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First published online January 23, 2003; 10.1104/pp.013052 Plant Physiol, March 2003, Vol. 131, pp. 1191-1208 Analysis of the Small GTPase Gene Superfamily of Arabidopsis1Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, California 92521 (V.V., Z.Y.); Biology Department, Washington University, Campus Box 1137, One Brookings Drive, St. Louis, Missouri 63130 (A.C.H., E.N.); and Donald Danforth Plant Science Center, St. Louis, Missouri 63132 (E.N.)
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.
Small GTP-binding proteins are
molecular switches that are "activated" by GTP and
"inactivated" by the hydrolysis of GTP to GDP. The resulting cycles
of binding and hydrolysis of GTP by small GTP-binding proteins
represents a ubiquitous regulatory mechanism in eukaryotic cells.
Members of this class of proteins are among the largest families of
signaling proteins in eukaryotic cells. Their importance in cellular
signaling processes is underscored by their conservation throughout
evolution of eukaryotic organisms and by the presence of homologs that
perform related functions in cells of yeasts, humans, and plants.
Although the GTP-hydrolysis "core" of this class of regulatory
molecules is highly conserved, the surrounding domains are highly
variable and undergo conformational changes as these proteins switch
from GTP-associated to GDP-associated states. Eukaryotes have harnessed
this diversity of protein conformations, linked to the
nucleotide-associated state of the GTP-binding domain, to regulate a
myriad of cellular processes (for review, see Takai et al.,
2001 Structural and functional similarities between different members
of this large superfamily has led to establishment of five distinct
families: Ras, Rab, Rho, Arf, and Ran (Kahn et al.,
1992
Identification and Classification of Small GTPase Genes in Arabidopsis To identify small GTPase genes in the Arabidopsis genome, we first
compiled sequences previously documented in S. cerevisiae and humans as members of the small GTP-binding protein superfamily of regulatory proteins. These genes were classified into the five accepted families of small GTPases (Kahn et al., 1992 To classify Arabidopsis small GTPase genes into Ras, Rho, Rab, Arf, and
Ran families, theoretical protein sequences from these genes were
aligned to previously compiled lists of small GTPases of S. cerevisiae and humans using ClustalW (Thompson et al.,
1994
Rab GTPases Rab GTPases make up the largest family of the small
GTP-binding protein superfamily. Both in vivo and in vitro experiments have demonstrated the roles of this class of proteins in intracellular membrane trafficking. Furthermore, given the specific distributions of
Rab GTPases to different cellular membranes, it was hypothesized that
Rab GTPases would, in conjunction with SNARE proteins, provide specificity for membrane fusion events (for reviews, see
Stenmark and Olkkonen, 2001 Our analysis of the Arabidopsis genome (Arabidopsis Genome
Initiative, 2000
Correlation of Sequence Similarity with Localization in  ). 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 the
Pseudomonas 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 and
PRA3, was examined (Nagano et al., 1995).
Whereas PRA3 was constitutively expressed, PRA2
was 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 Members of the Rho GTPase family have emerged as key regulators of
the actin cytoskeleton in yeast and animal cells. Transduction of
signals from cell surface receptors, which ultimately result in
reorganization of the actin cytoskeleton, occurs through interaction of
Rho GTPases with regulatory proteins and downstream effector proteins
(Hall, 1998
Due to a slightly higher overall similarity with human RAC GTPases,
plant Rho-related GTPases have been identified as plant RAC GTPases
(Winge et al., 1997 Analysis of the Arabidopsis genome revealed the existence of 11 ROP
GTPases, which we have named AtROP (Fig. 3; Table II). Previous reports
have been inconsistent regarding the number of Rho-related GTPases
present in Arabidopsis, e.g. 13 in Lemichez et al.
(2001) 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 (Table
II) 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, see
Arellano 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, see
Ridley, 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
 -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 ADP-ribosylation factors (Arfs) were initially identified due to
their ability to stimulate the ADP-ribosyltransferase activity of
cholera toxin A (Moss and Vaughan, 1998
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 included
AtARFA1a, previously identified as AtArf1
(Regad et al., 1993), which has been localized to
peripheral Golgi stacks along with At -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
 -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.
; 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, -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
-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 -adaptin homology domain interacts with clathrin (for review, see
Boman, 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 -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 In animals, Ran GTPases together with their regulatory factors,
the Ran-binding proteins (RanBPs), RCC1 (a GEF, RanGEF) and RanGAP (a
GAP), play key roles in controlling nuclear processes throughout the
cell mitotic cycle (for review, see Clarke and Zhang,
2001 At the protein level, AtRAN1, AtRAN2, and AtRAN3 are nearly
identical (95%-96% of identity) differing only in their C-terminal regions, whereas AtRAN4 is more divergent with only 65%
identity to the other AtRAN sequences. All AtRan GTPases contain
sequence motifs involved in GTP binding/hydrolysis and an
effector-binding domain for interaction with RanGAPs. This
effector-binding motif is 100% identical in AtRAN1 to AtRAN3 and in
tomato and tobacco Ran GTPases (KKYEPTIGVEV) but diverges strikingly in
AtRAN4, with only five residues of 11 conserved. Moreover, although
other plant Ran GTPases possess conserved C-terminal acidic domains
(DDDDD/E), this sequence is absent in AtRAN4. In animals, this
acidic domain is necessary for interaction with Ran-binding proteins
(Haizel et al., 1997 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 ). 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.
From these studies and observations, it is clear that small GTPases represent an important and diverse set of regulatory molecules in plants, with Arabidopsis containing members of the Rab, Rho, Arf, and Ran classes of small GTPases. In many cases, plant small GTPases appear to maintain similar functions as their animal and yeast counterparts. The identification of additional families of putative GAP and GEF proteins for most of these families further highlights the highly conserved nature of these regulatory molecules throughout evolution of eukaryotes. However, plants contain some unique variations on this conserved regulatory mechanism. Although small GTPases related to Rab, Rho, Arf and Ran GTPases were identified in Arabidopsis, Ras GTPases were not detected. This, in principal, concurs with the evolution of eukaryotic regulatory systems. The notable absence of plant Ras GTPases correlates with lack of Tyr kinase receptors, which act upstream of Ras signaling in animals. Plant Rop GTPases segregate as a distinct subfamily within the Rho GTPase family. While maintaining conserved functions in regulation of actin cytoskeleton and cellular signaling pathways, newly established roles in ABA-signaling events and interactions with plant RLKs highlight potential plant-specific variations of the functions of these Rop GTPases. Plant Rab GTPases segregate into distinct subfamilies that appear to be organized around the types of compartments upon which they are localized. Increased numbers of Arabidopsis Rab GTPases in subfamilies with predicted functions in vacuolar and post-Golgi trafficking may indicate plant-specific elaborations of these pathways. Plant Arf GTPases and Ran GTPases functionally complement mutations of their respective counterparts in yeast. However, plants contain multiple, novel ArfGEFs and ArfGAPs, probably with novel functions. Also, AtRAN4 is significantly different in sequences conserved throughout Ran GTPases from plants, yeast, and animals, perhaps indicating novel roles during plant growth and development. A wealth of knowledge of the mechanisms of action of small GTPases and of some of their important activator proteins (GEFs) and inhibitor proteins (GAPs) will clearly serve as a strong basis for understanding how these conserved regulatory mechanisms have been modified to carry out plant-specific processes.
We thank Warren Lathe III for initial help with phylogenetic analysis of the small GTPases and Jannie Santos-Serna for help with the tables and figures. We also thank Tony Sanderfoot, Daniel Schachtman, and Christiane Wobus for critical reading of the manuscript, and Ian Moore for helpful comments regarding Rab GTPase nomenclature.
Received August 14, 2002; returned for revision September 3, 2002; accepted November 19, 2002. 1 This work was supported by the National Aeronautics and Space Administration (grant no. NAG2-1525 to E.N.), by the U.S. Department of Agriculture (grant no. 2000-01586), and by the Department of Energy (grant no. DE-FG03-00ER15060 to Z.Y.).
* Corresponding author; e-mail enielsen{at}danforthcenter.org; fax 314-587-1381.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.013052.
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ABSTRACT
INTRODUCTION
