Cell and chloroplast 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) Chloroplasts are endosymbiotic organelles of cyanobacterial ori- cumulate large chloroplasts with similar morphology to MinD gin. It seems reasonable to assume that cell division and organelle 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 Bacillus subtilis, 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 protein 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 bacteria (13). Except the MIS antisense plants revealed chloroplasts arrested in the late already mentioned FtsZ homologues and the chloroplast MinC stages of division. Chloroplasts showed clearly separated and and MinE proteins, homologues of other bacterial cell division distinct multiple thylakoid systems, whereas the final organelle proteins have not been found in the Arabidopsis genome (3), fission remained unaccomplished. Inactivation of a cyanobacterial suggesting that chloroplast division involves different or addi- ͞ ͞ gene 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 plastid-dividing ring coordinated? Which com- division. ponents are involved in possible nuclear control of chloroplast division? ivision of higher plant 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 organisms 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 tubulin-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 genes 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 endosymbiont, 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 plastids of cells leads to a cell cycle 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 cytoskeleton (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 fission, 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 Genetics, Rudjer Bosˇkovic´ Institute, P.O. Box 1016, Hr-10000 Zagreb, Croatia. E-mail: PLANT BIOLOGY 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