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Journal of Experimental Botany, Vol. 54, No. 390, pp. 2053±2063, September 2003 DOI: 10.1093/jxb/erg230 RESEARCH PAPER Identi®cation and localization of a b-COP-like protein involved in the morphodynamics of the plant Golgi apparatus Isabelle Couchy, Susanne Bolte, Marie-TheÂreÁse Crosnier, Spencer Brown and BeÂatrice Satiat-Jeunemaitre* Laboratoire de Dynamique de la Compartimentation Cellulaire, Institut des Sciences du VeÂgeÂtal, CNRS UPR2355, 91198 Gif-sur-Yvette cedex, France Received 18 March 2003; Accepted 29 May 2003 Abstract Introduction This paper examines the molecular machinery involved in membrane exchange within the plant endomembrane system. A study has been undertaken on b-COP-like proteins in plant cells using M3A5, an antibody raised against the conserved sequence of mammalian b-COP proteins. In mammalian cells, b-COP proteins are part of a complex named the coatomer, which probably recruits some speci®c areas of the endomembrane system. Immuno¯uorescence analyses by confocal laser scanning microscopy showed that b-COP-like proteins marked predominantly the plant Golgi apparatus. Other proteins known to be part of a potential machinery for COPI vesicle formation (g-COP, b¢-COP and Arf1 proteins) were immunolocalized on the same membraneous structures as b-COP. Moreover, b-COP and other COPI antibodies stained the cell plate in dividing cells. It is further shown that, in maize root cells, and in contrast to observations upon mammalian cells, the drug Brefeldin A (BFA) does not induce the release of b-COP and Arf1 proteins from the Golgi membrane into the cytosol. These data clearly demonstrate that the antibody M3A5 is a valuable marker for studies on traf®cking events in plant cells. They also report for the ®rst time the location of COP components in plant tissue at the light level, especially on a model well known for secretion, i.e. the maize root cells. They also suggest that the membrane recruitment machinery may function in a plant-speci®c way. In all eukaryotic cells, the Golgi apparatus (GA) is a central organelle in the secretory processes. In higher plant cells, the GA is characterized by more than a hundred Golgi stacks dispersed throughout the cytoplasm (SatiatJeunemaitre and Hawes, 1992). These stacks consist of membrane-bounded cisternae, organized in a polarized manner and surrounded by populations of vesicles (Robinson et al., 1998; Dupree and Sherrier, 1998; Hawes et al., 1999). The recent visualization of Golgi stacks in vivo has underlined its tremendous ability to exchange membranes and proteins with other components of the endomembrane system in mammalian cells rapidly (Cole et al., 1996; Simpson et al., 2001; Presley et al., 2002) as in plant cells (Boevink et al., 1998; NebenfuÈhr et al., 1999, 2000; Saint-Jore et al., 2002). The intensity of this membrane exchange contrasts with the apparent structural stability of the plant GA. A fundamental question is to understand the mechanisms by which the Golgi morphology, its spatial distribution and function are acquired, preserved and regulated concomitantly with membrane ¯ow. There is a previous report on a family of Rho proteins which could be involved in the spatial distribution of the plant GA (Couchy et al., 1998). This paper focuses on proteins that may be involved in the regulation of the Golgi membrane exchanges. The integrity of the GA implies maintenance both of lipid composition in its bounding membranes and of the protein complement for processing, packaging and targeting biosynthetic molecules. Therefore, in parallel with the secretory membrane ¯ow which goes through the GA, cells have to operate a complex membrane recycling machinery Key words: b-COP, Brefeldin A, coatomer, Golgi apparatus, plant cells. * To whom correspondence should be addressed. Fax: +33 1 69 82 33 55. E-mail: [email protected] Journal of Experimental Botany, Vol. 54, No. 390, ã Society for Experimental Biology 2003; all rights reserved 2054 Couchy et al. to preserve and regulate the membrane economy of the Golgi. Occurrence of a general retrograde/recycling pathway throughout the plant GA was initially suggested by studies on endocytosis in plant cells (Tanchak et al., 1984; Hawes et al., 1996), where internalized cationized ferritin was observed within Golgi cisternae. Such retrograde transport may use a speci®c population of transport vesicles. Protein-coated vesicles, different from clathrin-coated vesicles, have been described in the vicinity of the GA (Hawes et al., 1996; Pimpl et al., 2000). They share some structural features with the mammalian COPI-coated vesicles. These latter vesicles are thought to be able selectively to co-opt and transport membrane proteins and soluble molecules in a retrograde direction and, some believe, in an anterograde direction as well through mammalian Golgi stacks (Nickel and Wieland, 1997; Harter, 1999). They are described as 75 nm vesicles, covered with a 10 nm protein coat (COP = Coat Protein), made of seven peptides (a-COP, b-COP, b¢-COP, d,-COP, e-COP, g-COP, and z-COP) (Kreis et al., 1995). These peptides are present in the cytosol as a protein complex, termed the coatomer. The coatomer is recruited on the Golgi membrane by a small GTP binding protein, Arf1 (ADP-ribosylation factor) (Nickel and Wieland, 1997). The question is whether the `COPI-like vesicles' observed in plant cells share some protein identity and function with their mammalian homologues, and how they are involved in the morphodynamics of the plant Golgi apparatus. Genes coding for homologues of most of these mammalian proteins have now been identi®ed in plant cells (Memon et al., 1993; Regad et al., 1993; Szopa and MuÈller-RoÈber, 1994; Kahn, 1995; Andreeva et al., 1998; Takeuchi et al., 2002; Pasqualato et al., 2003). To study COPI protein homologues in plant tissues further, antibodies raised against mammalian b-COP and b¢-COP proteins have been used for biochemical and immunocytochemical studies. Immunolocation of the targeted epitopes was observed by confocal laser scanning microscopy (CLSM). In order to identify putative protein partners co-localizing with the epitopes recognized by b-COP and b¢-COP antibodies, comparative immuno¯uorescence studies using antibodies raised against Arf and g-COP homologues cloned from A. thaliana (Pimpl et al., 2000) were performed. Potential association of these proteins with the plant GA was studied by co-localization experiments using a Golgi marker (JIM84: Horsley et al., 1993). Brefeldin A (BFA), a potent disrupting agent for plant Golgi morphology (Satiat-Jeunemaitre and Hawes, 1992; Satiat-Jeunemaitre et al., 1996) and, in mammalian cells, an inhibitor of COPI coat recruitment onto Golgi membranes was used to investigate the dynamic association and the cycling of these proteins with Golgi membranes. Materials and methods Plant material Nicotiana tabacum Bright Yellow-2 (BY-2) suspension-cultured cells were grown in the dark at 26 °C with 100 rpm and subcultured every 7 d at 5/80 ml, in a Murashige and Skoog medium supplemented with sucrose and thiamine. For immuno¯uorescence studies and protein extracts, cells were sampled 3 d after subculturing (middle of their exponential phase of growth). Maize caryopses (Zea mays, LG20.80, Limagrain, France) were immersed in tap water for 3 h and then allowed to germinate in Petri dishes on moist ®lter paper in the dark at 26 °C. Root apices were excised from 3-d-old shoots. Mung bean (Vigna radiata) seeds were immersed for 4 h in distilled water and allowed to germinate on moistened ®lter paper in plastic boxes at 26 °C in darkness; biochemical analysis and immunocytolocalization studies on the epidermal strips were performed from the growing part of 3-d-old hypocotyl (5 mm below the hook). Pollen tubes from Nicotiana sylvestris were grown for 3 h at room temperature in a medium containing 10% sucrose, 0.01% boric acid, and calcium nitrate 3 mM. BFA treatment Brefeldin A (BFA, Sigma) stock was 20 mg ml±1 in dimethylsulphoxide (DMSO). Roots were immersed in 100 mg ml±1 aqueous BFA at 26 °C in darkness and ®xed after 1 h as previously described (Satiat-Jeunemaitre and Hawes, 1992). To treat the suspension cultures, BFA was added at a ®nal concentration of 100 mg ml±1 (Couchy et al., 1998). Parallel analyses were made upon cells treated with DMSO at the same concentration. Antibodies The b-COP antibodies (mouse monoclonal M3A5) were initially kindly provided by the late TE Kreis (Universite de GeneÁve, Suisse). They are now commercially produced by Sigma. In order to identify putative protein partners co-localizing with the epitopes recognized by b-COP antibodies, a panel of antibodies were used in co-localization studies. Anti g-COP antibody and anti-Arf1p, raised against homologues cloned from A. thaliana, were kindly provided by D Robinson (Heidelberg, Germany); anti-b¢-COP antibody by R Pepperkok (Heidelberg, Germany). A rat monoclonal antibody JIM84 (IgM) was used as neat supernatant as a Golgi marker (Horsley et al., 1993). Secondary antibodies were purchased either from Sigma (antirabbit, anti-mouse, or anti-rat IgGs conjugated with FITC, respectively used at 1:60, 1:40 and 1:40) or Interchim (anti-mouse, anti-rat, or anti-rabbit IgG conjugated with Cy3, used at 1:800). After washing, cells were mounted in Citi¯uor antifade-agent. Images were collected either with a Sarastro 2000 (Molecular Dynamics) or a Leica upright laser scanning confocal microscope TCS SP2 (Leica Microsystems, Heidelberg, Germany). Different ¯uorochromes were detected sequentially frame-by-frame with the acousto-optical tunable ®lter system (AOTF) using laser lines 488 and 543 nm. The images were coded green (¯uorescein-isothiocyanate) and red (cyanidine3.18) giving yellow co-localization in merged images. Oil objectives used were 403 NA 1.25 and 633 NA 1.30, giving resolution of ~200 nm in the XY-plane and 400 nm along the Z-axis (pinhole 1 Airy unit). Images were processed using Adobe Photoshop (Adobe Systems). Protein extraction 5 g of material were ground in a mortar with 6 ml of homogenization buffer (0.5 M sorbitol, 10 mM KH2PO4, 2 mM salicylhydroxamic acid (a chaotropic agent), 5% (w/v) polyvinylpyrrolidone 40 000 COPI components in plant cells 2055 MW (PVP40); 10 mM EGTA, pH 8.2, in the presence of protease inhibitors: 1 mM PMSF, 10 mM leupeptin, 10 mM E-64, and 1 mM pepstatin A (Boehringer). Cell lysates were spun 15 min at 15 000 g. The supernatant was then spun 1 h at 150 000 g. This new supernatant containing soluble proteins was kept for analysis. The pellet containing microsomes was suspended in 0.5 M sorbitol and 10 mM KH2PO4, pH 8.2, and kept for analysis. Immunoblot analysis Protein samples were treated by a modi®ed SDS-lysis buffer (Laemmli, 1970) containing 5 mM dithiothreitol (DTT), heated at 100 °C for 3 min, and separated on a 10% or 12% SDSpolyacrylamide gel at 25 mA in a Bio-Rad system. Relative molecular mass markers were run in parallel with the protein extracts (unstained markers, 14±97.4 kDa; stained markers 20±105 kDa, Bio-Rad). Gels were either stained with Coomassie Brilliant Blue (Serva) or proteins on gels were transferred to nitrocellulose sheets (0.45 mm; Schleicher and Schuell) by the method of Towbin et al. (1979), and stained with Ponceau S (Sigma) in 1% acetic acid to verify equal loading in each lane. After destaining in water, the sheets were blocked with a solution containing 5% milk powder, 10 mM TRIS-buffered saline, TBS 0.1 M, pH 7.4 and 0.05% (v/v) Tween 20 for 90 min prior to incubation with the primary antibody overnight at 4 °C in the buffer (M3A5, 1:50; Arf1p, 1:1000; g-COP, 1:1000; b¢-COP, 1:200 and 1:50; JIM84, 1:10). Primary antibodies were detected by two methods. (a) Using alkaline phosphatase conjugated anti-rabbit, antirat IgG or anti-mouse antisera (Promega) at 1:5000 dilution. Colour development was carried out by standard nitro-blue tetrazolium/ BCIP procedures. (b) Chemiluminescence, where primary antibodies were detected using horseradish peroxidase (HRP) conjugated anti-rat (Santa-Cruz), anti-rabbit (Amersham) or anti-mouse (Santa-Cruz) antibodies at 1:5000. The chemiluminescence reaction was detected using a Kodak ®lm. Fig. 1. Detection of b-COP-like-proteins by immunoblotting of protein extracts from BY-2 cells (A), mung bean hypocotyls (B), or Nicotiana sylvestris pollen tubes (C). (A, B) 50 mg of proteins were loaded; (C), 15 mg of proteins were loaded. Gel, 10% SDS-PAGE; S, soluble proteins; M, membraneous proteins; T, total extracts. Membrane probed with mouse anti-b-COP (M3A5) antibody (1:50) and revealed by HRP-coupled secondary antibody. These results are typical of repetitions. The positions of prestained commercial molecular markers are shown to the left, and the deduced molecular mass of an essential band is shown to the right. (A) »100 kDa band is clearly detected in all plant extracts. Results 625), recognized a polypeptide of ~100 kDa (Fig. 1). That is the expected molecular mass for b-COP. The speci®c activity of antigens was higher in the soluble than the membrane fractions, being stronger in the soluble fraction (Fig. 1A). This 100 kDa band was also identi®ed in protein extracts from mung bean hypocotyls (Fig. 1B) and from Nicotiana sylvestris pollen tubes (Fig. 1C), suggesting that a b-COP homologue was present in various plant cell types. The idea was to look for other COPI components, in particular, the proteins g-COP (98 kDa) and b¢-COP (102 kDa). Western blots on BY-2 protein extracts were therefore realized with the corresponding antibodies. Blotting with the plant g-COP antibody (Pimpl et al., 2000) revealed a unique band of ~100 kDa (Fig. 2A). Similarly to what was previously described for b-COP, the band was broader in the soluble fraction, suggesting a higher protein concentration. These results are in accordance with data obtained for cauli¯ower (Pimpl et al., 2000). The b¢-COP antibodies used here did not cross-react on western blots with various plant extracts, probably due to an insuf®cient loading of proteins. The dif®culty of clearly detecting speci®c plant proteins on western blots using other types of b¢-COP antibodies has been outlined by Contreras et al. (2000). Western blot analysis on various plant extracts, using antibodies against COPI components M3A5 antibody stains Golgi stacks, the cell plate and the plasma membrane In western blots of BY-2 cell protein extracts, the antibody M3A5, raised against a peptide sequence in the C-terminal moiety of the mammalian b-COP protein (24 AA, 620± Immunolocalization experiments were performed either on maize root squashes, mung bean epidermal strips or BY-2 suspension cells with substantially similar results. In Immuno¯uorescence staining Immunostaining procedures were performed on partially digested tissues or cells as described in Couchy et al. (1998) or SatiatJeunemaitre and Hawes (2001). Brie¯y, biological material (i.e. BY-2 cells, 3 mm root apices, mung bean epidermal strips) was ®xed with paraformaldehyde 3% in PBS, pH 6.9. Cell walls were partially digested by incubation in enzyme solution: 1% cellulase R10 (Onozuka), 1% pectinase (Sigma) in PBS, pH 6.9. Immuno¯uorescence was performed on Vectabond coated multiwell slides (Vector Laboratories), after having layered cells or gently squashed root apices on the slides. Cell membranes were permeabilized with 0.5% Triton X-100 for 20 min and washed with buffer. Non-speci®c binding was blocked by 1% bovine serum albumin (BSA). Primary antibodies were applied overnight at 4 °C. Slides were rinsed with a stream of PBS supplemented with 1% ®sh gelatine (Sigma), and then secondary antibodies conjugated with ¯uorochrome were applied for 1 h at room temperature and in darkness. After a 1 h wash comprising ®ve baths of PBS supplemented with 1% ®sh gelatin, slides were either mounted with Citi¯uor AF1 or retained for a second immunostaining series. 2056 Couchy et al. In control cells, similar 1 mm organelles were stained by JIM84 (Figs 4A, 5A) and M3A5 antibodies (Fig. 5B). Merged pictures showed a clear co-localization of the two antibodies (Fig. 5C). The cell plate of dividing cells was also stained by the two antibodies (Fig. 5D±F). Similar results were obtained in BY-2 tobacco cells, except that the plasma membrane was never stained by either of the two antibodies. These results con®rmed that b-COP-like proteins are associated with Golgi membranes. BFA effects on the reorganization of M3A5 immunostaining pattern Fig. 2. Western blot with anti-g-COP and anti-Arf1p on BY-2 cell protein extracts. 50 mg of proteins were loaded. S, soluble proteins; M, membraneous proteins; Gel, 10% SDS-PAGE. Membrane probed with rabbit anti-g-COP and rabbit anti-Arf1p antibodies (1:1000) and revealed by HRP-conjugated secondary antibody. These results are typical of repetitions. The positions of prestained commercial molecular markers are shown to the left, and deduced molecular masses of essential bands are shown to the right.(A) Anti-g-COP antibody revealed a unique band of ~100 kDa, both in M and S fractions, with a higher density in the S fraction. (B) Arf1p antibody revealed a major protein band at 20 kDa as expected, and a light band at 40 kDa, which may represent a dimer. interphase cells, the M3A5 antibody revealed numerous ~1 mm structures dispersed throughout the whole cell (Fig. 3A, B). In dividing cells, the M3A5 antibody decorated the new cell plate as well (Fig. 3C), and the plasma membrane may appear slightly stained (not shown). To check the consistency of the immunolabelling throughout the root tissue, methacrylate sections of the whole root were immunostained with M3A5. The punctate ¯uorescent pattern was found in each cell type (Fig. 3D). Staining of the plasma membrane was also clearly observed. The observed variability of plasma membrane staining by M3A5 in maize root squash techniques was observed for other antibodies known to stain the root plasma membrane such as JIM84 (compare Fig. 4A and Fig. 5). It may be related to the 0.5% Triton treatment needed to permeabilize the membranes. This M3A5 ¯uorescent pattern resembled in many respects the typical immunostaining of plant Golgi stacks by JIM84 (Satiat-Jeunemaitre and Hawes, 1992). Indeed, JIM84 staining revealed a ¯uorescent punctuated pattern of ~1 mm structures throughout the cytoplasm and a plasma membrane staining (Figs 4A, 5A). This resemblance suggested either an association or a co-localization of b-COP with Golgi stacks. To check this, double immunostaining of maize root cells with JIM84 (a Golgi marker, Horsley et al., 1993) and M3A5 antibody was performed. Observations were made on both control (Fig. 5A±F) and BFA-treated cells (Fig. 5G±I). BFA induced typical Golgi morphological changes in maize root cells. After 1 h of BFA treatment (100 mg ml±1), the 1 mm Golgi stacks stained with JIM84 coalesced in the cell to form larger ¯uorescent domains typical of BFA compartments (Figs 4B, 5G). When BFA-treated cells were immunostained with the b-COP antibody M3A5 (Fig. 5H), the ¯uorescent pattern was changed in the same manner as that observed for Golgi membranes with JIM84 (Fig. 5G): membranes carrying the epitopes coalesced in two or three ¯uorescent areas. Dual immunostaining patterns showed that the proteins recognized by M3A5 were associated with Golgi membranes and they were not released upon BFA treatment into the cytosol, as already suggested by our biochemical data (Fig. 5I). Immunostaining pattern of other COPI components machinery in maize root cells g-COP and b¢-COP location was investigated by immuno¯uorescence. In maize root cells, antibodies raised against plant g-COP and mammalian b¢-COP proteins stained punctate structures similar to M3A5 or JIM84 (Fig. 6A, B). They also stained the cell plate (Fig. 6B; and data not shown), but not the plasma membrane. These staining patterns suggest that both of these proteins may be associated with the Golgi apparatus. This hypothesis was further reinforced by the two proteins' behaviour after BFA treatment: epitopes recognized either by g-COP or b¢-COP antibodies were gathered in ¯uorescent aggregates recalling BFA compartments (Fig. 6C, D), as were epitopes recognized by the b-COP antibody. However, small differences in Atg-COP and b¢-COP staining were observed when compared to M3A5 (anti-b-COP) staining. Both stained weaker and their ¯uorescent patterns were more irregular than those of JIM84 or M3A5. The location of Arf1p (p=plant), another potential protein partner for b-COP, was also investigated in this study. An antibody against Arf1p cloned from Arabidopsis thaliana (Pimpl et al., 2000) was tested on maize roots and BY-2 cells. The speci®city of the antibody was checked on western blots. It revealed a major protein band at 20 kDa as expected, and a minor band at 40 kDa (Fig. 2B), which may represent a dimer (Pasqualato et al., 2003). COPI components in plant cells 2057 Fig. 3. Immuno¯uorescence studies with M3A5 antibody. In maize root cells (A, C, D) as in BY-2 cells (B), a group of 8 cells, a punctate pattern throughout the cytoplasm was revealed by the anti-b-COP antibody, within numerous structures of ~1 mm. (C) In dividing cells, M3A5 antibody decorated the cell plate. (D) This ¯uorescent pattern was not dependent upon cell type or position, as seen on this root section (methacrylate embedding). Plasma membrane was slightly stained on methacrylate sections, and in some root cells. Bar units in mm. Fig. 4. BFA effects on Golgi distribution in maize: immuno¯uorescence by JIM84. Golgi staining in maize root cells by JIM84. (A) The Golgi apparatus is comprised of numerous units dispersed throughout the cytoplasm. Plasma membrane is usually stained. (B) After BFA treatment (100 mg ml±1 for 1 h), typical BFA compartments formed from Golgi membranes; plasma membrane remained positive. Bar units in mm. In maize root cells, immunostaining with Arf1p antibody gave a punctate pattern (Fig. 7A). Double immunostaining with JIM84 con®rmed that Arf1p was partly associated with Golgi membranes (Fig. 7D±F). Moreover, it also appeared to be associated with other membraneous structures: a stained radiating network can be seen, with 2058 Couchy et al. Fig. 5. Double immunolocalization of epitopes recognized by JIM84 (anti-Golgi) and M3A5 (anti-b-COP) in control (A±F) and BFA-treated (G± I) maize root. When roots were probed with both JIM84 (A, D, G) and M3A5 (B, E, H), a clear co-localization of the antigens within the cell was observed at the level of the Golgi units (C, F, I) both in interphase cells (A±C), in dividing cells (D±F) as well as in BFA-treated cells at the level of the BFA compartments (G±I). Co-localization at the plasma membrane was also detected in some cases (F). Bar units in mm. staining of the plasma membrane and a light staining of the nuclear membrane (Fig. 7A, E); the cell plate was also stained in dividing cells (Fig. 7B). BFA treatment induced a redistribution of Arf1p into two or three ¯uorescent compartments (Fig. 7C). Double immunostaining con®rmed that Arf1p was mostly associated with BFA compartments where Golgi membranes concentrate (Fig. 7G±I), even in dividing cells (Fig. 7J±L). These results suggest that, in maize roots, epitopes recognized by b¢-COP, g-COP and Arf1p antibodies are indeed associated with Golgi membranes as is the b-COPlike protein. The four proteins behave in the same way: redistribution concomitant with Golgi membranes into BFA compartments and decoration of the cell plate. Discussion The aim of this work was to identify a speci®c tool to study plant b-COP proteins, to examine their cellular location and BFA reactivity, and to question the role of these proteins in the morphodynamics of the plant Golgi apparatus. For mammalian cells, it is claimed that the coatomer is involved in the recruitment of Golgi membrane areas, forming COPI vesicles. COPI-coated structures have been shown to be involved in the bi-directional membrane exchange between the endoplasmic reticulum (ER) and Golgi complex, and within the Golgi complex (Nickel and Wieland, 1997; Orci et al., 1997; Harter, 1999). It is unclear whether these models, derived from genetic, biochemical and morphological dissection of mammalian and yeast cells, also pertain to plant cells. In favour of this later claim lie the facts that: (i) pro®les of budding COPlike vesicles have been described on Golgi membranes (Hawes et al., 1996; Pimpl et al., 2000); (ii) homologues of COPI vesicular transport machinery have been identi®ed in various databases or cloned (Regad et al., 1993; Andreeva et al., 1998; Movafeghi et al., 1999; Pimpl COPI components in plant cells 2059 Fig. 6. Immuno¯uorescence study with plant g-COP and mammalian b¢-COP antibodies in control and BFA-treated maize root. (A, C) Staining with anti-g-COP antibody. (A) In control cells, a punctate pattern was observed within the cytoplasm. (C) After BFA treatment (100 mg ml±1, 1 h), ¯uorescence was redistributed into two or three zones recalling Golgi-derived BFA compartments. (B, D) Staining with anti-b¢-COP antibody. (B) Numerous ~1 mm structures were revealed in the cytoplasm, as also the cell plate in dividing cells. No staining of the plasma membrane was observed. (D) After BFA treatment, ¯uorescence was redistributed into areas recalling Golgi-derived BFA compartments. et al., 2000); and (iii) g-COP and Arf1p, two components of COPI vesicle formation (Kreis et al., 1995), have been localized on plant Golgi membranes (Pimpl et al., 2000; Ritzenthaler et al., 2002; Takeuchi et al., 2002). However, plant cell compartmentation, plant Golgi apparatus organization, functions, and reactivity to pharmacological agents are different from those of other eukaryotic cells (Satiat-Jeunemaitre et al., 1996; MeÂrigout et al., 2002) and, therefore, the role of putative COP vesicles in plant Golgi morphodynamics has still to be de®ned. Plant b-COP is associated with Golgi membrane dynamics These biochemical and immuno¯uorescence studies suggest that the M3A5 antibody recognizes a b-COP-like protein in plant cells. The hypothesis was further reinforced by MALDI-TOF mass spectrometry analysis of the proteins immunoprecipitated with M3A5, showing signi®cant homology with rat b-COP (data not shown; Couchy, 2001). Such `b-COP like' protein, having strong similarities with the rat b-COP, has recently been cloned in A. thaliana (GenBank accession number AL161259.2). In addition, it was shown by immunolabelling that putative b-COP, b¢-COP, g-COP, and Arf1 proteins are localized on Golgi membranes of maize root cells. These data complement the analysis of Contreras et al. (2000) on rice extracts where all COPI subunits seem to coimmunoprecipitate with antibodies against b¢-COP. They are also in agreement with the in vitro and in vivo data recently obtained with other plant cells (Pimpl et al., 2000; Ritzenthaler et al., 2002; Takeuchi et al., 2002). Therefore, all the potential machinery for COPI vesicle formation (i.e. coatomer complex and Arf1 proteins) are co-detected on Golgi membranes, suggesting a role for COPI vesicles in plant Golgi morphodynamics. Whether the function for COPI vesicles essentially concerns the 2060 Couchy et al. Fig. 7. Immuno¯uorescence study with anti-Arf1p antibody and Golgi marker JIM84 on maize root cells, in control and BFA-treated cells. In interphase cells (A, cell on the right; E), the anti-Arf1p antibody stains 1 mm structures dispersed throughout the cytoplasm, but also labels the nuclear membrane and the plasma membrane. Moreover, in dividing cells (B, telophase), staining revealed the cell plate. After BFA treatment, the anti-Arf1p antibody was redistributed into ¯uorescent aggregates; plasma membrane remained positive (C, H, K). Double localization with the Golgi marker JIM84 (D, G, J) and Arf1p (E, H, K) shows a clear co-localization between the recognized epitopes as seen on the merged images (F, I, L), in control cells (F) as in BFA-treated cells (I, L). This co-localization concerns plasma membrane and the cell plate as well. recycling of Golgi membrane components through a retrograde movement (trans to cis cisternae recycling pathway) or not has still to be studied. Furthermore, it may be noted that the vesicular or tubular form of coatomercoated structures is, in fact, still the subject of vigorous debate beyond the scope of this discussion, but suggesting that the roles for the coatomer in Golgi membrane transformation has still to be precisely established. Is there an additional function for b-COP proteins in plant cells? Besides Golgi staining, two novel staining features with COPI antibodies were described in this study: in dividing cells, proteins recognized by Arf1, b-COP, b¢-COP, and g-COP antibodies were localized on cell plates. Moreover, in maize root cells, b-COP and Arf1p antibodies also COPI components in plant cells lightly stained the plasma membrane. Proteins recognized by anti-b-COP have been found in a maize plasma membrane fraction (P Moreau et al., personal communication). Arf1 is not found on the plasma membrane of suspension cells (Ritzenthaler et al., 2002; Takeuchi et al., 2002). However, Arf1p was found in several membrane fractions of yeast cells, suggesting an association with multiple membrane compartments as well as an enrichment at the plasma membrane (Yahara et al., 2001). Does the presence of Arf on the plasma membrane or other membraneous fractions bear any relation to the COPI machinery? The fact that both Arf1 and b-COP proteins are localized on the cell surface and that all components of the COPI machinery observed in this study stain the new cell plate suggest additional functions for COPI components in plant cells. It has been shown that Golgi stacks accumulate near the growing edge of the cell plate (NebenfuÈhr et al., 2000). (i) The localization of COPI components on the cell plate may represent an ultimate step of the recycling movement by COPI vesicles between the new plasma membrane being formed (made of Golgi-derived membranes) and the Golgi stacks (part of the trans-to-cis cisternae recycling pathway). This continuous cycling of COPI vesicles would ensure a retrieval of Golgi membrane domains from the newly forming plasma membrane and would help the plasma membrane to acquire its identity. (ii) The cell plate is known to be rich in clathrin coated vesicles, involved in endocytosis, i.e. recycling events between plasma membrane and endosomal compartments (Samuels et al., 1995). The staining of the cell plate by COPI antibodies may be linked to these endocytic events. In animal and yeast cells there is a clear involvement of Arf1 in endocytosis (Gaynor et al., 1998) and a function for b-COP in the endocytotic pathway has been suggested as well (Aniento et al., 1996). As a whole, b-COP may be part of the molecular machinery recycling Golgi proteins, but also determining the identity of the new cell surface. Molecular implications of the reactivity of Golgi and coatomer components to Brefeldin A The reactivity of the plant Golgi to BFA varies according to the plant cell type studied and can differ from what is usually described for mammalian cells (Satiat-Jeunemaitre et al., 1996; Ritzenthaler et al., 2002; MeÂrigout et al., 2002; Saint-Jore et al., 2002). Is this diversity in Golgi reactivity to BFA mirrored by differential behaviour of COPI type vesicles? What might be learnt from these BFA data concerning the function of COPI components? In mammalian and yeast cells, the prime molecular target for BFA is a guanine nucleotide exchange factor (GEF) responsible for the conversion of Arf-GDP to ArfGTP (Donaldson et al., 1992; Pasqualato et al., 2003). Therefore, in the presence of BFA, Arf is not attached to 2061 the membranes, and the recruitment of the coatomer to the membrane mediated by Arf anchoring is no longer achieved. Consequently, b-COP proteins are released into the cytosol within minutes of treatment and, later, most of the Golgi apparatus is redistributed within the ER compartment (see review in Scales et al., 2000; Presley et al., 2002). So, in that case, alteration of the Golgi morphology appears to be directly caused by the release of Arf and COP components into the cytosol. In maize root cells, the situation may be different: BFA induced a reorganization of the Golgi in a tissuespeci®c manner, as Golgi markers formed aggregates that were previously named `BFA compartments' (SatiatJeunemaitre and Hawes, 1993). These BFA compartments have been observed in various root materials (Wee et al., 1998; Baldwin et al., 2001; Jin et al., 2001). They are made of Golgi-derived vesicles and accumulated secretory products (Satiat-Jeunemaitre and Hawes, 1993; SatiatJeunemaitre et al., 1996). Arf1p, b-COP, g-COP, and b¢-COP proteins stay associated (or continue to cycle) with the Golgi-derived membranes trapped in these BFA compartments. Therefore, in contrast to what has been described for mammalian cells, they do not appear to be released into the cytosol. Regarding g-COP staining, the results on maize root cells slightly differ from those decribed for the BY-2 cell line, where g-COP was dissociated from the Golgi membrane after 5 min of BFA treatment (Ritzenthaler et al., 2002). This difference in g-COP reactivity to BFA is probably due to tissue organization, as in Arabidopsis roots it is also distinct from observations upon BY-2 cells (Geldner et al., 2003). Further studies are needed to understand g-COP behaviour and function in maize root cells better. The observations that substantial amounts of Arf1 remain associated with the Golgi stacks after BFA has already been reported in biochemical studies with other plant material (Fig. 9 in Ritzenthaler et al., 2002; Fig. 7 in Pimpl et al., 2000), and in immunocytological studies in transgenic BY-2 cell lines (Fig. 10 in Ritzenthaler et al., 2002). Surprisingly, this fact has sometimes been neglected when discussing BFA action. For instance it is claimed that `one of the ®rst detectable effects of BFA treatment of BY-2 cells is the nearly complete loss of COPI coat proteins (as judged by Atg-COP and AtArf1) from Golgi stacks, an observation that is identical to that from BFAtreated mammalian cells' (Ritzenthaler et al., 2002). This claim implies that `BFA causes the release of AtArf1 and COPI from the GA, with a subsequent alteration of the GA morphology' (see quotation in Takeuchi et al., 2002). By contrast, for maize roots, data suggest that alteration of GA morphology is not subsequent to AtArf1 release. Based on b-COP and Arf1 immunolocalization after BFA treatment, it is deduced that, in maize root cells, the Golgi apparatus was reorganized even when a signi®cant amount of Arf1p 2062 Couchy et al. and b-COP remained attached to the membranes. This situation contrasts with what happens in animal cells, where Arf release occurs in 15±30 s after BFA, concomitantly or before the Golgi reorganization (Presley et al., 2002). The failure of BFA to release b-COP and Arf proteins from the Golgi does not necessarily mean that the drug's target or its mechanism of action differ from that in animals. However, it suggests that additional molecular targets may be involved in the disruption of plant Golgi dynamics in response to BFA. BFA may well have multiple molecular actions in plants, as it also inhibits the synthesis of speci®c lipids of the plant Golgi (MeÂrigout et al., 2002). An alteration of membrane lipids by BFA may alter the anchoring of Arf1p, and/or the steady state of coatomer attachment/recycling to the membrane. Acknowledgements We are very grateful to Dr Reiner Pepperkok (Heidelberg) for providing antibodies against b'-COP, and to Professor David Robinson (Heidelberg) for providing antisera against g-COP and Arf1p. 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