Plant RABs: Role in Development and in Abiotic and Biotic Stress Responses

Endosomal trafficking plays an integral role in various eukaryotic cellular activities and is vital for higher-order functions in multicellular organisms. RAB GTPases are important proteins that influence various aspects of membrane traffic, which consequently influence many cellular functions and responses. Compared to yeast and mammals, plants have evolved a unique set of plant-specific RABs that play a significant role in their development. RABs form the largest family of small guanosine triphosphate (GTP)-binding proteins, and are divided into eight sub-families named RAB1, RAB2, RAB5, RAB6, RAB7, RAB8, RAB11 and RAB18. Recent studies on different species suggest that RAB proteins play crucial roles in intracellular trafficking and cytokinesis, in autophagy, plant microbe interactions and in biotic and abiotic stress responses. This review recaptures and summarizes the roles of RABs in plant cell functions and in enhancing plant survival under stress conditions.     

Unique mechanism of plant endocytic/vacuolar transport pathways

The post-Golgi traffic network in plant cells is highly complex, which is correlated with the large number of genes related to this function. RABs and SNAREs are key regulators of tethering and fusion of transport vesicles to target membranes, and the numbers of these regulators have also expanded in plant lineages. In addition to this increase in the net number of genes, plants also seem to have evolved new gene families tailored to fulfill plant-unique functions. In this article, we summarize recent progress in studies on plant-unique RABs and SNAREs functioning in post-Golgi trafficking, with a special focus on the endocytic pathway. Takashi Ueda is the recipient of the BSJ Award for Young Scientist, 2007.     

Diversification of the RAB Guanosine Triphosphatase Family in Dicots and Monocots

RAB guanosine triphosphatases （GTPases） are key regulators of vesicle trafficking and are essential to the growth and development of all eukaryotic cells. During evolution, the RAB family has expanded in different patterns to facilitate distinct cellular, developmental and physiological adaptations. Yeast has only 11 family members, whereas mammalian RABs have expanded to 18 RAB subfamilies. Plant RABs have diversified primarily by duplicating members within a single subfamily. Plant RABs are divided into eight subfamilies, corresponding to mammalian RAB1, RAB2, RAB5, RAB6, RAB7, RAB8, RAB11 and RAB18. Functional diversification of these is exemplified by the RAB1 ls, orthologs of which are partitioned into unique cell compartments in plants where they function to transport vesicles during localized tip growth. Similarly, the RAB2 family in grasses is likely involved in vesicle secretion associated with wall expansion, as determined by analysis of over-expression mutants. We propose that dicots and monocots have also diverged in their RAB profiles to accommodate unique cellular functions between the two groups. Here we present a bioinformatics analysis comparing the RAB sub-families of rice, maize and Arabidopsis. These results will guide future functional studies to test for the role of diversification of subfamilies unique to monocots compared to dicots.     

Molecular Analysis and Localization of CaARA7 a Conventional RAB5 GTPase from Characean Algae

RAB5 GTPases are important regulators of endosomal membrane traffic. Among them Arabidopsis thaliana ARA7/RABF2b is highly conserved and homologues are present in fungal, animal and plant kingdoms. In land plants ARA7 and its homologues are involved in endocytosis and transport towards the vacuole. Here we report on the isolation of an ARA7 homologue (CaARA7/CaRABF2) in the highly evolved characean green alga Chara australis. It encodes a polypeptide of 202 amino acids with a calculated molecular mass of 22.2 kDa and intrinsic GTPase activity. Immunolabelling of internodal cells with a specific antibody reveals CaARA7 epitopes at multivesicular endosomes (MVEs) and at MVE‐containing wortmannin (WM) compartments. When transiently expressed in epidermal cells of Nicotiana benthamiana leaves, fluorescently tagged CaARA7 localizes to small organelles (putative MVEs) and WM compartments, and partially colocalizes with AtARA7 and CaARA6, a plant specific RABF1 GTPase. Mutations in membrane anchoring and GTP binding sites alter localization of CaARA7 and affect GTPase activity, respectively. This first detailed study of a conventional RAB5 GTPase in green algae demonstrates that CaARA7 is similar to RAB5 GTPases from land plants and other organisms and shows conserved structure and localization.     

Arabidopsis RABA1 GTPases are involved in transport between the trans‐Golgi network and the plasma membrane,and are required for salinity stress tolerance

RAB GTPases are key regulators of membrane traffic. Among them, RAB11, a widely conserved sub‐group, has evolved in a unique way in plants; plant RAB11 members show notable diversity, whereas yeast and animals have only a few RAB11 members. Fifty‐seven RAB GTPases are encoded in the Arabidopsis thaliana genome, 26 of which are classified in the RAB11 group (further divided into RABA1–RABA6 sub‐groups). Although several plant RAB11 members have been shown to play pivotal roles in plant‐unique developmental processes, including cytokinesis and tip growth, molecular and physiological functions of the majority of RAB11 members remain unknown. To reveal precise functions of plant RAB11, we investigated the subcellular localization and dynamics of the largest sub‐group of Arabidopsis RAB11, RABA1, which has nine members. RABA1 members reside on mobile punctate structures adjacent to the trans‐Golgi network and co‐localized with VAMP721/722, R‐SNARE proteins that operate in the secretory pathway. In addition, the constitutive‐active mutant of RABA1b, RABA1b^Q72L , was present on the plasma membrane. The RABA1b ‐containing membrane structures showed actin‐dependent dynamic motion . Vesicles labeled by GFP–RABA1b moved dynamically, forming queues along actin filaments. Interestingly, Arabidopsis plants whose four major RABA1 members were knocked out, and those expressing the dominant‐negative mutant of RABA1B, exhibited hypersensitivity to salinity stress. Altogether, these results indicate that RABA1 members mediate transport between the trans‐Golgi network and the plasma membrane, and are required for salinity stress tolerance.     

Systematic analysis of SNARE molecules in Arabidopsis: dissection of the post-Golgi network in plant cells

In all eucaryotic cells, specific vesicle fusion during vesicular transport is mediated by membrane-associated proteins called SNAREs (soluble N-ethyl-maleimide sensitive factor attachment protein receptors). Sequence analysis identified a total of 54 SNARE genes (18 Qa-SNAREs/Syntaxins, 11 Qb-SNAREs, 8 Qc-SNAREs, 14 R-SNAREs/VAMPs and 3 SNAP-25) in the Arabidopsis genome. Almost all of them were ubiquitously expressed through out all tissues examined. A series of transient expression assays using green fluorescent protein (GFP) fused proteins revealed that most of the SNARE proteins were located on specific intracellular compartments: 6 in the endoplasmic reticulum, 9 in the Golgi apparatus, 4 in the trans-Golgi network (TGN), 2 in endosomes, 17 on the plasma membrane, 7 in both the prevacuolar compartment (PVC) and vacuoles, 2 in TGN/PVC/vacuoles, and 1 in TGN/PVC/plasma membrane. Some SNARE proteins showed multiple localization patterns in two or more different organelles, suggesting that these SNAREs shuttle between the organelles. Furthermore, the SYP41/SYP61-residing compartment, which was defined as the TGN, was not always located along with the Golgi apparatus, suggesting that this compartment is an independent organelle distinct from the Golgi apparatus. We propose possible combinations of SNARE proteins on all subcellular compartments, and suggest the complexity of the post-Golgi membrane traffic in higher plant cells.     

TBC1D14 sets the TRAPP for ATG9

Amino acid withdrawal induces the formation of autophagosomes, which results in dozens of these large double-membrane vesicles appearing in the starved cell within 10–15 min, and the initiation of autophagy. This vesicle-mediated response clearly requires an adequate supply of membrane and a tight molecular regulation creating a substantial challenge for the cell in terms of vesicle trafficking pathways. Several membrane sources, which contribute to autophagosome initiation and formation, have been identified including the ER, Golgi, plasma membrane, mitochondria and recycling endosomes. How contributions from these organelles are regulated is an intensive area of study. Members of several families of membrane traffic regulators, including small GTPases, such as RAB proteins, and their regulators, SNARE proteins and BAR domain-containing proteins, have recently been shown to support autophagosome formation.     

Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20 degrees C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.     

Structural Mechanisms for Regulation of Membrane Traffic by Rab GTPases

In all eukaryotic organisms, Rab GTPases function as critical regulators of membrane traffic, organelle biogenesis and maturation, and related cellular processes. The numerous Rab proteins have distinctive yet overlapping subcellular distributions throughout the endomembrane system. Intensive investigation has clarified the underlying molecular and structural mechanisms for several ubiquitous Rab proteins that control membrane traffic between tubular-vesicular organelles in the exocytic, endocytic and recycling pathways. In this review, we focus on structural insights that inform our current understanding of the organization of the Rab family as well as the mechanisms for membrane targeting and activation, interaction with effectors, deactivation and specificity determination.     

The autophagic roles of Rab small GTPases and their upstream regulators: A review

Macroautophagy is an evolutionarily conserved degradative process of eukaryotic cells. Double-membrane vesicles called autophagosomes sequester portions of cytoplasm and undergo fusion with the endolysosomal pathway in order to degrade their content. There is growing evidence that members of the small GTPase RAB protein family—the well-known regulators of membrane trafficking and fusion events—play key roles in the regulation of the autophagic process. Despite numerous studies focusing on the functions of RAB proteins in autophagy, the importance of their upstream regulators in this process emerged only in the past few years. In this review, we summarize recent advances on the effects of RABs and their upstream modulators in the regulation of autophagy. Moreover, we discuss how impairment of these proteins alters the autophagic process leading to several generally known human diseases.     

Plant TGNs: dynamics and physiological functions

In all eukaryotic cells, a membrane trafficking system connects the post-Golgi organelles, including the trans-Golgi network (TGN), endosomes, and vacuoles. This complex network plays critical roles in several higher-order functions in multicellular organisms. The TGN, one of the important organelles for protein transport in the post-Golgi network, functions as a sorting station, where cargo proteins are directed to the appropriate post-Golgi compartments. The TGN has been considered to be a compartment belonging to the Golgi apparatus, located on the trans side of the Golgi apparatus. However, in plant cells, recent studies have suggested that the TGN is an independent, dynamic organelle that possesses features different than those of TGNs in animal and yeast cells. In this review, we summarize recent progress regarding the dynamics and physiological functions of the plant TGN.     

Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane

Small GTP-binding proteins of the rab family have been implicated as regulators of membrane traffic along the biosynthetic and endocytic pathways in eukaryotic cells. We have investigated the localization and function of rab8, closely related to the yeast YPT1/SEC4 gene products. Confocal immunofluorescence microscopy and immunoelectron microscopy on filter-grown MDCK cells demonstrated that, rab8 was localized to the Golgi region, vesicular structures, and to the basolateral plasma membrane. Two-dimensional gel electrophoresis showed that rab8p was highly enriched in immuno-isolated basolateral vesicles carrying vesicular stomatitis virus-glycoprotein (VSV-G) but was absent from vesicles transporting the hemagglutinin protein (HA) of influenza virus to the apical cell surface. Using a cytosol dependent in vitro transport assay in permeabilized MDCK cells we studied the functional role of rab8 in biosynthetic membrane traffic. Transport of VSV-G from the TGN to the basolateral plasma membrane was found to be significantly inhibited by a peptide derived from the hypervariable COOH-terminal region of rab8, while transport of the influenza HA from the TGN to the apical surface and ER to Golgi transport were unaffected. We conclude that rab8 plays a role in membrane traffic from the TGN to the basolateral plasma membrane in MDCK cells.     

Coordination between RAB GTPase and phosphoinositide regulation and functions

Membrane trafficking relies on dynamic changes in membrane identities that are determined by the regulation of distinct RAB GTPases and phosphoinositides. RABs and phosphoinositides both act to spatiotemporally recruit effectors of membrane remodelling, including sequential RAB and phosphoinositide activities. New ideas on coordinated regulation of specific RABs and phosphoinositides, achieved by direct physical and functional interactions between their regulatory enzymes, are emerging as a central mechanism to ensure precision and fidelity of membrane trafficking.     

Dynamic reorganization of the endomembrane system during spermatogenesis in <Emphasis Type="Italic">Marchantia polymorpha</Emphasis>

The processes involved in sexual reproduction have been diversified during plant evolution. Whereas charales, bryophytes, pteridophytes, and some gymnosperms utilize motile sperm as male gametes, in other gymnosperms and angiosperms the immotile sperm cells are delivered to the egg cells through elongated pollen tubes. During formation of the motile sperms, cells undergo a dynamic morphological transformation including drastic changes in shape and the generation of locomotor architecture. The molecular mechanism involved in this process remains mostly unknown. Membrane trafficking fulfills the exchange of various proteins and lipids among single membrane-bound organelles in eukaryotic cells, contributing to various biological functions. RAB GTPases and SNARE proteins are evolutionarily conserved key machineries of membrane trafficking mechanisms, which regulate tethering and fusion of the transport vesicles to target membranes. Our observation of fluorescently tagged plasma membrane-resident SNARE proteins demonstrated that these proteins relocalize to spherical structures during the late stages in spermiogenesis. Similar changes in subcellular localization were also observed for other fluorescently tagged SNARE proteins and a RAB GTPase, which acts on other organelles including the Golgi apparatus and endosomes. Notably, a vacuolar SNARE, MpVAMP71, was localized on the membrane of the spherical structures. Electron microscopic analysis revealed that there are many degradation-related structures such as multi-vesicular bodies, autophagosomes, and autophagic bodies containing organelles. Our results indicate that the cell-autonomous degradation pathway plays a crucial role in the removal of membrane components and the cytoplasm during spermiogenesis of Marchantia polymorpha. This process differs substantially from mammalian spermatogenesis in which phagocytic removal of excess cytoplasm involves neighboring cells.     

Two syntaxin homologues in the TGN/endosomal system of yeast.

Intracellular membrane traffic is thought to be regulated in part by SNAREs, integral membrane proteins on transport vesicles (v-SNAREs) and target organelles (t-SNAREs) that bind to each other and mediate bilayer fusion. All known SNARE-mediated fusion events involve a member of the syntaxin family of t-SNAREs. Sequence comparisons identify eight such proteins encoded in the yeast genome, of which six have been characterized. We describe here the remaining two, Tlg1p and Tlg2p. These have the expected biochemical properties of t-SNAREs, and are located in separable compartments which correspond to a putative early endosome and the yeast equivalent of the TGN, respectively. They co-precipitate with the v-SNARE Vti1p, which is implicated in Golgi-endosome traffic and, remarkably, binds to five different syntaxins. Tlg1p also binds the plasma membrane v-SNARE Snc1p. Both Tlg1p and Tlg2p are required for efficient endocytosis and to maintain normal levels of TGN proteins. However, neither is required for intra-Golgi traffic. Since no further syntaxins have been identified in yeast, this implies that the Golgi apparatus can function with a single syntaxin, Sed5p.     

New insights into the role of Arabidopsis RABA1 GTPases in salinity stress tolerance

RAB11 GTPases, widely conserved members of RAB small GTPases, have evolved in a unique way in plants; plant RAB11 has notable diversity compared with animals and yeast. Recently, we have shown that members of RABA1, a subgroup in Arabidopsis RAB11 group, are required for salinity stress tolerance. To obtain a clue to understand its underlying mechanism, here we investigate whether RABA1 regulates sodium transport across the plasma membrane and accumulation in the vacuole. The results indicate that the raba1 quadruple mutant is not defective in the import and intracellular distribution of sodium, implying that RABA1 members are involved in a more indirect way in the responses to salinity stress.     

The regulation of endosome-to-Golgi retrograde transport by tethers and scaffolds

Retrograde transport between endosomes and the trans-Golgi network (TGN) is essential for the recycling of membrane proteins which are involved in a range of biological processes. A variety of machinery components have been identified at the TGN which regulate endosome-to-TGN transport, including small G proteins, SNAREs, tethering factors and scaffold molecules. The challenge is to understand how these regulatory components orchestrate not only the specific docking and fusion of retrograde membrane carriers with the TGN, but also maintain the integrity of this highly dynamic compartment to ensure efficient delivery and export of cargo. Here we review recent advances in defining the form and function of tethers and scaffolds in the regulation of the retrograde transport pathways.     

Post-Golgi protein traffic in the plant secretory pathway

The Golgi apparatus in plants is organized as a multitude of individual stacks that are motile in the cytoplasm and in close association with the endoplasmic reticulum (ER) (Boevink et al. in Plant J 15:441–447, 1998). These stacks operate as a sorting centre for cargo molecules, providing modification and redirection to other organelles as appropriate. In the post-Golgi direction, these include vacuole and plasma membrane, and specialized transport routes to each are required to prevent mislocalization. Recent evidence in plant cells points to the existence of post-Golgi organelles that function as intermediate stations for efficient protein traffic, as well as to the influence of small GTPases such as Rabs and ARFs on post-Golgi trafficking. This review focuses on the latest findings on post-Golgi trafficking routes and on the involvement of GTPases and their effectors on the trafficking of proteins in the plant secretory pathway. Sally L. Hanton and Loren A. Matheson have contributed equally to this work.     

A membrane trafficking pathway regulated by the plant-specific RAB GTPase ARA6

Endosomal trafficking plays an integral role in various eukaryotic cell activities and serves as a basis for higher-order functions in multicellular organisms. An understanding of the importance of endosomal trafficking in plants is rapidly developing, but its molecular mechanism is mostly unknown. Several key regulators of endosomal trafficking, including RAB5, which regulates diverse endocytic events in animal cells, are highly conserved. However, the identification of lineage-specific regulators in eukaryotes indicates that endosomal trafficking is diversified according to distinct body plans and lifestyles. In addition to orthologues of metazoan RAB5, land plants possess a unique RAB5 molecule, which is one of the most prominent features of plant RAB GTPase organization. Plants have also evolved a unique repertoire of SNAREs, the most distinctive of which are diverse VAMP7-related longins, including plant-unique VAMP72 derivatives. Here, we demonstrate that a plant-unique RAB5 protein, ARA6, acts in an endosomal trafficking pathway in Arabidopsis thaliana. ARA6 modulates the assembly of a distinct SNARE complex from conventional RAB5, and has a functional role in the salinity stress response. Our results indicate that plants possess a unique endosomal trafficking network and provide the first indication of a functional link between a specific RAB and a specific SNARE complex in plants.