The plant S -adenosyl- L -methionine:Mg-protoporphyrin IX methyltransferase is located in both envelope and thylakoid chloroplast membranes Maryse A. Block, Arun Kumar Tewari, Catherine Albrieux, Eric Mare  chal and Jacques Joyard Laboratoire de Physiologie Cellulaire Ve  ge  tale, CNRS/CEA/Universite  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 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 in the chloroplast 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]. Envelope membranes 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 in both the thyla- koids and the envelope membranes [4]; the subunits of the Mg chelatase are present in large amounts in the stroma but become associated with the envelope 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 in the envelope [7]. POR w as 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 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. The methyltransferase has not yet been identi®ed in plants but has bee n clearly recognized in purple bacteria [10,11] and in 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 the plant protein within chloroplast membranes and 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 in the 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) in the 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 and chloroplast 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 ), and chloroplast 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 in the 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. The envelope 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) and the pellet was washed twice by centrifugation in chloroplast 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). In the 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 The plant Mg-protoporphyrin IX methyltransferase (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 IX and 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 both the 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 the plant 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 in the 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 dierent 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 and the position in the align- ment of the three classical motifs for Ado-met dependent methyltransferase is 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 The plant Mg-protoporphyrin IX methyltransferase (Eur. J. Biochem. 269) 243 containing an additional domain of a bout 40 amino acids, which are not involved in the methyltransferase 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 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 en velope and t hylakoids were clearly separated, t he MgP IX MT ac tivity was detected in both t ype s of mem- branes: the envelope and the 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 in the envelope was 2.5-fold higher than in the thylakoids. In puri®ed Arabidopsis chloroplast membranes, a similar distribution of the activity was found between envelope and 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 in the envelope 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 and the white bars correspond to the same construct but with insert in the 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 in the 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 the envelope [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 located and active in both chloroplast membrane systems, i.e. envelope and thylakoids, and that the protein has the same size in both 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 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 IX MT, suggesting t hat the transmem- brane prediction was not reliable. Therefore, we analyzed the association of the MgP IX MT with the envelope and with the t hylakoid 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 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 the plant 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 in chloroplast membranes. (A) Analysis of s pinach c hloroplast envelope and 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 chloroplast envelope and 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 buer 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 columnandwashedwithbuerStoremoveE37,anAdo-metmeth- yltransferase present in the envelope [23]. We veri®ed that eluted E37 hadnoMgP IX MT activity. DEAE bound protein fraction ( 2) was then eluted in buer 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 in the puri®ed envelope fraction. Ó FEBS 2002 The plant Mg-protoporphyrin IX methyltransferase (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 in the 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, in the N-terminal end of the m ature p lant protein, we found a very h ydrophobic region possibly i nvolved in the 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 in both 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 the envelope membranes than in the 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 envelope and 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 the envelope should be the most important. In Euglena,MgP IX MT was reported to be ® rmly attached to chloroplast membranes [30]. However 15± 25% of the activity was also r ecovered i n a supernatant t hat 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 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 membranes and 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 envelope and 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 in the 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 in the 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 the envelope to the thylakoids would be required to supp ly the thylakoid MgP IX MT with substrate. Localization of MgP IX MT 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 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 in the envelope and in the 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  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. In Plant 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  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. 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