Rice small GTPase Rab11 is required for intracellular trafficking from the trans-Golgi-network to the plasma membrane and/or prevacuolar compartments

Abstract

Members of the Rab family of small GTPases play important roles in membrane trafficking along the exocytic and endocytic pathways, their function being dependent on their localization. Here, we show the vital roles of the Oryza sativa Rab11 (OsRab11) in intracellular trafficking using a dominant-negative mutant approach based on a protoplast-trafficking assay. GTP-binding and GTP hydrolysis assays revealed that OsRab11 is a small GTP-binding protein complementing △ypt32/ypt31. Green fluorescent protein (GFP)-OsRab11 has been localized to both the trans-Golgi-network (TGN) and the endosomes/prevacuolar compartments (PVC) in Arabidopsis protoplasts. The protoplast transformation with the dominant-negative mutant OsRab11(S28N) revealed that the trafficking of plasma membrane marker proteins [H⁺-ATPase-GFP and Ca²⁺-ATPase8-GFP (ACA8-GFP)] and central vacuole marker proteins [Arabidopsis aleurain-like protein (AALP-GFP) and sporamin (Spo-GFP)] was inhibited. Moreover, overexpression of Arabidopsis AtRha1 did not recover the trafficking inhibition of marker proteins from the central vacuole. These results strongly indicate that OsRab11 localizes to either the TGN or the PVC, and plays a significant role in the intracellular trafficking from the TGN to the PM and/or to the PVC in planta.

Introduction

The importance of intracellular vesicular trafficking has been documented extensively in eukaryotic cells (Rothman and Wieland 1996; Jahn and Sudhof 1999; Guo et al. 2000). Membrane trafficking includes many processes such as vesicle formation (budding), motility, docking, and fusion all of which are controlled by several protein factors (Derby and Gleeson 2007; Lowe and Barr 2007). For example, the Rab proteins (members of the Rab GTPase family) are major regulators of vesicular trafficking (Fukuda 2008).

The Rab protein family is the largest family within the Ras superfamily of monomeric small GTPases (Novick and Zerial 1997). To date, more than 60 members of the Rab have been identified in several organisms (Zuk and Elferink 1999; Allan et al. 2000). These proteins are localized at different organelles and they are pivotal in the vesicular trafficking pathway and in other signaling pathways (Chen et al. 1993; Dugan et al. 1995; Batoko et al. 2000). For instance, the human Rab1 regulates the protein transport of calcium-sensing receptors from the Endoplasmic Reticulum (ER) to the Golgi (Zhuang et al. 2010). The tobacco pollen-predominant Rab2 plays a role in vesicle trafficking between the ER and the Golgi body in elongating pollen tubes and, thus, contributes to pollen tube growth (Cheung et al. 2002). The Arabidopsis Rha1 (AtRha1, a Rab5 homolog) localizes to the PVC and it is involved in the trafficking of soluble cargo from the PVC to the central vacuole (Sohn et al. 2003; Lee et al. 2004). The rice OsRab7 is localized to the vacuolar membrane and it is thought to be involved in the vesicular trafficking to the vacuole (Nahm et al. 2003). More recently, another rice Rab protein, OsRab11 has been identified as a protein involved in the regulation of the JA-mediated signaling pathway (Hong et al. 2013). Hong et al. (2013) reported that OsRab11 interacts with the 12-oxo-phytodienoate-10,11-reductase8 (OPR8) increasing the NADPH oxidation activity of this protein. Plants overexpressing OsRab11 show increased resistance to pathogens due to a greater expression of JA-responsive genes (Hong et al. 2013). In addition, it has also been shown that the rice GTPase-activating protein 1 (GAP1) is a positive regulator of the OsRab11 protein and, together, they play a role in vesicular trafficking from the TGN to the PM or to the central vacuole (Heo et al. 2005). Despite all these vital roles, the detailed function of OsRab11 remains to be elucidated.

In this study, we describe the basic GTP-binding and GTP hydrolysis activities of the recombinant OsRab11 protein and investigate the biological role of OsRab11 in intracellular vesicular trafficking using an Arabidopsis protoplast-based trafficking assay. We conclude that OsRab11 plays a vital role in the vesicular trafficking from the TGN to the PM and/or to the PVC.

Materials and methods

Strains, growth conditions

The genotypes of the yeast strains S. cerevisiae YTH3 (MATa/MATα LEU2/leu2 ura3/ura3 ypt32::URA3 HIS/his3 ypt31::HIS3 TRP1/trp1) was used for the complementation assay (Benli et al. 1996). The strain was transformed with recombinant pYES2 plasmid containing T7-OsRab11 cDNA under control of the gal1 promoter. Haploid progeny were produced by streaked on YPG(gal) medium (1.25 % yeast extract, 2 % peptone, 0.1 % galactose, and 2 % agar) for 5 days.

Purification of GST-fused OsRab11 in E. coli

The OsRab11 cDNA was subcloned into the pGEX-2T vector which produces GST-fusion protein. The recombinant construct was transformed into the E. coli BL21strain. Transformed cells were inoculated into 5 mL of LB medium containing 50 μg/mL Ampicillin. The cultures were grown at 28 °C overnight and then poured into 500 mL of fresh LB medium. When an A600 of 0.9 was reached, isopropylthio-ß-galactoside was added to the cultures to a final concentration of 1 mM and the incubation was continued for another 3 h at 28 °C for induction. GST-fusion proteins were purified using glutathione agarose (Sigma.USA) according to each manufacturer’s instructions.

GTP-binding assay

The [³⁵S] GTPγS binding activity of full-length OsRab11 protein was measured by the rapid filtration technique (Seo et al. 1997). 0.5 μg of the purified recombinant proteins was incubated at 30 °C in 200 μL of 50 mM Tris–Cl buffer, pH 8.0, containing 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM NaN₃, 0.2 μM [³⁵S]GTPγS, and 10 mM MgCl₂. The reaction was stopped by addition of 2 mL of ice-cold buffer (20 mM Tris–Cl, pH 8.0, 100 mM NaCl, and 25 mM MgCl₂). Aliquots were taken at indicated times, vacuum-filtered onto nitrocellulose membrane (Millipore, USA) which had been rinsed with 3 mL of ice-cold buffer, and membrane-bound material was washed rapidly three times with 3 mL of ice-cold buffer. The washed membranes were dried and quantified for radioactivity by liquid scintillation counting.

GTP hydrolysis assay

GTPase assays were performed using PEI-cellulose TLC plates and was carried out according to the method of Seo et al. (1997) with a slight modification. Briefly, the reaction was performed at 30 °C in a buffer containing 20 mM Na·HEPES, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 1 mM MgCl₂, 1 mM NaN₃, 33 nM [α-32P]GTP, and 0.5 μg of OsRab11 protein. Aliquots of 10 μL were sampled at appropriate time intervals and the same volume of 0.5 M EDTA (pH 8.0) was added to stop the reaction. Samples (2 μL) were spotted onto PEI-cellulose TLC plates, which were then developed in 0.5 M KH2PO4, pH 3.4 solution. After drying, the plate was exposed to an X-ray film.

Transient expression assay

OsRab11 cDNA was fused upstream of the green fluorescent protein (GFP) cDNA under the control of the CaMV 35S promoter (pUC::GFP). Transient expression of GFP- and RFP-fused constructs in Arabidopsis protoplasts was performed according to the method described by Sohn et al. (2003). Briefly, recombinant plasmids were introduced by polyethylene glycol-mediated transformation into Arabidopsis protoplasts that had been prepared from leaf tissues. Expression of the fusion constructs was monitored after transformation, and observed by fluorescence microscopy (Olympus AX70 TR, Olympus. JAPAN).

Immunohistochemistry

Immunohistochemistry assay was referred to Sohn et al. 2003 (Sohn et al. 2003). Briefly, protoplasts coexpressing GFP-OsRab11 and AtPEP12a-Hemagglutinin (HA) were placed onto poly-l-lysine-coated slide glasses and fixed with 2 % paraformaldehyde in a fixing buffer (10 mM HEPES pH7.5, 125 mM NaCl, 125 mM CaCl₂, 2.5 mM maltose, 5 mM KCl) for 1 h at room temperature. The fixed cells were incubated with rat monoclonal anti-HA (Roche, Switzerland) at 4 °C overnight and washed with TSW buffer five times. Subsequently, the cells were incubated with TRITC-conjugated goat anti-rat IgG (Zymed) as the second antibody.

Generation of OsRab11(S28N) mutant construct

The mutant OsRab11(S28N) was constructed by PCR using the following primers: forward primer 5′-CCGGCGTCGGCAAGAACAACCTCCTCTCCCGCT-3′and reverse primer 5′-AGCGGGAGAGGAGGTTGTTCTTGCCGACGCCGG-3′. The T7 epitope tag was added to the N-terminus of OsRab11(S28N) mutant. XbaI/XhoI digestion fragments of the sequence were ligated into the same restriction sites of the pUC vector which has 35S cauliflower mosaic virus promoter. HA epitope tag was added to the N-terminus of OsRab11 wild type and Rha1 and XbaI/XhoI digestion fragments of these sequences were ligated into the same vector.

Western blot analysis

To prepare cell extracts from protoplasts, protoplasts expressing T7-OsRab11(S28N), HA-OsRab11 or HA-Rha1 were lysed by repeated freeze–thaw cycles and then centrifuged at 6500×g for 5 min at 4 °C in a microcentrifuge to remove cell debris (Sohn et al. 2003). The total protein extracts were separated on a 12 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel, transferred onto a nitrocellulose membrane using a semi-dry electro-blotter, and incubated with each of the indicated antibodies.

Results

OsRab11 is a small GTP-binding protein homologous to the yeast Ypt31p

A large number of Rab GTPases have been identified in plant cells. Most of these proteins play important roles in plant development and in the control of environmental stresses (Cheung et al. 2002; Woollard and Moore 2008; Hong et al. 2013). In a recent study, we showed that OsRab11 is involved together with OPR8 in the JA-mediated defense signaling pathway (Hong et al. 2013). Here, we further characterize the intracellular trafficking function of OsRab11 as a small GTPase. First, to determine whether OsRab11 is a true GTP-binding protein, the biochemical properties of OsRab11 were assayed. A purified recombinant GST-fusion protein was used to measure both the GTP-binding capacity and the GTPase activity of OsRab11 (Fig. 1B, C). During the GTP-binding test, the amount of [γ-³⁵S]GTP bound to the GST-OsRab11 protein was suddenly increased when the cofactor Mg²⁺ was present in the reaction buffer (Fig. 1B). After this initial sudden increase, the amount of [γ-³⁵S]GTP bound to OsRab11 was stabilized (Fig. 1B). Next, poly(ethyleneimine)-cellulose thin-layer chromatography (PEI-cellulose TLC) (Seo et al. 1997) was used to determine the GTPase activity of OsRab11. In 2 h, more than 50 % of the [α-³²P]GTP-labeled OsRab11 was converted to [α-³²P]GDP-labeled OsRab11 protein by the recombinant OsRab11 protein (Fig. 1C), indicating that OsRab11 is indeed a small GTP-binding protein.

Fig. 1

Purification and biochemical activities of OsRab11. (A) Purified GST and GST-fused OsRab11 proteins were separated on 12 % SDS-PAGE gel. (B) Time course of [γ-³⁵S]GTP binding to OsRab11. 1 µg of GST or GST-OsRab11 protein were incubated with 200 nM[γ-³⁵S]GTP at 30 °C in the presence of Mg²⁺. Bound [γ-³⁵S]GTP levels were determined by rapid filtration assay. (C) GTP hydrolysis activity of OsRab11. 100 uL reaction mixtures containing 20 pmol of [α-³²P]GTP-loaded OsRab11 proteins were incubated for zero, 1 h and 2 h separately. Each 2 uL of removed samples was separated by TLC. (D) Complementation test of △ypt32/ypt31 mutant cells using OsRab11. Cells were grown at 25 °C to mid-log phase and shifted to 37 °C for 1 h in YPG(Gal) media before lysis. Western blot analysis of CPY was performed with total protein extracts from each cell

Finally, to address whether OsRab11 is involved in vesicular trafficking from the TGN to the PM or to the central vacuole, a complementation assay was also performed using yeast △ypt31/ypt32 mutant cells which partially missort vacuolar proteinase carboxypeptidase Y (CPY) (Luo and Gallwitz, 2003). Usually, CPY is synthesized at the endoplasmic reticulum (ER) and then transported to the vacuole through the Golgi body (Tsukada et al. 1999). Thus, the CPY synthesis process produces three CPY forms: a 67-kDa ER core-glycosylated form (p1CPY), a 69-kDa Golgi-modified form (p2CPY), and a 61-kDa mature form (mCPY) found in the vacuole (Tsukada et al. 1999). Unlike W303 wild-type yeast cells, the Golgi-glycosylated p2CPY is partially missorted in the △ypt31/ypt32 null mutant cells (Fig. 1D lane 2). To determine whether OsRab11 could rescue the CPY missorting in the △ypt31/ypt32 null mutant cells, we used T7 tagged-OsRab11 to transform △ypt31/ypt32 null mutant cells and observed the secretion forms of CPY by Western blotting. As expected, the missorted unprocessed Golgi form (p2CPY) disappeared in the transformed cells suggesting that OsRab11 complements the yeast Ypt31p function and, that consequently, it is involved in vacuolar protein transport (Fig. 1D lane 3).

Subcellular localization of the OsRab11 protein

The Rab proteins are localized to different organelles and, consequently, they are involved in different steps of the intracellular trafficking (Chavrier et al. 1990; Morimoto et al. 1991). Therefore, observation of OsRab11 localization in plant cell is important to address the involvement of OsRab11 in vesicular trafficking. To confirm the subcellular localization of OsRab11, a protoplast transient assay was performed. Arabidopsis protoplasts were transformed with free GFP or GFP fused to OsRab11(GFP-OsRab11) and the GFP signal was observed by fluorescence microscopy. Free GFP was observed in the cytosol and nucleus, whereas protoplasts transformed with GFP-OsRab11 exhibited cytosol staining and punctate staining patterns consistent with a Golgi or a PVC localization (Heo et al. 2005). To examine the exact localization of the punctate staining signal of GFP-OsRab11, GFP-OsRab11 was co-transformed with AtVti1a-RFP, a TGN marker (Heo et al. 2005) in Arabidopsis protoplasts. Although the green and red punctate staining signals from GFP-OsRab11 and AtVtila-RFP overlapped well, there were still many punctate signals from GFP-OsRab11 which did not overlap with RFP signals (Fig. 2B). Considering that most Rab proteins have been shown to play roles in vesicle budding and vesicle fusion at both donor and target compartments (Chavrier et al. 1990; Allan et al. 2000; Prekeris et al. 2000), the punctate staining signals of GFP-OsRab11 were further assessed using AtPEP12p, a PVC marker (da Silva et al. 1997; Bassham and Raikhel 1998). AtPEP12p was tagged with the HA epitope to detect its expression with the anti-HA antibody. We investigated the localization of AtPEP12p-HA in Arabidopsis protoplasts co-transformed with GFP-OsRab11 and AtPEP12p-HA. AtPEP12p-HA was detected by immunohistochemistry using anti-HA antibody. Red punctate signals of AtPEP12p-HA closely overlapped GFP-OsRab11 signals (Fig. 2C). Also, few of GFP signals of OsRab11 did not colocalize with AtPEP12p-HA. Taken together, these results strongly support a vital role of OsRab11 in the intracellular vesicular trafficking at the TGN and/or PVC.

Fig. 2

Subcellular localization of GFP-OsRab11. (A) The localization patterns of GFP-OsRab11 in protoplasts. Protoplasts were transformed with GFP-OsRab11. Cells were incubated for 36 h, and then observed by fluorescent microscopy. (B) Co-localization of GFP-OsRab11 with AtVti1a-RFP. GFP-OsRab11 was co-transformed with AtVti1a-RFP in protoplasts and the signals of GFP-OsRab11 was examined. Yellow color indicates the overlap between green and red fluorescent signals. (C) Protoplasts transformed with GFP-OsRab11 and AtPEP12p-HA were fixed and stained with anti-HA antibody. GFP signals of GFP-OsRab11 were observed directly. The white-colored arrows in (B) and (C) indicate the overlapped GFP and RFP signals. Bar = 10 µm

OsRab11(S28N) inhibits the PM trafficking of reporter proteins

OsGAP1 is a GTPase-activating protein which is specifically required for OsRab11 function. Consequently, the trafficking of PM and central vacuolar marker proteins is inhibited in OsGAP1 mutants, whereas overexpression of OsRab11 relieved this inhibition (Heo et al. 2005). This raised the possibility of OsRab11 being involved in the vesicular trafficking of proteins from the PM and the central vacuole. To confirm this, an Arabidopsis protoplast-based trafficking assay was carried out using a dominant-negative mutant [OsRab11(S28N)] (Hong et al. 2013) and two PM reporter proteins (H⁺-ATPase-GFP and ACA8-GFP). Most cells transformed with H ⁺-ATPase-GFP alone showed a GFP signal in the PM; however, in the presence of T7-OsRab11(S28N), more than half of the cells showed signals in the cytosol (Figs. 3A, 3C). To validate the inhibition of PM protein trafficking in the presence of T7-OsRab11(S28N), a second PM reporter protein (ACA8-GFP) was co-transformed together with T7-OsRab11(S28N) in Arabidopsis protoplasts. As expected, PM localization of ACA8-GFP was severely decreased in cells transformed with both ACA8-GFP and T7-OsRab11(S28N) when compared to cells transformed only with ACA8-GFP (Fig. 3B, C). These results show that the dominant-negative mutant OsRab11(S28N) inhibits the trafficking of both H⁺-ATPase-GFP and ACA8-GFP reporter proteins to the PM.

Fig. 3

OsRa11(S28N) inhibits the trafficking of H⁺-ATPase-GFP and ACA8-GFP to the plasma membrane. (A) Protoplasts were transformed with H⁺-ATPase-GFP alone (a, b) or with T7-OsRab11(S28N) (c, d), and (B) with ACA8-GFP alone (a, b) or with T7-OsRab11(S28N) (c, d). Cells were incubation for 36 h, and then observed by fluorescent microscopy. Two types of protoplasts with GFP signals at the PM and the cytosol stains were observed. Bar = 10 µm. (C) Trafficking efficiency of H⁺-ATPase-GFP and ACA8-GFP to the PM. The whole population of transformed protoplasts was divided into two groups based on GFP signal localization: protoplasts with the PM staining and cytosol staining pattern. A minimum of 100 transformed protoplasts in each group was counted

OsRab11(S28N) inhibits the vacuolar trafficking of reporter proteins

As previously mentioned, we assumed that OsRab11 is involved in vacuolar protein trafficking as well as in PM protein trafficking. To test whether this is indeed the case, the vacuolar proteins AALP-GFP and Spo-GFP were selected as reporter proteins and co-transformed with T7-OsRab11(S28N) in Arabidopsis protoplasts. Most cells transformed with either AALP-GFP or Spo-GFP alone showed a staining pattern coinciding in more than 75 % of the area with the central vacuole with only little ER or non-vacuole staining (Fig. 4A–C). However, the majority of cells co-transformed with both vacuole reporter proteins and T7-OsRab11(S28N) showed a cytosol staining pattern with only little ER or non-vacuole staining, and only less than 20 % of the stained area coincided with the central vacuole (Fig. 4A–C). This result suggests that OsRab11(S28N) inhibits the targeting of both AALP-GFP and Spo-GFP to the central vacuole probably by competing with endogenous wild-type Rab11 protein. To confirm this, wild-type HA-OsRab11 was co-transformed with T7-OsRab11(S28N) in Arabidopsis protoplasts expressing the PM or the central vacuole reporter proteins (Fig. 5). In the transformation with only Rab11 (S28N), the frequency of the protoplasts showing PM staining pattern was about less than 38 %, whereas the frequency in the co-transformation with HA-Rab11 increased to about 60 % (Fig. 5A, C). Similarly, in the transformation with only Rab11 (S28N), the frequency of the protoplasts showing vacuole staining pattern was about less than 20 %, whereas the frequency in the co-transformation with HA-Rab11 increased to about 55 % (Fig. 5A, B), providing further support for a role of OsRab11 in the intracellular trafficking from the TGN to the PM and/or to the central vacuole.

Fig. 4

OsRa11(S28N) inhibits the trafficking of AALP-GFP and Spo-GFP to the central vacuole. (A) Protoplasts were transformed with AALP-GFP alone or with T7-OsRab11(S28N), (B) and with Spo-GFP alone or with T7-OsRab11(S28N). Cells were incubated for 36 h, and then observed by fluorescent microscopy. Three types of GFP signal pattern were observed, namely a central vacuole pattern (a), an ER or non-vacuole pattern (b), and a cytosol pattern (c). Bar = 10 µm. (C) The efficiency of AALP-GFP or Spo-GFP trafficking to the central vacuole. The GFP signal patterns in a minimum of 70 randomly selected transformed protoplasts were assessed and classified as central vacuole, ER-non-vacuole, or cytosol staining patterns. The trafficking efficiency of these proteins was calculated as described in Fig. 3(C)

Fig. 5

The overexpression of OsRab11 rescues the inhibitory effect of the OsRab11(S28N) mutant. (A) Protoplasts expressing T7-OsRab11(S28N) were co-transformed with HA-OsRab11 along with H⁺-ATPase-GFP or ACA8-GFP. (B) Protoplasts expressing T7-OsRab11(S28N) were co-transformed with HA-OsRab11 or HA-Rha1 along with AALP-GFP or Spo-GFP. After incubating the cells for 36 h, the targeting efficiencies of the reporter proteins were calculated as described in Fig. 3(C). (C, D) In vivo expression levels of T7-OsRab11(S28N), HA-OsRab11, or HA-Rha1. Total protein extracts were prepared from protoplasts transformed with various constructs and analyzed by protein gel-blot analysis using monoclonal anti-T7 and HA

Most central vacuole proteins synthesized at the ER are transported to the Golgi apparatus and moved to the TGN after the maturation process (Sohn et al. 2003). Matured vacuole proteins at the TGN are finally transported to the central vacuole through PVC/endosome. As shown above, the vacuolar trafficking inhibition by OsRab11(S28N) is very similar to that of the AtRha1(S24N) mutant (Sohn et al. 2003). AtRha1 participates in the intracellular trafficking of the cargo from the PVC to the Vacuole. The trafficking inhibition of vacuole proteins by T7-OsRab11(S28N) was not rescued by the introduction of HA-AtRha1 (Fig. 5B and 5D). Therefore, these results support that OsRab11 participates in the trafficking of the cargo from the TGN to the PVC.

Discussion

It has previously been reported that plant Rab proteins are major components in intracellular membrane trafficking and, consequently, they are involved in a variety of signal transduction pathways such as defense, stress signaling pathways, and plant development (Vernoud et al. 2003; Grosshans et al. 2006; Agarwal et al. 2008). In this study, we further characterize one of these proteins, OsRab11, and show its relevance in intracellular trafficking.

Searching for OsRab11 homology in Arabidopsis database (https://www.arabidopsis.org/cgi-bin/Blast/TAIRblast.pl) showed high homology to Rab11/RabA proteins such as RabA1a, RabA1b, RabA1c, and RabA1d with 79, 83, 85, and 86 % identity and 92, 93, 94, and 94 % similarity (Supplemental Fig. 1), and in yeast database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed high homology to Rab/Ypt31 with 65 % identity and 80 % similarity and to Rab/Ypt32 with 60 % identity and 74 % similarity (Supplemental Fig. 2), suggesting that OsRab11 is a close homolog of the Arabidopsis Rab11/RabA proteins and the yeast Ypt31/32 proteins which regulate an early step of the exocytic pathway in yeast (Jedd et al. 1997). Ypt31p plays a role in the fusion of recycling vesicles at the trans-Golgi compartment (Jedd et al. 1997) and depletion of Ypt31p has shown to inhibit invertase secretion and vacuolar protein maturation (Benli et al. 1996). In our study, we found that the missorting of vacuolar proteinase CPY (Fig. 1D) in yeast △ypt31/ypt32 null mutant cells is rescued by OsRab11 complementation, supporting a role of OsRab11 in intracellular secretion pathway and/or vacuolar trafficking similar to that of Ypt31/32.

The vacuolar trafficking pathway is also well characterized in Arabidopsis and AtRha1 has been identified as an important component in this pathway using the dominant-negative mutant approach (Sohn et al. 2003). Dominant-negative Rab proteins have shown to prevent the activity of the endogenous wild-type proteins by competing for their effector proteins (Tisdale et al. 1992). For example, the dominant-negative mutant AtRha1(S24N) strongly inhibits the vacuolar trafficking of marker proteins (Sohn et al. 2003). This result is very similar to the inhibition of vacuolar trafficking caused by the OsRab11(S28N) mutant (Fig. 5). Sohn et al. (2003) also reported that vacuolar cargo proteins are likely to be transported to the central vacuole through the PVC. AtRha1 is thought to be localized to the PVC and maybe tonoplast (Sohn et al. 2003). Whereas GFP-OsRab11 localizes to the TGN and/or to the PVC when expressed transiently in Arabidopsis protoplasts (Fig. 2). In cells expressing OsRab11(S28N) the vacuolar cargo proteins were mainly accumulated in the cytosol and to a lesser degree on the ER or non-vacuole region (Fig. 4), this is in contrast to what was observed in AtRha1(S24N) expressing cells and, thus, it suggests that OsRab11 may not play a role in the trafficking of vacuolar cargo protein from the PVC to the central vacuole. As an alternative, OsRab11 might be involved in the transport of vacuolar cargo proteins from the TGN to the PVC. In order to test this hypothesis, Arabidopsis protoplasts were co-transformed with wild-type AtRha1 and OsRab11(S28N). In these protoplasts, the trafficking inhibition of vacuolar cargo protein caused by OsRab11(S28N) was not recovered by wild-type AtRha1, suggesting that OsRab11 and AtRha1 might be involved in different stages of the transport of the vacuolar cargo proteins.

Taken together, the biochemical and the in vivo functional characteristics of OsRab11 show that this protein is a rice homolog of the Rab11/Ypt31 subfamily, and it is required for the intracellular vesicular trafficking from the TGN to the PM and/or to the vacuole.

Recently, we found that transgenic plants expressing OsRab11(S28N) is very insensitive to environmental stresses due to trafficking inhibition of proteins involved in each stresses (data not shown). It will be interesting to investigate the relationship between trafficking by Rab protein and plant response to environmental stresses. Nevertheless, in order to improve our understanding of the roles played by OsRab11 protein in rice, further characterization of its regulatory factors is required.