Analysis of the Small GTPase Gene Superfamily of Arabidopsis

Correlation of Sequence Similarity with Localization in
Different Species

Rab GTPase functions have been studied extensively in yeast and mammalian systems (for review, see Stenmark and Olkkonen, 2001). Individual members of the Rab GTPase family localize to different intracellular compartments where they regulate vesicle trafficking events (for review, see Simons and Zerial, 1993). S. cerevisiae only contains 11 Rab GTPases. Because of the cellular similarities between eukaryotic cells, we used the known distribution and function of these Rab GTPases as a basis for classification of plant Rab GTPases. S. cerevisiae Rab GTPases, termed yeast protein transport (YPT) proteins, are localized to the endoplasmic reticulum (ER), the Golgi apparatus and trans-Golgi network (TGN), the endosomal/prevacuolar compartments, and vacuoles in S. cerevisiae. They also regulate retrograde trafficking of proteins from Golgi to ER and polarized secretion (for review, see Lazar et al., 1997).

The correlation between sequence similarity and regulation of membrane trafficking through related compartments appears to be a conserved feature in the Rab GTPase family (Fig. 2). When functions of mammalian Rab GTPases sharing significant similarity to yeast counterparts have been studied, they regulate membrane trafficking through compartments with related function. For example, the human Rab8 GTPase is targeted to the protein secretion pathway, and displays high sequence similarity to Sec4p/Ypt2p, which regulates protein secretion in S. cerevisiae. Rab8 complements the ypt2 mutant (Sec4p homolog) in fission yeast (Schizosaccharomyces pombe;Huber et al., 1993a, 1993b). Rab8 and Sec4p cosegregate in the same subfamily in our phylogenetic analysis (Fig. 2). Functions for most AtRAB GTPases have not yet been established. However, when plant Rab GTPase function is known, these isoforms also cosegregate in subfamilies containing their mammalian and yeast counterparts (discussed below). In particular, studies of Rab GTPases that are members of five of the eight subfamilies highlighted by our phylogenetic analysis have been performed and published (AtRABA,Inaba et al., 2002; AtRABB, Cheung et al., 2002; AtRABD, Batoko et al., 2000; AtRABF,Ueda et al., 2001; AtRABH, Bednarek et al., 1994). In every case, their localization and/or function in membrane trafficking correlate with their segregation in subfamilies for yeast and mammalian Rab GTPases whose functions not only are the same, but also are well established. We have therefore indicated the compartments to which mammalian and yeast homologs are localized, and suggest that this may prove a useful initial framework for classification of AtRAB GTPases (Fig. 2).

Rab GTPases of the Endocytic Trafficking Pathway

Endocytosis is the primary means by which proteins and macromolecules too large to pass through the plasma membrane enter the cell. During endocytosis, regions of the plasma membrane with associated cargo molecules are invaginated and pinch off into transport vesicles, which then fuse with endocytic compartments. In addition, many proteins targeted to lysosomal and/or vacuolar compartments are sorted in endosomal compartments.

When compared with other eukaryotic Rab GTPases, three members of the AtRABF subfamily show highest similarity to human Rab5 and yeast Ypt51p family members (Fig. 2). In mammals, Rab5 GTPases localize to early endosomes and regulate fusion of clathrin-coated vesicles to early endosomes and fusion between early endosomes (Bucci et al., 1992). In yeast, Ypt51p family members similarly regulate membrane trafficking through prevacuolar compartments (Horazdovsky et al., 1994;Singer-Krüger et al., 1995). Both AtRABF2a (E. Nielsen, unpublished data) and AtRABF2b (Ueda et al., 2001) localize to compartments observed upon uptake of the fluorescent styryl dye, FM 4–64. Interestingly, the third Arabidopsis member of this subfamily, AtRABF1, appears to be a novel, plant-specific Rab GTPase (Bolte et al., 2000;Ueda et al., 2001). AtRABF1 lacks the traditional carboxy-terminal-CAAX motif for posttranslational isoprenylation but is myristoylated at the amino terminus (Ueda et al., 2001). Despite these differences, AtRABF1 colocalizes with AtRABF2b on the plant endosomal compartment where it may provide novel regulatory functions (Ueda et al., 2001).

The AtRABG subfamily contains eight members with significant similarity to human Rab7 and Ypt7p (Fig. 2). In mammals, Rab7 regulates transport of cargo from early endosomes to late endosomes and lysosomes (Feng et al., 1995; Mukhopadhyay et al., 1997). In yeast, Ypt7p localizes to vacuoles and regulates homotypic fusion between vacuolar compartments (Price et al., 2000). In plants, vacuoles are used as storage organelles in addition to having lytic roles, and single cells can contain multiple vacuole types (Paris et al., 1996; Vitale and Raikhel, 1999). This increased complexity appears to be reflected in the large number of members in this Rab GTPase subfamily in plants.

Rab GTPases of the Biosynthetic Trafficking Pathway

Newly synthesized proteins enter the secretory pathway by translocation through ER membranes. Subsequent transport from the ER to Golgi complexes involves recruitment of cargo proteins into vesicle transport intermediates. The AtRABD subfamily contains five members that display significant similarity with mammalian Rab1 and Ypt1p Rab GTPases (Fig. 2). In mammals, Rab1 GTPase isoforms localize to ER, ER Golgi intermediate compartment, and Golgi compartments and regulate ER-to-Golgi membrane trafficking steps (Tisdale et al., 1992; Nuoffer et al., 1994). In yeast,YPT1 is an essential gene required for ER-to-Golgi trafficking events (Segev et al., 1988). In plants, transient expression of a dominant-negative mutant of AtRABD2a resulted in accumulation of a secreted GFP marker in an intracellular compartment reminiscent of the ER and inhibited movement of Golgi complexes along cytoskeletal elements (Batoko et al., 2000). Arabidopsis contains three AtRABB subfamily members related to the human Rab2 GTPase. Rab2 GTPases are found associated with ER Golgi intermediate compartment membranes and COP-I transport vesicles in mammalian cells (Tisdale et al., 1992). The function and localization of the AtRABB subfamily members as yet remains uncharacterized in plants.

Recycling of ER-resident proteins, such as the KDEL-receptor Erd2p and Sec23p/Sec24p cargo receptor proteins requires the specific enrichment of these proteins in transport vesicles and subsequent delivery back to ER membranes (for review, see Kirchhausen, 2000). In mammals, Rab6 GTPases regulate this process (Martinez et al., 1997; White et al., 1999). Five isoforms of this subfamily of Rab GTPases (AtRABH) are present in the Arabidopsis genome. Of these, the AtRABH1b homolog, previously identified as AtRAB6, was shown to complement a yeast ypt6 mutation, although the function of this Rab GTPase in plants was not determined (Bednarek et al., 1994).

Rab GTPases Involved in Polarized Secretion

Arabidopsis contains five Rab GTPases that cosegregate with yeast and mammalian isoforms that regulate polarized secretion (AtRABE subfamily; Fig. 2). In yeast, Sec4p regulates membrane trafficking to the daughter cell bud site (Salminen and Novick, 1987;Goud et al., 1988). In plants, members of this Rab GTPase subfamily may have roles in cellular responses to bacterial pathogens. In tomato (Lycopersicon esculentum), yeast two-hybrid screening for plant proteins that interact with thePseudomonas sp. avirulence factor, avrPto, identified a RabE subfamily member with significant similarity to mammalian Rab8 (Bogdanove and Martin, 2000). This interaction only occurred in absence of the tomato resistance protein, Pto (Bogdanove and Martin, 2000), raising the possibility that in susceptible plants, avrPto may interfere with membrane trafficking pathways regulated by this RabE homolog. In an intriguing possibility, the authors suggest this Rab GTPase might regulate polarized secretion of antimicrobial compounds and/or components involved in mounting cellular responses to attack by bacterial pathogens.

A Plethora of Post-Golgi Rab GTPases in Plants

One of the most striking features evident upon examination of the AtRAB GTPases is the large number of AtRABA subfamily members (Fig. 2). This subfamily contains Rab GTPases from mammals and yeast that regulate TGN membrane trafficking pathways. In mammals, human Rab11A and Rab11B isoforms localize to recycling endosomes (Ullrich et al., 1996), and in epithelial cells, mammalian Rab11A is critical for exit of internalized proteins from apical recycling endosomes (Calhoun et al., 1998;Duman et al., 1999). Yeast Ypt31p and Ypt32p Rab GTPases appear to function during exit of membrane traffic from trans-Golgi cisternae (Benli et al., 1996; Jedd et al., 1997).

The Arabidopsis genome contains 26 distinct AtRABA subfamily members. Whether this multitude of genes provides distinct functions or represents functionally redundant gene families remains to be determined. Antisense inhibition of RABA subfamily GTPases in tomato results in complex developmental abnormalities and delayed fruit ripening (Lu et al., 2001). In pea (Pisum sativum), expression of two RABA GTPases, PRA2 andPRA3, was examined (Nagano et al., 1995). Whereas PRA3 was constitutively expressed, PRA2was specifically up-regulated in the stem-elongation zone of dark-grown seedlings, a region in which rapid plant cell expansion is observed (Nagano et al., 1995). More recent experiments indicated that Pra2 GTPase localizes predominantly to Golgi and possibly endosomal compartments, whereas Pra3 appears on TGN and/or prevacuolar compartments (Inaba et al., 2002). These RABA isoforms were suggested to play a role in delivery of new cell wall components to the plasma membrane. Interestingly, identification of effector proteins for RABA GTPases may further support roles for this class of GTPases in stem elongation in dark-grown seedlings because one effector protein for this Rab GTPase was identified as a cytochrome p450 involved in biosynthesis of brassinosteroids (Kang et al., 2001). Plant cells display a high degree of polarized deposition of primary and secondary cell wall components, in some cases, adjacent walls of a cell display differential localization of antigenic determinants (Lynch and Staehelin, 1992). The delivery of hemicelluloses, integral cell wall proteins, and probably even delivery of the cellulose synthase complex to the plasma membrane likely occur via post-Golgi membrane trafficking pathways. Could the large numbers of Rab GTPases in the AtRABA subfamily reflect the increased complexity associated with the highly polarized deposition of cell wall material observed in plants?

Rab-Interacting Proteins

Rab GTPases cycle between an inactive GDP-bound form located in the cytosol and an active GTP-bound form that is membrane associated. For most Rab GTPases, membrane association is promoted by posttranslational lipid modification. Stabilization of the lipid-modified Rab GTPase in cytosol occurs through association with Rab GDP-dissociation inhibitor proteins (RabGDI). Three RabGDI homologs (AtRabGDI1-AtRabGDI3; see supplemental data) are present in Arabidopsis. Two of these, AtRabGDI1 and AtRabGDI2 were identified by complementation of yeast sec19 mutants, indicating functional conservation of activity between yeast and plants. Although AtRabGDI1 was ubiquitously expressed, AtRabGDI2 displayed higher expression levels in suspension cultures cells and in roots (Ueda et al., 1996, 1998;Zarsky et al., 1997). AtRabGDI1-AtRabGDI3 homologs all share significant similarity (more than 77% identity). Interestingly, two RabGDI transcripts were determined to be up-regulated during early stages of fungal infection in rice (Oryza sativa; Kim et al., 1999), however the exact roles of these proteins during early plant defense responses to fungal infection remain unknown. A fourth related protein, AtREP1 displays similarity to the AtRabGDI homologs, but appears somewhat diverged (27% identity, 44% similarity), instead displaying higher similarity to mammalian REP-1 (Alexandrov et al., 1994). In mammals, REP-1 associates with newly synthesized Rab GTPases and presents them for posttranslational lipid modification.

GTP/GDP exchange is essential for Rab GTPase function. Upon binding to its target membrane, the Rab GTPase is converted from Rab:GDP to Rab:GTP through the action of RabGEF proteins. RabGEFs have been identified for several Rab GTPases in mammals and yeast (Horiuchi et al., 1997; Wada et al., 1997;Walch-Solimena et al., 1997; Hama et al., 1999; Iwasaki and Toyonaga, 2000;Siniossoglou et al., 2000). In general, significant levels of sequence similarity have not been observed between RabGEFs for different Rab GTPases. This suggests that despite having similar guanine exchange functions, different RabGEF proteins may have diverse evolutionary origins. However, in one notable exception to this trend, Vps9p, the RabGEF for the yeast Rab GTPase, Ypt51p, and Rabex-5, which catalyzes nucleotide exchange on mammalian Rab5, shows significant sequence similarity (Horiuchi et al., 1997;Hama et al., 1999). The Arabidopsis genome contained two sequences with significant similarity to the Vps9p and Rabex-5 RabGEF sequences. We designated these RabGEFs AtVPS9A and AtVPS9B (supplemental data). No Arabidopsis sequences with significant primary amino acid similarities could be detected for non-Vps9p-like RabGEFs.

As opposed to RabGEFs, which activate Rab GTPases, RabGAPs inactivate Rab GTPases by accelerating the slow intrinsic Rab GTPase activity. The first Rab-specific GAP proteins, Gyp6p, Gyp7p, and Gyp1p, were identified in yeast (Strom et al., 1993;Vollmer and Gallwitz, 1995; Vollmer et al., 1999). These proteins all contained six conserved sequence motifs within their catalytic domains (Albert et al., 1999). In other proteins, such as RN-tre and GAPCenA proteins, presence of these motifs successfully predicted their RabGAP activity (Cuif et al., 1999; Lanzetti et al., 2000). Analysis of the Arabidopsis genome revealed 20 proteins with all or most of the RabGAP “catalytic core” motifs, which we have named AtGYP proteins (supplemental data). All AtGYP proteins contained a conserved Arg residue critical for RabGAP activity (Albert et al., 1999) and at least five of the six “catalytic core” motifs. Although additional sequences containing portions of the RabGAP “catalytic core” were detected, these were not included because they (a) were missing two or more conserved motifs, and (b) did not contain essential conserved residues. Apart from the RabGAP motif, AtGYP family members contain no other detectable protein domains and displayed significant sequence diversity. In animals, two RabGAP proteins have been found associated with noncatalytic partner proteins (Nagano et al., 1998;Lanzetti et al., 2000). It seems likely that other RabGAP proteins may also be found in association with partner proteins.

In the last several years, many Rab effector proteins have been characterized. Because Rab GTPases are highly conserved and function similarly in different organisms, one might expect that Rab effector proteins also be conserved. But this is not apparently the case (Zerial and McBride, 2001). The structural heterogeneity of Rab effectors make it unlikely that plant Rab effectors can be identified by sequence similarity alone. However, some established Rab effectors, such as lipid kinases (Christoforidis et al., 1999), are present in Arabidopsis. In addition, in several cases, zinc-finger domains are associated with Rab effector protein functions (Christoforidis et al., 1999; Simonsen et al., 1998), and proteins with these domains are present in Arabidopsis (Jensen et al., 2001; Heras and Drobak, 2002). Identification of plant Rab effector proteins clearly is likely to uncover specialized proteins whose activities have been exclusively tailored for plant-specific transport systems.

Rop GTPases Are Multifunctional Signaling Molecules

Rho GTPases play central roles in a wide range of cellular processes, many of which are associated with the actin cytoskeleton. In mammalian cells, RHO proteins control assembly of actin stress fibers and focal adhesion complexes, RAC proteins regulate accumulation of actin filaments responsible for lamellipodia formation, and CDC42 is involved in formation of actin-containing microspikes called filopodia (Machesky and Hall, 1997; Kaibuchi et al., 1999). In S. cerevisiae, CDC42p and RHO1p regulate polarization of the actin cytoskeleton and control establishment and maintenance of cell polarity (Adams et al., 1990;Johnson and Pringle, 1990; Yamochi et al., 1994). More recently, Rho GTPases were found to influence microtubule dynamics and organization (Wittmann and Waterman-Storer, 2001). Rho GTPases also play important roles in a multitude of other processes (for reviews, see Erickson and Cerione, 2001; Ridley, 2000; Settleman, 2001).

The expression and localization patterns of the 11 AtROP GTPases (TableII) is consistent with their involvement in complex signaling networks in plants (Yang, 2002). Given the conspicuous absence of Ras-family GTPases and other RHO, RAC, and CDC42 subfamily members, it was hypothesized that the ROPs might replace these GTPases in plant signaling (Li et al., 1998; Winge et al., 2000). Overexpression of wild-type ROP GTPase genes and expression of mutants either deficient for GTPase activity (constitutively active; CA) or that can only bind GDP (dominant negative [DN]) have provided evidence for the above hypothesis (for reviews see, Yang, 2002; Zheng and Yang, 2000a).

Mammalian and fungal Rho GTPases regulate the establishment of cell polarity and influence cell morphogenesis (for review, seeArellano et al., 1999). Plant ROP GTPases appear to have retained this function. Three AtROP genes,AtROP1, AtROP3, and AtROP5, are expressed in pollen and may be functionally redundant during pollen tube growth. AtROP1 and AtROP5 are preferentially localized to the plasma membrane in apical regions of the pollen tube and control tip growth (Kost et al., 1999; Li et al., 1999). AtROP2 and AtROP4 localize to tips of elongating root hairs (Molendijk et al., 2001; Jones, 2002). CA mutants of AtROP2, AtROP4, and AtROP6 either caused isotropic growth or increased length in root hairs of Arabidopsis, whereas DN-AtROP2 mutants inhibited root hair elongation, indicating that AtROP GTPases also control tip growth during root hair development (Molendijk et al., 2001; Jones, 2002). In both root hairs and pollen tubes, AtROP GTPases control tip growth by modulating the formation of both the dynamic fine tip F-actin and a tip-focused cytosolic calcium gradient (Li et al., 1999;Fu et al., 2001; Fu and Yang, 2001;Molendijk et al., 2001; Jones, 2002). AtROP GTPases also regulate formation of cell shape by controlling assembly of dynamic cortical F-actin in cells that do not undergo tip-based growth such as epidermal cells (Fu and Yang, 2002).

In mammals, RAC GTPases control production of reactive oxygen species by directly associating with and regulating the activity of plasma membrane-associated NADPH oxidase complexes (for review, seeRidley, 1995). In Arabidopsis, oxygen deprivation rapidly and transiently activates ROP GTPases, resulting in H₂O₂ accumulation and alcohol dehydrogenase gene expression (Baxter-Burrell et al., 2002). ROP GTPase-dependent H₂O₂ production is blocked by treatment with DPI, an inhibitor of the plasma membrane NADPH oxidase (Kawasaki et al., 1999; Baxter-Burrell et al., 2002). These results are consistent with the suggestion that plant ROP GTPases may regulate the plasma membrane NADPH oxidase complex.

AtROP GTPases are also involved in signal transduction pathways mediated by the plant hormone, abscisic acid (ABA). ABA promotion of seed dormancy was enhanced and inhibited respectively by DN-Atrop2 and CA-Atrop2 expression in Arabidopsis (Li et al., 2001).Lemichez et al. (2001) showed that AtROP6 was implicated during the negative regulation of stomatal closure, and studies using loss of function mutants have revealed that AtROP9 and AtROP10, two putative ERA1 targets, act as general negative regulators of ABA responses (Zheng et al., 2002). ERA1 encodes a β-subunit of protein farnesyltransferase involved in the negative regulation of ABA responses both in guard cell movement and seed dormancy.

Plant ROP GTPases may also regulate the accumulation of and/or responses to brassinolide and auxin. CA-AtROP2 plants exhibit diverse morphologies that resemble auxin- or brassinolide-overproduction mutants, whereas DN-AtROP2 plants show opposite phenotypes (Li et al., 2001). Readers are referred to several recent reviews for more detailed discussion of the function of the ROP family GTPases in these processes (Valster et al., 2000; Zheng and Yang, 2000a, 2000b; Fu and Yang, 2001; Yang, 2002).

Rop-Interacting Proteins

Several types of Rho GTPase regulators are known in animals: GAPs and GDIs (guanine nucleotide dissociation factors) that act as negative regulators of Rho GTPase function, and GEFs, which activate these GTPases through exchange of GDP for GTP (see introduction and “RAB GTPases” section).

In most fungal and animal RhoGEFs, GDP-GTP exchange activity typically is localized in Dbl-homology domains (Cerione and Zheng, 1996). Surprisingly, plants lack proteins containing obvious Dbl-homology domains. This suggests that plants may have evolved different mechanisms to activate the GDP-bound ROP GTPases. One possible mechanism of activation may be through direct association of ROP GTPases with plant receptor-like kinases (RLKs). A plant ROP GTPase was co-immunoprecipitated with the plant RLK, CLAVATA1 (Trotochaud et al., 1999). Therefore, plant ROP GTPases could be activated by RLKs directly, although the functional significance of ROP-RLK association remains to be determined.

Three RhoGDI homologs are present in Arabidopsis. AtRhoGDI1 is expressed in all tissues examined and has been shown to interact specifically with AtROP GTPases (Bischoff et al., 2000), but so far, no functional data is available. Using the yeast two-hybrid method, Wu et al. (2000) identified a family of Rop-specific, Rho GAPs called RopGAPs. Arabidopsis contains six RopGAPs, all with an N-terminal Cdc42/Rac-interactive binding (CRIB) motif located adjacent to a conserved RhoGAP catalytic domain. In yeast and animals, CRIB domains mediate GTP-dependent interaction between Rho GTPases and their effector proteins, but are not found in conjunction with RhoGAP domains. In plants, presence of the CRIB domain is critical for the Rop-specific regulation of GAP activity (Wu et al., 2000). RopGAP1 localizes in the apical PM region in pollen tube and acts as a negative regulator of AtROP1 function during pollen tube growth (G. Wu and Z. Yang, unpublished data). A role for RopGAP4 in Arabidopsis responses to oxygen deprivation has been revealed using a ropgap4 knockout mutant (Baxter-Burrell et al., 2002).

No obvious homologs of animal and yeast Rho effector proteins, except for phosphatidyl-inositol-phosphate kinases, are present in the Arabidopsis genome. Because the CRIB motif is a hallmark structure of many CDC42/RAC effectors, plant proteins containing these domains make attractive Rop GTPase effector protein candidates. Arabidopsis contains a novel plant-specific family of 11 putative ROP GTPase effector proteins, Rop-interactive CRIB motif-containing proteins (RICs; Wu et al., 2001). RIC1 associates with AtROP1 in a nucleotide-dependent manner, and this association is dependent upon the CRIB domain. Because RIC family members display little sequence similarity with other proteins or even with one another (apart from the CRIB domain), it seems unlikely that the variable regions of RICs function as enzymes to regulate downstream events. RICs could act as adaptor proteins linking Rop GTPases to effector proteins, which in turn would activate specific downstream events. Such a linker function would allow the generation of a greater functional diversity for the Rop GTPase switch and would present a novel mechanism for the activation of G protein effectors (Wu et al., 2001;Yang, 2002).

Arf GTPases Recruit Coat Proteins to Transport Vesicles

Arf GTPases play important roles during membrane trafficking steps in eukaryotic cells (for review, see Chavrier and Goud, 1999). In all cases, Arf GTPases act to recruit cytosolic coat proteins to sites of vesicle budding. Three types of protein coats have been described for transport vesicles, COP-I, COP-II, and clathrin coats (Kirchhausen, 2000), and formation of these coats are attributed to two distinct subsets of the Arf GTPase family. Arf GTPases recruit COPI and clathrin protein coats to transport vesicles, whereas a specific subset of the Arf GTPase family, the Sar1p GTPases, recruit COP-II coats.

The best understood mechanism by which Arf GTPases recruit coat proteins to transport vesicles is in S. cerevisiae, where recruitment of COP-II protein coat by Sar1p has been extensively studied (for review, see Kirchhausen, 2000). Sar1p GTPases regulate formation of COP-II coated vesicles carrying cargo from the ER to the cis-Golgi compartment (Wieland and Harter, 1999). In Arabidopsis, three Sar1p homologs have been identified (Fig. 4). Because these GTPases cosegregated with animal and yeast Sar1 GTPases, we have named them AtSARA1a, AtSARA1b, and AtSARA1c, respectively (Fig. 4; Table III). AtSARA1a, previously identified as AtSAR1, was shown to functionally complement mutants in S. cerevisiae (d'Enfert et al., 1992), and AtSARA1a expression was correlated to levels of secretion activity from ER membranes in plant cells, because blockage of transport from the ER resulted in up-regulation of mRNA levels for AtSARA1a (Bar-Peled et al., 1995).

In S. cerevisiae, two Arf GTPases, Arf1p and Arf2p, are functionally interchangeable and act to recruit COP-I coat proteins to transport vesicles during transport of cargo from cis-Golgi compartments back to the ER (Cosson and Letourneur, 1997; Gaynor and Emr, 1997). The yeast Arf1p and Arf2p GTPase functions are distinct from that of Sar1p, which acts in ER-to-Golgi trafficking (Barlowe et al., 1994). In Arabidopsis, 12 Arf GTPase isoforms that cosegregated with members of the Arf GTPase subfamily were identified (Table III; Fig. 4). It should be noted, however, that we also observed mammalian and yeast Arl GTPase sequences within this grouping. Six AtARF GTPases cosegregated with human Arf1 and Arf3 sequences (AtARFA subfamily). This includedAtARFA1a, previously identified as AtArf1(Regad et al., 1993), which has been localized to peripheral Golgi stacks along with Atγ-COP, an Arabidopsis homolog of the COP-I coat protein complex (Ritzenthaler et al., 2002). Six additional Arabidopsis sequences cosegregated with the Arf GTPase subfamily (AtARFB-AtARFD), however, no significance could be ascribed to these groupings outside that of the AtARFA subfamily, which cosegregated with a distinct subset of mammalian Arf GTPases.

Arl GTPases: Regulatory Proteins with Mystery Functions

Six of the 21 Arabidopsis Arf GTPases segregate in groups that contain neither Sar1p nor Arf GTPase subfamily members (Fig. 4). As such, they have been classified as Arl GTPases. Little is known of the function of these GTPases, although at least one member provides essential functions as demonstrated in fruitfly (Tamkun et al., 1991). In plants, mutation of the TITAN5 gene, which corresponds to AtARLC1, results in dramatic alterations of mitosis and cell cycle control during seed development (McElver et al., 2000). The authors speculated that this gene might play a role in membrane trafficking steps necessary for proper cell plate deposition during cytokinesis in developing embryos (McElver et al., 2000). However, much remains to be determined regarding the role(s) of these proteins in any eukaryotic organism.

Arf GTPase-Interacting Proteins

Like other small GTPases, Arf GTPases cycle between an active, membrane-bound form when associated with GTP, and an inactive, predominantly cytosolic form when bound to GDP. Similarly, this GTPase cycle is regulated by GEFs (ArfGEFs) and GAPs (ArfGAPs;Donaldson and Jackson, 2000). However, unlike Rho and Rab GTPases, Arf GTPases do not require GDIs to catalyze their delivery to membranes. Whereas Rab and Rho GTPases are lipid modified at their carboxy terminus, Arf GTPases are myristoylated at their amino terminus. Due to this myristoylation, GDP-bound Arf GTPases display a weak interaction with membranes (Franco et al., 1995). Activation of the membrane-associated Arf GTPase through GEF-catalyzed exchange of GDP for GTP causes conformational changes resulting in stabilization of the Arf GTPase-membrane interaction (Antonny et al., 1997).

Much of the research into Arf GTPase function has focused upon their roles in coat protein recruitment during vesicle formation (see above), and several proteins that interact with Arf GTPases during these processes such as Sec12p and Sec23p, display Sar1p-specific GEF and GAP activities, respectively (Springer et al., 1999). However, Arf GTPases also have other roles such as alteration of membrane lipid compositions, and actin remodeling upon various membrane compartments of the cell (Donaldson, 2000). Distinct ArfGEF or ArfGAP proteins regulate these additional activities.

To date, all ArfGEFs possess a Sec7 domain (Donaldson and Jackson, 2000). This 200-amino acid domain is sufficient to catalyze GDP for GTP exchange on Arf GTPases in vitro (Chardin et al., 1996). Arabidopsis encodes eight proteins containing Sec7 domains. One of these ArfGEFs, GNOM, was identified in mutant screen for Arabidopsis with defective body organization in embryos (Mayer et al., 1991, 1993). Polarized distribution of the auxin-efflux carrier, PIN1, was disrupted in gnom mutants, indicating that this ArfGEF is involved in trafficking of PIN1 proteins during development (Steinmann et al., 1999; Geldner et al., 2001). Little is known of the functions of the other seven Arabidopsis ArfGEF proteins. However, given the central role of the ArfGEF GNOM during organization of the early embryo, this class of proteins is likely to fulfill important roles in growth and development in plants.

In animals, ArfGAPs are a diverse family of multidomain proteins (Donaldson, 2000) that contain a zinc-finger motif and a conserved Arg residue within the ArfGAP catalytic domain (Cukierman et al., 1995; Randazzo et al., 2000). Arabidopsis contains 15 proteins with ArfGAP domains, and we have termed these Arf GAP domain (AGD) proteins. We grouped the AtAGD proteins into four distinct classes based on phylogenetic analysis (data not shown) and overall domain organization (Fig.5). The first class of AtAGD proteins, consisting of AtAGD1-AtAGD4, is a novel, plant-specific family of putative Arf-GAP proteins. These proteins all contain pleckstrin homology (PH) domains, and two or three ankyrin repeat domains in addition to the AGD (Fig. 5). Presence of PH domains in these AtAGD proteins raises the possibility that they may coordinate their ArfGAP activity with phospholipid signaling events as PH domains bind to phosphoinositides (for review, see Lemmon et al., 2002). In addition, AtAGD1, AtAGD2, and AtAGD3 contain amino-terminal Bin1-amphiphysin-Rvs167p/Rvs161p (BAR) domains. BAR domains are found in adaptor proteins implicated in numerous biological functions including actin regulation and synaptic vesicle endocytosis (Wigge and McMahon, 1998; Balguerie et al., 1999). However, presence of BAR domains in ArfGAP proteins appears to be plant specific. Class 2 AtAGD proteins (AtAGD5–AtAFGD10; Fig. 5) contain AGDs at the amino terminus and no other discernible protein domains. These proteins show similarity both at sequence level and in overall domain organization to Golgi-localized ArfGAPs in mammals and in yeast (Cukierman et al., 1995;Poon et al., 1999). Class 3 AtAGD proteins (AtAGD11–AtAGD13; Fig. 5) are distinguished by the presence of a centrally located C2 domain. C2 domains bind a range of ligands, such as phospholipids, phosphoinositides, and other proteins, in calcium-dependent fashion (Sutton et al., 1995;Essen et al., 1996; Shao et al., 1997). In Class 4 AtAGD proteins (AtAGD14 and AtAGD15; Fig. 5), the AGD comprises almost the entire open reading frames of these proteins. In AtAGD14, the AGD is followed by an apparent membrane-spanning α-helix, suggesting this ArfGAP could be an integral membrane protein. As in mammals, plant ArfGAPs have diversified into a large family of proteins. Several classes of the AtAGD proteins contain additional domains, and these may act to coordinate the timing of ArfGAP activity within the cell or may serve functions beyond simply acting as negative regulators of Arf GTPases. Understanding the mechanisms by which temporal and spatial activities of plant ArfGAPs are regulated will be an interesting area for future investigation.

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Fig. 5.

ArfGAP proteins of Arabidopsis. AtAGD proteins were identified using BLASTP (Altschul et al., 1997) with the AGD of ASAP1 (GenBank accession no. NP_060952). Domains within the AtAGD sequences were detected using SMART (Letunic et al., 2002). AGI numbers of AtAGD1 to AtAGD15are as follows: At5g61980 (AtAGD1), At1g60680 (AtAGD2), At4g13300 (AtAGD3), At1g10870 (AtAGD4), At5g54310 (AtAGD5), At3g53710 (AtAGD6), At2g37550 (AtAGD7), At4g17890 (AtAGD8), At5g46750 (AtAGD9), At2g35210 (AtAGD10), At3g07490 (AtAGD11), At4g21160 (AtAGD12), At4g05330 (AtAGD13), At1g08680 (AtAGD14), and At3g17660 (AtAGD15).

In yeast and animals, increasing attention is being paid to the roles of Arf GTPases in lipid modification. Mammalian and yeast Arf GTPases activate phosphatidylinositol 4-phosphate, 5-kinases (Honda et al., 1999; Walch-Solimena and Novick, 1999). Arabidopsis also contains phosphatidylinositol 4-phosphate, 5-kinase proteins, but whether AtARF GTPases interact with these remains unknown. A new class of Arf GTPase effector proteins that facilitate membrane trafficking at the TGN has recently been described. These Golgi-localizing, γ-adaptin ear homology, Arf-binding (GGA) proteins contain multiple domains: an amino-terminal VHS (Vps27, Hrs, and STAM) domain, followed by the GAT (GGA1 and TOM proteins) region and the γ-adaptin homology domain at the carboxy terminus. The VHS domain is thought to mediate interactions with cargo molecules, whereas the GAT domain is responsible for association with Arf GTPases, and the γ-adaptin homology domain interacts with clathrin (for review, seeBoman, 2001). Five GGA-like proteins are present in the Arabidopsis genome. Intriguingly, although these proteins contain the amino-terminal VHS and GAT domains, they appear to lack the carboxy-terminal γ-adaptin homology domain. Given the apparent central role of GGA proteins during sorting of cargo proteins and recruitment of vesicle coat proteins, it will be interesting to determine the role of this class of proteins in plant Arf GTPase functions.

Ran GTPases Establish Nuclear Compartment Identity

In interphase cells, Ran GTPases direct nucleocytoplasmic transport. RanGAP and RanBP2 (which stimulate the intrinsic GTPase activity of the Ran GTPase) are cytoplasmic proteins, whereas RCC1 (which generates Ran:GTP) is confined to the nucleus. These distributions ensure that Ran GTPases are bound with GTP in the nucleus and GDP in the cytoplasm. The restriction of Ran:GTP to the nuclear interior has been proposed to be crucial for establishing directional transport of proteins and RNA through the nuclear pore (Moore and Blobel, 1993; Izaurralde et al., 1997). Ran GTPases also play important roles during cellular mitosis (Kalab et al., 1999; Wilde and Zheng, 1999). Association of RCC1 with chromatin results in localized generation of Ran:GTP in the vicinity of chromosomes, which in turn promotes microtubule spindle assembly (Carazo-Salas et al., 1999;Ohba et al., 1999; Wiese et al., 2001). At the end of mitosis, the cycling of Ran:GDP and Ran:GTP induces nuclear envelope reassembly, probably by controlling vesicle binding and fusion (Hetzer et al., 2000; Zhang and Clarke, 2000).

Functions of Arabidopsis Ran GTPases and
Associated Proteins

AtRAN1 to AtRAN3 are ubiquitously expressed during development, with higher levels in meristematic tissues and developing embryos (Haizel et al., 1997). Overexpression of plant Ran GTPases suppressed cell cycle defects in mutant fission yeast, indicating that they functioned similarly to their yeast homologs (Ach and Gruissem, 1994; Merkle et al., 1994; Haizel et al., 1997). In animals, Exportin-1 is the nuclear export receptor for proteins carrying nuclear export signals (NES). Exportin-1 binds NES-containing proteins and Ran GTPases cooperatively, and this triple complex undergoes nuclear export (Fornerod et al., 1997). In Arabidopsis, AtRAN1 interacts with AtXPO1, an Arabidopsis Exportin-1 homolog, and a NES-containing protein, AtRanBP1a (Haizel et al., 1997;Haasen et al., 1999). This suggests the nuclear export machinery may be functionally conserved in plants (Haasen et al., 1999). Antisense expression of an additional AtRanBP isoform in Arabidopsis, AtRanBP1c, enhanced primary root growth, suppressed lateral root growth and rendered transgenic roots hypersensitive to auxin (Kim et al., 2001). The authors proposed a model where AtRanBP1c plays key roles in delivery of nuclear proteins responsible for suppression of auxin action and regulation mitosis in root tips. However, direct evidence is still required to clarify the role(s) of Ran GTPases and RanBPs in plant growth and development.

The mammalian RanGEF, RCC1, contains seven tandem repeats of a 50- to 60-amino acid domain, and these repeats constitute the majority of the protein. Arabidopsis contains 18 proteins containing one to five RCC1 domains (see supplemental data). One of these, uvr8, is a mutant hypersensitive to UV-B (Kliebenstein et al., 2002). The UVR8 protein contains five RCC1 repeats but interestingly does not posses nuclear localization sequences conserved in animal and yeast RCC1 homologs (Kliebenstein et al., 2002). Further work is necessary to determine whether UVR8, or other Arabidopsis proteins with RCC1 domains act as RanGEFs.

Two RanGAP sequences have been identified in Arabidopsis: AtRanGAP1 (accession no. AF2214559) and AtRanGAP2 (accession no. AF214560). Both AtRanGAPs complemented yeast RanGAP mutants (Pay et al., 2002), suggesting that these proteins are functional orthologs of the yeast RanGAP. AtRanGAP-GFP fusions associate with the nuclear envelope, and this localization is dependent upon a unique N-terminal domain (Rose and Meier, 2001). AtRanGAP1 localization is consistent with a role for AtRAN GTPases in nucleocytoplasmic transport. However, whether plant Ran GTPases function to regulate these transport steps or different plant Ran GTPases, especially the divergent AtRAN4, have distinct functions remains undetermined.