Imke Lang

Cornelia Göbel

Andrea Porzel

Ingo Heilmann

Ivo Feussner

A Lipoxygenase with Linoleate Diol Synthase Activity from Nostoc sp. PCC 7120

Keywords: α-linolenic acid, AOS, allene oxide synthase, CP-HPLC, chiral phase-HPLC, diHODE, dihydroxy octadecadienoic acid, GC, gas chromatography, GLA, γ-linolenic acid, H(P)ETE, hydro Bifunctional enzyme, Cyanobacteria, Oxylipins, Prokaryotic lipoxygenase

Synopsis

The dioxygenation of polyunsaturated fatty acids in plants is mainly catalyzed by members of the lipoxygenase enzyme family. Lipoxygenase products may be further metabolized and are known as signalling substances in plant development and in responses to wounding and pathogen attack. In contrast to the situation in eukaryotes, information on the relevance of lipidperoxide metabolism in prokaryotic organisms is scarce. Therefore, we aimed to analyze lipoxygenases and oxylipin patterns of cyanobacterial origin. A search of the genomic sequence of the cyanobacterium, Nostoc sp. PCC 7120, suggested an open reading frame encoding a putative lipoxygenase named NspLOX that harboured an amino-terminal extension. Individual analysis of recombinant carboxy-terminal domain revealed enzymatic activity as a linoleate (9R)-lipoxygenase. Analysis of the full-length NspLOX-protein however, revealed linoleate diol synthase activity, generating (10E,12E)-9,14-dihydroxy-10,12-octadecadienoic acid as the main product from linoleic acid and (10E,12E,14E)-9,16dihydroxy-10,12,14-octadecatrienoic acid as the main product from α-linolenic acid substrates, respectively, with α-linolenic acid as preferred substrate. The enzyme exhibited a broad pH optimum between pH 7 and 10. Soluble extracts of Nostoc sp. contain more 9lipoxygenase-derived hydroperoxides in sonified than in non-sonified cells, but products of full-length NspLOX were not detectable under the conditions used. As no other lipoxygenaselike sequence was identified in the genome of Nostoc sp. PCC 7120, the results presented suggest that (9R)-lipoxygenase-derived oxylipins may represent the endogenous products of NspLOX. Based on the biochemical data of NspLOX we suggest that this bifunctional enzyme may represent a more ancient way to control the intracellular amount of oxylipins in this cyanobacterium.

Short title Linoleate Diol Synthase from Nostoc sp.

INTRODUCTION

Eukaryotic algae have developed mechanisms involving lipidperoxidation by which they defend themselves against abiotic and biotic stresses [START_REF] Potin | Biotic interactions of marine algae[END_REF]. One of these mechanisms relates to the induction of the so-called lipoxygenase (LOX) pathway [START_REF] Potin | Biotic interactions of marine algae[END_REF][START_REF] Pohnert | Diatom/copepod interactions in plankton: The indirect chemical defense of unicellular algae[END_REF]. This metabolic pathway is well-characterized from flowering plants and involved in the production of hormones and defensive metabolites [START_REF] Blée | Impact of phyto-oxylipins in plant defense[END_REF]. The products synthesized by the LOX-pathway are collectively named oxylipins [START_REF] Hamberg | Oxylipin pathway to jasmonates: biochemistry and biological significance[END_REF]. The first committed step of the pathway is the LOX reaction and the resulting oxidation products serve as substrates for the synthesis of a large variety of oxylipins that are synthesized in plants by at least six different enzyme families [START_REF] Liavonchanka | Lipoxygenases: Occurrence, functions and catalysis[END_REF]. LOX enzymes represent a family of non-heme iron-containing fatty acid dioxygenases, which catalyze the stereospecific insertion of molecular oxygen into polyunsaturated fatty acids (PUFAs), leading to the formation of hydroperoxy fatty acids [START_REF] Brash | Lipoxygenases: Occurrence, functions, catalysis, and acquisition of substrate[END_REF]. LOXs are widely distributed in nature and occur ubiquitously in plants and mammals [7,[START_REF] Kühn | The diversity of the lipoxygenase family -Many sequence data but little information on biological significance[END_REF]. In recent years successful cloning of genes encoding LOXs from coral, moss, and bacteria, has been reported and expands our current knowledge on the occurrence of this class of enzymes [START_REF] Brash | Purification and molecular cloning of an 8R-lipoxygenase from the coral Plexaura homomalla reveal the related primary structures of Rand S-lipoxygenases[END_REF][START_REF] Koeduka | Cloning of lipoxygenase genes from a cyanobacterium, Nostoc punctiforme, and its expression in Eschelichia coli[END_REF][START_REF] Lang | Oxylipin formation in Nostoc punctiforme (PCC73102)[END_REF][START_REF] Senger | A multifunctional lipoxygenase with fatty acid hydroperoxide cleaving activity from the moss Physcomitrella patens[END_REF][START_REF] Vidal-Mas | Cloning and expression of a lipoxygenase from Pseudomonas aeruginosa 42A2[END_REF]. However knowledge on the biological function of the enzymes in prokaryotes is still scarce.

In plants, LOXs are classified with respect to their positional specificity of fatty acid oxygenation against linoleic acid (LA). LA is oxygenated either at carbon atom 9 (9-LOX) or at C-13 (13-LOX) of the hydrocarbon backbone, which leads to the formation of 9hydroperoxy-and 13-hydroperoxy derivatives of LA (9-and 13-HPODE), respectively. In mammals LOXs are classified according to their positional specificity of arachidonic acid (AA) oxygenation. This PUFA can be oxygenated by LOXs at six different positions: C-5 (5-LOX), C-8 (8-LOX), C-9 (9-LOX), C-11 (11-LOX), C-12 (12-LOX) and C-15 (15-LOX), respectively [START_REF] Kühn | Structural biology of mammalian lipoxygenases: Enzymatic consequences of targeted alterations of the protein structure[END_REF]. All of the LOX-derived fatty acid hydroperoxides are formed with high chiral purity [START_REF] Brash | Lipoxygenases: Occurrence, functions, catalysis, and acquisition of substrate[END_REF]. The prototypical LOXs produce fatty acid hydroperoxides in S configuration, however, a few enzymes have been described that produce R-configurated products [START_REF] Brash | Purification and molecular cloning of an 8R-lipoxygenase from the coral Plexaura homomalla reveal the related primary structures of Rand S-lipoxygenases[END_REF]15,16].

Interestingly, in the cyanobacterium Anabaena flos-aquae f. flos aquae two different hydroxy fatty acids in R-configuration have been isolated, (9R)-HPODE and (9R)-hydroperoxy linolenic acid ((9R)-HPOTE), suggesting cyanobacteria may contain as well a (9R)-LOX [17].

As the corresponding cyanobacterial enzyme has not been identified in Anabaena, a search of the accessible genomic sequence of the related filamentous cyanobacterium, Nostoc sp. PCC 7120, was conducted in order to identify open reading frames with similarity to known LOX-sequences. A single sequence was identified and named NspLOX, which was found to encode a LOX harbouring a large amino-terminal extension and exhibits highest similarity to a peroxidase-LOX fusion-protein from the coral Plexaura homomalla [START_REF] Koljak | Identification of a naturally occurring peroxidase-lipoxygenase fusion protein[END_REF]. The previously reported coral enzyme consists of two distinct domains that have been demonstrated individually to exhibit different catalytic activities, the amino-terminal domain as an allene oxide synthase (AOS), and the carboxy-terminal domain as an (8R)-LOX [START_REF] Boutaud | Purification and catalytic activities of the two domains of the allene oxide synthase-lipoxygenase fusion protein of the coral Plexaura homomalla[END_REF]. In case of the coral fusion-protein the LOX domain converted AA to its corresponding (8R)hydroperoxide ((8R)-HPETE), while the AOS-domain metabolized (8R)-HPETE to an unstable allene oxide which hydrolyzes spontaneously into α-and γ-ketols and cyclopentanone derivatives. The LOX protein from N. sp. PCC 7120 has a domain structure similar to that of its coral equivalent, suggesting a common origin in evolution.

EXPERIMENTAL Algae Cultures

For the characterisation of NspLOX Nostoc sp. PCC 7120 (obtained as SAG 25.82 from the Sammlung für Algenkulturen in Goettingen (SAG)) was cultivated in liquid BG 11 medium according to http://www.epsag.uni-goettingen.de/html/sag.html at 20 °C in 1 l conical flasks shaking at 120 rpm. For the analysis of oxylipins in vivo two additional strains, PCC 7120 P and PCC 7120 A, were obtained from Prof. Peter Wolk, Michigan State University, East Lansing, MI, USA and Prof. James Golden, Texas A&M University, College Station, TX, USA, respectively and cultivated under the identical conditions. After 3 weeks of cultivation, cells were harvested by centrifugation at 3220 x g for 10 min and pellets were stored at -20 °C.

Isolation and Expression of Separate Domains and Entire Protein of NspLOX

Genomic DNA was isolated from approximately 50 mg of frozen cell material from N. sp. SAG 25.82 using the NucleoSpin ® Plant kit (Macherey-Nagel) according to manufacturer's instructions. The gene fragment encoding the LOX-domain was isolated by PCR using the primer-combination LOXFNdeI (5´-ACATATGCAGTATTTGTATGGAAGTAAAGG-3´)/LOXRBglII (5´-AAGATCTCTAAATGTTGATACTCATCATGAG-3´). The DNAsequence encoding the putative peroxidase domain was isolated by PCR using the primercombination KatFNdeI (5´-ACATATGGATTTGAATACTTACTTGAAG-3´)/KatRBglII (5´-AAGATCTTTTTCCAGGCAAATCATCCTTAC-3´). Amplicons were cloned into pGEM-T (Promega) and moved as NdeI/BglII fragments into the expression vector pET15b, introducing an amino-terminal His-Tag (Novagene). The constructs were transformed into E. coli Rosetta(DE3) cells. . The entire gene encoding NspLOX was amplified using the primercombination KatFNdeI (5´-ACATATGGATTTGAATACTTACTTGAAG-3´)/LOXRBglII (5´-AAGATCTCTAAATGTTGATACTCATCATGAG-3´). The fragment initially obtained from SAG 25.82 contained an insertion of 1300 bp within the amino-terminal domain, which was removed by PCR mutagenesis as follows. The DNA-stretches encoding the individual amino-terminal and carboxy-terminal domains were amplified with chimeric primers flanking the insertion site: KatFNdeI (5´-ACATATGGATTTGAATACTTACTTGAAG-3´)/ChimR (5´-GTAAACGAATCTGGATTGCGTAAGGCTGCCTCCAC-3´) for exon 1 and ChimF (5´ GTGGAGGCAGCCTTACGCAATCCAGATTCGTTTAC-3´)/LOXRBglII (5´-AAGATCTCTAAATGTTGATACTCATCATGAG-3´) for exon 2. DNA-fragments were then combined for the fusion reaction. After 12 PCR cycles an aliquot (1:200) of the fused fragments was used as a template for amplification of the fusion product with primers KatFNdeI and LOXRBglII. The obtained NspLOX fragment was purified using the DNA and Gel Band Purification Kit (GE Healthcare) according to manufacturer's instructions.

Subsequently, the fragment was cloned directly into the expression system pCR ® T7/TOPO ® NT vector (Invitrogen), introducing an amino-terminal His-Tag, and this construct was transformed into E. coli BL21(DE3) cells.

Expression cultures were grown in 30 ml LB medium at 37 °C until they reached OD 600 0.6 [START_REF] Bertani | Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli[END_REF]. Then 50 µl of 1 M IPTG was added and the cultures were further incubated at 16 °C over night. E. coli expression cultures were harvested by centrifugation at 3220 x g at 4 °C for 20 min.

Activity Assays

For activity assays the pellets of expression cultures were resuspended in 2 ml lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 10% (w/w) Glycerol, 0.1% (v/v) Tween20). Cells were disrupted by 2 pulses of 20 s of sonification on ice and then centrifuged at 3220 x g for 10 min at 4 C. A 500 µl aliquot of the supernatant was added to 1.5 ml of 50 mM Tris buffer, pH 8.0. Reactions were started by adding 250 µg of fatty acids, and were allowed to proceed for 30 min on ice. Carboxy-terminal portion: Hydroperoxides formed were reduced to their corresponding hydroxides with 2 ml of 50 mM SnCl 2 , dissolved in methanol. After acidification to pH 3.0 with glacial acetic acid, fatty acids were extracted and analyzed as described [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF]. Amino-terminal portion: The activity was either tested with 612 kBq of radiolabelled [1-14 C]-(9S)-HPODE or with (9R)-HPODE. The reaction was stopped by adding 2 ml diethyl ether and 1 ml water. After acidification to pH 3.0 with glacial acetic acid the samples were centrifuged (3220 x g, 5 min, 4 °C), the upper phase was used for further analysis and the products were analyzed as described [START_REF] Lang | Oxylipin formation in Nostoc punctiforme (PCC73102)[END_REF]. NspLOX: The reaction and analysis conditions were as for the LOX domain.

For the determination of the pH optimum of the carboxy-terminal portion and the fulllength protein, pellets of expression cultures were used. The activity was determined by monitoring LA dependent O 2 consumption with an oxygen electrode (Rank Borther, Bottisham). The reaction mixture contained 900 µl of either 50 mM Tris/HCl or 50 mM sodium borate buffer ranging from pH 4.0 to 12.0, and 100 µl of supernatant in a total volume of 1 ml. Reactions were started by adding 10 µl of 10 mM sodium linoleate.

NspLOX Activity Assay in the

O

In the first case 1.5 ml of 50 mM Tris HCl, pH 8.0, were degassed and buffers were enriched with 18 O 2 (Campro Scientific) by continuous gas flow through the liquid for 5 min. The supernatant of an expression culture was added to an equal volume of 18 O 2 -saturated buffer.

The mixture was incubated for 30 min on ice with 250 µg LA as substrate. During incubation the reaction mix was further enriched with 18 O 2 by continuous gas flow through the liquid (~10 ml). The reaction was stopped by adding 2 ml diethyl ether, and LOX-reaction products were analysed as described [START_REF] Lang | Oxylipin formation in Nostoc punctiforme (PCC73102)[END_REF].

In the second case the pellet of an expression culture was resuspended in 500 µl H 2 18 O (Chemotrade) and the cells were disrupted via sonification as described. The crude extract was centrifuged at 3220 x g for 5 min at 4 °C and the supernatant transferred into a 15 ml glass tube. The reaction was started by adding 250 µg LA and allowed to proceed for 30 min on ice. The reaction was stopped by adding 2 ml diethyl ether, and LOX-reaction products were analysed as described [START_REF] Lang | Oxylipin formation in Nostoc punctiforme (PCC73102)[END_REF].

Determination of Oxylipins Formed under In Vivo Conditions

Three 100 ml cultures of each cyanobacterial strain (PCC 7120, PCC 7120 A, and PCC 7120 P) were harvested and the pellets each resuspended in 1 ml of 50 mM Tris HCl, pH 8.0.

Two samples of each strain were stressed by sonification for 40 s on ice (about 20% of cells were lysed by this treatment). Then to one stressed sample of each strain either 250 µg LA or 5.5 MBq of radio-labelled [1-14 C]-LA was added. Samples were incubated for 30 min at room temperature, and reaction products were extracted and analysed as described below.

Analysis of Fatty Acids, Hydroxy and Hydroperoxy Fatty Acids

For the analysis of the fatty acid profile of N. sp. the lyophilised algal material was extracted by adding 405 µl of methanol/toluol 2:1 (v/v) followed by homogenising the cells for 30 s. An internal standard (10 µg of tripentadecanoate) was added. Transesterification of lipid bound fatty acids to their corresponding methyl esters was accomplished by adding 150 µl sodium methoxide. After 20 min shaking at room temperature the fatty acid methyl esters were extracted twice with 500 µl n-hexane and 500 µl 1 M NaCl. The hexane phases were transferred into a 1.5 ml tube, dried under streaming nitrogen, redissolved in 10 µl acetonitrile and analysed as described [START_REF] Lang | Oxylipin formation in Nostoc punctiforme (PCC73102)[END_REF]. Dihydroxy fatty acids were separated from monohydroxy FAs by a RP-HPLC gradient with a combination of the following solvents: A: 50:50:0.1 acetonitrile:water:acetic acid (v/v/v) and B: 100:0.1 acetonitrile:acetic acid (v/v). The gradient elution profile was as follows starting with a flow rate of 0.18 ml/min: 0-10 min, 100% A; 10-20 min from 100% A to 0% A and 100% B; 20-22 min flow rate increasing from 0.18 ml/min to 0.36 ml/min; 22-27 min 100% B; 27-32 min from 100% B to 100% A, 32 min-35 min 100% A. Oxylipin analysis was performed as described [START_REF] Lang | Oxylipin formation in Nostoc punctiforme (PCC73102)[END_REF].

Mass-Spectrometric Identification of Compounds

Reaction products were further analyzed using a MS Surveyor HPLC system equipped with a photo diode array detector and coupled to a LCQ Advantage electrospray ionisation ion trap mass spectrometer (Thermo Finnigan). The NspLOX protein products were separated by RP-HPLC (EC 250/2 Nucleodure 100-5 C18ec column, 250 x 2.1 mm, 5 µm particle size, Macherey-Nagel). Solvents and also the gradient were the same as above. The mass spectrometer was operated in negative mode with the source voltage set to 4 kV and a capillary voltage of 27 V of a temperature of 300 °C. In full scan mode, scans were collected between m/z values of 50 and 400, according to [START_REF] Hughes | Allene oxide synthase from Arabidopsis thaliana (CYP74A1) exhibits dual specificity that is regulated by monomer-micelle association[END_REF]. To gain more structural information mono-and dihydroxy fatty acids prepared by HPLC were methylated with diazomethane (Sigma) and silylated with bis-(trimethylsilyl)trifluoroacetamide (Pierce) and analyzed by GC/MS as described [START_REF] Hoffmann | A small membrane-peripheral region close to the active center determines regioselectivity of membrane-bound fatty acid desaturases from Aspergillus nidulans[END_REF].

Structural Analysis by NMR

The 1D and 2D NMR spectra were recorded on a Varian Inova 500 spectrometer at 499.799 MHz

( 1 H) using CDCl 3 as deuterated solvent. 1 H and 13 C chemical shifts are referenced to internal TMS (δ 1 H 0 ppm), and internal CDCl 3 (δ 13 C 77.0 ppm), respectively.

Western-Blot Analysis and Immunodetection

In order to detect recombinant proteins in bacterial extracts proteins were expressed with amino-terminal His-Tags. Proteins were separated by SDS-PAGE, transferred from the gel to a nitrocellulose membrane and as primary antiserum an anti-tetra-His (Qiagen) was used.

Phylogenetic analysis

Phylogenetic tree analysis was performed on deduced amino acid sequences of selected LOXs and NspLOX using phylip 3.5 (Department of Genome Sciences, University of Washington).

RESULTS

Isolation of NspLOX Protein and Its Domains

Database searches and alignments of available LOX amino acid sequences revealed a sequence harbouring two putative enzymatic domains (Acc. no. NP_478445 in the published genome sequence of the cyanobacterium N. sp. PCC 7120). The deduced open reading frame was termed NspLOX. The amino acid sequence of NspLOX exhibited an identity of 34% to an AOS-LOX fusion-protein from the coral Plexaura homomalla (Acc. no. AAC47743) [START_REF] Koljak | Identification of a naturally occurring peroxidase-lipoxygenase fusion protein[END_REF].

In addition, amino acid sequence comparisons were performed individually for each portion of the protein, revealing 40% sequence identity of the amino-terminal domain to a protein with unknown function from O. sativa (Acc. no. XP 472850). With 16% identity, the LOXactive site domain located at the carboxy-terminal part showed only little similarity to the arachidonate (8R)-LOX domain of the fusion-protein from P. homomalla [START_REF] Koljak | Identification of a naturally occurring peroxidase-lipoxygenase fusion protein[END_REF].

Phylogenetic analysis of the amino acid sequence of the full-length protein with other known LOX-sequences showed that NspLOX could be grouped with the family of LOXs and was closely related to other sequences identified in prokaryotic organisms (Figure 1). The prokaryotic branch was subdivided into two subgroups, with NspLOX and a hypothetical protein from Myxococcus xanthus forming a separate clade. It is interesting to note that the prokaryotic enzymes are more closely related to LOXs from mammals and coral than to LOXs from plants.

An alignment of the deduced amino acid sequence of the amino-terminal portion revealed high similarity to peroxidase-like sequences from O. sativa, the coral P. homomalla [START_REF] Koljak | Identification of a naturally occurring peroxidase-lipoxygenase fusion protein[END_REF] and the bacterium Sulfitobacter sp. EE-36. All sequences share conserved amino acids implicated in forming the active site of a peroxidase, either in heme binding or as catalytic residues. In the AOS domain of the coral fusion-protein, three residues, Thr-66, His-67 and Asn-137, are important for coordinating the distal face of the heme group; the heme's proximal-side At the position first described by Borngräber and co-workers, a phenylalanine (Phe-163) is found instead of an alanine or isoleucine in both plant LOXs (LOX2 At2 and PpLOX1;

Figure 2, bo). These amino acids together differ remarkably from the reported determinants for positional specificity of plant LOXs [START_REF] Hornung | Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis[END_REF], precluding accurate prediction of positional specificity of the LOX-domain. In addition, an arginine residue reported to determine inverse substrate orientation in plant LOXs is missing as well in NspLOX (Figure 2, ho) [START_REF] Hornung | Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis[END_REF].

However, the amino acid residue determining the stereospecificity of LOXs (Coffa site, Figure 2, cof) is an alanine residue, suggesting that this enzyme is an S-specific LOX [START_REF] Coffa | A single active site residue directs oxygenation stereospecificity in lipoxygenases: Stereocontrol is linked to the position of oxygenation[END_REF].

To isolate the NspLOX protein and its putative domains, gene specific primers were used to amplify the full-length protein and the separate domains from genomic DNA of N. sp. PCC The activity of the carboxy-terminal portion was tested with free fatty acid substrates and the methyl ester of LA. The enzyme showed activity with all free substrates and converted LA, αlinolenic acid (ALA) and γ-linolenic acid (GLA) predominantly to the corresponding 9hydroperoxides. The corresponding ratios of the different regioisomers formed are shown in Figure 3. AA was mainly converted to 11-hydro(pero)xy-5,8,12,14-eicosatetraenoic acid (11-H(P)ETE). With LA methyl ester no activity was detectable. CP-HPLC revealed that the main enantiomer of the hydroxide formed was in R configuration (Figure 3, left inset). Therefore, the carboxy-terminal portion of NspLOX was identified as a linoleate (9R)-LOX, as the analysis of endogenous fatty acids of N. sp. showed no C20 fatty acid which could be a potential substrate for the carboxy-terminal portion of NspLOX (Table 1).

The pH-optimum of the LOX-domain was investigated by monitoring the oxygen consumption during substrate conversion. The highest activity of the LOX-domain was between pH 7.0 and pH 10.0, and the activity decreased dramatically above pH 10.5.

Furthermore, we aimed to determine the preferred substrates of the LOX domain by incubating the supernatant of E. coli cell lysates with a mixture of LA, ALA, GLA and AA.

ALA was the preferred substrate of the LOX-domain (70% substrate conversion), whereas GLA, AA and LA were converted to a lesser extent of about 54%, 46%, and 42%, respectively.

The Amino-terminal Portion of NspLOX Does Not Exhibit Detectable Enzymatic

Activity on its Own.

To test whether the amino-terminal portion of NspLOX had enzymatic activity on its own the supernatants of lysates of E. coli cells expressing the fragment were incubated with one product of the LOX-reaction, (9R)-HPODE and as a negative control with (13S)-HPODE. In addition the reduced hydroperoxides (9R)-HODE and (13S)-HODE were tested. With all tested substrates, including [1-14 C]-9-HPODE, no activity was detectable. However, the protein was formed as verified by Western Blot analysis (data not shown).

Production of Dihydroxylated Fatty Acids by NspLOX.

Next we aimed at characterizing the activity of the full-length NspLOX protein. NspLOX was tested by incubation with free fatty acids (LA, ALA, GLA and AA) as substrates and subsequent product analysis by reverse phase HPLC, HPLC/MS and GC/MS. HPLC-analysis showed, that conversion of LA resulted in one main product peak and several smaller peaks (Figure 4). Besides the intermediate product of the LOX domain, (9R)-HPODE eluting at about 24 min and absorbing at 234 nm, all additional newly formed substances were more polar than the monohydroperoxide of the LOX reaction and had absorption maxima at 232 nm (Figure 4), whereby the peaks eluting later than 24 min were present in the empty vector control as well. By HPLC/MS-analysis the major three products were identified as dihydroxide isomers of the fatty acid substrates (Table 2). Additional GC/MS analysis of silylated product derivatives allowed the identification of the positions of the hydroxyl groups and double bonds, and the main product of LA conversion was identified as 9,14-dihydroxy-10,12-octadecadienoic acid by GC/MS and LC/MS analysis (9,14-diHODE; Figure 5A, Table 2). The mass spectrum (Figure 5A) was characterized by diagnostic ions at m/z 439 However, from the UV absorption at 232 nm an E,E configuration was assigned. In addition the double bond configuration of 9,14-diHODE was confirmed via NMR-analysis (Table 3).

1 H and 13 C chemical shifts are in good agreement with those of (7E,9E)-hexadeca-7,9-diene-6,11-diol but distinct from those of (7Z,9Z)-hexadeca-7,9-diene-6,11-diol [START_REF] Tolstikov | A convenient synthesis of symmetrically functionalized 1,3-dienes by palladium(II)-catalyzed homocoupling of 1-alkenylstannanes[END_REF]. The two other main products were identified as (11E,13E)-9,10-dihydroxy-11,13-octadecadienoic acid given in Table 2. Three additional minor products were not identified so far (Figure 4, marked by stars).

For the conversion of ALA, GLA and AA we obtained an equivalent HPLC pattern: one main product and several side products. The main products were (10E,12E,14E)-9,16dihydroxy-10,12,14-octadecatrienoic acid (9,16-diHOTE) for ALA, (6Z,10E,12E)-9,14dihydroxy-6,10,12-octadecatrienoic acid (9,14-diHOTE) for GLA and (5Z,8Z,12E,14E)-11,16-dihydroxy-5,8,12,14-eicosatetraenoic acid [START_REF] Lang | Oxylipin formation in Nostoc punctiforme (PCC73102)[END_REF] for AA. The most prominent ions in the mass spectra of 9,16-diHOTE were as follows in m/z and ion structure: Thus, the NspLOX seemed to catalyze the conversion of the fatty acid substrate in a two step reaction: First the carboxy-terminal portion (LOX domain) converted LA to (9R)-HPODE in a reaction depending on molecular oxygen. In a second step, (9R)-HPODE or an unstable metabolite that derived there from was metabolized to (10E,12E)-9,14-diHODE by the full-length protein in a reaction introducing an oxygen atom not derived from the peroxy group of (9R)-HPODE but from water.

Analysis of Endogenous Oxylipins in N. sp. PCC 7120.

So far, we have described the in vitro characterization of NspLOX isolated from the N. sp.

strain PCC 7120. Analysis of the isolated sequences showed, that it harbours an insertion in form of a transposon in the NspLOX sequence. In order to compare the dependency of cyanobacterial oxylipin formation on the NspLOX protein, two additional N. sp. strains, PCC 7120 A and P collectively named wildtype (wt) strains, were obtained, which do not harbour a disruptive insertion in the NspLOX-gene. The three strains showed the same growth behaviour and analysis of lipid as well as fatty acid patterns revealed that there were no obvious differences between cells containing a functional and dysfunctional gene for NspLOX.

Considering that the insertion in the NspLOX gene presumably results in the translation of an inactive NspLOX enzyme, oxylipin metabolism of the cyanobacterial strains harbouring the insertion in NspLOX may be compromised. The potential differences in oxylipin metabolism were exploited to compare the "wildtype-allele" (wt) strains PCC 7120 A and P with the "mutant-allele" (mt) strain PCC 7120 with respect to their oxylipin profiles. As it is known from previous work that the LOX-pathway is induced upon ultrasonification in algae and mosses [START_REF] Lang | Oxylipin formation in Nostoc punctiforme (PCC73102)[END_REF][START_REF] Pohnert | Phospholipase A 2 activity triggers the wound-activated chemical defense in the diatom Thalassiosira rotula[END_REF][31][START_REF] Stumpe | Biosynthesis of C9-aldehydes in the moss Physcomitrella patens[END_REF], cyanobacterial cultures were subjected to ultrasonification, and the effects on oxylipin metabolism were monitored.

In all samples free hydro(pero)xy dienoic fatty acids were measured. Although we detected C16-and C18-derived PUFAs in wt and mt-strains (Table 1), only C18-PUFAderived hydroxides were detected in untreated and treated cells, and no C16-PUFA-derived oxylipins were present. In addition, traces of fatty acid hydroperoxides were detected. The analysis of free oxylipins in the wt strains showed, that the most abundant hydroxy fatty acids were 9-HODE and 9-HOTE that derived from 9-LOX activity, since both substances showed a clear preponderance of the R-enantiomer (0.3-1.0 nmol/g f.w.; Figure 6A, grey bars, shown for PCC 7120 P; inset shows the CP-HPLC analysis of 9-HODE). The amount of the hydroxy fatty acids increased upon sonification, in particular that of 9-hydroxy fatty acids, which showed an almost ten-fold increase (9.1-11.7 nmol/g f.w., Figure 6A, grey vs. black bars).

The amount of the other hydroxy fatty acids (13-HODE, 13HOTE, 12-HOTE and 16-HOTE) increased only slightly to about 0.8 nmol/g f.w.. In comparison, the mt strain contained similar amounts of hydroxy fatty acids between 0.1 and 0.5 nmol/g f.w. (Figure 6B), and there was only a slight preference for the 9-hydroxy fatty acid isomers. In addition, only a threefold induction of oxylipin formation was observed upon sonification with no preference for a hydroxy fatty acid isomer (Figure 6B). These data indicated an induction of (9R)-LOX activity in the wt strains upon sonification and the absence of LOX activity in the mt strain, most likely due to the insertion of a disruptive transposase insert into the NspLOX-gene.

Interestingly, neither in wt nor in mt strains any dihydroxylated fatty acids products of NspLOX were detected under the conditions used, contrasting with the results from the in vitro tests with the recombinant enzyme.

Since we failed to detect any dihydroxy fatty acids that may derive from the activity of NspLOX we aimed to measure extractable enzyme activity that may derive from NspLOX.

Therefore we analyzed cell extracts obtained from sonified and non sonified mt and wt strains either after incubation with LA or [1-14 C]-labelled LA. In the untreated samples, formation of neither monohydroxylated nor dihydroxylated fatty acids was detected. However, stressinduced formation of (9R)-HODE was found in wt strains. Again, neither formation of 9,14-diHODE nor that of any other dihydroxy fatty acid was detected. The results for the two wt strains were similar, whereas for the mt-strain no oxylipins were found.

DISCUSSION

The NspLOX protein identified in the present study is a candidate for a cyanobacterial enzyme generating (9R)-LOX products with a possible function in stress responses of cyanobacteria. Lipidperoxidation in the better-characterized flowering plants is mainly a controlled process catalyzed by enzymes of the LOX enzyme family [START_REF] Liavonchanka | Lipoxygenases: Occurrence, functions and catalysis[END_REF], and products originating from the LOX reaction can be further metabolized in the LOX pathway. Plant products of the LOX-pathway have demonstrated roles as signalling substances in plant development or during plant responses to wounding or pathogen attack [33,[START_REF] Wasternack | Jasmonates: An update on biosynthesis, signal transduction and action in plant stress response, growth and development[END_REF].

The data presented here support the presence of enzymatic oxylipin production in prokaryotes, which suggests an early advent of oxylipin signalling in evolution, however, clear differences to plant oxylipin metabolism are also obvious. While LOXs can be ubiquitously found among higher plant species, our analysis shows that yet only two (Figure 1). N. punctiforme strain PCC 73102 was first analyzed as the most promising candidate, since its genome harbours two genes encoding different LOX enzymes being more related to plant LOXs than the third one PCC 7120, which is described in this report.

Recently, the first two candidates were positively identified as linoleate 13-LOXs [START_REF] Koeduka | Cloning of lipoxygenase genes from a cyanobacterium, Nostoc punctiforme, and its expression in Eschelichia coli[END_REF][START_REF] Lang | Oxylipin formation in Nostoc punctiforme (PCC73102)[END_REF] and it is tempting to assume that these enzymes may resemble the evolutionary precursors of plastidial 13-LOXs found in plants [START_REF] Liavonchanka | Lipoxygenases: Occurrence, functions and catalysis[END_REF].

In this study we analyzed a third candidate representing a LOX protein with a large amino-terminal extension from the cyanobacterium N. sp. PCC 7120, termed NspLOX.

Amino acid sequence comparisons indicate substantial similarity between NspLOX and the AOS-LOX fusion-protein from the coral P. homomalla [START_REF] Koljak | Identification of a naturally occurring peroxidase-lipoxygenase fusion protein[END_REF]. The coral enzyme converts AA to (8R)-HPETE by its LOX domain, which is then further metabolized by the peroxidase domain of the same protein to an unstable allene oxide which immediately hydrolyzes to the corresponding α-ketols [START_REF] Koljak | Identification of a naturally occurring peroxidase-lipoxygenase fusion protein[END_REF]. Despite of a comparable protein domain structure we could not detect fatty acid-derived ketols as products of NspLOX, and observed instead formation of fatty acid dihydroxides (Figs. 4 and5).

Diagnostic differences or similarities within the structure of a LOX are mainly found around the active site residues of the protein [START_REF] Liavonchanka | Lipoxygenases: Occurrence, functions and catalysis[END_REF][START_REF] Kühn | Structural biology of mammalian lipoxygenases: Enzymatic consequences of targeted alterations of the protein structure[END_REF]. The iron binding site of plant LOXs is formed by a central histidine-rich region that is conserved in NspLOX [START_REF] Siedow | Plant lipoxygenase -structure and function[END_REF], with the exception of a long carboxy-terminal deletion in its sequence. Due to the deletion the fifth ligand does not align properly to the other sequences, but is most likely represented by Ile-429 as in other plant LOXs (Figure 2). Since structural modelling is not possible due to low sequence similarities to the available data sets of LOX1 and LOX3 from soybean [START_REF] Boyington | The three-dimensional structure of an arachidonic acid 15-lipoxygenase[END_REF][START_REF] Skrzypczak-Jankun | Structure of soybean lipoxygenase L3 and a comparison with its L1 isoenzyme[END_REF] and the rabbit reticulocyte 15-LOX [38], it remains open whether Ile-429 has access to the active site of the LOX. Additional determinants conserved in evolution between NspLOX and other LOXs from plants and mammals [START_REF] Sloane | A primary determinant for lipoxygenase positional specificity[END_REF][START_REF] Hornung | Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis[END_REF][START_REF] Borngräber | Phenylalanine 353 is a primary determinant for the positional specificity of mammalian 15-lipoxygenases[END_REF] have been found to be involved in the substrate-and regiospecificity of LOXs. The amino acid residues determining the stereospecificity of all LOXs characterized so far (Coffa site) suggested for the NspLOX to be an S-specific LOX [START_REF] Coffa | A single active site residue directs oxygenation stereospecificity in lipoxygenases: Stereocontrol is linked to the position of oxygenation[END_REF]. In contrast to this prediction, we were able to demonstrate that NspLOX acts as a (9R)-LOX (Figure 3 and6), to our knowledge representing the first LOX deviating in specificity from that indicated by the Coffa site. Thus, our results indicate that either more than one amino acid residue in the active site may be involved in determining the stereospecificity of LOXs or that in this case an amino acid with a larger side chain would lead to (9S)-LOX activity. Moreover, our analysis on substrate specificity of NspLOX indicates that the fusionprotein "counts" from the methyl end of the substrate to insert the first oxygen molecule.

In addition to the carboxy-terminal LOX domain the NspLOX protein contained an amino-terminal extension with similarity to peroxidase enzymes and especially resembling catalases. Catalases are specialized peroxidases that metabolize hydrogen peroxide.

Indications to possible functions of the amino-terminal part come from the investigation of the related coral AOS-LOX fusion-protein from the coral P. homomalla [START_REF] Koljak | Identification of a naturally occurring peroxidase-lipoxygenase fusion protein[END_REF]. In the peroxidase domain of NspLOX a number of highly conserved residues at the distal face of the heme group are characteristic for peroxidases (Thr-40, His-41 and Asn-118) and may be related to enzymatic function although we failed to show the amino-terminal part of the enzyme is active on its own.

NspLOX is localized on the Gamma plasmid of the bacterium and, thus, it is tempting to assume that it was taken up by horizontal gene transfer from other bacteria. The similar gene structure of PhAOS-LOX and NspLOX and the notion, that many corals either live in symbiosis with algae so called zooxanthellae or are parasited by cyanobacteria may suggest a common algal origin of such enzymes. However, since only a restricted search in published genomes of bacteria and microalgae is currently possible, considerations about their origin must remain speculative at this point. However, the positive analysis of endogenous oxylipins Surprisingly, neither the products derived from the linoleate diol synthase activity of NspLOX nor those of additional enzymes metabolizing the 9-LOX-derived fatty acid hydroperoxides, as known from the so-called LOX pathway in higher plants, were found so far (Figure 6) and, thus, such enzymes seem to be absent from N. sp. PCC 7120. Besides the gene encoding NspLOX, no other open reading frames were found in the genome of N. sp.

PCC 7120 that may encode additional LOXs or other enzymes of the LOX pathway as it is known from higher plants. It should be noted that the appearance of (9R)-HOTE/HODE is not unusual in cyanobacteria of the genus Anabaena, as has been reported before [17], and that (9R)-HOTE/HODE has been shown to occur in the eukaryotic alga, Ulva conglobata [START_REF] Akakabe | Enantioselective formation of (R)-9-HPODE and (R)-9-HPOTrE in marine green alga Ulva conglobata[END_REF],

while dihydroxy fatty acids similar in structure to those formed in vitro by NspLOX have been described to occur in red algae [START_REF] Hamberg | Biosynthesis of vicinal dihydroxy fatty acids in the red alga Gracilariopsis-lemaneiformis -identification of a sodium-dependent 12-lipoxygenase and a hydroperoxide isomerase[END_REF].

Based on the biochemical data and phylogenetic relationships among enzymes studied so far, it appears that in evolution the use of bi-or multifunctional enzymes may represent a more ancient way to form and to control the intracellular amount of oxylipins, whereas metabolic pathways involved in the formation of more complex oxylipins, such as jasmonates or green leaf-volatiles, may have emerged at a later stage during evolution together with the rise of more complex and multicellular organisms. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

© 2007 The Authors Journal compilation © 2007 Biochemical Society three determinants for substrate and regiospecificity (bo, according to [START_REF] Borngräber | Phenylalanine 353 is a primary determinant for the positional specificity of mammalian 15-lipoxygenases[END_REF], sl, according to [START_REF] Sloane | A primary determinant for lipoxygenase positional specificity[END_REF], ho, according to [START_REF] Hornung | Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis[END_REF]). ADLPRD----------------IPGDLFTPAATAYWELLNNYVEQGL PhAOS-LOX 829 CFKEVNLQDYHLPNALKKRGVDDPSK---------LPGFYYRDDGLALWEAIETFIGE --------PYIYLLPERIPNGTAI------- Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071277 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071277residues are occupied by Arg 349 and Tyr 353[START_REF] Koljak | Identification of a naturally occurring peroxidase-lipoxygenase fusion protein[END_REF]24]. In the amino-terminal domain of NspLOX the residues that may coordinate the distal face of a heme group are conserved as Thr-40, His-41 and Asn-118. In contrast, the residues on the proximal face of the heme group differ from those of the coral enzyme, and instead of Arg-349 a glycine is situated at the corresponding position (Gly-323), while the position of Tyr-353 corresponds to His-327 with the neighbouring amino acid a tyrosine (Tyr-328).The alignment of the carboxy-terminal LOX-domain of NspLOX with known LOX sequences showed, that the residues involved in iron binding within the active site (His-170, His-175, His-342 and Asn-346) are highly conserved and identical to those from plant LOXs (Figure2, marked by stars). However, due to a long carboxy-terminal deletion in the sequence of the LOX domain the fifth ligand did not align properly to the other sequences but was suggested to be represented by an isoleucine (Ile-429) as for other plant LOXs (Figure2, li).At the position of the three determinants that may be involved in regiospecificity NspLOX contains different amino acids in comparison to the other LOX sequences from flowering plants[START_REF] Sloane | A primary determinant for lipoxygenase positional specificity[END_REF][START_REF] Hornung | Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis[END_REF][START_REF] Borngräber | Phenylalanine 353 is a primary determinant for the positional specificity of mammalian 15-lipoxygenases[END_REF]. At the site described first by Sloane and co-workers, a serine (Ser-229) is found instead of a phenylalanine in both plant LOXs (LOX2 At2 and PpLOX1; Figure2, sl).

  7120. The DNA-fragment encoding the full-length fusion-protein had a complete open reading frame of 2322 bp, encoding a protein of 774 amino acids with a molecular mass of 70.9 kDa. The analysis of the three obtained fragments via gel electrophoresis showed, that the amino-terminal domain as well as the full-length protein were about 1300 bp longer than expected from the published genomic sequence of the NspLOX gene. Sequence analysis of the two fragments in question revealed the presence of a large transposon insertion within the region encoding the amino-terminal domain, causing a frame shift. This insertion had highest similarity to the putative transposase gene IS891N, identified in Nostoc ellipsosporum (Acc.no. U48694). The insertion was removed via PCR mutagenesis to allow expression of the intact NspLOX protein. To investigate the catalytical activities of full-length protein and the two separate portions, we made three corresponding constructs, which were overexpressed in E. coli (DE3) expression strains.The NspLOX Carboxy-terminal Portion Exhibits Activity as a Linoleate (9R)-LOX.

14 S

 14 t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071277 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071277

( 9 ,

 9 10-diHODE) and (9E,12E)-8,11-dihydroxy-9,12-octadecadienoic acid (8,11-diHODE) by mass spectrometry. The corresponding ions in m/z, ion structure and relative intensities are

  437 [M + -31, loss of OCH 3 ] + , 378 [M + -90; loss of Me 3 SiOH] + , 337 [M + -131, loss of CH 3 -CH 2 -CH-(OMe 3 Si)] + , 259 [Me 3 SiO-CH-(CH 2 ) 7 COO-CH 3 ] + , 209 [CH 3 -CH 2 -CH-(OMe 3 Si)-CH=CH-CH=CH-CH=CH] + , 191 [intra molecular fragment: (OMe 3 Si) 2-CH] + , 131 [CH 3 -CH 2 -CH-(OSiMe 3 )] + . For 9,14-diHOTE the following ions in m/z and ion structure could be found: 313 [M + -155, loss of CH 2 -CH=CH-(CH 2 ) 4 -COOCH 3 ] + , 256 [Me 3 SiO-CH-CH=CH-CH=CH-CH-OSiMe 3 ] + , 223 [CH 3 -(CH 2 ) 2 -CH=CH-CH=CH-CH=CH-CH=OMe 3 SiO] + , 191 [intra molecular fragment: (OSiMe 3 ) 2-CH] + and 159 [CH 3 -(CH 2 ) 3 -CH-(OSiMe 3 )] + . The most prominent ions in the mass spectra of 11,16-diHETE were as follows in m/z and ion structure:313 [Me 3 SiO-CH-CH=CH-CH=CH-CH-(OMe 3 Si)-(CH 2 ) 3 -CH 3 ] + , 283 [Me 3 SiO-CH-CH 2 -CH=CH-CH 2 -CH=CH-(CH 2 ) 3 -COOCH 3 ] + , 256 [Me 3 SiO-CH-CH=CH-CH=CH-CH-OMe 3 Si] + , 211 [CH 3 -(CH 2 ) 3 -CH-(OMe 3 Si)-CH=CH-CH=CH] + , 191 [intra molecular fragment: (OMe 3 Si) 2-CH] + , 159 [CH 3 -(CH 2 ) 3 -CH-(OMe 3 Si)] + .Several trapping experiments were performed, but no allene oxides were detected. The formation of dihydroxy fatty acids started immediately after addition of either LA or ALA to the reaction mix. (9R)-HPO(D/T)E and dihydroxides were detectable as early as 20 s after substrate addition, with 9,14-diHO(D/T)E being the major product. Furthermore, the intermediate (9R)-HPO(D/T)E was present over the whole time course of 120 min, at approximately constant amounts. The pH-optimum of NspLOX was similar to that of the LOX domain alone, the highest activity being observed in the pH range between 7 and 10.5.Since the carboxy-terminal portion was identified as a (9R)-LOX, we anticipated that the oxygen at C-9 derives from dioxygen. To investigate the origin of the second oxygen at C-14, we performed activity tests in the presence of either 18 O 2 or H 218 O. Reaction products were analyzed by GC/MS and LC/MS, allowing for the unequivocal identification of diagnostic mass fragments. The mass spectra of 9,14-diHODE for the experiment with18 O 2 revealed that only the first oxygen atom derived from18 O 2 , whereas the second oxygen atom remained unlabelled (Figure5B,18 O-containing fragments are marked by stars). The fragments containing the C-9 atom with the first hydroxy group, deriving from the LOX reaction, contained the labelled oxygen atom: m/z 472, m/z 441 [M + -31, loss of OCH 3 ] + , m/z 415 [M + -57, loss of CH 3 -(CH 2 ) 3 ] + , m/z 382 [M + -90; loss of Me 3 SiOH at C-9] + , m/z 261 (Me 3 Si[ 18 O]-CH-(CH 2 ) 7 COO-CH 3 ] + and m/z 225 [CH 3 -(CH 2 ) 2 -CH=CH-CH=CH-CH=CH-CH=[ 18 O]SiMe 3 ] + . In contrast, the obtained mass fragments of 9,14-diHODE containing the second hydroxy group at C-14 were only labelled, when LA was converted in the presence of H 2 18 O (Figure 5C, 18 O-containing fragments are marked by stars). The mass spectra showed mass ions at m/z 441(M + -31, loss of OCH 3 ), m/z 325 [M + -147; loss of Me 3 SiOH from C-9 and CH 3 -(CH 2 ) 3 ] + and m/z 161 (CH 3 -(CH 2 ) 3 -CH-([ 18 O]Me 3 Si)] + .

S t a g e 2

 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071277 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071277 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society cyanobacterial genomes harbour putative open reading frames that may encode for LOXs

  detected in N. sp. PCC 7120 may give first hints for a potential physiological function of 21 S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071277 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071277 NspLOX in this cyanobacterium. Analysis of oxylipin profiles in N. sp. PCC 7120 indicates that this organism may primarily form oxylipins that derive from the (9R)-LOX activity of NspLOX at least under conditions used in this study. So far nothing is known about the function of 9-LOX-derived hydro(pero)xy C18-PUFAs in cyanobacteria, a class of compounds recently discussed to be involved in triggering cell death in pathogen-infected plant cells [39-41] and possibly involved in the regulation of root development [42].

22 S t a g e 2

 222 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071277 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071277 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071277 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071277 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071277 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071277 Schneider, C., Keeney, D. S., Boeglin, W. E. and Brash, A. R. (2001) Detection and cellular localization of 12R-lipoxygenase in human tonsils. Arch. Biochem. Biophys. 386, 268-74 S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071277 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071277 S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071277 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071277 S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 21 Nov 2007 as manuscript BJ20071277 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071277

FIGURE LEGENDS Figure 1

 LEGENDS1 FIGURE LEGENDS

Figure 2

 2 Figure 2 Partial amino acid sequence alignment of LOX-active site of the NspLOX

Figure 3

 3 Figure 3 Conversion of LA, ALA and AA by the carboxy-terminal part of NspLOX. The

Figure 4

 4 Figure 4 Product profile of NspLOX conversion of LA. The reaction was carried out in

Figure 5

 5 Figure 5 Identification of products of NspLOX conversion of LA. A, Mass spectrum and

Figure 6

 6 Figure 6 Free oxylipins of two cyanobacterial strains. The oxylipin levels were determined

Figure 1 LOX1 35 S

 135 Figure 1

Figure 2 NspLOX

 2 Figure 2

  Figure 3

Figure 5 A 9 39 S

 5939 Figure 5

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  Figure 6

Presence either of 18 O 2 or H 2 18

  [M + -31, loss of OCH 3 ] + , m/z 413 [M + -57, loss of CH 3 -(CH 2 ) 3 ] + , m/z 380 [M + -90; loss of Me 3 SiOH] + , m/z 323 [M + -147; loss of CH 3 -(CH 2 ) 3 and Me 3 SiOH] + , m/z 259 [Me 3 SiO-CH-(CH 2 ) 7 COO-CH 3 ] + , m/z 223 [CH 3 -(CH 2 ) 2 -CH=CH-CH=CH-CH=CH-CH=OMe 3 SiO] + , m/z 211 [CH 3 -(CH 2 ) 3 -CH-(OMe 3 Si)-CH=CH-CH=CH] + , m/z 191 [intra molecular fragment: (OMe 3 Si) 2 -CH] + , m/z 159 [CH 3 -(CH 2 ) 3 -CH-(OMe 3 Si)] + and m/z 73 [Me 3 Si + ]. The massspectrometric analysis left the double bond configuration of 9,14-diHODE unresolved.

Table 1 Fatty acid composition of N. sp. SAG 25.82.

 1 The lipids were extracted from lyophilized cells, esterified fatty acids were transmethylated and GC/FID analysis of fatty acid methyl esters isolated from the cultures was performed as described under materials and methods. All fatty acids were characterized by co-elution of authentic standards. Amount of each fatty acid was expressed as relative ratio of all esterified fatty acids.

	Fatty acid	% Fatty acid of total fatty acids
	16:0	19.5
	16:1 (9Z)	21.5
	16:3 (4Z,7Z,10Z)	2.2
	18:1 (9Z)	7.8
	18:1 (11Z)	4.4
	18:2 (9Z,12Z)	14.0
	18:3 (9Z,12Z,15Z)	30.6

Table 2 Overview of the dihydroxy fatty acids formed in the reaction of the NspLOX protein with different fatty acid substrates.

 2 The analysis of products was done with HPLC, HPLC/MS and GC/MS. For each substance the absorption maximum [nm] and fragment sizes [m/z] are given. In case of GC/MS spectra the relative intensities of the fragments are given in brackets.

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	LA ALA GLA AA	9,14-diHODE 232 nm (conj. diene) 139, 171, 225, 293, 311 159 (9.5), 191 (69.4) 211 (2.9), 223 (20.6), 259 (5.2), 311 (1.4), 323 (13.1), 380 (85.5), 413 (0.8), 439 (6.2) 470 (0.12) 9,16-diHOTE 232 nm (conj. triene: 260/268/281) 137, 171, 251, 309 131 (38.7), 191 (5.9) 209 (3.5), 259 (11.6), 311 (3.9), 337 (8.7), 468 (1.4) 9,14-diHOTE 232 nm (conj. diene) 137, 171, 251, 309 159 (6.8), 191 (100), 223 (11.5), 256 (1.2), 313 (17.1), 468 (0.01) 11,16-diHETE 232 nm (conj. diene) 167, 249, 335 159 (5.1), 191 (100), 211 (0.7), 256 (2.1) 283 (0.18), 313 (14.5), 494 (0.02) S t a g e 2 ( a ) P O S T -P R I N T 9,10-diHODE 8,11-diHODE HPLC nm 232 nm (conj. diene) 232 nm (conj. diene) LC/MS m/z (fragment ions, [M-H] -) 139, 171, 201, 293, 311 No MS/MS GC/MS m/z (rel. intensity) 109 (9.3), 155 (18.6), 211 (17.7), 259 (100), 360 (0.1), 470 (0.01) 129 (34.9), 143 (3.3), 155 (9.3), 199 (100), 285 (0.27), 327 (0.36), 470 (0.03) 9,13-diHOTE HPLC nm 232 nm (conj. diene) LC/MS m/z (fragment ions, [M-H] -) 137, 171, 239, 309 GC/MS m/z (rel. intensity) 243 (6.5), 259 (100), 299 (4.3), 401(0.07), 468 (0.04) HPLC nm LC/MS m/z (fragment ions, [M-H] -) GC/MS m/z (rel. intensity) HPLC nm LC/MS m/z (fragment ions, [M-H] -) GC/MS m/z (rel. intensity)
		30

Table 3 Selected NMR data for 9,14-diHODE.

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		H	δ 1 H [ppm]	δ 13 C [ppm] b
		8, 15	1.54 a	n.d.
		9, 14	4.15	72.7
		10, 13	5.70	136.4
		11, 12	6.20	129.8
	a	1 H-chemical shifts of 1 H, 1 H COSY 2D NMR correlation peaks
	b	13 C-chemical shifts of 1 H, 13 C HSQC 2D NMR correlation peaks
	n.d. not detected because of poor signal/noise ratio

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

The authors are grateful to Dr. Fredi Brühlmann, Geneva, Dr. Maike Lorenz, Goettingen, and Prof. Thomas Friedl, Goettingen, for continuous support and stimulating discussions, to Prof.

Peter Wolk, East Lansing, MI, USA and Prof. James Golden, College Station, TX, USA for providing us with additional strains of PCC 7120, Prof. Klaus Raschke, Goettingen, for providing us with H 2 18 O, to Dr. Michael Stumpe, Goettingen, who prepared radio-labeled [1-14 C]-(9S)-HPODE, and to Dr. Juergen Schmidt, Halle/Saale, for his help with the identification of mass fragments. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number NP_478445. This work was supported by Firmenich SA (Geneva, Switzerland).


Joseph D Dukes

Judith D Richardson

Ruth Simmons

Paul Whitley

A DOMINANT NEGATIVE ESCRT-III PROTEIN PERTURBS CYTOKINESIS AND TRAFFICKING TO LYSOSOMES

Keywords: Abscission, Cytokinesis, Endosome, ESCRT, Midbody Charged multivesicular body protein, CHMP, Early endosomal autoantigen 1, EEA1, Endosomal sorting complex

In eukaryotic cells, the completion of cytokinesis is dependent on membrane trafficking events to deliver membrane to the site of abscission. Golgi and recycling endosomal derived proteins are required for the terminal stages of cytokinesis. Recently, protein subunits of the Endosomal Sorting Complex Required for Transport (ESCRT) that are normally involved in late endosome to lysosome trafficking have also been implicated in abscission. Here we report that a subunit, Charged Multivesicular Body Protein-3 (CHMP3), of ESCRT-III localises at the midbody. Deletion of the C-terminal autoinhibitory domain of CHMP3 inhibits cytokinesis. At the midbody, CHMP3 does not co-localise with Rab11 suggesting that it is not present on recycling endosomes. These data combined provide compelling evidence that proteins involved in late endosomal function are necessary for the end stages of cytokinesis.

INTRODUCTION

Proteins that make up the endosomal sorting complex required for transport (ESCRT) 1 are involved in the sorting and trafficking of membrane proteins into multivesicular bodies (MVBs). Current models of ESCRT function place the ESCRT proteins into three complexes, ESCRT-I, II and III (reviewed in [START_REF] Nickerson | A concentric circle model of multivesicular body cargo sorting[END_REF][START_REF] Hurley | The ESCRT complexes: structure and mechanism of a membrane-trafficking network[END_REF][START_REF] Babst | A protein's final ESCRT[END_REF][START_REF] Slagsvold | Endosomal and non-endosomal functions of ESCRT proteins[END_REF]). ESCRT-I is thought to be involved in the recognition of ubiquitylated cargo membrane proteins that are to be sorted into MVBs. ESCRT-II and ESCRT-III have been implicated in further protein sorting and invagination of the endosomal membrane away from the cytoplasm to form MVBs. Once assembled on membranes, removal of ESCRT proteins by an AAA-ATPase called Vps4 is required in order for ESCRT proteins to carry out multiple rounds of sorting [START_REF] Babst | The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function[END_REF]. The latter stages of MVB formation are not currently well understood, however it is known that the ultimate fate of cargo proteins that are sorted into MVBs is usually degradation in lysosomes.

Perturbing the function of the ESCRT machinery in mammalian cells by protein knockdown or expression of dominant negative proteins such as an ATPase defective Vps4 (Vps4 E235Q ) results in intracellular accumulation of cargo proteins that fail to get degraded in lysosomes [START_REF] Bache | The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor[END_REF][START_REF] Doyotte | Depletion of TSG101 forms a mammalian "Class E" compartment: a multicisternal early endosome with multiple sorting defects[END_REF][START_REF] Fujita | A dominant negative form of the AAA ATPase SKD1/VPS4 impairs membrane trafficking out of endosomal/lysosomal compartments: class E vps phenotype in mammalian cells[END_REF].

In addition to their role in sorting of membrane proteins for destruction in lysosomes, ESCRT proteins have been implicated in membrane virus budding (reviewed in [START_REF] Morita | Retrovirus budding[END_REF]), mRNA trafficking [10] and cytokinesis [START_REF] Morita | Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis[END_REF][12][START_REF] Spitzer | The Arabidopsis elch mutant reveals functions of an ESCRT component in cytokinesis[END_REF]. The involvement of ESCRT proteins in cytokinesis is particularly interesting as evidence is accumulating from model organisms that endocytosis and many proteins involved in endocytic pathways such as dynamin, clathrin and Rab11 are also essential for the successful completion of cytokinesis [START_REF] Albertson | Membrane traffic: a driving force in cytokinesis[END_REF][START_REF] Feng | Furrow-specific endocytosis during cytokinesis of zebrafish blastomeres[END_REF][16][17][18].

Cytokinesis is the separation of one cell into two daughter cells following mitosis.

During the final stages of cytokinesis in animal cells a midbody is formed which is a thin membrane tether-like structure connecting the two daughter cells together. The end of cytokinesis is typified by the abscission of this structure at the midbody, which is an event heavily reliant on membrane dynamics. A model for abscission that seems to unify the seemingly different mechanisms of cytokinesis in animals and plants, involves the trafficking of membrane vesicles to the midbody and phragmoplast, respectively [START_REF] Albertson | Membrane traffic: a driving force in cytokinesis[END_REF]19,20]. At the mid-body, in animal cells, these vesicles have been suggested to fuse homotypically and heterotypically with the plasma membrane causing abscission at this region and separation of the two daughter cells [21]. The source of the membrane vesicles required for abscission is seemingly complex, as proteins usually associated with Golgi, early stages of endocytosis and endosomal recycling all appear to be important in the process [START_REF] Albertson | Membrane traffic: a driving force in cytokinesis[END_REF]. The recent discovery that ESCRT proteins, which are generally considered as functioning in late endosomal membrane trafficking, are required for cytokinesis adds additional complexity to the source of membranes present at the site of abscission. An ESCRT-I protein (TSG101), an ESCRT related protein (Alix) and ESCRT-III proteins (CHMPs 2, 4 and 5) are all present at the midbody during the final stages of cytokinesis in mammalian cells [START_REF] Morita | Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis[END_REF]12]. Furthermore, interfering with the expression, by knockdown or overexpression, of TSG101, Alix and other ESCRT proteins results in impaired cytokinesis [START_REF] Morita | Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis[END_REF]12]. Thus, it seems that a functional ESCRT machinery is required for the late stages of cytokinesis.

Following on from this work, we wanted to investigate whether the ESCRT-III protein CHMP3 is present at the midbody and functionally required for cytokinesis in animal cells. To address this point we utilised a dominant negative derivative of charged multivesicular body protein 3 (CHMP3), an ESCRT-III protein. We and others have shown that full length CHMP3-FLAG and -GFP fusion proteins are cytosolic, do not noticeably affect endosome morphology and do not inhibit membrane trafficking to lysosomes or HIV particle production when expressed in cultured mammalian cells [22][START_REF] Shim | Structure/function analysis of four core ESCRT-III proteins reveals common regulatory role for extreme C-terminal domain[END_REF][24]. However, truncated CHMP3-fusion proteins, with the C-terminal autoinhibitory domain removed, become membrane associated and act as dominant negative proteins in that they dramatically alter endosome morphology, prevent trafficking to the lysosome and inhibit virus budding [22][START_REF] Shim | Structure/function analysis of four core ESCRT-III proteins reveals common regulatory role for extreme C-terminal domain[END_REF][24][START_REF] Yan | mVps24p functions in EGF receptor sorting/trafficking from the early endosome[END_REF].

We now show that as well as perturbing endocytic trafficking to the lysosome a dominant negative CHMP3 derivative (CHMP3 1-179 GFP) localises to the mid-body of dividing mammalian cells and dramatically inhibits cytokinesis.

DNA Manipulations and Constructs

A cDNA fragment encoding amino acid residues 1-179 of rat CHMP3 was cloned into the BglII/HindIII sites of pEGFP-N1 (Clontech) as a BglII/HindIII fragment to create a vector for the expression of the fusion protein CHMP3 1-179 GFP. Site directed mutagenesis was performed using the Quikchange method (Stratagene). The mutant protein CHMP3 1-179 GFP M1 had amino acids R24, K25, and R28 of CHMP3 changed to S, A and N respectively. All other constructs have been described previously [24,26].

Antibodies

Mouse monoclonal anti-β-tubulin antibodies (2.1) were purchased from Sigma, antiubiquitin (FK2) from Biomol and anti-Rab11a from BD Biosciences. The rabbit anti-EEA1 (early endosome antigen 1) was a gift from Dr Michael Clague (University of Liverpool, Liverpool, U.K.), and the rabbit anti-CI-M6PR was a gift from Dr Paul Luzio (University of Cambridge, Cambridge, U.K.). Species-specific fluorophore (Alexa Fluor TM 546)-conjugated anti-IgG secondary antibodies were all purchased from Molecular Probes.

Cell culture and Transfections

Cos-7 and HeLa cells were maintained at 37 o C, 5% CO 2 in DMEM media supplemented with 10% (v/v) fetal calf serum, 2mM L-glutamine, 100units/ml penicillin and 100µg/ml streptomycin. Cells were plated onto 13mm coverslips in 24-well plates (Nunc) and grown until approximately 60% confluent when they were transfected with Trans-IT (Mirus) according to manufacturer's instructions.

Immunofluorescence and Multinucleation Counts

24 h post-transfection cells were fixed with 4% (w/v) paraformaldehyde for 20 min and permeabilised using methanol at -20 o C for 5 min and then blocked with 10% newborn calf serum. Primary and secondary antibodies were diluted in 2% NCS-PBS (2% newborn calf serum in PBS) and cells were incubated with primary antibodies for ~ 2 h at 18 o C and ~1 h for secondary antibodies. Cells were washed five times for 5 min with 2% NCS-PBS following all antibody incubations. Stained cells were then mounted in Mowiol (Calbiochem, San Diego, CA, U.S.A.) and examined on a Zeiss LSM510 laser scanning confocal microscope and appropriate images taken. For cell multinucleation counts, transfected cells were counted on a coverslip and scored for either a single nucleus or multiple (two or more) nuclei. Cells with continuous plasma membrane and connected by tethered tubulin "bridges" between them were defined as multinucleated, provided both "cells" contained nuclei.

S t a g e 2 ( a ) P O S T -P R I N T EGF Degradation Assay

HeLa cells were seeded on 13 mm coverslips 24 h prior to transfection and grown to 60-80% confluency. Cells were transfected using TransIT reagent as described, with CHMP3 1-179 GFP or CHMP3 1-179 GFP M1 and allowed to express the constructs for 24 h. The media was replaced, following a wash in warm PBS, with DMEM containing 1% BSA (no FBS). Cells were serum starved in this media for 16 h and then incubated with 500ng/ml Alexa Fluor TM 555-conjugated EGF (Invitrogen) for 2 min or 60 min. Following EGF stimulation for given times, cells were washed twice with cold PBS and then fixed in PFA, stained with DAPI for 30 min and mounted onto slides using Mowiol. Slides were examined on a Zeiss LSM510Meta laser scanning confocal microscope.

RESULTS

The Dominant Negative Protein CHMP3 1-179 is Present at the Midbody During Cytokinesis.

Recent work has shown that TSG101, an ESCRT-I component, localises to Flemming bodies during the late stages of cytokinesis and that its knockdown by siRNA inhibits cytokinesis at abscission [12]. In the same study it was shown that ESCRT-III components may also have a role to play in abscission. In another study the ESCRT-III proteins CHMP2, 4 and 5 have been localised to the midbody of dividing cells [START_REF] Morita | Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis[END_REF]. In order to investigate this further and determine whether other ESCRT-III components are also present at the midbody during the final stages of cytokinesis we made use of a dominant negative CHMP3 construct (CHMP3 1-179 GFP). Transient transfection of this dominant negative truncated form of CHMP3 resulted in a swollen vacuolar phenotype typified by CHMP3 1-179 GFP bound to large vacuolar structures in Cos-7 (Fig. 1) and HeLa cells (not shown). These structures were endosomal in origin as they contained both early and late endosomal markers (EEA1 and M6PR) (Fig. 1a-f). Ubiquitylated proteins also accumulated on the CHMP3 1-179 GFP containing endosomes (Fig. 1g-i). It is possible that the accumulated ubiquitinylated proteins are cargoes destined for lysosomal degradation, however we cannot rule out the possibility that they are ubiquitylated cytosoplasmic proteins recruited to membranes or even ESCRT components. In an EGF degradation assay fluorescent EGF accumulated intracellularly after 60 minutes incubation with EGF in CHMP3 1-179 GFP expressing cells but disappeared almost completely from neighbouring untransfected cells (Fig. 2d-f). These results indicate that the CHMP3 1-179 GFP fusion protein is dominant negative, as its expression blocks trafficking of EGF to the lysosome and prevents its degradation.

In cells fixed during the late stages of cytokinesis, the midbody was observed to contain CHMP3 1-179 GFP protein apparently present on membrane vesicles (Fig. 3a-c). The CHMP3 1-179 GFP specifically localises to the central region of the midbody, where there is S t a g e 2 ( a ) P O S T -P R I N T lack of β-tubulin staining. This distribution was observed in both Cos-7 cells (Fig. 3a-c) and HeLa cell lines (Fig. 3d-f), indicating that this phenomenon is not cell type specific. CHMP3 1- 179 GFP was often seen distributed along the microtubules in the midbody channel (not shown) suggesting that vesicles may be being transported along microtubules towards the midbody.

Interestingly, although Rab11, an endosomal protein required for abscission [27], was present in the midbodies of dividing cells, it did not seem to co-localise with CHMP3 1-179 GFP (Fig. 3g-i).

Dominant Negative CHMP3 Perturbs Cytokinesis.

HeLa cells were transfected with constructs for the expression of CHMP3 1-179 GFP, GFP-Vps4 E235Q and GFP-Vps4 WT or a GFP control. They were then fixed and immunostained for β-tubulin and treated with DAPI to stain nuclei. Transfected cells were quantified under the fluorescence microsope for the percentage of multinucleate cells. Multinucleate cells were defined as cells connected by a continuous plasma membrane to another cell, thus containing two or more nuclei (Fig. 4b). Of the GFP control transfected cells, 12% were multinucleate (Fig. 4a). The percentage of multinucleate cells was dramatically increased (to 48%) in CHMP3 1-179 GFP expressing cells. This indicates that CHMP3 1-179 GFP acts as a dominant negative protein in cytokinesis in addition to lysosomal trafficking. In agreement with a previous study [12], the positive control, GFP-Vps4 E235Q blocked cytokinesis, while GFP-Vps4 WT had very little effect. GFP-Vps4 E235Q appears to block cytokinesis at a late stage, similar to CHMP3 1-179 GFP, as it is also enriched on vesicles at the midbody of dividing cells (data not shown). The ATPase activity of Vps4 which is required to disassemble ESCRT-III from membranes seems therefore to be required for abscission. GFP-Vps4 WT , which does not block cytokinesis, is not enriched at the midbody.

In a recent study, Muziol et al [28] showed that the dominant negative effect of a truncated CHMP3 construct on viral budding could be abrogated by mutating 3 basic amino acids (R24, K25, and R28). We mutated these same amino acids to create CHMP3 1-179 GFP M1 and expressed this protein in mammalian cells. Unexpectedly CHMP3 1-179 GFP M1 associated with endosomal membranes (Fig. 5a,b), caused the intracellular accumulation of ubiquitylated proteins (Fig. 5c), internalised EGF (Fig. 2g-h) and blocked cytokinesis (Fig. 4 and5d). Thus, mutations that abrogate the dominant negative effect of truncated CHMP3 on viral budding do not block the dominant negative effect of truncated CHMP3 on trafficking to the lysosome or cytokinesis.

DISCUSSION

Vesicular membrane traffic is important for the successful completion of cytokinesis in animal cells and in plants [START_REF] Albertson | Membrane traffic: a driving force in cytokinesis[END_REF]19,20]. In plants, membrane vesicles provide material for cell plate formation and in animals membrane trafficking is required for midbody channel closure. The source of membranes that are targeted to the midbody channel is seemingly complex, with Golgi associated proteins involved in exocytosis and proteins that control endocytic recycling being required for late stages of cytokinesis. Recently, three studies, one in plants, and the others in mammalian cells have implicated that proteins best characterised as being involved in MVB biogenesis are also required for cytokinesis [START_REF] Morita | Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis[END_REF][12][START_REF] Spitzer | The Arabidopsis elch mutant reveals functions of an ESCRT component in cytokinesis[END_REF].

In this study we show that a dominant negative ESCRT-III protein, CHMP3 1-179 GFP localises to the midbody in dividing cells. As TSG101, an ESCRT-I component localises to the Flemming body [12], a phase dense structure involved in abscission, and the ESCRT-III proteins CHMP2, 4 and 5 localise to the midbody of dividing cells [START_REF] Morita | Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis[END_REF], our data strengthens the hypothesis that a complete ESCRT machinery may be present at the midbody. We further show that expression of CHMP3 1-179 GFP inhibits cytokinesis, as seen by a large increase in the percentage of multinucleate cells compared to controls. This indicates that the ESCRT-III machinery is not simply passively present at the site of abscission but is also functionally required.

So what is the role of the ESCRT machinery in abscission? It has been suggested that the ESCRT machinery may be involved in cytokinesis at the step of membrane fission as it is likely that ESCRT protein function in cytokinesis, virus budding and MVB formation is mechanistically conserved [12]. To support this, ESCRT proteins, in particular those in ESCRT-III are required for a late stage, possibly fission, in virus budding [START_REF] Von Schwedler | The protein network of HIV budding[END_REF][START_REF] Strack | AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding[END_REF][START_REF] Martin-Serrano | Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins[END_REF]. This process is topologically similar to the separation (budding) of two cells and the scission of inwardly budded vesicles from the limiting membrane of late endosomes to form MVBs. However, it has recently been shown that CHMP3 is not absolutely required for intralumenal vesicle formation at late endosomes [START_REF] Bache | The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor[END_REF]. Therefore it has been proposed that CHMP3 is important for the fusion of multivesicular endosomes with lysosomes [START_REF] Bache | The ESCRT-III subunit hVps24 is required for degradation but not silencing of the epidermal growth factor receptor[END_REF] and not fission of inwardly budded endosomal vesicles. This raises the intriguing possibility that CHMP3 may be involved not in membrane fission but in vesicle fusion events that take place at the site of abscission [START_REF] Albertson | Membrane traffic: a driving force in cytokinesis[END_REF]21,32]. A further possibility is that the ESCRT machinery is involved in the function of recycling endosomes that are required for cytokinesis [27,33,34]. Several studies have shown that perturbation of ESCRT function results in a defect in endosomal recycling [START_REF] Doyotte | Depletion of TSG101 forms a mammalian "Class E" compartment: a multicisternal early endosome with multiple sorting defects[END_REF][START_REF] Fujita | A dominant negative form of the AAA ATPase SKD1/VPS4 impairs membrane trafficking out of endosomal/lysosomal compartments: class E vps phenotype in mammalian cells[END_REF]. However, although we observe some co-localisation of CHMP3 1-179 GFP with Rab11 on endosomes in the cytoplasm, there is little overlap of CHMP3 1-179 GFP with Rab11 at the midbody (Fig. 3g-i). This suggests that there are at least two distinct populations of endosomal membranes, late and recycling endosome derived membranes, in addition to Golgi In order to investigate whether membrane association of CHMP3 is required for localisation to the midbody and inhibition of cytokinesis, we assessed the effect of the mutant CHMP3 1-179 GFP M1 on cytokinesis. CHMP3 with the same 3 positively charged amino acid residues mutated had previously been shown to lose both its ability to associate with membranes and its dominant negative effect on viral budding [28]. CHMP3 1-179 GFP M1 localised at the midbody (Fig. 5d) and inhibited cytokinesis, although to a slightly lesser extent than the non-mutated protein (Fig. 4). On further analysis it was observed that unexpectedly CHMP3 1-179 GFP M1 clearly associated with endosomal membranes and also perturbed trafficking to the lysosome as indicated by the accumulation of ubiquitylated cargo proteins and internalised EGF in cells expressing CHMP3 1-179 GFP M1 (Fig. 2g-i and5). Thus, it was not possible to determine whether membrane association is required for localisation to the midbody and inhibition of cytokinesis. In their study, Muziol et al [28] observed plasma membrane association of truncated CHMP3-GFP constructs, whereas we (Fig. 1 and data not shown) and others [START_REF] Shim | Structure/function analysis of four core ESCRT-III proteins reveals common regulatory role for extreme C-terminal domain[END_REF] do not, except at very high expression levels (data not shown). We have previously shown that CHMP3 binds to the endosomal lipid phosphatidylinositol 3,5bisphosphate in vitro and have argued, as have others, that over-expressed, truncated CHMP3 may associate with membranes via endosomal specific lipids rather than protein-protein interactions [START_REF] Shim | Structure/function analysis of four core ESCRT-III proteins reveals common regulatory role for extreme C-terminal domain[END_REF]24]. It will be interesting to determine whether endosome-specific lipids are required for abscission as establishment of specialised lipid composition seems to be important in cytokinesis [START_REF] Albertson | Membrane traffic: a driving force in cytokinesis[END_REF]. It is difficult to explain the discrepancies in membrane association of the CHMP3 mutant constructs between our study and Muziol et al [28], but it is possible that cytokinesis and trafficking to the lysosome are more sensitive to ESCRT-III perturbation than viral budding.

In summary, we have shown that a dominant negative ESCRT-III protein, CHMP3 1- 179 GFP localises to the midbody and inhibits cytokinesis at a late stage. It is likely that the ESCRT machinery is involved in abscission but further studies will be required to resolve the detailed mechanism of the role of CHMP3 in cytokinesis.

FIGURE LEGENDS

Fig. 1 Transient expression of CHMP3 1-179 GFP results in localisation to early and late endosomal compartments as well as impaired trafficking. Cos-7 cells were transfected (ai) with the CHMP3 1-179 GFP fusion construct using Trans-IT transfection reagent according to manufacturer's instructions. Cells were fixed 24 h after transfection and immunostained with anti-EEA1 (a-c), anti-CI-M6PR (d-f) and anti-ubiquitin (g-i), followed by Alexa Fluor 546conjugated anti-rabbit (a-f) and anti-mouse (g-i) IgG secondary antibodies. Fluorescence corresponding to CHMP3 1-179 GFP is shown in a, d and g (green).

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071296 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071296 EXPERIMENTAL PROCEDURES

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071296 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071296 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071296 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071296 derived membranes, present at the site of abscission. The complexity of components present at the midbody that are necessary for abscission highlights the sophistication of the mechanisms required for the final stage of cytokinesis. Much work is still required to understand these final stages.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071296 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071296 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

  Fluorescence corresponding to EEA1, CI-M6PR and ubiquitin is shown in b, e and h respectively (red). Images of merged fluorescence are shown in c, f and i (yellow fluorescence indicates co-localisation). Insets are magnifications of boxed areas. Note: neighbouring untransfected cells do not accumulate ubiquitin (h).

Fig. 2

 2 Fig. 2 Dominant negative CHMP3 perturbs endo-lysosomal trafficking of EGF. HeLa cells were transfected with appropriate constructs and allowed to express for 24 h until serumcontaining medium was removed and replaced with DMEM containing 1% BSA. Cells were serum starved overnight for 16 h and then incubated with 500ng/ml Alexa-Fluor ® 555conjugated EGF for 2 min or 60 min at 37 o C prior to processing for confocal microscopy. Panels a-c show two transfected cells and a single untransfected cell after 2 min of EGF stimulation, with EGF (red) found largely at the plasma membrane of the cells. Panels d-f and g-i show the results of CHMP3 1-179 GFP and CHMP3 1-179 GFP M1 (green) expression respectively on EGF (red) degradation after a 60 min stimulation. Note the untransfected cells denoted by '*' in panels d-i.

Fig. 3

 3 Fig.3CHMP31-179 GFP localises to the mid-body in late stages of cytokinesis. Cos-7 (a-c, g-i) and HeLa (d-f) cells were transfected with CHMP3 1-179 GFP as outlined previously. Cells were fixed 24 h post-transfection and immunostained with anti-β-tubulin (a-f) and anti-Rab11A (g-i) antibodies followed by Alexa Fluor 546-conjugated anti-mouse IgG secondary antibodies. Fluorescence corresponding to CHMP31-179 GFP is shown in a, d and g (green). Fluorescence corresponding to β-tubulin and Rab11A is shown in b+e and h respectively (red). Images of merged fluorescence are shown in c, f and i. Insets are magnifications of boxed areas.

Fig. 4

 4 Fig. 4 Dominant negative ESCRT component mutants disrupt proper cytokinesis. HeLa cells were seeded on 13 mm coverslips 24 h prior to transfections. Cells were then transiently S t a g e 2 ( a ) P O S T -P R I N T

Fig. 5

 5 Fig. 5 Expression of mutant CHMP3 1-179 -GFP M1 results in early and late endosomal localisation as well as disruption of endosomal trafficking and localisation to the midbody in cytokinesis. Cos-7 (a-c) and HeLa (d) cells were transfected with the CHMP3 1- 179 GFP M1 fusion construct as outlined previously. Cells were fixed 24 h post-transfection and immunostained for anti-EEA1 (a), anti-CI-M6PR (b), anti-ubiquitin (c) and anti-β-tubulin (d) followed by Alexa Fluor 546-conjugated IgG anti-rabbit (a-b) and anti-mouse (c-d) secondary antibodies. Fluorescence corresponding to CHMP3 1-179 GFP M1 is shown in a-d left-hand insets (green). Fluorescence corresponding to EEA1, CI-M6PR, ubiquitin and β-tubulin is shown in a-d right-hand insets respectively (red). Main panel images show merged fluorescence, with yellow indicating co-localisation.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2007 The Authors Journal compilation © 2007 Biochemical Society

THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071296 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS This work was supported by the Wellcome Trust (project grant 070085 to P.W) and the BBSRC (Ph.D studentship to J.D.D). We thank Dr David Tosh for critical reading of the manuscript. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 12 Dec 2007 as manuscript BJ20071296 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20071296

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.