Distinct Localization of Two Closely Related Ypt3/Rab11 Proteins on the Trafficking Pathway in Higher Plants*

Ypt/Rab proteins are Ras-related small GTPases that act on the intracellular membrane through the trafficking pathway, and their function depends on their localization. Approximately 25 genes encoding Ypt3/Rab11-related proteins exist in Arabidopsis , but the reason for the presence of many genes in plants remains unclear. Pea Pra2 and Pra3, members of Ypt3/Rab11, are closely related proteins. Because possible orthologs are conserved among dicots, they can be studied to determine their possible localization. Biochemical analysis revealed that these proteins were localized on distinct membranes in pea. Furthermore, using green fluorescent protein-Pra2 and green fluorescent protein-Pra3 fusion proteins, we demonstrated that these proteins are distinctively localized on the trafficking pathway in tobacco Bright Yellow 2 cells. Pra2 was predominantly localized on Golgi stacks and endosomes, which did not support the localization of Pra2 on the endoplasmic reticulum (Kang, J. G., Yun, J., Kim, D. H., Chung, K. S., Fujioka, S., Kim, J. I., Dae, H. W., Yoshida, S., Takatsuto, S., Song, P. S., and Park, C. M. (2001) Cell 105, 625–636). In contrast, Pra3 was likely to be localized on the trans Golgi network and/or the prevacuolar compartment. We concluded that Pra2 and Pra3 proteins are distinctively localized on the trafficking pathway. This finding suggests reductase, assayed methods previously onto membrane,

genome has been sequenced (3). Interestingly, despite the small size of its genome, Arabidopsis has more than 50 Ypt/Rab GTPases. Ypt/Rab GTPases are subdivided into subgroups, one of which is Ypt3/Rab11. Yeast and human genomes have two (Ypt31 and Ypt32) and three (Rab11A, Rab11B, and Rab25) genes belonging to Ypt3/Rab11, respectively, whereas Arabidopsis has more than 25 related genes (Fig. 1). Usually, proteins of the Rab subgroup with a similar structure have been believed to have similar functions in yeast and human. However, it has been recently shown that proteins belonging to one subgroup do not always have similar functions (1). The reason that plants have so many Ypt3/Rab11 proteins is still not known. Probably, each Ypt3/Rab11 protein in higher plants has a different localization and function, but evidence is lacking because there have been only a few reports of Ypt3/Rab11 localization in higher plants (4).
Pea Pra2 and Pra3 proteins are members of the Ypt3/Rab11 family (5,6). Both have some common features as follows. 1) The expression of these genes is negatively regulated by light mediated by photoreceptor phytochromes (7)(8)(9)(10). 2) Both proteins are mainly expressed in the etiolated epicotyls (8). 3) Their primary structures are closely related, and the proteins are present on the adjacent branches of the phylogenetic tree ( Fig. 1 and Ref. 5). Sequence similarity searches revealed that the possible orthologs of Pra2 and Pra3 proteins were found in the Arabidopsis genome (GenPept accession numbers AAF97325 and BAB11663, respectively) ( Fig. 1) and in Lotus japonicas (11) and that Pra2 and Pra3 proteins are probably conserved among dicots. It is interesting to examine whether these similar proteins, Pra2 and Pra3, are localized on distinct subcellular membranes engaging in different functions.
The mechanism of light-regulated expression of the PRA2 gene has been extensively studied (9,10,12), but its precise function remains unclear. A recent report (13) postulated that the Pra2 protein is a mediator for the cross-talk between light and brassinosteroids in the etiolation process. In that report, Kang et al. (13) claims that Pra2 is localized on the endoplasmic reticulum (ER) 1 and regulates the activity of the DDWF1 protein, which belongs to the P450 family and catalyzes the hydroxylation step of brassinosteroids by direct interaction on the ER. Transgenic analysis led them to conclude that Pra2 and its orthologs are regulators of brassinosteroid biosynthesis. However, Pra2 is a Rab GTPase, and experiments using yeast cells suggest that Pra2 has a function as a Rab GTPase and is likely to be involved in vesicle transport (6). It is important to answer the question of whether the Pra2 protein is localized on the ER and regulates DDWF1 activity without participating in vesicle transport.
In this report, we investigated the subcellular localization of the Pra2 and Pra3 proteins. Using biochemical and cytological methods, we have shown that the Pra2 protein is predominantly localized on Golgi and endosomes, whereas the Pra3 protein is likely to be localized on the trans-Golgi network and/or prevacuolar compartment. Our study yielded no evidence that Pra2 was localized on the ER. Our results indicate that Pra2 and Pra3 were distinctively localized on the trafficking pathway. Furthermore, they suggest that functional diversification takes place in the plant Ypt3/Rab11 family.

EXPERIMENTAL PROCEDURES
Phylogenetic Analysis-Ypt3/Rab11 proteins of Arabidopsis were collected using either the TBLASTN program against the complete genome sequence of Arabidopsis or the SMART web tool (14). Arabidopsis proteins are distinguished by their GenPept accession numbers. If one protein has more than one accession number, only a representative one is shown. Related proteins from yeast, human, and other plant species are also included. First, multiple alignments were created using the program ClustalX version 1.8 (15) and refined by visual observation. Non-aligned regions were excluded from the alignments. Second, a tree was calculated by the neighbor-joining method using refined sequences. ScYpt1 was defined as an out-group in the tree.
Sucrose Density Gradient Centrifugation-In the experiment described in Fig. 2, the growing zone of etiolated pea epicotyl (1-cm section from the top of the hook, 30 g) was collected and homogenized in a homogenization buffer consisting of 50 mM glycylglycine-NaOH, pH 7.5, 2 mM EDTA, 1 mM DTT, 0.25 M sucrose, 1.5% Polyclar AT, and a protease inhibitor mixture (Complete TM , Roche Molecular Biochemicals). After cutting into pieces in a buffer, the sample was homogenized with a homogenizer (Hitachi). The homogenate was filtered through two layers of Miracloth and then centrifuged at 6,000 ϫ g for 10 min. The supernatant was overlaid on a cushion buffer containing 50 mM glycylglycine-NaOH, pH 7.5, 1 mM DTT, 1 mM EDTA, and 50% (w/v) sucrose. The sample was then centrifuged at 100,000 ϫ g for 30 min. The interface layer (microsomes) between the upper and lower layers was collected, and the sucrose concentration of the sample was adjusted to 18% (w/w). This microsomal fraction was finally applied to the linear sucrose density gradient (14 ml) of 42-19% (w/w). The gradient was buffered in 50 mM glycylglycine-NaOH, pH 7.5, 1 mM DTT, and 1 mM EDTA. The sample was centrifuged at 100,000 ϫ g for 2 h and then separated into 0.3-ml fractions. Aliquots (180 l) of fractions were mixed with a 4ϫ SDS-PAGE loading buffer (60 l), boiled for 3 min, and then stored at Ϫ20°C. For the enzyme assay, the rest of the samples were stored at Ϫ80°C until use.
The experiments described in Fig. 3 were carried out as described above with the exception of some modifications to a buffer. Pea epicotyls were homogenated in a buffer containing 50 mM glycylglycine-NaOH, pH 7.5, 1 mM EDTA, 1 mM MgCl 2 , 1 mM DTT, 0.25 M sucrose, 1.5% Polyclar AT, and a protease inhibitor mixture, Complete TM . The 6,000 ϫ g supernatant was overlaid on a cushion buffer containing 50 mM glycylglycine-NaOH, pH 7.5, 1 mM DTT, 1 mM EDTA, 1 mM MgCl 2 , and 50% (w/v) sucrose. After 100,000 ϫ g centrifugation, the interface layer was collected, and the sucrose density was adjusted to 10% (w/w). This microsomal fraction was finally applied to the linear sucrose density gradients (14 ml) of 38 -15% (w/w). The gradient was buffered in 50 mM glycylglycine-NaOH, pH 7.5, and 1 mM DTT containing either 1 mM EDTA (Fig. 3A) or 1 mM MgCl 2 (Fig. 3B). After centrifugation, samples were collected as described above.
Construction of Plasmids and Transformation-Green fluorescent protein (GFP) fusion genes were constructed as follows. The desired cDNA was amplified by Pfu DNA polymerase (Stratagene) subcloned into the EcoRV site of a pZErO-2.1 vector (Invitrogen) and digested with restriction enzymes. Digested fragments were subcloned into the BglII-EcoRI sites of a pMAT-137 vector (a gift from Dr. K. Matsuoka) containing the GFP gene. The fusion gene was driven by the cauliflower mosaic virus 35S promoter-nopaline synthase terminator system. As a result, the desired gene was fused to the C terminus of GFP. The resulting plasmid was transformed into tobacco Bright Yellow 2 (BY-2) cells using Agrobacterium tumefaciens (16).
Red fluorescent protein (RFP) fusion genes were also constructed using a similar method. The desired fragment was subcloned into a pUC vector carrying the RFP gene (DsRed, CLONTECH). The fusion gene was driven by the cauliflower mosaic virus 35S promoter-nopaline synthase terminator system. The particle bombardment device was the same as that described previously (9). After bombardment, cells were incubated for 18 h and observed by a confocal laser-scanning microscope.
Microscopic Analysis and Trace of Endocytosis-Unless otherwise stated, living cells were directly observed using an MRC-1024 confocal laser-scanning microscope as described previously (17).
Staining by FM4 -64 was carried out as follows (18,19). The cells were digested for 2 h with an enzyme solution that contained 1% cellulase Y-C and 0.1% pectolyase Y-23. After washing in a fresh medium containing 0.4 M mannitol, FM4 -64 was added to a final concentration of 50 M, and cells were incubated for 10 min. The cells were then harvested and resuspended in a fresh medium. After incubation for the indicated time, cells were observed using a confocal laserscanning microscope.
Measurements of Enzyme Activities and Immunoblotting-Enzyme activities, such as inosine diphosphatase and NADPH-cytochrome c reductase, were assayed by the methods described previously (20,21). For immunoblotting, samples were subjected to SDS-PAGE, blotted onto a nitrocellulose membrane, and then probed with specific antibodies. The antibodies used in this study have been used and cited in the following studies: Pra2 and Pra3 (8), vacuolar-type ATPase (22), and plasma membrane-type ATPase (23). Pra2 and Pra3 proteins were detected by an LAS-1000 plus luminoimage analyzer (Fuji, Japan) using a SuperSignal WestFemto detection kit (Pierce), whereas plasma membrane-type ATPase and vacuolar-type ATPase were detected using an immunostaining HRP-1000 kit (Konica, Japan).

Pra2 and Pra3 Proteins Are Localized on Distinct Subcellular Membranes in Pea-Because the function of Ypt/Rab
GTPase depends on its localization, it is necessary to confirm the localization of Pra2 and Pra3 proteins on subcellular membranes. First, we performed biochemical analysis using the growing zone of the etiolated pea epicotyl where both Pra2 and Pra3 were the richest. A crude microsomal fraction was fractionated on a sucrose density gradient buffered in glycylglycine-NaOH (Fig. 2). The distributions of markers in Fig. 2 were similar to those in the previous report (24). Pra2 was strongly observed in fractions 39 -45, whereas Pra3 was enriched in fractions 33-39 (Fig. 2), indicating that these proteins are localized on distinct subcellular membranes in pea. Because both the ER and Golgi were distributed over the light density area under this condition, we could not predict the precise localization of Pra2 and Pra3.
It is important to identify the localization of Pra2, because a recent report claims that Pra2 is localized on the ER in onion epidermal cells (13). To demonstrate protein localization on the ER, "Mg 2ϩ -shift" experiments are usually performed. The distribution of the ER is disrupted and shifted to a heavier density area in the presence of Mg 2ϩ but not in its absence. We examined the effects of 3 mM MgCl 2 on pea epicotyl membranes (25) and found that this method was not suitable for the analysis of pea epicotyl membranes, because the distribution of various membranes as well as that of the ER was altered. We employed a method developed by Lord et al. (26) with some modifications. A microsomal fraction homogenized in a buffer containing both 1 mM EDTA and 1 mM MgCl 2 was analyzed by sucrose density gradient centrifugation in the presence of either 1 mM EDTA or 1 mM MgCl 2 . Under the presence of EDTA, four kinds of membranes, ER, Golgi, vacuolar, and plasma membranes, were differently distributed (Fig. 3A). Western blot analysis of Pra2 and Pra3 showed that their patterns were different from those of the membrane markers (Fig. 3A). In the presence of 1 mM MgCl 2 , the ER marker distributed over various fractions without remarkable disruption of Golgi (Fig. 3B). Under such a condition, the patterns of Pra2 and Pra3 were similar to those shown in Fig. 3A, suggesting that the membranes on which Pra2 and Pra3 were localized are not the ER, which contradicts the result of the study on onion epidermal cells. However, this information was insufficient to conclude the localization of Pra2 and Pra3.
Pra2 and Pra3 Proteins Are Distinctively Localized on the Trafficking Pathway in Tobacco BY-2 Cells-To further examine the localization of Pra2 and Pra3 proteins, we next tried to conduct cytological analysis using tobacco BY-2 cells. We made stable transformants of tobacco BY-2 cells expressing GFP-Pra2 or GFP-Pra3 proteins and observed the fluorescence of GFP using a confocal laser-scanning microscope. Both GFP-Pra2 and GFP-Pra3 were localized around the perinuclear cytosol with bright-dot staining (Fig. 4, A and B). The dot structure was a representative pattern of Golgi that has been observed in previous reports (27,28). To confirm whether the dot structure is actually Golgi, we examined the effect of brefeldin A (BFA). BFA treatment (10 g/ml) caused the redistribu- tion of GFP-Pra2 to the ER (Fig. 4C, arrowheads). The results indicate that a part of GFP-Pra2 is localized on Golgi sensitive to BFA. BFA treatment could not cause the redistribution of GFP-Pra3 to the ER (Fig. 4D), again confirming that Pra2 and Pra3 are localized on distinct intracellular membranes. Because BFA treatment cannot cause the redistribution of the protein localized in the trans-Golgi network (TGN) to the ER (29), Pra3 might be localized on the TGN.
To confirm the probable localization of Pra3 on the TGN, we introduced a plasmid carrying the AtVTI11-RFP fusion gene into BY-2 cells expressing GFP-Pra3 by particle bombardment. AtVTI11 is believed to be localized on the TGN and the prevacuolar compartment in plant cells (30). In Arabidopsis cells, fluorescent AtVTI11 has been used as a marker of the pathway from the TGN to the prevacuolar compartment (31). Thus, we constructed an AtVTI11-RFP fusion gene and introduced it into tobacco cells expressing GFP-Pra3. As shown in Fig. 5, most of the red signals derived from AtVTI11-RFP overlapped with the green signals of GFP-Pra3 and turned yellow (Fig. 5, merged). The results of our BFA experiments and those shown in this figure suggest that Pra3 is likely to be localized on the TGN and/or the prevacuolar compartment.
Although Pra2 and Pra3 are localized around Golgi, the appearance of an unspecified structure after BFA treatment (Fig. 4C, arrows) suggested that Pra2 was localized on other intracellular membranes. Thus, we also examined the possible localization of Pra2 and Pra3 on the endocytic pathway using FM4 -64, which develops a red color. FM4 -64 has been used as an endocytic tracer that travels from the plasma membrane to the vacuolar membrane via the endosomes in yeast (18), Arabidopsis (19), and tobacco BY-2 cells (31). After a 60-min chaseperiod, the red signals derived from the endosomes overlapped with the green signals of GFP-Pra2 and turned yellow (Fig. 6A,  arrowheads), indicating that a part of Pra2 is localized on the endosomes as well as on Golgi. After 3 h, FM4 -64 arrived at the vacuolar membranes and showed that this drug could be used as a tracer for endocytosis in BY-2 cells (Fig. 6, C and D). In the case of the GFP-Pra3 transformant (Fig. 6B), only a small number of yellow signals was observed, suggesting that Pra3 is not predominantly localized on the endosomes.
To finally confirm the distinct localization between Pra2 and Pra3, we introduced an RFP-PRA3 fusion gene into cells expressing the GFP-Pra2 protein by particle bombardment. As shown in Fig. 7, GFP-Pra2 was apparently distinguishable from RFP-Pra3, again indicating the distinct localization of Pra2 and Pra3 in a cell. Although the artificial localization of fluorescent proteins cannot be completely excluded, reconfirmation of distinct Pra2 and Pra3 localization in BY-2 cells as well as in pea led us to conclude that fluorescent-tagged proteins are likely to be targeted to membranes precisely in BY-2 cells.
Our results from biochemical analysis and those shown in this figure clearly indicate that closely related Pra2 and Pra3 proteins are distinctively localized on the trafficking pathway in both pea and a model system, that of the tobacco BY-2 cell. Pra2 is predominantly localized on Golgi and endosomes, whereas Pra3 is likely to be localized on the TGN and/or the prevacuolar compartment. Our results were not consistent with those of a previous report (13), according to which the Pra2 was localized on the ER.

DISCUSSION
In this study, we examined the subcellular localization of two closely related plant Ypt3/Rab11 GTPases, Pra2 and Pra3. A biochemical analysis demonstrated that these proteins are localized on distinct membranes in pea (Fig. 2). Using GFP fusion proteins, Pra2 was shown to be predominantly localized on Golgi and endosomes, whereas Pra3 was likely to be localized on the trans-Golgi network and/or prevacuolar compartment .
Although the Golgi localization of GFP-Pra2 seems to be inconsistent with the results of biochemical analysis, it is well known that the distribution of inosine diphosphatase, a general marker of Golgi, does not fit that of the Golgi protein such as glucuronyltransferase, which is localized on a specific compartment (32). The distinct localization of GFP-Pra2 and RFP-Pra3 (Fig. 7) supports the idea that both proteins are likely to be targeted to membranes precisely in BY-2 cells as observed in pea (Fig. 2), although the possibility of artificial GFP-Pra2 localization on Golgi cannot be completely excluded. We hypothesize that Pra2 is likely to be localized on the specific subcompartment of Golgi and that Pra2 and the Golgi marker were thus observed differently on a sucrose gradient.
This study clearly shows for the first time that functional diversification of Ypt3/Rab11 takes place in plants. In yeast, it has been believed that two isoforms (Ypt31 and Ypt32) have almost identical functions (33,34). In mammal, Rab11 and Rab25 are expressed in different cells, but their functions were shown to be similar using polarized cells (35). However, our results clearly showed that closely related Pra2 and Pra3 are distinctively localized on the trafficking pathway in the same cell, indicating that they have distinct functions. Such func- tional diversification of plant Ypt3/Rab11 is probably caused by the specific function of the plant Golgi apparatus. In plant cells, matrix polysaccharides such as hemicelluloses are synthesized in Golgi and transported to the cell wall via Golgi-derived vesicles (36); plant cells have to prepare the machinery for delivery of cell wall components and biosynthetic enzymes from Golgi to the plasma membrane. Judging from the fact that Ypt3/Rab11 diversification takes place only in plants, one reason for Ypt3/Rab11 diversification in higher plants might be the necessity of cell wall biogenesis, which is unique to plants. Recent reports also led us to hypothesize other possibilities (37). For example, the inhibition of vesicle trafficking affects the polarized distribution of the efflux carrier of auxin, a plant hormone (37). The implication of vesicle trafficking in such plant hormone transport suggests that Ypt3/Rab11 might be more involved in diverse phenomena in plants than we expected.
Our results also support the proposition of the functional difference of the Ypt3/Rab11 family among different organisms. Ypt31/32 in yeast seemed to be localized on the intra-Golgi (33), whereas Rab11 in human was localized on the endosomes (38,39), providing grounds for proposing a functional difference between Ypt3 and Rab11 (1). However, this is still controversial because no t-SNAREs, a partner of Ypt/Rab, were localized on intra-Golgi stacks in yeast (40). Our finding of Pra2 localization on both endosomes and Golgi suggests that Pra2 protein has a distinct function from both yeast Ypt3 and human Rab11. The result that Pra2 could not complement either temperature-or cold-sensitive ypt31 mutants (6) is consistent with this idea. In addition to Ypt3/Rab11, unique diversification within the Rab5 family also takes place. Arabidopsis has Ara6 GTPase, which resembles Rab5 best but has a unique structure (19). Ueda et al. (19) demonstrated that Ara6 and Ara7, a putative authentic ortholog of human Rab5, are differently regulated by the distribution, expression, and localization levels (19). Their findings and our results encourage us to investigate the functional difference of Ypt/Rab GTPase between plants and other organisms.
A recent report (13) showed that Pra2 is localized on the ER and regulates an essential step of brassinosteroid biosynthesis by interacting with DDWF1, which belongs to the P450 family and catalyzes the hydroxylation step of brassinosteroid biosynthesis. Although our finding did not support the existence of Pra2 on the ER (Fig. 3), there is a possibility that Pra2 is a multifunctional protein. A minor fraction of Pra2 might be localized on the ER or be recruited to the ER by additional factors where Pra2 can interact with DDWF1. However, phylogenetic analysis of DDWF1 relatives showed that a gene orthologous to pea DDWF1 is not present in the current data base of the Arabidopsis genome (data not shown). In addition, possible DDWF1 orthologs in tobacco were induced by the inoculation of a phytopathogen (41) or the treatment of a fungal elicitor (42), suggesting that these genes might be involved in the resistance to phytopathogens. The report claimed that suppression of NtRab11D, a proposed ortholog of Pra2 in tobacco, is responsible for reduced brassinosteroid biosynthesis (3). However, our phylogenetic analysis showed that NtRab11D is a Pra3 ortholog (Fig. 1). In view of these facts, further studies would be required to demonstrate that Pra2 is a multifunctional protein that regulates brassinosteroid biosynthesis by interacting with DDWF1.
In summary, we demonstrated that closely related Pra2 and Pra3 proteins are distinctively localized on the trafficking pathway. Our results suggest that functional diversification takes place in the plant Ypt3/Rab11 family.