Localization and trafficking of an isoform of the AtPRA1 family to the Golgi apparatus depend on both N- and C-terminal sequence motifs

Prenylated Rab acceptors (PRAs) bind to prenylated Rab proteins and possibly aid in targeting Rabs to their respective compartments. In Arabidopsis, 19 isoforms of PRA1 have been identified and, depending upon the isoforms, they localize to the endoplasmic reticulum (ER), Golgi apparatus and endosomes. Here, we investigated the localization and trafficking of AtPRA1.B6, an isoform of the Arabidopsis PRA1 family. In colocalization experiments with various organellar markers, AtPRA1.B6 tagged with hemagglutinin (HA) at the N-terminus localized to the Golgi apparatus in protoplasts and transgenic plants. The valine residue at the C-terminal end and an EEE motif in the C-terminal cytoplasmic domain were critical for anterograde trafficking from the ER to the Golgi apparatus. The N-terminal region contained a sequence motif for retention of AtPRA1.B6 at the Golgi apparatus. In addition, anterograde trafficking of AtPRA1.B6 from the ER to the Golgi apparatus was highly sensitive to the HA:AtPRA1.B6 level. The region that contains the sequence motif for Golgi retention also conferred the abundance-dependent trafficking inhibition. On the basis of these results, we propose that AtPRA1.B6 localizes to the Golgi apparatus and its ER-to-Golgi trafficking and localization to the Golgi apparatus are regulated by multiple sequence motifs in both the C- and N-terminal cytoplasmic domains.     

OsPRA1 plays a significant role in targeting of OsRab7 into the tonoplast via the prevacuolar compartment during vacuolar trafficking in plant cells

In yeast and mammals, the Yip/PRA1 family of proteins has been reported to facilitate the delivery of Rab GTPases to the membrane by dissociating the Rab–GDI complex during vesicle trafficking. Recently, we identified OsPRA1, a plant Yip/PRA1 homolog, as an OsRab7-interacting protein that localizes to the prevacuolar compartment, which suggests that it plays a role in vacuolar trafficking of plant cells. Here, we show that OsPRA1 is essential for vacuolar trafficking and that it has molecular properties that are typical of the Yip/PRA1 family of proteins. A trafficking assay using Arabidopsis protoplasts showed that the point mutant OsPRA1_(Y94A) strongly inhibits the vacuolar trafficking of cargo proteins, but has no inhibitory effect on the plasma membrane trafficking of H⁺-ATPase-GFP, suggesting its specific involvement in vacuolar trafficking. Moreover, OsPRA1 was shown to be an integral membrane protein, suggesting that its two hydrophobic domains may mediate membrane integration, and its cytoplasmic N- and C-terminal regions were found to be important for binding to OsRab7. OsPRA1 also interacted with OsVamp3, implying its involvement in vesicle fusion. Finally, we used a yeast expression system to show that OsPRA1 opposes OsGDI2 activity and facilitates the delivery of OsRab7 to the target membrane. Taken together, our results support strongly that OsPRA1 targets OsRab7 to the tonoplast during vacuolar trafficking.     

Prenylated Rab acceptor protein is a receptor for prenylated small GTPases

Localization of Ras and Ras-like proteins to the correct subcellular compartment is essential for these proteins to mediate their biological effects. Many members of the Ras superfamily (Ha-Ras, N-Ras, TC21, and RhoA) are prenylated in the cytoplasm and then transit through the endomembrane system on their way to the plasma membrane. The proteins that aid in the trafficking of the small GTPases have not been well characterized. We report here that prenylated Rab acceptor protein (PRA1), which others previously identified as a prenylation-dependent receptor for Rab proteins, also interacts with Ha-Ras, RhoA, TC21, and Rap1a. The interaction of these small GTPases with PRA1 requires their post-translational modification by prenylation. The prenylation-dependent association of PRA1 with multiple GTPases is conserved in evolution; the yeast PRA1 protein associates with both Ha-Ras and RhoA. Earlier studies reported the presence of PRA1 in the Golgi, and we show here that PRA1 co-localizes with Ha-Ras and RhoA in the Golgi compartment. We suggest that PRA1 acts as an escort protein for small GTPases by binding to the hydrophobic isoprenoid moieties of the small GTPases and facilitates their trafficking through the endomembrane system.     

Analysis of the small GTPase gene superfamily of Arabidopsis

Small GTP-binding proteins regulate diverse processes in eukaryotic cells such as signal transduction, cell proliferation, cytoskeletal organization, and intracellular membrane trafficking. These proteins function as molecular switches that cycle between "active" and "inactive" states, and this cycle is linked to the binding and hydrolysis of GTP. The Arabidopsis genome contains 93 genes that encode small GTP-binding protein homologs. Phylogenetic analysis of these genes shows that plants contain Rab, Rho, Arf, and Ran GTPases, but no Ras GTPases. We have assembled complete lists of these small GTPases families, as well as accessory proteins that control their activity, and review what is known of the functions of individual members of these families in Arabidopsis. We also discuss the possible roles of these GTPases in relation to their similarity to orthologs with known functions and localizations in yeast and/or animal systems.     

Activation of the Rab7 GTPase by the MON1-CCZ1 Complex Is Essential for PVC-to-Vacuole Trafficking and Plant Growth in Arabidopsis

Rab GTPases serve as multifaceted organizers during vesicle trafficking. Rab7, a member of the Rab GTPase family, has been shown to perform various essential functions in endosome trafficking and in endosome-to-lysosome trafficking in mammalian systems. The Arabidopsis thaliana genome encodes eight putative Rab7 homologs; however, the detailed function and activation mechanism of Rab7 in plants remain unknown. Here, we demonstrate that Arabidopsis RABG3f, a member of the plant Rab7 small GTPase family, localizes to prevacuolar compartments (PVCs) and the tonoplast. The proper activation of Rab7 is essential for both PVC-to-vacuole trafficking and vacuole biogenesis. Expression of a dominant-negative Rab7 mutant (RABG3f^T22N) induces the formation of enlarged PVCs and affects vacuole morphology in plant cells. We also identify Arabidopsis MON1 (MONENSIN SENSITIVITY1) and CCZ1 (CALCIUM CAFFEINE ZINC SENSITIVITY1) proteins as a dimeric complex that functions as the Rab7 guanine nucleotide exchange factor. The MON1-CCZ1 complex also serves as the Rab5 effector to mediate Rab5-to-Rab7 conversion on PVCs. Loss of functional MON1 causes the formation of enlarged Rab5-positive PVCs that are separated from Rab7-positive endosomes. Similar to the dominant-negative Rab7 mutant, the mon1 mutants show pleiotropic growth defects, fragmented vacuoles, and altered vacuolar trafficking. Thus, Rab7 activation by the MON1-CCZ1 complex is critical for vacuolar trafficking, vacuole biogenesis, and plant growth.     

Crystal structure of inactive form of Rab3B

Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 Å resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.     

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 that functional diversification takes place in the plant Ypt3/Rab11 family.     

Small, monomeric G proteins in plants

The superfamily of small, monomeric GTP-binding proteins, in Arabidopsis thaliana comprising 93 members, is classified into four families: Arf/Sar, Rab, Rop/Rac, and Ran families. All monomeric G proteins function as molecular switches that are activated by GTP and inactivated by the hydrolysis of GTP to GDP. GTP/GDP cycling is controlled by three classes of regulatory protein: guanine-nucleotide-exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine-nucleotide-dissociation inhibitors (GDIs). Proteins of Arf family are primarily involved in regulation of membrane traffic and organization of the cytoskeleton. Arf1/Sar1 proteins regulate the formation of vesicle coat at different steps in the exocytic and endocytic pathways. Rab GTPases are regulators of vesicular transport. They are involved in vesicle formation, recruitment of cytoskeletal motor proteins, and in vesicle tethering and fusion. Rop proteins serve as key regulators of cytoskeletal reorganization in response to extracellular signals. Several data have also shown that Rop proteins play additional roles in membrane trafficking and regulation of enzymes activity. Ran proteins are involved in nucleocytoplasmic transport.     

Genome-Wide Identification,Phylogeny and Expression Profile of Vesicle Fusion Components in Verticillium dahliae

Vesicular trafficking plays a crucial role in protein localization and movement, signal transduction, and multiple developmental processes in eukaryotic cells. Vesicle fusion is the final and key step in vesicle-mediated trafficking and mainly relies on SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the regulators including SM (Sec1/Munc18) family proteins, Rab GTPases and exocyst subunits. Verticillium dahliae is a widespread soil fungus that causes disruptive vascular diseases on a wide range of plants. To date, no genes involved in vesicular fusion process have been identified and characterized in V. dahliae. The recent publication of the draft genome sequence of V. dahliae allowed us to conduct a genome-wide identification, phylogeny and expression profile of genes encoding vesicular fusion components. Using compared genomics and phylogenetic methods, we identified 44 genes encoding vesicle fusion components in the V. dahliae genome. According to the structural features of their encoded proteins, the 44 V. dahliae genes were classified into 22 SNAREs (6 Qa-, 4 Qb-, 6 Qc-, 1 Qbc- and 5 R-types), 4 SM family proteins, 10 Rab GTPases and 8 exocyst proteins. Based on phylogeny and motif constitution analysis, orthologs of vesicle fusion component in filamentous fungi were generally clustered together into the same subclasses with well-supported bootstrap values. Analysis of the expression profiles of these genes indicated that many of them are significantly differentially expressed during vegetative growth and microsclerotia formation in V. dahliae. The analysis show that many components of vesicle fusion are well conserved in filamentous fungi and indicate that vesicle fusion plays a critical role in microsclerotia formation of smoke tree wilt fungus V. dahliae. The genome-wide identification and expression analysis of components involved in vesicle fusion should facilitate research in this gene family and give new insights toward elucidating their functions in growth, development and pathogenesis of V. dahliae.     

The functions of Rab GTPases in plant membrane traffic

Rab GTPases are important determinants of membrane identity and membrane targeting. Higher plants have evolved a unique set of Rab GTPases that presumably reflects the specific demands of plant cell trafficking. In recent years, significant progress has been made in identifying Rab GTPases involved in endosome organisation, cytokinesis and in post-Golgi traffic to the plasma membrane and vacuoles. These include members of the Rab-F1, Rab-F2, Rab-A1, Rab-A2 and Rab-A4 subclasses. Some important regulators or effectors have also been identified for Rab-F, Rab-A1 and Rab-A4 proteins. However, uncertainties remain about the trafficking pathways that connect the compartments in the trans-Golgi/prevacuolar/endosomal system and there is still little or no insight into the functions of several major subclasses within the Rab GTPase family.     

PRA1 inhibits the extraction of membrane-bound rab GTPase by GDI1

Rab is a family of small Ras-like GTPases regulating intracellular vesicle transport. We have previously reported that prenylated Rab acceptor or PRA1 interacts with Rab GTPases and vesicle-associated membrane protein (VAMP2). Structural prediction programs suggest that PRA1, with its two extensive hydrophobic domains, is likely to be an integral membrane protein. However, subcellular fractionation and immunocytochemical analyses indicated that PRA1 is localized both in the cytosol and tightly associated with the membrane compartment. The membrane-bound form can be partially extracted with physiological buffer and urea, suggesting that PRA1 is an extrinsic membrane protein. Deletion of the carboxyl-terminal domain resulted in a protein that behaved as an integral membrane protein, indicating that this domain plays an essential role in maintaining PRA1 in a soluble state. PRA1 can also bind weakly to GDP dissociation inhibitor (GDI), a protein involved in the solubilization of membrane-bound Rab GTPases. Addition of PRA1 inhibited the extraction of membrane-bound Rab3A by GDI, suggesting that membrane localization of Rab GTPases is dependent on the opposing action of PRA1 and GDI. The binding of Rab and VAMP2 to PRA1 is mutually exclusive such that Rab3A can displace VAMP2 in a preformed VAMP2-PRA1 complex.     

Plant Rabs: Characterization,Functional Diversity,and Role in Stress Tolerance

Rab proteins form the largest family of small guanosine triphosphate (GTP)-binding proteins. The Rab family in plants is divided into eight subfamilies, Rab1, Rab2, Rab5, Rab6, Rab7, Rab8, Rab11, and Rab18. Phylogenetic analyses of amino acid sequence of Rab GTPases suggest their segregation into subfamilies on the basis of their localization and/or function in membrane trafficking. The Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane-trafficking pathways. The Rab proteins show highly conserved structural features with a great functional versatility. They play an important role in regulating hormone signaling during fruit ripening and apical dominance, brassinosteroid biosynthesis, pollen and nodule development, and in response to both abiotic and biotic stresses.     

Saccharomyces cerevisiae Pra1p/Yip3p interacts with Yip1p and Rab proteins.

The regulation of membrane traffic involves the Rab family of Ras-related GTPases, of which there are a total of 11 members in the yeast Saccharomyces cerevisiae. Previous work has identified PRA1 as a dual prenylated Rab GTPase and VAMP2 interacting protein [Martinic et al. (1999) J. Biol. Chem. 272, 26991-26998]. In this study we demonstrate that the yeast counterpart of PRA1 interacts with Rab proteins and with Yip1p, a membrane protein of unknown function that has been reported to interact specifically with the Rab proteins Ypt1p and Ypt31p. Yeast Pra1p/Yip3p is a factor capable of biochemical interaction with a panel of different Rab proteins and does not show in vitro specificity for any particular Rab. The interactions between Pra1p/Yip3p and Rab proteins are dependent on the presence of the Rab protein C-terminal cysteines and require C-terminal prenylation.     

Rab 蛋白调控胞内囊泡运输

细胞内各个细胞器之间通过囊泡的膜转运是真核细胞存在的基本。Rab蛋白确保了转运蛋白被运输至正确的目的地。Rab蛋白是小GTP酶中的一大家族,它通过募集其效应物蛋白,其中包括接头蛋白,栓系因子,激酶,磷酸酶以及动力蛋白等,调控了细胞膜的选取,囊泡出芽,去包被,转运以及膜融合等过程。本文主要从Rab蛋白循环着手,依次论述了Rab蛋白在囊泡出芽,去包被,转运和膜融合等过程中起到的作用,从而使读者对Rab蛋白能有一个更加系统的了解。     

An Arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase.

The Arabidopsis Ara proteins belong to the Rab/Ypt family of small GTPases, which are implicated in intracellular vesicular traffic. To understand their specific roles in the cell, it is imperative to identify molecules that regulate the GTPase cycle. Such molecules have been found and characterized in animals and yeasts but not in plants. Using a yeast system, we developed a novel method of functional screening to detect interactions between foreign genes and identified this Rab regulator in plants. We found that the expression of the ARA4 gene in yeast ypt mutants causes exaggeration of the mutant phenotype. By introducing an Arabidopsis cDNA library into the ypt1 mutant, we isolated a clone whose coexpression overcame the deleterious effect of ARA4. This gene encodes an Arabidopsis homolog of the Rab GDP dissociation inhibitor (GDI) and was named AtGDI1. The expression of AtGDI1 complemented the yeast sec19-1 (gdi1) mutation. AtGDI1 is expressed almost ubiquitously in Arabidopsis tissues. The method described here indicates the physiological interaction of two plant molecules, Ara4 and GDI, in yeast and should be applicable to other foreign genes.     

Regulation of mTORC1 by the Rab and Arf GTPases

The mammalian target of rapamycin (mTOR) is a key cell growth regulator, which forms two distinct functional complexes (mTORC1 and mTORC2). mTORC1, which is directly inhibited by rapamycin, promotes cell growth by stimulating protein synthesis and inhibiting autophagy. mTORC1 is regulated by a wide range of extra- and intracellular signals, including growth factors, nutrients, and energy levels. Precise regulation of mTORC1 is important for normal cellular physiology and development, and dysregulation of mTORC1 contributes to hypertrophy and tumorigenesis. In this study, we screened Drosophila small GTPases for their function in TORC1 regulation and found that TORC1 activity is regulated by members of the Rab and Arf family GTPases, which are key regulators of intracellular vesicle trafficking. In mammalian cells, uncontrolled activation of Rab5 and Arf1 strongly inhibit mTORC1 activity. Interestingly, the effect of Rab5 and Arf1 on mTORC1 is specific to amino acid stimulation, whereas glucose-induced mTORC1 activation is not blocked by Rab5 or Arf1. Similarly, active Rab5 selectively inhibits mTORC1 activation by Rag GTPases, which are involved in amino acid signaling, but does not inhibit the effect of Rheb, which directly binds and activates mTORC1. Our data demonstrate a key role of Rab and Arf family small GTPases and intracellular trafficking in mTORC1 activation, particularly in response to amino acids.     

The Rab GTPase family

The Rab family is part of the Ras superfamily of small GTPases. There are at least 60 Rab genes in the human genome, and a number of Rab GTPases are conserved from yeast to humans. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane traffic pathways. In the GTP-bound form, the Rab GTPases recruit specific sets of effector proteins onto membranes. Through their effectors, Rab GTPases regulate vesicle formation, actin- and tubulin-dependent vesicle movement, and membrane fusion.     

Small GTPases in vesicle trafficking

Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H(2)O(2) production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking.     

Subcellular Localization and Functional Analysis of the Arabidopsis GTPase RabE

Membrane trafficking plays a fundamental role in eukaryotic cell biology. Of the numerous known or predicted protein components of the plant cell trafficking system, only a relatively small subset have been characterized with respect to their biological roles in plant growth, development, and response to stresses. In this study, we investigated the subcellular localization and function of an Arabidopsis (Arabidopsis thaliana) small GTPase belonging to the RabE family. RabE proteins are phylogenetically related to well-characterized regulators of polarized vesicle transport from the Golgi apparatus to the plasma membrane in animal and yeast cells. The RabE family of GTPases has also been proposed to be a putative host target of AvrPto, an effector protein produced by the plant pathogen Pseudomonas syringae, based on yeast two-hybrid analysis. We generated transgenic Arabidopsis plants that constitutively expressed one of the five RabE proteins (RabE1d) fused to green fluorescent protein (GFP). GFP-RabE1d and endogenous RabE proteins were found to be associated with the Golgi apparatus and the plasma membrane in Arabidopsis leaf cells. RabE down-regulation, due to cosuppression in transgenic plants, resulted in drastically altered leaf morphology and reduced plant size, providing experimental evidence for an important role of RabE GTPases in regulating plant growth. RabE down-regulation did not affect plant susceptibility to pathogenic P. syringae bacteria; conversely, expression of the constitutively active RabE1d-Q74L enhanced plant defenses, conferring resistance to P. syringae infection.