JXB Advance Access originally published online on June 25, 2008
Journal of Experimental Botany 2008 59(11):3087-3098; doi:10.1093/jxb/ern162
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RESEARCH PAPER |
The POK/AtVPS52 protein localizes to several distinct post-Golgi compartments in sporophytic and gametophytic cells
1INRA UR254, Station de Génétique et d'Amélioration des Plantes, Institut Jean-Pierre Bourgin, Centre de Versailles-Grignon, F-78026 Versailles, France
2Laboratoire de Dynamique de la Compartimentation Cellulaire, Institut des Sciences du Végétal, CNRS UPR2355, F-91198 Gif sur Yvette, France
3Department of Biological Sciences, University of Durham, South Road, Durham DH1 3LE, UK
* To whom correspondence should be addressed. E-mail: bonhomme{at}versailles.inra.fr
Received 11 February 2008; Revised 28 March 2008 Accepted 13 May 2008
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Abstract |
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The organization and dynamics of the plant endomembrane system require both universal and plant-specific molecules and compartments. The latter, despite the growing wealth of information, remains poorly understood. From the study of an Arabidopsis thaliana male gametophytic mutant, it was possible to isolate a gene named POKY POLLEN TUBE (POK) essential for pollen tube tip growth. The similarity between the predicted POK protein sequence and yeast Vps52p, a subunit from the GARP/VFT complex which is involved in the docking of vesicles from the prevacuolar compartment to the Golgi apparatus, suggested that the POK protein plays a role in plant membrane trafficking. Genetic analysis of Arabidopsis mutants affecting AtVPS53 or AtVPS54 genes which encode putative POK partners shows a transmission defect through the male gametophyte for all lines, which is similar to the pok mutant. Using a combination of biochemical approaches and specific antiserum it has been demonstrated that the POK protein is present in phylogenetically divergent plant species, associated with membranes and belongs to a high molecular weight complex. Combination of immunolocalization studies and pharmacological approaches in different plant cells revealed that the POK protein associates with Golgi and post-Golgi compartments. The role of POK in post-Golgi endomembrane trafficking and as a member of a putative plant GARP/VFT complex is discussed.
Key words: Arabidopsis mutants, GARP/VFT complex, Golgi and post-Golgi compartments, pollen, pollen tube
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The plant endomembrane system is the place of intense membrane trafficking events between distinct though highly dynamic compartments, that sustain key cell development processes such as growth, differentiation, or adaptation to external cues/stresses (for reviews see Surpin and Raikhel, 2004; Geldner and Jürgens, 2006; Robatzek, 2007). Major membrane compartments (endoplasmic reticulum (ER), Golgi apparatus (GA), prevacuolar compartment (PVC)/endosomes, and vacuoles), and their associated protein machineries required for vesicle formation and cargo recruitment, docking and fusion processes (e.g. COP (coat proteins), Rab GTPases, SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptor)), are collectively conserved among eukaryotic cells (for reviews see Sanderfoot and Raikhel, 2003; Jürgens, 2004; Képès et al., 2005). Nevertheless, the plant endomembrane system also shows specific features, according to different cell organization and needs. For instance, two main types of vacuoles have been described in plant cells (Paris et al., 1996; Marty, 1999), protein storage vacuoles (PSVs), and lytic vacuoles. Moreover, several types of intermediate compartments between the Golgi and the vacuoles have been reported, including prevacuolar compartments (PVC), multivesicular bodies (MVB), and endosomes (for reviews see Hawes and Satiat-Jeunemaitre, 2005; Mo et al., 2006). The exact mapping and functioning of these compartments, described on both secretory (anterograde) and endocytic (retrograde) pathways (Mo et al., 2006), is still being characterized. Identification of post-Golgi compartments is often based on the location of specific markers/proteins: a type of PVC compartment is labelled by a fluorescent tagged AtRabF2b GTPase protein (Kotzer et al., 2004) or using the m-Rabmc antiserum (Bolte et al., 2004); meanwhile distinct endosomal compartments, named after the localized proteins, have been identified by Geldner et al. (2003, the GNOM compartment, see below), and Jaillais et al. (2006, the SNX1 compartment). Protein machineries associated with the dynamics of the plant cell endomembrane system also show plant-specific features, although displaying sequence homologies with yeast or mammalian components. The GNOM protein cited above is an ARF-GEF protein (Guanine-nucleotide exchange factor for ADP-ribosylation factor GTPase), required for polar auxin transport, and vesicle recycling from endosomes to the plasma membrane (Geldner et al., 2003). Teh and Moore (2007) and Richter et al. (2007) concomitantly showed that the closest homologue to GNOM in Arabidopsis, GNOM-like 1, has kept the ARF-GEF function at Golgi membranes common to all eukaryotes, while the GNOM gene has acquired an additional plant-specific role in endosome trafficking. Another case highlighting specific features of the plant endomembrane system is the SNARE protein family, the key components of the vesicle docking and membrane fusion processes. Whereas 26 and 39 SNARE genes are found in the yeast and human genomes, respectively, up to 60 genes have been identified in land plants so far (reviewed in Sanderfoot, 2007). SNARE proteins from Arabidopsis have been localized in transient assays to different endomembrane compartments (ER, GA, and several post-Golgi compartments) and a few cases of multiple localization patterns have been observed (Uemura et al., 2004; Chatre et al., 2005). Moreover, additional and/or plant-specialized roles, in relation to their sedentary life cycle and their ability to respond to environmental changes have been shown (e.g. in shoot gravitropism, Kato et al., 2002; osmotic stress response, Zhu et al., 2002; or pathogen resistance, Collins et al., 2003; for a review see Sutter et al., 2006).
The examples above and other published work (reviewed in Jürgens, 2004; Surpin and Raikhel, 2004) illustrate that both direct and reverse genetics strategies in Arabidopsis thaliana have been used to establish the function of the molecular components of the plant membrane trafficking system. Using these approaches, we recently hypothesized that the plant AtVPS52/POK protein could play an essential role in plant-specific membrane trafficking events (Lobstein et al., 2004). Indeed, following a screen for Arabidopsis male gametophytic mutants, a number of T-DNA insertion lines showing a transmission defect that was related to altered pollen development were isolated and characterized (Bonhomme et al., 1998; for a review see Guermonprez et al., 2006). In a line that was named poky pollen tube (pok), mutant pollen tubes remain very short compared to the wild type, as a consequence of hampered or much reduced tip-growth (Lobstein et al., 2004). The mutated POK gene is homologous to the yeast VPS52 gene which encodes one of the subunits of the GARP (Golgi-associated retrograde protein)/VFT (Vps Fifty Three) complex (Conibear and Stevens, 2000; Conibear et al., 2003). This large tetrameric complex, composed of Vps51/52/53/54p, is required for retrograde transport from the vacuolar/late endosome compartment to the Golgi apparatus (Conibear and Stevens, 2000). Genes encoding homologues of the three Vps52p, 53p, and 54p subunits have been found in plant and animal genomes (Walter et al., 2002; Lobstein et al., 2004), and a human GARP/VFT complex has been reported (Liewen et al., 2005). In Arabidopsis, the observation of the pok mutant phenotype suggests that the AtVPS52/POK protein, as part of a putative GARP/VFT complex, is involved in membrane trafficking events that are essential for pollen tube apical growth. Moreover, it has been shown that the POK gene is expressed in most plant tissues, although at higher levels in roots and buds indicating that its role is not restricted to tip growth. Transient co-expression of a POK:GFP fusion with a Golgi marker:RFP in onion epidermal cells indicated an association of the POK protein with the Golgi membrane (Lobstein et al., 2004).
In this study, we argue for the involvement of the POK protein in post-Golgi endomembrane trafficking. Characterization of Arabidopsis mutant lines affecting the three genes encoding potential plant GARP subunits shows that all of them exhibited a male transmission defect, like the original pok mutant. A specific polyclonal antiserum was generated against the POK protein and used for both biochemistry and immunocytochemistry experiments. Combining observations from confocal laser scanning microscopy and pharmacological experiments, the association of POK with at least the Golgi apparatus and a prevacuolar compartment is demonstrated, suggesting the involvement of POK in membrane trafficking between these two compartments. A third unknown POK-labelled compartment was observed in the cells. Finally, these results permitted the question of the function of a putative GARP/VFT complex in both sporophytic and gametophytic plant cells to be addressed.
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Materials and methods |
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Plant material
The Arabidopsis pok mutant was in ecotype Wassilewskija as described in Bonhomme et al. (1998). Other vps mutants originated from the SALK Institute (in Columbia accessions, vps52-2, vps53-1, and vps 54-1, -2, and -3).
Arabidopsis tissues were collected from 8-week-old Arabidopsis plants, grown as described in Bonhomme et al. (1998), except for roots that were collected from hydroponically grown seedlings. Pollen was collected by differential low speed centrifugation in 10% sucrose. Arabidopsis suspension cells were grown at 25 °C for 4 d. Cells were maintained in 100 ml of liquid growth medium containing 4.6 g l–1 MS salts with vitamins (Sigma, France) and 30 g l–1 sucrose, at 25 °C with gentle agitation in the light. Immunocytochemistry observations were made on 4-d-old culture.
Maize caryopses (Zea mays, LG20.80, Limagrain, France) were grown as in Satiat-Jeunemaitre and Hawes (1992).
BY-2 tobacco (Nicotiana tabacum) suspension-cultured cells were grown as described in Couchy et al. (1998), and immunocytochemistry observations performed on 3-d-old cells (middle of the exponential phase of growth). Tobacco pollen grains were germinated in glass vials in 2 M NiCa2+, 10% sucrose, 0.01% H3BO3, for 3 h at room temperature under gentle agitation (20 rpm).
Daffodil (Narcissus pseudonarcissus) pollen grains were germinated in vitro as described by Heslop-Harrison and Heslop-Harrison (1992).
Mutant isolation and analysis
Insertion mutants in AtVPS genes (SALK lines) were selected using the SIGnAL ‘T-DNA Express’ Arabidopsis gene mapping tool (http://signal.salk.edu/cgi-bin/tdnaexpress; Alonso et al., 2003) and obtained from the Nottingham Arabidopsis Stock Centre. For each line, the NASC identification number is indicated between brackets.
T-DNA insertion locus were checked using the following primers in combination with LBsalk1 (5'-CATCAAACAGGATTTTCGCC): for vps52-2 (N55433 [GenBank] ), 5'-CTTTTGCAAGGGTCATGATGG; for vps53-1 (N547230), 5'-TCCTCGGTTTCTTGACTAGC; for vps54-1 (N536485), 5'-AAAAATCAGATTCGAGCGAT; for vps54-2 (N5062261), 5'-ATTACTTCCAAGTCGGGTCT; for vps54-3 (N580006), 5'-CTTCGCCTGCTTCTTCTTCTCT.
To estimate the male and female transmission ratios, progenies from the crosses were genotyped by PCR, using the primers described above, following rapid DNA extraction as described in Loudet et al. (2002). PCR conditions were standard (30 s 94 °C, 1 min annealing, 2 min 72 °C, 30 cycles).
The pollen phenotype was checked using Alexander's stain, as previously described by Procissi et al. (2001).
POK antiserum design
A polyclonal antiserum has been raised in rabbits against the first 212 amino acids of the predicted POK protein by Biogenes (Berlin). The POK cDNA (Lobstein et al., 2004) was subcloned into pCR BluntII TOPO vector (Invitrogen) and digested by SacI and BamHI. The resulting 630 bp cDNA fragment was cloned into expression vector pET-30c (Novagen, The Netherlands) and the final plasmid was transformed into E. coli BL21 cells (Novagen). Upon IPTG induction, the recombinant protein accumulated in the soluble fraction and was extracted in the binding buffer containing 50 mM TRIS–HCl, pH 8, 150 mM NaCl, and 5 mM imidazol. The resulting suspension was centrifugated at 10 000 g for 10 min. The supernatant was loaded on a 1 ml HiTrap chelating column (GE Healthcare) connected to the Äkta Prime system. The resin was washed with 20 ml binding buffer at a flow rate of 0.5 ml min–1 and the protein was eluted using a gradient of imidazol (20–500 mM). Fractions (0.5 ml) containing AtPOK were pooled and the total amount of recombinant protein was determined with the Biorad Protein assay kit according to the manufacturer's instructions.
Protein extractions and solubility tests
For western blot experiments plant material was ground in liquid nitrogen using a pestle and mortar, the tissue powder was resuspended in lysis buffer (50 mM TRIS, 100 mM NaCl, 5% glycerol, with protease inhibitors: 1 mM AEBSF, 1 µg ml–1 leupeptin) and incubated for 20 min at 4 °C with gentle agitation. Samples were then centrifugated for 15 min at 13 000 g, the supernatants were collected, and the protein concentration was measured in each supernatant using the Bradford assay.
For solubility tests, lysis buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 5% glycerol, 2 mM DTT supplemented with protease inhibitors) was added to the ground roots in liquid nitrogen and the suspension was filtered through Miracloth, then centrifugated at 26 000 g for 30 min in a TLA100.2 Beckman rotor. The supernatant containing soluble proteins according to Schein (1990) was collected, and the pellet was incubated for 30 min in lysis buffer supplemented with 1 M NaCl, 200 mM NaCO3 pH 11, or 2% SDS. Solubilized membrane proteins (S) were recovered in the supernatant after the second centrifugation at 48 000 rpm for 30 min. The remaining pellet (P) was resuspended in Laemmli buffer (Laemmli, 1970).
SDS-PAGE and immunoblot analysis
Protein extracts, prepared by boiling samples for 10 min in Laemmli buffer (Laemmli, 1970) containing 2.5% 2-β-mercaptoethanol, were separated in 10% SDS-PAGE (Bio-Rad system). Proteins were blotted on to PVDF membrane (Immobilon, Millipore, Billerica, USA) previously activated with 100% methanol, in a Trans-Blot SD semi-dry transfer-cell (Bio-rad, Hercules, USA), between Whatman 3MM papers wetted with transfer buffer (10% methanol (v/v), 120 mM Glycine, 15.6 mM TRIS) as described by the manufacturer. Membranes were dried at room temperature, then blocked with TRIS-buffered saline (TBS) containing 5% (w/v) fat-free dry milk powder for 1 h. Membranes were incubated in a 1:1000 dilution of POK antiserum or preimmune serum for 2 h at room temperature, then washed three times for 10 min in TBS-T (TBS, 0.1% Tween 20), then incubated in a 1:10 000 dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (Sigma) for 30 min. After six washes for 5 min in TBS-T, secondary antibodies were revealed by chemiluminescence with the Lumiglo kit (KPL, Gaithersburg, USA) according to the manufacturer's protocol.
Gel filtration assays
1 g of Arabidopsis root was gound and resuspended in 600 µl of lysis buffer (50 mM TRIS pH 8, 100 mM NaCl, 1 mM DTT with protease inhibitor). The sample was centrifugated at 3000 g for 10 min, followed by 100 000 g for 30 min, at 4 °C. Gel filtration chromatography was performed using a fast liquid chromatography system (Akta-purifier, Amersham Biosciences) with a high resolution Superdex 200 column. Column equilibration and chromatography were performed with lysis buffer. The collected supernatant (0.5 ml) was loaded onto the column and separated at flow rate of 0.5 ml min–1. Fractions (0.5 ml) were collected and probed with the POK antiserum after SDS–PAGE. The column was calibrated using the gel filtration size standard kit (Bio-Rad): thyroglobulin (670 kDa), 
-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B-12 (1.35 kDa).
BFA treatments
Brefeldin A (BFA, Sigma) was used at 100 µg ml–1 (stock solution 20 µg ml–1 in dimethylsulphoxide). Control samples were incubated in corresponding concentrations of DMSO. Drug treatments were performed by immersing the maize roots in the treating solutions at 22 °C for 1 h.
Immunolocalization
POK rabbit polyclonal antiserum and preimmune serum were used at a dilution of 1:500.
Golgi markers [polyclonal anti-plant Lewis antibodies, Fitchette-Lainé et al. (1997) and JIM84 monoclonal antibody, Satiat-Jeunemaitre and Hawes (1992)] were used at 1:1000 dilution and undiluted, respectively. m-Rabmc rabbit polyclonal antiserum (Bolte et al., 2004) was used at a dilution of 1:250.
The following secondary antibodies were used according to the manufacturers’ instructions: Anti-rabbit F(ab’)2 fragment coupled to fluorescein isothiocyanate (FITC), anti-rabbit F(ab’)2 fragment coupled to cyanine3 (Jackson Immunochemicals, USA), anti-rabbit IgG coupled to FITC (Sigma, France), anti-rabbit IgG coupled to Cy3 (Sigma, France), anti-rabbit IgG coupled to Alexa 546 (Molecular Probes, USA), and anti-rat IgG coupled to FITC (Sigma, France).
The double labelling with two rabbit primary antibodies was performed as described in Bolte et al. (2004).
Indirect immunofluorescence labelling procedures in Arabidopsis cells, BY-2 cells, and maize root squashes were performed as previously described (Satiat-Jeunemaitre and Hawes, 2001; Bolte et al., 2004).
Pollen samples from daffodils and Nicotiana tabacum pollen tubes were processed for immunolocalization as described, respectively, in Smertenko et al. (2001) and Satiat-Jeunemaitre and Hawes (2001).
Confocal microscopy and image processing
Slides were observed and images were collected with a spectral confocal microscope SP2 (Leica Microsystems) equipped with an Ar laser and a HeNe laser, with the exception of the daffodil images that were obtained with a Zeiss 510 confocal microscope also equipped with both lasers. The fluorochromes were detected using laser lines 488 nm (FITC) and 543 nm (Alexa 546, Cy3). The images were pseudocoloured in green in the case of FITC and in red in the case of Alexa 546 and Cy3; colocalization appeared in yellow on the merged images. The oil objectives used were x63 (Numerical aperture 1.30), giving a resolution of approximatively 200 nm in the XY-plane and 400 nm along the Z-axis (pinhole 1 Airy unit). Each image shown represents either a single focal plan or a projection of individual images taken as a Z series. For the specimens labelled with different fluorochromes, images corresponding to each fluorochrome were acquired sequentially. Images were processed using Adobe Photoshop Software.
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Results |
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Mutations in Arabidopsis genes encoding putative homologues of Vps52p partners
It has been shown that both AtVPS53 and AtVPS54 genes, encoding putative homologues of yeast Vps52p partners have similar transcription profiles, and, like POK, are expressed in most plant tissues, with higher levels detected in roots and buds (Lobstein et al., 2004). Therefore, it is suspected that those three putative proteins, POK (AtVPS52), AtVPS53, and AtVPS54, could associate in a plant GARP-like complex. It is postulated that mutations in genes encoding subunits of the same complex would show similar phenotypes. In order to compare their phenotype with the pok/vps52p original mutant, T-DNA insertion mutants affecting Atvps53 or Atvps54 genes (vps53-1, and vps54-1, -2, and -3), as well as a mutant allelic to pok (vps52-2) were selected through reverse genetics (Fig. 1). As for pok, only hemizygous plants were observed for four out of the five vps mutations, that segregated 1:1 versus wild-type plants in selfing progeny (Table 1), suggesting that those were also gametophytic mutations. In the selfed progeny of the vps54-3 line, however, 10% of homozygous plants were found. The study of the transmission ratio of the T-DNA insertions in the vps lines in crossing progeny confirmed that these were gametophytic mutations. All vps mutants showed a transmission defect of the mutation when used as male parent on wild-type plants (Table 1). The transmission ratios through the pollen were quite similar in vps52-2, vps53-1, and vps54-1 and -2, varying from 18.5% to 25%. For the vps54-3 allele, the transmission ratio of the mutation reached 40%, consistent with the observation of homozygous progeny. These figures, when compared to the 0.5% of male transmission of the original pok mutant (Bonhomme et al., 1998), suggest a weaker effect of the mutations on the male gametophyte. Transmission of the vps mutations through the female gametophyte was normal, as for pok, except for the vps54-1 mutant (Table 1). Using this line as female in crosses with wild-type plants, 14% of the progeny carried the mutant allele.
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The phenotype of the vps mutants was further described by staining anthers with Alexander stain, in order to check for mature pollen viability. As for the pok mutant, the mature pollen of the vps mutants was identical to wild-type pollen, that is to say all pollen grains were normally stained (Table 1), suggesting that the observed transmission defects through the male gametophyte resulted from later defects in pollen development, presumably at pollen tube germination and/or growth.
As no homozygous progeny was obtained for the vps mutants, except for the vps54-3 allele whose insertion lies close to the end of the VPS54 gene, it is supposed that these male gametophytic mutants might also be embryo-lethal. Siliques from hemizygous vps mutants showed lower seed set compared with wild type (Table 1). For all lines but one (vps54-1), the average number of seeds was consistent with the conclusion that they were homozygous lethal (a quarter of missing seeds versus wild type). In the vps54-1 allele, the seed set was estimated as less than half of the wild-type seed set; this result is expected, as this mutation also exhibited a female gametophytic effect. The vegetative development of all vps mutants, including homozygous vps54-3 progeny, was similar to that of wild-type plants (not shown). Characterization of mutant lines in all three POK/AtVPS52, AtVPS53, and AtVPS54 genes thus indicated that all were male gametophytic, in agreement with the hypothesis that all three encoded VPS proteins would be involved in the same process.
The POK protein is found in most organs of Arabidopsis, and in all tested plant species
To investigate POK protein expression, the antiserum raised against the first 212 amino acids of the POK protein was used for western blot analyses of several plant protein extracts. In all tested Arabidopsis protein extracts, from pollen, buds, leaves, and roots, the POK antiserum recognized a major 80 kDa band on immunoblots consistent with the 80 kDa predicted molecular weight for the POK protein (Fig. 2A, lower arrow). Immunoblots using preimmune serum showed no signal (Fig. 2A, lane Pre). The presence of lower molecular weight bands in root extracts was interpreted as protein degradation. Extracts from the hemizygous pok mutant showed, in addition to the expected 80 kDa main band, a lighter 120 kDa band observed in both buds and roots samples (Fig. 2A, right panel, upper arrow). This might correspond to the product of the translational GUS fusion that occurred in the pok mutant following the T-DNA insertion (Lobstein et al., 2004), as the POK antiserum was raised against the N-terminal part of the protein. A major 80 kDa band was also observed in protein extracts from maize roots, daffodil buds, and BY-2 tobacco suspension cells (Fig. 2B), indicating that POK is conserved amongst plant species. Two bands were detected in the daffodil bud extract, the 80 kDa band corresponding to the full-length POK and one below which is likely to be the result of protein degradation.
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The POK protein is part of a large protein complex
To test the incorporation of the POK protein into a complex, gel filtration chromatography was performed using Arabidopsis root protein extracts in native conditions. Following gel filtration, proteins from the different fractions were separated by SDS-PAGE and analysed by western blotting using the POK antiserum (Fig. 2C). The POK signal at 80 kDa was detected in fractions for which high molecular weight complexes (higher than 158 kDa) were eluted, therefore suggesting that the POK protein could be a subunit of a large complex.
The POK protein is associated with membranes
The western blotting of soluble and membrane protein fractions from Arabidopsis roots detected POK in both fractions (data not shown), suggesting that at least part of the POK protein pool was probably associated with a membrane compartment. In order to test the strength of this association, solubility tests were performed on the membrane fraction, using salt (NaCl), alkaline (NaCO3), and detergent (SDS) treatments (Fig. 2D). The POK protein remained associated with the membrane fraction and remained in the pellet following salt and alkaline treatment, but could be extracted by SDS (Fig. 2D), indicating a strong association of POK with a membrane compartment.
The POK protein accumulates in intracellular compartments in both sporophytic and gametophytic tissue
To study the subcellular localization of POK, immunolocalization experiments were performed using the POK antiserum on different tissues and plant species (Fig. 3). No labelling was obtained with the preimmune serum, confirming the specificity of the antiserum (Fig. 3D). On Arabidopsis suspension cells, the antiserum revealed an intracellular punctate pattern, with no labelling of the plasma membrane. In these interphase cells, labelled dots of approximatively 1 µm diameter were homogeneously distributed in the cytoplasm (Fig. 3A). A similar punctate pattern was observed in maize root interphase cells (Fig. 3B) and BY-2 suspension (Fig. 3C). POK localization was also punctate in pollen (Fig. 4). except that spatial distribution of the POK-positive dots seemed to vary depending upon the pollen tube growth stage.
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In daffodil ungerminated pollen grains, the punctate POK labelling appeared homogeneously distributed in the cytoplasm (Fig. 4A) occasionally showing accumulation in the probable pollen tube emergence site (Fig. 4A, arrow). Two patterns of POK staining were observed in the elongating pollen tubes. Dots of 1 µm diameter were either found all over the growing tube (Fig. 4B), or concentrated in the tip of the cell, with poor labelling of the rest of the tube (not shown). As in sporophytic cells, plasma membrane labelling was never observed. Similar variations of POK labelling were also observed in tobacco pollen tubes: the POK staining was either accumulated in the tip (Fig. 4C) or dispersed in the pollen tube cell (Fig. 4D–E).
To summarize, immunolocalization experiments demonstrated that the POK protein is found as a punctate signal, in plant pollen grains and pollen tubes, but also in sporophytic cells from all tested species.
Identification of POK associated compartments
To identify the POK-labelled intracellular compartments in both sporophyte and gametophyte cells, double labelling experiments were performed using the POK antiserum and well-characterized compartment markers: the anti plant Lewis antibody (Lea) for Golgi apparatus (Fitchette-Lainé et al., 1997) and the anti-m-Rabmc antibody as a prevacuolar compartment (PVC) marker (Bolte et al., 2000).
In maize root cells, immunolabelling with Lea antibody/FITC (Fig. 5A, middle panel) reveals a typical Golgi apparatus localization pattern in plant cells. Golgi stacks are dispersed in an homogenous manner throughout the cytoplasm. In addition, labelled epitopes are also observed on the plasma membrane, a common feature of the Lewis epitope in maize root cells (Fitchette-Lainé et al., 1997; Boutté et al., 2006). When cells were double-labelled with this Lea antibody and the POK antiserum, only part of POK-labelled compartments (Fig. 5A, left panel) co-localized with the Lea-labelled compartments (Fig. 5A, right panel, yellow spots). This suggested that POK protein localizes at least on two endomembrane organelles, one being the GA, and another to be identified (Fig. 5A, right panel, red spots).
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The possibility that this second population could be prevacuolar compartments was addressed next. The PVC marker m-Rabmc antibody revealed a punctate pattern throughout the cytoplasm of maize root cells (Fig. 5B middle panel), identified as the PVC (Bolte et al., 2004). Double labelling with the POK antiserum (Fig. 5B, left panel) showed that a minor population of compartments were labelled by both POK and m-Rabmc antiserum (Fig. 5B, right panel, yellow spots).
Similar observations were made in male gametophytic cells: double immunolabelling with m-Rabmc and POK antiserum in tobacco pollen tubes showed that POK labelling (Fig. 5C, left panel) also partially co-localized with structures labelled by the m-Rabmc antiserum (Fig. 5C, right panel). Interestingly, POK/m-Rabmc overlaying dots were more numerous in the tip region, though the very end of the pollen tube was preferentially stained with m-Rabmc antiserum (Fig. 5C, right panel).
These data show that POK protein associates with distinct endomembrane compartments. The fact that POK protein is only partially associated with Golgi and PVC compartments suggests that POK antiserum might label a third compartment, not recognized by the Lewis nor m-Rabmc antiserum. Unfortunately, attempts to triple-localize POK antiserum with Golgi and PVC markers did not provide conclusive enough results to assess POK partitioning within the endomembrane system and Brefeldin A (BFA) was used to investigate the nature of the POK-labelled compartments.
In maize roots, GA and PVC react differently to treatment with BFA, a drug known to block secretion and induce alterations of the Golgi immunofluorescence labelling pattern (Boutté et al., 2006). Following BFA treatment, the 1 µm labelled Golgi stacks (Fig. 6A) coalesce to form larger fluorescent domains called BFA compartments (Fig. 6C, and see also Satiat-Jeunemaitre and Hawes, 1994; Boutté et al., 2006), while m-Rabmc labelled compartments exhibit no aggregation (Boutté et al., 2006). When BFA-treated maize cells were immunostained with the POK antiserum (Fig. 6D), the fluorescent POK pattern showed no major change. The dots did not coalesce in a larger compartment (Fig. 6D versus Fig. 6B) indicating that in BFA-treated maize root cells, POK protein remains associated with BFA-insensitive compartments such as the PVC. In Arabidopsis suspension cells, however, the POK immunolabelling pattern is altered upon BFA treatment: larger fluorescent domains are observed, suggesting a preferential association of the POK protein with the GA (not shown).
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Altogether, immunofluorescence labelling data show that the POK protein is localized on several compartments of the endomembrane system, mainly Golgi and post-Golgi compartments. Indeed, the punctate labelling patterns strongly resemble intermediate compartments of the secretory or endocytic pathway, such as trans Golgi network (TGN), MVB/PVC, or endosomal populations. These observations also outline the versatility of the POK partitioning within the Golgi and post-Golgi compartments, probably depending on the dynamics of the membrane trafficking in the observed cells.
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Discussion |
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POK localizes to several compartments of the post-Golgi endomembrane system in both gametophytic and sporophytic cells
Alignments of POK and other members of the GARP/VFT complex protein sequences had previously demonstrated their evolutionary conservation in worm, fly, and mammals (Walter et al., 2002; Lobstein et al., 2003; Liewen et al., 2005) suggesting that a similar protein complex exists in plants. Phenotypic analysis of Arabidopsis mutants in putative GARP/VFT complex components agree with this prediction. The reduced transmission frequency and the pollen phenotype indicate that both AtVPS53 and AtVPS54, like the POK gene, are important for pollen tube germination and/or growth. No homozygous plants were obtained for the four strongest vps alleles (our results, and Lee et al., 2006 for two vps53 mutant lines), and lower numbers of seed set in siliques of selfed plants were observed. Since the transmission ratios of vps mutant pollen were not null in crosses with wild-type plants, the absence of homozygotes suggest that the encoded proteins play an essential role at the earliest stages of embryo development. Variations of the transmission bias as well as of the seed set between the different vps mutants might simply be due to the different strengths of the mutations in the different alleles of the same gene and/or the functional contribution of the different Arabidopsis VPS proteins in the complex. In yeast, the vps51p subunit for which no homologous protein has been found in plants nor mammals, would have a specific regulatory role (Conibear et al., 2003), while the vps54p subunit would participate in complex stability through its N-terminal domain on the one hand, and to its specific localization through its C-terminus on the other hand (Quenneville et al., 2006).
Solubility tests in Arabidopsis conclude that POK is present in the membrane pellet and that it could be made soluble by SDS treatment. This suggests an attachment to the membrane, although the bioinformatic analyses do not predict any membrane binding domains in the POK sequence. The yeast GARP/VFT complex has been shown to be peripherally associated with TGN membranes, with the vps52p subunit being particularly involved in protein–protein interactions because of its insolubility in 1% Triton X-100 (Conibear and Stevens, 2000). The components of the human GARP/VFT complex have also been described as non-cytosolic despite the absence of predicted transmembrane domains (Liewen et al., 2005).
The localization of POK was analysed in gametophytic cells from daffodil and tobacco, and sporophytic cells from maize and Arabidopsis using a specific antiserum. In both types of cells the POK protein was detected in vesicle-like structures, agreeing with its association with membranes. Although the distribution of POK was quite homogenous in the cytoplasm of somatic cells, its localization in pollen grains and pollen tubes depended upon the stage of pollen tube germination. Both endocytic and secretory activities are believed to sustain rapid pollen tube tip growth, in the apex of the cell (de Graaf et al., 2005; and for reviews see Campanoni and Blatt, 2006; Cole and Fowler, 2006). POK is thus found in pollen tube zones where these pathways are highly active. Short pollen tubes in the pok mutant (Lobstein et al., 2004) would then indicate that POK-related membrane trafficking is essential for pollen tube tip growth possibly because it delivers membrane material required for this process. On the other hand, observation of the POK-labelling pattern in somatic cells, and the lethality of POK-depleted embryos highlight an essential function for POK in sporophytic cells.
Transient expression of POK:GFP constructs in onion cells (Lobstein et al., 2004) and immunolocalization experiments using the POK antiserum (this study) showed that POK accumulates in vesicle-like structures that partly localized to the GA. Moreover, in both maize somatic cells and tobacco pollen tubes, part of the POK protein pool also resides in the PVC, which was identified using an m-Rabmc antiserum. Finally, POK also localized with other unknown vesicles. These multiple POK-labelled compartments have not been observed in plants expressing POK:GFP chimeras (Lobstein et al., 2004), although the endogenous POK gene promoter had been used to drive the expression. It is conceivable that the GFP protein partly interfered with the POK targeting and/or location which does happen for other membrane trafficking elements (Uemura et al., 2004). A similar differential distribution on distinct immunolabelled endomembrane compartments has already been described for membrane trafficking factors in sporophytic cells, for example, the AtVTI11/13 Arabidopsis SNARE proteins, found in the Golgi, but also in two other compartments, the PVC and the vacuole (Uemura et al., 2004). A second example is the RabGTPase AtRabF2b, that colocalizes with both Golgi ST-YFP and PVC PS1-GFP markers in tobacco leaf epidermal cells (Kotzer et al., 2004). In all cases, the multiple distribution of the protein is associated with a specific task between the labelled compartments. It is hypothesized that the POK protein cycles between post-Golgi compartments, including at least the PVC and GA.
What would be the role of a plant GARP/VFT complex?
Our results suggest that POK associates with a putative plant GARP/VFT complex possibly involved in retrograde transport from post-Golgi compartment(s) to the Golgi. Our assumptions are based on (i) the related phenotypes of the pok and other vps mutant lines, (ii) the association of POK with several endomembrane compartments, at least the GA and the PVC, (iii) the involvement of POK in a high molecular weight complex, and (iv) the similarity between the predicted POK protein sequence and yeast Vps52p. The association of the POK protein and therefore of the GARP complex would be highly dynamic, depending upon cell needs and/or physiology. Partners of POK in this complex remain to be identified, but AtVPS53 and AtVPS54 proteins are the first candidates to be tested.
Characterization of protein complexes involved in the regulation of plant membrane trafficking is far from complete. So far, two putative molecular complexes have been reported as post-Golgi trafficking factors in plant cells, the exocyst and the retromer. In yeast and mammals, the exocyst is required for targeted exocytosis (for review see Hsu et al., 2004). Genes encoding all eight subunits of a putative plant exocyst have been found in the Arabidopsis genome, some of them belonging to large gene families (Elias et al., 2003). Mutant plants in three of the eight putative subunits have been isolated and among those, Arabidopsis mutants in the AtEXO70A1 gene (Synek et al., 2006) and maize mutants in the SEC3 gene (rth1, Wen et al., 2005) show quite comparable sporophytic phenotypes, in particular, a reduced length of polar-growing cells consistent with a role in exocytosis. These mutants, however, show no visible alteration of pollen tube growth. Synek et al. (2006) propose gene redundancy as a possible explanation for the normal phenotype of the male gametophyte in sec3 and exo70A1 mutants. Interestingly, Arabidopsis mutants in the AtSEC8 gene show altered transmission through the pollen as a consequence of defective pollen tube germination and growth, that presumably prevented the observation of homozygous sec8 mutants (Cole et al., 2005) as observed for the pok and other vps mutants.
The retromer complex is a pentameric unit, conserved in yeast and mammals, and involved in the retrograde route from the PVC/endosomes to the Golgi (for a review see Seaman, 2005), thus performing a similar role to GARP/VFT complex (Conibear et al., 2003). An Arabidopsis mutant in the putative VPS29 subunit encoding gene has been reported and this incorrectly sorts seed storage proteins (maigo1 mutant; Shimada et al., 2006). Moreover, the putative subunits VPS26, VPS29, and VPS35 from Arabidopsis have been immunolocalized in the multivesicular bodies/PVC compartment (Oliviusson et al., 2006). Jaillais et al. (2007) provided evidence of a role of this retromer subcomplex of three proteins in cell polarity establishment during sporophyte development. A function for the other two retromer components, homologues of yeast vps17p and vps5p, is still to be determined in plants. The putative GARP complex would therefore share some characteristics with the retromer complex; mutant plant phenotypes suggest, however, that they would act on distinct though parallel pathways within cells, regulating some of the retrograde transport towards the Golgi apparatus.
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We are grateful to Jean-Luc Gallois for critical reading of the manuscript, and helpful discussions. Part of the work was funded by the EU human potential program, TIPNET HPRN-CT-2002–00265.
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