full-length cDNA of the MgP}xMT under T7 promoter was linearized downstream of the inserted DNA fragment using the Hindi restriction site Transcription and translation were carried out in vitro with T7 polymerase and wheat germ extract (TNT Coupled Wheat Germ Extract System, Promega) and [ÌS]methionine (37 TBqmmol, Amer- sham) The import was performed at room temperature for 15 min on 10-day-old pea chloroplasts (15 ug chlorophyll) with 5 wL of translated protein in 100 uwL of import buffer (330 mm sorbitol, 50 mm Hepes/KOH pH 7.6, 3 mm MgSO,, 10 mm methionine, 20 mm Kgluconate, 10 mm NaHC0Os, 2% BSA and 3 mm ATP) Th intact chloroplasts were then purified by centrifugation through a 40% (v/v) Percoll layer (300 uL) and the pellet was washed twice by centrifugation in chloroplast washing medium (330 mm sorbitol, 50 mm Hepes/KOH and 3 mm MgCl) The pellet was either resuspended in SDS/PAGE loading buffer or in

100 uL washing medium containing 0.5mm CaCl, and 0.03 mgmL7! thermolysin (Boehringer) In the case of thermolysin treatment, chloroplasts were incubated for

20 min on ice, then 10mm EDTA was added and

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242 M.A Block et al (Eur J Biochem 269)

chloroplasts were immediately pelleted and resuspended in

SDS/PAGE sample buffer Proteins were analyzed by

fluorography on a 12% acrylamide gel under films

(Amersham MP) at —70 °C for 1 week

Assay of MgP|xMT activity

MegP\xMT activity was measured in an assay mixture

containng 20mm Mops pH 7.8, 3 mm MgCl, 1 mm

dithiothreitol, 25 mm NaCl with the addition of 50 um

Mg-protoporphyrin [IX and of 10 um, S-[CH3-°H]Ado-met

(1 TBqmmol”), prepared just before the use by adding

cold Ado-met and labeled Ado-met and neutralizing with

BaCOs, as described previously [22] Incubation was

carried out with 5—20 ug protein im 24 HL of reaction

mixture at 30°C for 1-10 min The reaction was then

stopped by adding 1 mL of ice cold H,O Extraction of

tetrapyrroles was performed as described previously [10]

Proteins were precipitated by adding 3 mL 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 was resuspended in 500 uL of 0.125 m NH,OH and

1.5 mL of acetone Centrifugation was performed as

described above and both the supernatants were com-

bined together and extracted with 7.5 mL of hexane, then

again with 2.5 mL of hexane and finally with 3 mL of

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 sufficient 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 of peroxide free diethyl ether Ether

solution was then dried under argon and resuspended in

100 pL of solvent A (methanol/5 mm aqueous tetrabuty-

lammonium phosphate, 7 : 3, v/v) and stored at —20 °C

for further analysis with HPLC The HPLC was per-

formed on a Lichrospher 100 RP-18 (5 um) RT 125-4

Merck column preequilibrated with solvent A Elution

(1 mLmin ”) was maintained with this solvent for first

3 min then was carried out with solvent B (methanol/H;O,

7: 3, v/v) for 45 min Elution was followed by measuring

the absorbance at 420 nm and radioactivity by scintillation

detection (Berthold HPLC radioactivity monitor) in pre-

sence of QuicksZint 302 (PerkinElmer) In these conditions

we verified that Mg-protoporphyrin [X eluted at 10 min,

protoporphyrin [X at 12 min and methyl Mg-protoporph-

yrin [IX at 15 min after injection Further, each fraction

was tested for typical room temperature fluorescence

emission spectrum after excitation at 420 nm A weak

radioactive peak was usually detected 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 IX

Photolabeling with Ado-met

Photolabeling was carried out as described previously [23]

S-[CH;-*H]Ado-met (3.5 1m, 3 TBq:mmol!"!) was added to

15 wL of ice cold protein fractions The mixture was then

irradiated with UV for 15 min before the addition of SDS/

PAGE sample buffer After SDS/PAGE, labeled proteins

were analyzed by fluorography

Western blot analyses Arabidopsis MgP{xMT, containing a deletion of the first 160 amino acids (Al6l-protein) was expressed in E coli, as described above The inclusion bodies, enriched in the expressed protein, were purified and analyzed on an SDS/ PAGE gel The Al61-protein band was excised and used to obtain rabbit polyclonal antibodies (Elevage Scientifique des Dombes, Chatillon-sur-Chalaronne, France) Western blot analyses were performed using Arabidopsis, spinach or transformed £ coli subcellular fractions, as described previously [23] Proteins (15-50 pg) were separated on an SDS/12% polyacrylamide gel, as described previously [24] Nonspecific binding sites on the blot were blocked using

Tris/HCl 10 mm, pH 7.5, NaCl 9 g-L7', and nonfat dried

milk (50 g-L7') Arabidopsis MgP;xMT was detected with antibodies at a 1 : 500 dilution using secondary antibodies coupled to alkaline phosphatase or horse radish peroxidase For spinach fractions, a 1: 100 dilution was used Pre- immune sera gave no signal

Protein and chlorophyll determination Protein concentration was determined as described previously [25], using BSA as a standard Chlorophyll concentration was also measured as described previously [26]

RESULTS

Characterization of putative sequences for MgP|xMT

in plants

We analyzed nucleic databases starting from the sequence of Synechocystis sp MgPxMT (BAA 10812) and we identified several ORF sequences presenting a high alignment score with the Synechocystis protein [27], in A thaliana (CAB36750), in Oryza sativa (BAA84812) and in Nicoti- ana tabacum (AF213968) genomes The resulting putative plant proteins are very closely related (69% identity over

250 amino acids between Arabidopsis and rice proteins) A multiple alignment including the proteins from Arabidopsis and rice and the MgP;xMT sequences from Synechocystis and R capsulatus (P26236) shows that the plant proteins are

82 and 96 amino acids longer than the Synechocystis protein, respectively, containing a long N-terminal extension as compared to the prokaryotic 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 identified previously [28]

Identification of the arabidopsis MgP\xMT

To identify the activity associated with the protein encoded

by the A thaliana sequence, the 312 amino-acid protein (33 795 Da) corresponding to the full-length ORF was expressed in E coli MgPyxMT activity was assayed in the bacterial pellet by measuring the formation of [}H]methyl Mg-protoporphyrin [X from Mg-protoporphyrin IX and, S-[methyl-H]Ado-met After solvent extraction and HPLC purification, methyl Mg-protoporphyrin [X was further identified by room temperature fluorescence emission

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A

Fig 1 Comparison of different MgP);xMT of

was t ~~-MPFAPSLLSSSSSVSQFLPRFPNATRFENVTPRSRAATVVAASVTDLAGVDST REBAR 57

rokar r ryotic origin (A) Compar- ầ

P 0Karyoflc or eucaryot © ° St (A) Compa Os MARAAVSTAPLSRVHSPPPLTPRHPHSRHSRVGLLHPQRKALTTAAALPPAADLPPLSION 60 ison of the deduced amino acid sequences of scystis -~ -+-++++++++f -

cDNA encoding the MgP;xMT of Arabidopsis thọCa TrrrrrrTTTTT TT 30 TT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

thahiana (At;: AL035523.1), Oriza sativa (Os;

AP002542.2), Synechoecystis (Scystis; at TT TE.YVN.VLÀN s00) DE,RRRKLOAEEVGGGDKEVVREYFNSTGFERNRKIYGET-DEVN 116 L47126.1), and Rhodobacter lat Os PUN VV WWE NSH PE RRRRAQARAAGGGDKEAVRAYENSTGFERWRKIYGSATDGVN 120

củ) 060004161 683.18 scystis - MTNAALDDKTIVRDYFNSTGFDRWRRIYGD GQVN 34

(Rhoca or Rc; P26236) using CLUSTAL w (1.8) rhoca = - —— MPSDYAEIRNRVEHYFDRTATRAWARLTTAD-EKVS 35

multiple alignment program Stars indicate —

position of amino acids identical among the 161

four sequences and the position in the align- |

at RVQKDIRLGHAKTVENTMLMLTE D—RERW ACh MAO NCCCFREIMMSBESPAKEGATVSASDIS 175

ment of the three classical motifs for Ado-met os RVOLDIREGHARTVAATLSMLRDSP VguACLUAROACOCHCOIE MESES CMEASDIS 180

dependent methyltransferase is underlined scystis FVOKDIRVGHOOTVDSVVAWLVAD-GNLPG LG G616 06:01: 00207:(97:(/.L00 042519 93 with a gray bar The transit peptide cleavage rhoca KVROTVREGRDTMRAVHILSRILPDD~~~I.TGCRVMDAGCGTGLTTVELARRGADVVAVD1S 92 site for each plant protein as found by the aE

CHLOROP program is indicated by an arrow at AAMVAEAEMKAKAQLPSE-~ NLPKFEVNDLES-LTGKYDTVVCLDVLIHYPQNKADGM 230

Os AAMVSEAQRQAEAAAMAASDTFRMPRFEVRDLES-LEGKYDIVVCLDVLIHYPREEAKQM 239 Several putative transmembrane sequences scystis EKMVGEAQQKAQEVLAYG NQPTFMTQDLAQ-LGGKYDTVICLDVLIHYPTEEASAM 148 were determined by the TMPRED program rhoca _ POLIDTIAKDRLPPELRGK - VSFHVGDMADPALGQFDYVVAMDSLIYYRAPDIGRV 146 They are indicated in white letters on black -

background The amino terminus end of the at IAHLASLAEKRVILSFAPKTFYYDILKRIGELFPGPSKATRAYLHSEADVERALGKVGWK 290

bị đ Os IRHLASLAEKRVLISFAPRTLYFDFLKRVGELFPGPSKATRAYLHSERDIEDALRDAGWR 299

recombinant truncated proteins (A80-protein scystis ISHLASLADRRLILSFAPKTLGLTVLKKIGGLFPGPSKTTRAYQHKEADIRKILGDNGFS 208

and Al61-protein) 1s indicated by a box above rhoca LTELGKR IPG Watguayge.ee Wal MAFWWLGKLFPRSNRSPVMIPHALDKLQRHAGDSLIK 206

* kk Ok kek KKK *

the Arabidopsis sequence (B) Unrooted Es EI

phenogram drawn using an alignment of the at ISKRGLTTTQFYFSRLIEAVPM - 312

Os VANRGFISTQFYFAKLFEAVPIAAASQ 326

four sequences described above and of those scystis IARTCMTSTRFYYSRILEAVRS -—~ 230

of: Nicotiana tabacum (Nt; AF213968.1), rhoca IDR -VARGEYTISECLEYRP - 224

Heliobacillus mobilis (Hm; AF080002), ,

Rhodospirullum rubrum (Rr, AF202319.1),

Rubrivivax gelatinosus (Rg; AB034704), B

Rhodobacter spheroides (Rs; X81413.1)

The alignment was carried out over the 240 Synechocystis

C-terminal amino acids of each sequence At

according to [31] Distance is indicated in Nt Rs

PAM (probability of accepted mutation) O Rc

The plant proteins appear very close to each S

other and as well as to the cyanobacterial

protein whereas all types are relatively far Rg

from the proteins present in photosynthetic Rr

bacteria (purple bacteria or heliobacteria) Hm — 20 PAM

spectra Following IPTG induction, the MgP;xMT activity

was detected in the transformed bacteria (Fig 2A) Max-

imum MegPixMT activity was observed after 2 h of

induction whereas no activity was recorded in control

bacteria, 1.e bacteria transformed with either the plasmid

containing the inverted insert or containing soMGDI

cDNA, a control cDNA encoding a MGDG synthase from

spinach (Fig 2A,B) These results demonstrate that the

characterized Arabidopsis CDNA sequence encodes a func-

tional MgP;xMT When the bacterial proteins were

analyzed by SDS/PAGE and Coomassie staining, no

recombinant protein could be visualized Therefore,

although the recombinant protein was present in low

amounts 1n E coli, 1t was in a highly active form Expression

of truncated portions of the protein, [(a) A80-protein

missing the first 79 amino acids and similar in size to the

prokaryotic MgP;xMT; and (b) Al61-protein missing the

first 160 amino acids] led to high amounts of recombinant

proteins (Fig 2B) The Al61-protein was inactive, whereas

the A80-protein exhibited MgP;xMT activity Therefore, the

methyltransferase domain of the MgP;xMT 1s clearly contained after the 80 N-terminus amino acids of the full- length protein

MegPixMT 1s a chloroplast protein and therefore should contain an N-terminal chloroplast targeting sequence CHLOROP predicted that the Arabidopsis protein presents a chloroplast transit peptide with a peptidase cleavage site located between amino acids 39 and 40 leading to a putative mature protein of 29 500 Da Indeed, we analyzed whether the in vitro radiolabeled full-length precursor could be imported into isolated pea chloroplasts (Fig 3) We demonstrated that the precursor protein was processed into a mature protein with an apparent molecular mass of 31 kDa The processed protein was protected 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 protein suggesting that the CHLOROP predicted that the peptidase cleavage site 1s probably correct The mature Arabidopsis protein is therefore larger than the prokaryotic proteins,

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A

0 1 2 3 4 5h

Time after induction

MgP „ MT activity (pmol.min'1.mg'! pro

Fig 2 Analysis of the MgPxMT activity associated with the expression of recombinant proteins in E coli BL21(DE3) (A) MgPixMT activity of bacterial cultures after induction by | mm isopropyl thio-B-p-galactoside IPTG) The black bars correspond to bacteria transformed with pET Y3a plasmid containing full-length Arabidopsis MgP}xMT ORF insert under T7 promoter and the white bars correspond to the same construct but with insert in the opposite orientation Enzyme activity was measured in bacterial pellet obtained from 200 uL of culture (B) Comparison of MgP}xMT activity and protein composition of bacteria 2 h after of induction P corresponds to bacteria transformed with pET Y3a plasmid containing full- length Arabidopsis MgP}xMT ORF insert under T7 promoter, and R corresponds to the same construct but with insert in the opposite orientation P’ corresponds to the same bacteria as in P; 80 and 161 are bacteria transformed with pET Y3a plasmid containing A80 and A161 truncated Arabidopsis MgP}xMT ORF; M are bacteria transformed with pET Y3a plasmid containing spinach MGDG synthase cDNA [21] Positions of clearly overexpressed recombinant proteins are indicated by triangles

kDa

Fig 3 Import of the [PS]methionine-labeled Arabidopsis MgP\xMT

precursor protein into isolated pea chloroplasts Precursor protein or

chloroplast suspensions after in vitro import were analyzed by SDS/

PAGE, Coomassie staining and fluorography detection Lanes: P,

labeled precursor proteins (1 : 100 of what used for iv vitro import); I,

proteins after in vitro import; It, proteins after in vitro import and

subsequent limited thermolysin treatment of chloroplasts

containing an additional domain of about 40 amino acids,

which are not involved in the methyltransferase catalytic

activity

Localization of the MgP,xMT in chloroplasts

In purified spinach chloroplasts, we detected the MgPpxMT activity in membrane pellets whereas no activity was found

in the soluble fraction (stroma) We then analyzed MgP,xMT activity in chloroplast 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 envelope and thylakoids were clearly separated, the MegP;xMT activity was detected in both types of membranes: the envelope and the thylakoids (Fig 4A) We controlled that MgP;xMT activity was linear with protein amounts and time under our measurement conditions The MegP\xMT specific activity in the envelope was 2.5-fold higher than in the thylakoids In purified Arabidopsis chloroplast membranes, a similar distribution of the activity was found between envelope and thylakoids (Fig 4B) Western blot analysis of Arabidopsis chloroplast membranes using antibodies raised against the truncated recombinant Arabidopsis protein (A161-protein) detected a protein with a 31-kDa apparent molecular mass in the envelope fraction and in the thylakoids, whereas the distribution of the E37 envelope marker [29] was clearly restricted to the envelope (Fig 4B) In spinach chloroplast membranes, envelope and thylakoids, the Al6l-protein antibodies also detected a 31-kDa protein This size corresponds to the size of the processed protein after import In addition, in a fraction

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S

_—_— —

45 - —— ©

1" :

—s

B

A

&

U

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Fig 4 Localization of MgP¡yMT in chloroplast membranes (A) Analysis oŸ spinach chloroplast envelope and thylakoids fractions The fractions were analyzed by SDS/PAGE and Coomassie blue staining and MgP;xMT activity was compared to MGDG synthase activity as a marker of envelope membranes and to chlorophyll concentration as a marker of thylakoids (B) Analysis of A thaliana chloroplast envelope and thylakoids fractions MgP}xMT activity was 3 nmolh7!mg protein”! in envelope fraction and 0.47 in thylakoids fraction Western blot detection of MegP;xMT was compared to detection of E37, a marker of envelope membranes (E) chloroplast envelope; (T) thylakoids

derived from spinach envelope membrane and devoid of

E37, a major methyltransferase of the envelope [23]

removed by purification on a DEAE column, a 31-kDa

protein was photolabeled with radioactive Ado-met under

UV light (Fig 5) corresponding to the mature MgP;xMT

From these results, we conclude that the mature MgP;xMT

is located and active in both chloroplast membrane systems,

97—

4

14—

Fig 5 Photolabeling of envelope subfractions with Ado-met Spinach

chloroplast envelope was solubilized in buffer S (6 mm CHAPS, 1 mm

DTT, 50 mm Mops pH 7.8) at | mg protein mL" After centrifugation

at 100 000 g for 30 min, solubilized fraction (1) was loaded on a DEAE

column and washed with buffer S to remove E37, an Ado-met meth-

yltransferase present in the envelope [23] We verified that eluted E37

had no MgP;xMT activity DEAE bound protein fraction (2) was then

eluted in buffer S containing 0.3 m NaCl MgP;xMT activity was

92 pmol-min”'mL"! in fraction (1) and 50 pmolmin™'mL™ in frac-

tion (2) Each fraction (18 wL aliquot) was assayed for binding of

>H-Ado-met (3.5 um) by crosslinking under UV After SDS/PAGE and

Coomassie staining, labeled proteins were detected by fluorography A

labeled 31 kDa protein ( — ) is visible in the purified envelope fraction

i.e envelope and thylakoids, and that the protein has the same size in both fractions

Association of MgP,xMT with the membranes Analysis of the Arabidopsis MgP;xMT sequence by the TMPRED program predicted two transmembrane helices in the mature Arabidopsis protein, one located in the plant specific N-terminal extension (amino acids 54—72), which is very hydrophobic, and one in the methyltransferase 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 in the homologous Rhodobacter MgP{xMT, suggesting that the transmembrane prediction was not reliable Therefore, we analyzed the association of the MgP;xMT with the envelope and with the thylakoid membranes by ionic and alkaline extractions The same pattern of results was obtained for both envelope and thylakoids (Fig 6B) The protein was not released from the membrane by treatment with 1 Mm 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 leaflet of the membrane via lipid interaction As the plant specific N-terminal extension (amino acids 54-72) is very hydrophobic, and as the recombinant truncated protein (A80-protein) deleted of this zone was partially soluble in E coli (Fig 6C), 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 reports had proposed that a MgP;xMT might be associated with plastid membranes [14,15] In the present study, we show that an Arabidopsis MgP,xMT is encoded by a gene located on chromosome 4 One short intron (175 bp) is present in the

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0.8 } Ị i \ ‘ |

0.6 0.4

Amino acid position

1M 0.1M pH11 0.1M

Envelope

Coomassie

Ado-met binding

middle of the ORF, between the lysine 185 and alanine 186

codons We identified three different regions in the full-

length precursor 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 MgPrxMT from Synechocystis and a

more distant homology to the MgP}xMT 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}xMT site Thirdly,

between the two previous domains, in the N-terminal end of

the mature plant protein, we found a very hydrophobic

region possibly involved in the anchoring of the protein to

the membranes This part of the protein is absent in the

prokaryotic MgP}xMT

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;xMT activity, we can assume they do due

to their high similarity to A thaliana MgPyxMT

In spinach and A thaliana chloroplasts, the MgP;xMT

protein is present in an active form in both thylakoids and

envelope membranes Its size is apparently identical in both

types of membranes although we cannot exclude some

NaOH extracted thylakoid proteins

;S— pl —>10

Fig 6 Analysis of the association of MegPixMT with membranes (A) Hydropathy profile of Arabidopsis MgP}xMT The hydropathy was analyzed as described previously [32], using a 11 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;xMT with spinach chloroplast envelope or with Arabidopsis thylakoids by ionic and alkaline extractions (B1) Each fraction, soluble (S) and insoluble (1), obtained after treatment of 50 ng membrane protein was analyzed by Western-blot with anti-MgP;xMT antibodies (dilution

1 : 100 for envelope and 1 : 500 for thylakoids) (B2) MgP;xMT was detected by West- ern blot after two-dimensional analysis of NaOH solubilized proteins from Arabidopsis thylakoids (1 mg protein) The NaOH solubilized thylakoid MgP,;xMT presents an apparent molecular weight and an apparent isoelectric point close to those predicted by ChloroP for the mature protein (29.5 kDa and

pl = 5.7) (C) Analysis of the solubility of A80-protein ( > ) in E coli Expression of A80-protein was induced by IPTG for 2 h Frozen bacterial pellet was resuspended in

25 mm Mops pH 7.8, 1 mm MgCl and soni- cated on ice for 15 s Soluble (S) and insoluble (1) fractions were separated by centrifugation (100 000 g, 30 min) and analyzed by SDS/

= PAGE and Coomassie blue staining (C2) The

identity and activity of A80-protein were verified by UV photolabeling with Ado-met

limited modifications The strong association of MgP;xMT with either envelope or thylakoids membranes, and the very low degree of cross contamination of envelope and thylakoids preparations, support that MgP;xMT is present

in both types of membrane The specific activity is higher

in the envelope membranes than in the thylakoids Taking into account that the thylakoids network represents 50- to 100-fold more protein than the total envelope, the MegPixMT partition between envelope and thylakoids can be considered close to 1 : 30 in mature chloroplasts

On the other hand, during early development of chloroplasts, MgP;xMT activity from the envelope should be the most important

In Euglena, MgP}xMT was reported to be firmly attached

to chloroplast membranes [30] However 15-25% of the activity was also recovered in a supernatant that was probably enriched in chloroplast envelope considering the low density of envelope membranes

To understand the role of the additional N-terminal hydrophobic domain present in the plant MgP;xMT and absent from the prokaryotic MgPrxMT, it would be particularly interesting to know the localization of MegP,xMT in cyanobacteria We do not know if it strictly associates with membranes and with which membranes; the cell envelope or the thylakoids?

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The dual localization of MgP;xMT in chloroplasts may

be related to different roles of the protein in each

membrane For chlorophyll synthesis, the enzyme 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 in the thylakoids but

presumably involved in heme synthesis [4] and POR can

associate with the thylakoids [9] Finally, the final steps of

chlorophyll synthesis, such as addition and reduction of

geranylgeranyl chain, are located on the thylakoids [1]

Therefore, we cannot exclude the possibility that there are

two locations for chlorophyll synthesis in the chloroplast

or a motion of intermediate compounds between the two

types of membrane As the Mg chelatase does not seem to

associate with the thylakoids, a transfer of Mg-proto-

porphyrin IX from the envelope to the thylakoids would

be required to supply the thylakoid MgP;xMT with

substrate

Localization of MgP;xMT in the envelope may play a

role in plastidic signaling Mg-protoporphyrin [IX or its

methyl derivative are thought to have a signaling function

outside of the chloroplast and it is therefore important to

regulate their abundance in the chloroplast envelope

Kropat ef 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 [TX 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, accessibility of Mg-proto-

porphyrin [X to the cytoplasm was reported to be crucial to

drive light signaling [19]

Finally, it is possible that the two pools of MgP;xMT

located in the envelope and in the thylakoids play differ-

ential roles regarding chlorophyll biosynthesis and produc-

tion of plastidic signals responsible for controlling nuclear

gene expression The sorting of the single MgP;xMT in two

different locations also presents a problem that will require

further exploration

ACKNOWLEDGMENTS

We are grateful to Dr Stephane Ravanel for providing us with the

Arabidopsis RACE library This research work was supported in part

by a postdoctoral fellowship from the French Ministére de l’Education

Nationale, de la Recherche et de la Technologie (to A.K.T.)

SUPPLEMENTARY MATERIAL

The following material is available from http://www.ejbio

chem.com

Figure S1 Fluorescent spectra of tetrapyrroles compounds

issued from HPLC analysis

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