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Cell and division requires ARTEMIS

Hrvoje Fulgosi*†, Lars Gerdes*, Sabine Westphal*, Christel Glockmann*, and Ju¨ rgen Soll‡

*Botanisches Institut der Christian-Albrechts-Universita¨ t Kiel, Am Botanischen Garten 1-9, D-24118 Kiel, Germany; and ‡Botanisches Institut der Ludwig-Maximilians-Universita¨t Mu¨ nchen, Menzinger Strasse 67, D-80638 Munich, Germany

Edited by Roland Douce, Universite´ de Grenoble, Grenoble, France, and approved June 25, 2002 (received for review January 18, 2002) are endosymbiotic of cyanobacterial ori- cumulate large chloroplasts with similar morphology to MinD gin. It seems reasonable to assume that division and mutants (11). A unique feature of MinC and MinD is their ability division still share general principles, as shown for the FtsZ pro- to rapidly oscillate from one bacterial cell pole to another (12). teins. However, further components involved in this process are In , the MinCD complex appears to be stationary largely unknown. Here we describe ARTEMIS, a nuclear-encoded and equally distributed on both poles. The exact mechanisms of of chloroplast inner envelope membranes that is required MinCD recruitment at polar zones are unclear and underline the for organelle division. ARTEMIS consists of three distinct modules: general problem of how a cell can identify its midpoint (12). an N-terminal receptor-like region, a centrally positioned glycine- Almost equally elusive are the mechanisms of initial FtsZ rich stretch containing a nucleoside triphosphate-binding site, and tethering to the bacterial cytoplasmic membrane at very early a C-terminal YidC͞Oxa1p͞Alb3 protein translocase-like domain. stages of division. The cytoplasmic membrane protein ZipA has Analysis of Arabidopsis En-1 transposon mutants as well as ARTE- been proposed as an FtsZ receptor in (13). Except the MIS antisense revealed chloroplasts arrested in the late already mentioned FtsZ homologues and the chloroplast MinC stages of division. Chloroplasts showed clearly separated and and MinE , homologues of other bacterial cell division distinct multiple thylakoid systems, whereas the final organelle proteins have not been found in the Arabidopsis (3), fission remained unaccomplished. Inactivation of a cyanobacterial suggesting that chloroplast division involves different or addi- ͞ ͞ with sequence similarity to the YidC Oxa1p Alb3-like do- tional components. It remains to be clarified how chloroplast main of ARTEMIS resulted in aberrant cell division, which could be division proteins adhere to the inner envelope in a timely and a rescued by the Arabidopsis protein. ARTEMIS represents a so-far- spatially coordinated manner. How are constrictions of FtsZ unrecognized link between prokaryotic cell fission and chloroplast ring(s) and the -dividing ring coordinated? Which com- division. ponents are involved in possible nuclear control of chloroplast division? ivision of higher chloroplasts is a complex and still In recent years, different protein translocases have been Dpoorly understood process that combines mechanisms of identified in membranes of various subcellular compartments organelle constriction with the assembly and expansion of and organelles of eukaryotic cells and prokaryotic envelope membranes and the thylakoid network. Four nuclear- (14). These auxiliary molecules not only transport proteins from encoded proteins have so far been implicated in chloroplast one side of a membrane to another but also assist in protein division. FtsZ is a -like GTPase that assembles into a ring insertion into the lipid bilayer. Formation of cellular membranes structure at the bacterial cell midpoint and enables recruitment follows similar blueprints from prokaryotic organisms to eu- of other division proteins (1). Most of identified eukaryotic FtsZ karyotic organelles, and the conserved nature of certain protein are of cyanobacterial origin and are implicated in chlo- translocases supports a bacterial origin of both mitochondria and roplast division. In chromophyte and red algae, an additional chloroplasts (14). In mitochondria, Oxa1p protein was shown to ␣ -proteobacterial-related FtsZ is involved in mitochondrial di- be involved in the insertion of a subset of inner membrane vision (2). At least four FtsZ homologues encoded by two proteins from the mitochondrial matrix (15–18). Alb3, a homo- different gene families, FtsZ1 and FtsZ2, can be found in logue of Oxa1p, is a chloroplast protein involved in the insertion Arabidopsis (2, 3). Members of both families colocalize in the of light-harvesting antenna proteins into the thylakoid mem- chloroplast stroma and form a contractile ring(s) at the plastid brane (19). The deletion of Alb3 leads to defective thylakoid division site (3, 4). Apart from FtsZ ring(s), constriction of assembly (20). Both Oxa1p and Alb3 seem to originate from the chloroplasts during division involves at least one additional ring bacterial translocase YidC (21), which is associated with the structure located at the cytoplasmic surface of the outer enve- SecYEG trimeric complex (22). YidC assists in sorting of lope (5). This plastid-dividing (PD) ring is in red algae composed proteins that were previously believed to insert into the mem- of so-far-unidentified component(s) that are not related to FtsZ brane directly, without the aid of proteinaceous components. proteins (6). It has been postulated that the FtsZ ring-based Most eubacteria possess only one YidC homologue, but species system evolved from cyanobacterial , whereas the of Bacillus, Listeria, and Streptomyces contain an additional PD ring probably originates from the eukaryotic host cell (7). YidC-related protein (23). The function of this additional pro- Both systems appear to complement each other and are in tein is poorly investigated; however, its disruption in B. subtilis dynamic transition during a division process (7). In of cells leads to a arrest in the intermediate stage of spore moss Physcomitrella, FtsZ-GFP monomers polymerize to highly formation. The protein has accordingly been designated SpoIIIJ organized structures resembling (8). Accordingly, (stage III sporulation protein J) (24). Expression of spoIIIj is the network has been designated plastoskeleton, although its dispensable during vegetative growth; however, its sporulation- existence in plastids of other species has yet to be established. specific expression is crucial for efficient sporulation (25). Akin to bacterial , placement of the plastid division During vegetative growth, SpoIIIJ localizes to the cell mem- initiation site on the stromal surface of the inner envelope is brane, but in sporulating cells it accumulates at polar and probably mediated by MinD and MinE proteins (9–11). In engulfment septa (25). It remains unclear whether inactivation of bacteria, MinD forms a complex with MinC and prevents Z-ring formation at all potential division sites, except the mid-cell (12). Plants with altered MinD expression form large plastids unable This paper was submitted directly (Track II) to the PNAS office. to carry out the fission reaction (9–10). MinE, on the other hand, †To whom reprint requests should be sent at the present address: Department of Molecular prevents MinCD from inhibiting Z-ring formation at the proper , Rudjer Bosˇkovic´ Institute, P.O. Box 1016, Hr-10000 Zagreb, Croatia. E-mail: PLANT mid-cell site (12). Arabidopsis plants overexpressing MinE ac- [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.172032599 PNAS ͉ August 20, 2002 ͉ vol. 99 ͉ no. 17 ͉ 11501–11506 spoIIIj leads to a block in spore formation because of impaired and reselected by spraying 2-wk-old seedlings with 250 mg⅐lϪ1 of assembly of membrane proteins, either spore specific or unspecific. phosphinotricin in 0.1% Tween 20. Here we describe the identification of ARTEMIS, an addi- tional chloroplast homologue of the YidC͞Oxa1p͞Alb3 trans- Generation of Synechocystis Knockout and Complementation Lines. locase whose inactivation in the model plant Arabidopsis leads to Locus slr1471 was amplified by PCR from purified Synechocystis a specific defect in chloroplast division. ARTEMIS is an integral sp. PCC6803 genomic DNA by using oligonucleotides that inner envelope membrane protein that combines an N-terminal allowed cloning into BamHI͞KpnI sites of pBluescript to create region similar to receptor protein kinases with a YidC͞Oxa1p͞ pBSArt construct. The kanamycin-resistance cassette was in- Alb3-like C-terminal domain. Cyanobacterium Synechocystis serted into the HindIII site of pBSArt located within the slr1471 contains an ARTEMIS-related protein whose inactivation leads sequence. pBSArt was transformed into Synechocystis sp. to defects in cell division rather than general protein-targeting PCC6803 cells, and resistant colonies were selected on BG-11 deficiencies. This defect can be complemented by ARTEMIS plates supplemented with increasing concentrations of kanamy- ␮ ͞ from Arabidopsis, suggesting an evolutionary and functional cin (20 g ml end concentration). To ensure proper segregation, relationship between organellar and prokaryotic cell division. resistant colonies were screened through several rounds of replating on kanamycin-containing BG-11 medium. Efficiency Materials and Methods of segregation was tested by PCR with slr1471-specific oligonu- ⌬ Standard Methods. The RNA isolation for reverse – cleotides and genomic Southern blotting. 1471 cells were grown ␮ Ϫ2⅐ Ϫ1 PCR was made by using RNeasy Plant Kit (Qiagen, Hilden, autotrophically at 28°C in BG-11 under 80 mol m s white Germany). Hybridization of total RNA from Arabidopsis thali- light in flasks bubbled through with 2% CO2 in air. The promoter ana Col-0 was performed by using a 269-bp-long DNA probe region of the sll0617 gene (31) was PCR amplified with oligo- nucleotides fitted with PstI͞NdeI restriction sites and ligated corresponding to the region between nucleotides 1600 and 1869 ͞ of the art1 gene. Stringency of washing was 0.5 ϫ SSC, 0.1% SDS with a nucleotide sequence fitted with NdeI KpnI overhangs and corresponding to V538-R1013 stretch. The construct was in- at 65°C. DNA was isolated according to ref. 26. Total protein ͞ extracts were made by using extraction buffer (0.05 M Tris͞HCl, serted into PstI KpnI sites of the pBluescript and transformed ͞ ͞ ͞ ␤ into Synechocystis ⌬1471 cells. Transformants were selected on pH 6.8 0.05 M EDTA 1% SDS 0.1% -mercaptoethanol) and ␮ ͞ ␮ ͞ boiling at 95°C. Western transfer was made by using blotting BG-11 plates containing 20 g ml of kanamycin and 30 g ml buffer (25 mM Tris͞192 mM glycine͞10% Met-OH). of ampicillin. Photosynthesis and respiration rates were measured with a Results and Discussion Clark-type electrode at 28°C, with a cell density of 5 ␮gof Characterization of ARTEMIS Protein. In a search of components chlorophyll per milliliter. Respiration followed after a 5-min involved in chloroplast biogenesis, we have identified an Arabi- incubation in the dark; photosynthesis was measured at least Ϫ Ϫ dopsis protein (locus NP࿝173858 in GenBank) with a unique three times for 10 min at 1,400 ␮mol m 2⅐s 1. molecular structure. The 1,013-residue polypeptide (Fig. 1) Total protein extracts were made as described in ref. 27. Syn- encoded on 1 contains a COOH-terminal domain echocystis subfractionation was carried out according to ref. 28. similar to the Alb3 protein (predicted polytopic region) with conserved YidC translocase elements. The middle portion con- Protein Overexpression and Production of Antisera. DNA fragments tains a predicted ATP͞GTP-binding domain, whereas the NH - corresponding to amino acid stretches K537–K625 and G732– 2 ͞ terminal region resembles receptor domains of receptor protein Q802 were cloned into BamHI KpnI sites of pRSETA (Invitro- kinases. Northern blot analysis indicated a moderately abundant gen) vector and overexpressed in Escherichia coli BL21 cells. transcript of an estimated size of 3,550 bp in Arabidopsis plants Recombinant proteins were purified on Talon-Metal-Affinity (data not shown). We tested the hypothesis that this polypeptide Column (CLONTECH) and used as antigenes for immunization is located in chloroplasts, which is indicated by a putative of two different rabbits. ␣ArtA is raised against ␣ NH2-terminal chloroplast targeting signal (32). Antisera raised K537–K625 and ArtB against G732–Q802. against different parts of the protein were used to analyze various cellular and chloroplast subfractions. A single immunoreactive Identification of En-1 Transposon Lines. Transposon lines were band with an apparent molecular mass of 110 kDa was identified obtained from the ZIGIA collection (Max-Planck-Institut fu¨r exclusively in inner envelope membranes of chloroplasts (Fig. Zu¨chtungsforschung, Cologne, Germany) (29). The presence of 2A). The same 110-kDa protein band could be detected with an the En-1 transposon was tested by genomic Southern hybridiza- antibody raised against a conserved domain from E. coli Oxa1p tion. The position of the En-1 element in the art1 genomic (data not shown). Immunogold labeling of ultrathin sections (35) sequence was determined with PCR by using oligonucleotide from leaves further corroborated the chloroplast localization corresponding to the 18-bp stretch downstream of the art1 (Fig. 2B). Consequently, the protein was designated ARTEMIS Ј initiation codon and En-1-specific en205 (5 - (Arabidopsis thaliana envelope membrane integrase). Inner en- Ј AGAAGCACGACGGCTGTAGAATAGGA-3 ). A 508-bp velope vesicles were extracted by using different salt and urea amplified fragment was sequenced. Placement was reconfirmed treatments. In every case, we observed ARTEMIS exclusively in by PCR amplification from the opposite side using exon 3 reverse the insoluble membrane fraction (Fig. 2C), strongly suggesting oligonucleotide and En-1-specific en8130 (5Ј-GAGCGTCG- that ARTEMIS is an integral membrane protein. The membrane GTCCCCACACTTCTATAC-3Ј) and sequencing. association properties of ARTEMIS are comparable to those of some translocon at the inner envelope of chloroplast (Tic) Generation of ARTEMIS Antisense Lines. The DNA fragment cor- subunits (Fig. 2C). responding to the stretch between nucleotides 722 and 1329 was In ARTEMIS, the receptor part and the YidC͞Oxa1p͞Alb3- inserted in an antisense direction into SacI͞XbaI sites of pGPTV like domain are connected by a glycine-rich region harboring a phosphinotricin resistance binary vector (30). The construct was predicted ATP͞GTP-binding motif (P-loop) (Fig. 1B). To test introduced to Arabidopsis plants by Agrobacterium-mediated whether ARTEMIS can bind nucleoside triphosphates, deter- transformation by using vacuum infiltration. Transformants gent-solubilized inner envelope vesicles were incubated with were selected on 0.5 ϫ Murashige and Skoog medium supple- GTP-agarose matrix. Bound proteins were eluted with an excess mented with 10 mg͞ml of DL-phosphinotricin (Riedel-de Hae¨n, amount of either GTP or ATP, and ARTEMIS was detected by Seelze, Germany). Seeds of resistant plants were grown on soil immunoblot analysis. ARTEMIS could bind to the GTP matrix

11502 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.172032599 Fulgosi et al. Fig. 1. Deduced ARTEMIS protein sequence and comparison with related proteins. (A) Schematic representation of ARTEMIS three-domain structure. (B) Alignment of ARTEMIS deduced amino acid sequence (ART) with Lrrpk (A. thaliana, light repressible receptor , locus. NP࿝172061), Rlpk (A. thaliana, receptor-like protein kinase, locus AC003058), Alb3 (A. thaliana, albino3, locus AAB61458), and s1471 (Synechocystis sp. PCC6803, putative inner membrane protein slr1471, locus S75683). Residues conserved in all three sequences are shaded. A predicted nucleotide binding site is underlined. Continuation of amino acid sequence beyond the region of homology is indicated by three dots. Sequence alignment was performed by using the CLUSTAL method. and was efficiently eluted only with GTP but barely with ATP, mutant plants revealed extended, seemingly duplicated or trip- indicating a specificity for this nucleoside triphosphate (Fig. 3A). licated, yet undivided chloroplasts of irregular shapes (Fig. 4B). It was also possible to directly label ARTEMIS with [␣32-P] GTP Each half of a mutant chloroplast contained a wild-type-like (36) in isolated inner membrane vesicles, providing further thylakoid network with normally stacked membranes, indicating evidence of nucleoside triphosphate binding and possibly GTP- that thylakoid biogenesis is undisturbed, and that unlike the alb3 dependent regulation of ARTEMIS function (Fig. 3B). Al- (20), ARTEMIS does not influence light-harvesting though the predicted P-loop most likely participates in nucleo- complex protein insertion and photosystem II assembly. Other side triphosphate binding, other unidentified structural elements chloroplast division mutants are also able to carry out normal of ARTEMIS might be necessary for determination of specificity photosynthesis, providing additional evidence that thylakoid and strength of interaction. GTP could also assist in a possible assembly and function are not affected by the inability of homo- or heterooligomerization of ARTEMIS via the receptor chloroplasts to carry out the division (9, 10). Only at the domain. midpoint of the two undivided organelles were fewer membranes observed. They appeared deformed and pulled toward one side Arabidopsis ARTEMIS Mutants Have Defective Chloroplast Division. of the envelope membranes. In every case, envelope membranes We subsequently performed a screen in a population of maize were continuous and seemed unconstricted. Sometimes, chlo- En-1 transposon mutagenized Arabidopsis plants. The 7AR137 roplasts formed long filament-like structures (not shown). Ap- mutant contained a single En-1 element inserted into the second parently, constriction and formation of the envelope membrane exon of the art1 gene (Fig. 4A). The location of the En-1 furrow were not initiated in mutant plastids and were uncoupled insertion, combined with drastically reduced levels of ARTEMIS from thylakoid partitioning and elongation (Fig. 4B Lower Left). protein in mutant plants, indicated that 7AR137 is impaired in Even more, developing cotyledons contained tripolar chloro- ARTEMIS synthesis. The phenotype of 7AR137 did not signif- plasts, with three individual thylakoid systems (Fig. 4B Lower icantly differ from the wild type, although 7AR137 plants began Center). We have identified an F2-generation plant in which the PLANT BIOLOGY flowering about 4 days earlier on average. The ultrastructure of transposon element was no longer detectable in the art1 se-

Fulgosi et al. PNAS ͉ August 20, 2002 ͉ vol. 99 ͉ no. 17 ͉ 11503 comparable phenotypic characteristics as observed in the 7AR137 line (Fig. 4B Lower Right). We conclude that mutation in the art1 gene has a specific effect on late stages of plastoki- nesis, probably the positioning of constriction ring(s) and re- cruitment of the envelope-located division apparatus. Although some basic elements of the division machinery are conserved, e.g., FtsZ GTPase, from to chloroplasts (37), plastokinesis in algae and higher plants is likely more complex than bacterial binary fission. Together with division of the envelope membranes, the photosynthetic membrane net- work has to be separated. The genome of Arabidopsis contains several FtsZ genes (38), and the assembly of at least two distinct division rings has been documented in chloroplasts (39). Divi- sion site determining factors MinD and MinE have been shown to participate in positioning of the FtsZ ring in chloroplasts (9–11), but other factors involved in division have largely remained elusive. Our findings suggest that constriction and division of thylakoid membranes can be accomplished indepen- dently of envelope invagination. ARTEMIS seems to orchestrate these apparently independent division processes. Formation of Fig. 2. Subcellular localization and membrane association properties of tripolar chloroplasts and thylakoids curved to specific regions of ARTEMIS. (A) Immunoblot using ␣ArtB serum to probe mitochondrial (Mito), the inner membrane may be indicative of an improper and stromal (Stro), tylakoid membrane (Thyl), chloroplast outer envelope (OE), redundant placement of envelope division initiation sites or a and chloroplast inner envelope (IE) fractions resolved on SDS͞PAGE. Mito- failure to insert essential components of envelope biogenesis or chondria and chloroplast subfractions were isolated from pea (Pisum sativum) of chloroplast division into the envelope membrane via the as described (33, 34). The identity of various subfractions was tested by using YidC͞Oxa1p͞Alb3-like module of ARTEMIS. The apparently immunoblotting with antisera against: voltage-dependent anion-selective normal phenotype of thylakoid network and of envelope mem- channel (VDAC), large subunit of Rubisco (LSU), light harvesting complex branes suggests that ARTEMIS is not involved, at least not protein (LHCP), outer envelope protein of 21 kDa (OEP21), outer envelope protein of 16 kDa (OEP16), translocon at the inner envelope component of 110 directly, in general protein translocation into chloroplasts. It is, kDa (Tic110), and translocon at the inner envelope component of 55 kDa however, possible that ARTEMIS influences translocation of a (Tic55). (B) Immunogold labeling of leaf ultrathin sections with ␣ArtA. Gold specific subset of proteins that are necessary for chloroplast particles are highlighted with arrowheads. Env, envelope membranes; Stro, division or positioning of the organellar midpoint. stroma; Thyl, thylakoid membranes. (C) ARTEMIS is an integral inner envelope Consistent with its role in plastid division, ARTEMIS accu- protein. Purified inner envelopes were treated with 5 mM Hepes͞KOH, pH mulates in different plant organs (Fig. 3C). The highest concen- ͞ 7.6 1 M NaCl.0.5 M Na2CO3 or 4 M urea, as indicated and separated into tration is found in green tissues, although low but detectable soluble (S) and insoluble (P) protein fractions. ARTEMIS, Tic 110, and Tic 40 levels can be found in etiolated seedlings. Fully differentiated were determined by immunoblotting. roots and flowers, containing mostly and amylo- plasts, accumulate barely detectable levels of ARTEMIS protein (slightly higher accumulation in flowers could be a result of quence. This plant contained only wild-type-like chloroplasts but chloroplast contamination from the green tissue of the floral still contained the En-1 element, however, in a different region base). This indicates that ARTEMIS is relatively abundantly of the genome as tested by PCR and Southern hybridization. To expressed in organs containing actively dividing plastids, further substantiate these findings, we created transgenic Ara- whereas those organs with only few meristematic or dividing cells bidopsis plants harboring an antisense construct of art1. Electron accumulate only low amounts of ARTEMIS. microscopy of leaf ultrathin sections revealed plastids with Synechocystis ARTEMIS-Related Protein Is Involved in Cell Division. Chloroplasts have evolved from cyanobacterial ancestors, and it is reasonable to assume that certain mechanisms of cell division have been retained throughout evolution. However, only two (FtsZ and FtsI) of the nine E. coli cell division proteins identified so far have been detected in the Synechocystis PCC6803 genome (7). We have performed a search of the Synechocystis genome using the YidC͞Oxa1p͞Alb3-like domain of ARTEMIS as a query and have identified a locus, slr1471 (Fig. 1B), which encodes a putative plasma membrane protein of 384 residues. To verify the proposed localization, we have performed immunoblot analysis of various Synechocystis subfractions using the E. coli Fig. 3. Nucleoside triphosphate binding of ARTEMIS. (A) Fifty micrograms of antibody. A single immunoreactive band with apparent molec- inner envelope vesicles was solubilized with 1% TritonX-100, diluted 10 times ular mass of 43 kDa could be detected in the plasma membrane ͞ ͞ with binding buffer (20 mM Tris, pH 7.5 100 mM NaCl 5 mM MgCl2) and fraction (Fig. 5A), strongly indicating that slr1471 is indeed incubated for 20 min at 4°C with GTP-agarose resin. After washing with 40 located on the plasma membrane. To assess the function of the resin volumes of binding buffer, proteins were released with 10 mM ATP or slr1471 gene product, we created a ⌬1471 deletion mutant cell GTP. ARTEMIS was detected by immunoblotting. (B) Interaction of ARTEMIS line. Synechocysis ⌬1471 cells have a changed morphology and with [␣-32P]GTP. Chemical crosslinking was performed according to ref. 36. Protein–GTP complexes were immunoprecipitated with ␣ArtA antibody, re- frequently form tetrameric or even hexameric clusters evidently solved on SDS͞PAGE and detected by autoradiography. (C) Expression profil- arrested in late stages of division (Fig. 5B Lower). Cells also seem ing of ARTEMIS in different organs. Ten micrograms of total cellular proteins to initiate their fission unevenly, creating cells of irregular from various tissues (as indicated) was immunostained with ␣ArtA antiserum. shapes. As in chloroplasts, thylakoid membrane biogenesis was (Left) Positions of molecular weight standards. not affected. Measurements of photosynthetic activity indicated

11504 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.172032599 Fulgosi et al. Fig. 4. (A) Schematic representation of art1 gene in Arabidopsis mutant 7AR137. Position of En-1 element in the second exon is depicted. Exons are represented by black boxes; introns are indicated as lines. ATG, translation initiation codon; TAA, translation termination codon. (B) Ultrastructure of ARTEMIS mutant chloroplasts. Young dividing wild-type Arabidopsis chloroplast (WT), undivided chloroplasts of 7AR137 line (En-1), enlarged region of plastidal midpoint lacking envelope isthmus (Lower Left), triangular chloroplast of 5-day-old cotyledons (cot), and undivided chloroplast of antisense plants (Lower Right). Env, envelope membranes; Stro, stroma; Thyl, thylakoid membranes. (Bar ϭ 1 ␮m.) no significant differences between wild-type cells and ⌬1471 plemented by the YidC͞Oxa1p͞Alb3-like domain of Arabidopsis mutants (data not shown). In wild-type Synechocystis, only ARTEMIS, the C-terminal domain was fused to a 450-bp-long monomeric or dimeric cells undergoing normal division are promoter region of the sll0617 gene (31) to form a pVipp1art1 detectable. The morphological phenotype of cyanobacterial construct. We performed a rescue transformation of Synecho- ⌬1471 cells resembles those of undivided Arabidopsis chloro- cystis ⌬1471 by using pVipp1art1 plasmidal expression and found plasts. We conclude that altered morphology of ⌬1471 cells that cell division was restored, i.e., only monomeric and dimeric might be a result of an aberrant assembly of the cell division cells as in wild type were detected (Fig. 5B Upper Right). The machinery and not a general phenomenon caused by an im- YidC͞Oxa1p͞Alb3 module of ARTEMIS can thus complement proper membrane biogenesis. slr1471 deletion, suggesting that both proteins are evolutionarily and functionally related. ⌬ ARTEMIS Can Rescue 1471 Synechocystis Mutant. To determine The large NH2-terminal receptor-like domain of ARTEMIS, whether defects in the Synechocystis ⌬1471 line could be com- including the nucleoside triphosphate-binding site, has no sig-

Fig. 5. Subcellular localization of slr1471 and the ultrastructure of Synechocystis ⌬1471 cells. (A) Immunoblot analysis of outer membrane (OM), plasma membrane (PM), (Cyto), and thylakoid membrane (Thyl) fractions using antisera against the conserved domain of E. coli YidC (slr1471), Synechocystis porin protein SomA, a component of nitrate transporter NrtA, and a rod-linker protein of phycobilisomes Phye3A. (B Upper) overview image of wild-type

Synechocystis cells (WT), mutant cells forming cell clusters (⌬1471, indicated by arrowheads), and restored cell division of ⌬1471 cells rescued by pVipp1art1 PLANT BIOLOGY (Right). (Lower) Enlarged clusters of undivided ⌬1471 cells. (Bar ϭ 1 ␮m.)

Fulgosi et al. PNAS ͉ August 20, 2002 ͉ vol. 99 ͉ no. 17 ͉ 11505 nificant protein homologue in Synechocystis and has most likely ligand binding or homo-͞heterooligomerization. Addition of been fused with the YidC͞Oxa1p͞Alb3 domain on evolution of such a large regulatory domain onto a conserved YidC͞Oxa1p͞ chloroplasts as eukaryotic organelles. Interestingly, homologous Art core domain could have been evolutionarily necessary to sequences can be found in a number of plant transmembrane establish coordination between the host cell and the cyanobac- receptor protein kinases (Fig. 1B). These proteins are involved terial endosymbiont. in an array of cellular signaling events and can form homo- and heterooligomeric complexes with themselves or other receptors Conclusion (40). It is intriguing to speculate that the receptor domain of We propose that ARTEMIS evolved during endosymbiosis from ARTEMIS might be a protein module of eukaryotic origin to a single-domain single-function polypeptide involved in prokary- establish the nuclear control over organelle division, whereas the otic cell fission into a multidomain multifunctional polypeptide. YidC͞Oxa1p͞Art translocase domain assists in the integration Today, ARTEMIS most likely combines regulatory circuits and positioning of the division machinery into the inner enve- engaged between organelle and nucleus to control chloroplast lope. Several bacterial division proteins are transmembrane division with a protein translocase function for the integration of proteins and at least one of them, FtsQ, has been shown to membrane components into the inner envelope to enable chlo- interact with the YidC translocase (41). As in the case of receptor roplast division. kinases involved in development, the ligand(s) per- ceived by the receptor domain could be peptide(s) (40). It is also We thank B. Zeppenfeld and A. Daniel for excellent technical assistance. Antibody against Oxa1p was a kind gift of J. W. de Gier, Department of tempting to speculate that common ligands may orchestrate Biochemistry and Biophysics, the Arrhenius Laboratory for Natural plastid division with cell expansion and development. GTP most Sciences, Stockholm University, Stockholm, Sweden. The research was probably plays a regulatory role, as in the case of outer envelope supported by grants from the Deutsche Forschungsgemeinschaft, protein import receptor Toc34, or may provide a switch for Sonderforschungsbereich TR-1, and Fonds der Chemischen Industrie.

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11506 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.172032599 Fulgosi et al.