518
The Arabidopsis Rab GTPase family: another enigma variation
Stephen Rutherford* and Ian Moore†
The Arabidopsis genome sequence reveals that gene families
such as the Rab GTPase family, which encodes key
determinants of vesicle-targeting specificity, are considerably
more diverse in plants and mammals than in yeast. In
mammals, this diversity appears to reflect the complexity of
membrane trafficking. Phylogenetic analyses indicate that,
despite its large size, the Arabidopsis Rab family lacks most
of the Rab subclasses found in mammals. The Arabidopsis
Rab family has, however, undergone a distinct ‘adaptive
radiation’ that has given rise to proteins that may perform
plant-specific functions.
Addresses
Department of Plant Sciences, University of Oxford, South Parks Road,
Oxford OX1 3RB, UK
*e-mail: stephen.rutherford@plants.ox.ac.uk
†e-mail: ian.moore@plants.ox.ac.uk
Current Opinion in Plant Biology 2002, 5:518–528
1369-5266/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S1369-5266(02)00307-2
Abbreviations
BFA
Brefeldin A
ER
endoplasmic reticulum
GFP
green fluorescent protein
PIN1
PIN-FORMED1
PM
plasma membrane
SNARE
soluble-N-ethyl-maleimide sensitive fusion factor
attachment protein receptor
VTC
vesicular tubular cluster
Introduction
The plant endomembrane system comprises several
biochemically distinct membrane-bound organelles that
are linked by membrane traffic. More than 17% of all
Arabidopsis gene products are predicted to enter the
endomembrane system [1] and each must be transported
to its correct destination. That each compartment retains
its distinct molecular identity in the face of rapid membrane
flux [2–4,5•] is testament to the efficiency of the sorting
and targeting functions of the endomembrane compartments
and their associated vesicles. Gene sequencing suggests
that the core apparatus for the formation and targeting of
vesicles in plants is similar to that described for yeast [1].
One important group of genes encoding the Arabidopsis
‘SNARE’ proteins has been reviewed recently [6•] (SNARE
stands for soluble-N-ethyl-maleimide sensitive fusion
factor attachment protein receptor). In this review, we
focus on another important family, the Rab GTPase family,
and discuss the implications of the Arabidopsis Rab GTPase
complement for membrane traffic in higher plants.
Rab GTPases are members of the ras superfamily of
regulatory GTPases and are emerging as key regulators of
targeting specificity in eukaryotic membrane traffic [7–9].
In yeast and mammalian cells, they are known to regulate
the activity of the tethering factors and possibly also the
SNARE complexes. These two groups of proteins combine
to promote the initial docking and subsequent fusion of
specific vesicle and organellar membranes. Some Rab
GTPases also promote interactions between transport
vesicles and the cytoskeleton [7,8]. Individual members of
the Rab GTPase family appear to be responsible for
distinct vesicle-targeting events. In some cases, a single
Rab GTPase can act in two consecutive transport steps [10].
To perform these regulatory roles Rab GTPases interact
with a large array of regulatory and effector molecules that
couple the cycle of GTP-binding and GTP-hydrolysis to
the processes of vesicle formation, targeting, and docking
([7,8]; Figure 1a,b).
The plant Rab GTPase family has a distinct
composition
Although the Rab GTPase family clearly arose early in
eukaryotic evolution, the yeast, mammalian, invertebrate,
and angiosperm lineages have each elaborated quite distinct
complements of Rab proteins [7,9,11••]. The genome of
Schizosaccharomyces pombe encodes a basic complement of
seven Rab proteins, whereas Saccharomyces cerevisiae has
11 Rabs in eight functionally distinct subclasses, six of
which have homologues with varying degrees of functional
conservation in animals and S. pombe [7,9,11••]. It is possible
that these six Rab subclasses represent a minimal set of
eukaryotic Rab functions. Indeed, the available functional
evidence suggests that one member of this set can account
for the principal transport events between the major
organelles of the biosynthetic and endocytic transport
pathways (Figure 1c). In marked contrast, humans have at
least 60 different Rab proteins that are ascribed to about
40 different functional subclasses [7,11••,12•].
In the Arabidopsis genome sequence, we and others [11••]
have identified 57 loci that can encode Rab GTPases. 48 of
these are known as cDNAs or expressed sequence tags
(ESTs) or can be amplified from cDNA (I Moore, T Ueda,
unpublished data). The remaining nine loci retain the
characteristic structural and functional motifs of the Rab
family [12•,13], and for the time being we will consider
them as functional members of the family. Thus,
Arabidopsis apparently encodes 57 Rab GTPases; but the
more important and challenging question is ‘how many
different functional subclasses does this represent?’.
In phylograms, all plant Rab sequences can be grouped
into just eight clades [14]. These clades are related to the
six Rab subclasses that are common to yeasts and animals
plus Rab2 and Rab18, which are not present in yeast ([9];
see Figure 2 in which branches leading to these groups are
The Arabidopsis Rab GTPase family Rutherford and Moore
519
Figure 1
(a)
(c)
CC
Golgi
ER
GxxxxGKS/T
WDTAGQE
Ypt1
Rab1
D
GNKxD ETSAK
Rab2
B
Sec4
Various Rabs
E
(b)
Ypt6
Rab6
H
Ypt31/32
Rab11/Rab25
A
∆C
En/PV
En/Pv
Ypt7
Rab7
G
Vacuole
Rab18
C
Rab5
Ypt51/52/53
F
∆N
Current Opinion in Plant Biology
(a) Schematic representation of the approximately 200-amino-acid
sequence of a typical Rab GTPase. Green boxes indicate the positions
of sequence motifs that are conserved in Rab GTPases. These motifs,
indicated in single-letter code below each box, are involved in
nucleotide binding and hydrolysis. Mutation of the residues (shown in
red) has been used to generate dominant-negative and constitutively
active forms that exhibit altered nucleotide-binding or hydrolysis
characteristics; these can be used to investigate Rab function. Blue
boxes indicate the position of sequence motifs that are conserved
within each functional subclass but differ between subclasses, helping
to define the function and identity of Rab subclasses. Cysteine
residues (CC) that are geranylgeranylated to facilitate membrane
attachment are conserved near the carboxyl terminus. (b) A
representation of the backbone structure of mammalian Rab3a
(residues 19–191) in its GTP-bound conformation [52]. The conserved
nucleotide-binding motifs (green) fold to form a tight pocket around the
bound nucleotide (yellow) and magnesium ion (orange). The subclassspecific sequences (blue) form two distinct surfaces that facilitate the
interaction of Rab GTPases with a range of specific effector and
regulatory factors. These interactions usually depend on the
phosphorylation state of the bound nucleotide. Secondary structures
are shown in red. (c) The trafficking pathways of a generic eukaryotic
cell. The letters A to H represent probable sites of action of proteins in
the eight major clades of the Arabidopsis Rab phylogeny (see Figure 2).
Six of the Arabidopsis clades are conserved in animals and yeasts,
and the closest mammalian (red) and yeast (orange) subclasses are
indicated. The two clades that have no yeast homologues, B and C,
are shown in grey. En/Pv indicates a generic endosomal/prevacuolar
compartment. Compiled and modified from [7,8,12•,13,52] and from
the Brookhaven Protein Data Bank using Swiss PDB viewer 3.7 (b2).
indicated by capital letters A to H). If these eight groups
equate to the principal functional subclasses in the
Arabidopsis Rab family, then the Arabidopsis Rab repertoire
would comprise little more than the six basic Rab functions
that are common to all eukaryotic kingdoms.
containing distinct subclasses that share a common nearest
mammalian homologue.
Some of the eight Arabidopsis groups are, however,
substantially larger than the Rab subclasses in other
organisms. The extent of sequence dissimilarity between
protein sequences within single Arabidopsis groups can
be greater than that between mammalian Rab subclasses
that are known to have distinct trafficking functions
(e.g. between Rab2 and Rab4, which function in transport between endoplasmic reticulum [ER] and Golgi,
and between early endosomes and plasma membrane
[PM], respectively). Given this degree of sequence
diversity, we suspect that some of the eight groups that are
usually recognised in Arabidopsis are probably artificial,
Analysis of Rab sequence and function in yeast and
mammals has identified regions that define Rab subclass
specificity, probably by determining the interactions
between each Rab and its specific effectors and regulators
([8,12•,13,15]; Figure 1a,b). On the basis of overall
sequence similarity and conservation within these subfamilydetermining regions, a recent publication proposed rules
for determining whether two similar Rab sequences are
isoforms or members of distinct subclasses [12•]. When
applied to the Arabidopsis Rab family, these criteria
predicted that sequences in branches A, C, D, F, and G
should each be subdivided into two or more distinct
subclasses, designated A1–A6; C1 and C2; D1 and D2; F1
and F2; and G1, G2, and G3 [11••]. Hence, the family
would contain a total of 18 structural subclasses. With the
520
Cell biology
Figure 2
Arabidopsis Mammal
10 changes
*
RabF1
99
Rab22a
Rab22b
100 RabF2.a
*
RabF2.b
Rab5a
99
Rab5c
Rab5b
Ypt51
53
Ypt53
Ypt52
Rab21
Ypt10
Rab17
Rab24
*
70
RabF1
-
-
Rab22
C
RabD1
RabD2.c
RabD2.b
* 72 RabD2.a
87
Rab1a
97 Rab1b
Ypt1
D
Rab35
RabE1.c
98 RabE1.b
100
*
RabE1.a
RabE1.d
88 RabE1.e
E
Sec4
*
*
*
*
*
*
57
97
66
77
B
Rab39
? RabB1.b
RabB1.c
RabB1.a
Rab2
Rab4a
Rab4b
Rab14
-
-
Rab22, associated with endosomes
RabF2b, associated with putative endosomal compartments
RabF2
Rab5 Ypt51/52/53 Ypt5
Rab5, transport from PM to early endosomes and early endosome fusion
Rab21
Rab17
Rab24
-
Ypt10
-
-
Associated with apical PM in epithelial cells
Rab7
Ypt7
Ypt7
Ypt51, transport from PM to early endosomes and between endosomal compartments
RabG2
RabG2
*
RabG1 RabG1
RabG3.b
* 52
RabG3.a
*
RabG3.f
84 RabG3.e
*
RabG3
RabG3.d
61
RabG3.c
Rab7
84
Ypt7
Rab9
Rab32
Rab38
Rab29
Rab23
Rab20
Rab28
G
Rab34
Rab36
RabH1.d
*
RabH1.c
* RabH1.e
72
RabH1.a
Rab H1
82
RabH1.b
Rab6a
99
100 Rab6b
Ypt6
H
RabA1.d
RabA1.c
*
RabA1.b
RabA1.a
RabA1
72
RabA1.f
RabA1.g
RabA1.i
RabA1.h
RabA1.e
*
RabA2.c
RabA2.d
89
RabA2.b
RabA2.a
RabA2
* 100 Rab11a
Rab11b
*
*
100 Ypt31
*
Ypt32
Rab25
RabA4.d
RabA4.c
?
59
RabA4
RabA4.b
72
RabA4.e
RabA4.a
RabA3
RabA3
100
68
RabA6.a
RabA6
RabA6.b
RabA5.e
A
RabA5.d
RabA5
RabA5.c
93
RabA5.b
RabA5.a
Rab33a
Rab33b
*
Rab30
*
Rab19
100 RabC2.a
RabC2
92
RabC2.b
77
RabC1
RabC1
Rab18
F
S.
S.
cerevisiae pombe
RabF1, ARA6, associated with putative endosomal compartments
Rab17, transcytosis in epithelial cells
ScYpt7, late-endosome to vacuole and vacuole fusion
Rab7, late endosome to lysosome
Rab9
Rab32
Rab38
Rab29
Rab23
Rab20
Rab28
Rab34
Rab36
-
-
Rab6
Ypt6
Ryh1
Rab9, late endosome to trans-Golgi network
Ypt6, endosome to Golgi
Rab6A, retrograde traffic within the Golgi and from TGN and Golgi to ER
Rab6A′, retrograde traffic from early endosome to TGN
-
-
Rab11 Ypt31/32
-
Ypt3
Rab11, transport from recycling endosomes to PM, and between endosomes
and Golgi
Ypt31/32, transport from late Golgi and to endosomes and PM
Rab25
-
-
-
-
-
-
-
-
-
-
-
-
-
-
At RabA5.c (ARA4) localised to Golgi and adjacent vesicles
Rab33
Rab30
Rab19
-
-
Golgi associated
Golgi associated
-
-
-
Rab18
-
-
RabD1
-
-
-
RabD2
Rab1
Ypt1
Ypt1
-
Rab35
-
-
RabE1
Rab8
Sec4
Ypt2
Rab25, transport through recycling endosomes
PsRabA4 (Pra3) localised to post-Gogi prevacuolar compartments; distinct from PsRabA3
PsRabA3 (Pra2) localised to post-Gogi prevacuolar compartments; distinct from PsRabA4
Rab18 associated with endosomes, especially in epithelial cells (recycling to PM?)
ScYpt1, ER to Golgi transport and early intra-Golgi transport
Rab1, ER to intermediate-compartment/VTC and early intra-Golgi transport
RabD2a, ER to Golgi transport
Sec4, Golgi to PM (bud site)
Rab8a
Rab8b
Rab13
Rab10
Rab26
Rab37
Rab12
Rab3a
Rab3c
Rab3b
Rab3d
Rab27a
Rab27b
Rab15
Rab40a
Rab40b
Ypt2, Golgi to PM
Rab8, Golgi to basolateral PM in epithelial cells and regulated secretory
granule exocytosis
-
Rab13
Rab10
Rab26
Rab37
Rab12
-
-
Associated with PM at tight junctions in epithelial cells
Golgi associated
Associated with secretory granules
Associated with secretory granules
Golgi associated
-
Rab3
-
-
Regulated secretion in neurones and neuroendocrine cells
-
Rab27
-
-
Melanosome transport in melanocytes
-
Rab15
-
-
Antagonist of endocytosis at PM
-
Rab40
-
-
RabB1
Rab2
-
-
At RabB1b and tobacco homologue Golgi localised;
required for ER-Golgi traffic in pollen tubes
-
Rab4
-
-
Rab4, recycling from early endosome to PM
-
Rab14
Rab39
-
-
Rab14, phagosome associated
Rab2, maturation of VTC (recycling from VTC to ER?) and intra-Golgi traffic
Current Opinion in Plant Biology
The Arabidopsis Rab GTPase family Rutherford and Moore
521
Figure 2 legend
Phylogenetic analysis of the Rab GTPase families in Arabidopsis,
mammals, and yeast. This phylogram is based on maximum parsimony
of Arabidopsis (green), mammalian (red) and S. cerevisiae (orange)
Rab families. Arabidopsis sequences that are not known to be
transcribed are shown in grey. This tree was one of the four possible
shortest trees (the two nodes that varied in the other three trees are
indicated by question marks). The numbers on selected nodes are
bootstrap values (for 1000 replicates) given as a percentage.
Asterisks indicate selected nodes with bootstrap support of less than
50%. Branches leading to the eight major clades of Arabidopsis Rabs
are lettered A to H. Putative functional subclasses are identified by
colour banding. Subclasses that have putative orthologues in yeast,
mammals and Arabidopsis are indicated by blue bands. Subclasses
that have a clear orthologue in Arabidopsis and mammals but not
yeast are in yellow bands (when a branch of the Arabidopsis family is
proposed to contain two or more paralogous subclasses, the yeast
and mammalian subclasses are grouped with the Arabidopsis subclass
that has the greatest sequence similarity). Red bands indicate
mammalian subclasses that have no clear orthologue in plants or
yeast (alternating light and dark shading is used simply to highlight
the subclass boundaries). Green bands highlight putative subclasses
that have no clear counterpart in yeast or mammals. The names of
Arabidopsis, yeast and mammalian Rab subclasses are indicated to
the right of the tree. In this review, we have adopted the classification
system that was used for Arabidopsis in a recent comprehensive
summary of Rab GTPase families in organisms whose genomes have
been completely sequenced [11••]. This hierarchical classification
recognises the phylogenetic structure of the plant Rab GTPase family,
and we have found that it can be applied successfully to other dicot
species. This classification therefore helps to clarify the relationships
between plant Rab GTPases, which have hitherto been named
according to a great number of different ad hoc systems. Functional
information about selected subclasses has been compiled from
various published sources.
exception of G1–G3, these proposed subclasses are well
supported by our phylogenetic analysis, as indicated in
Figure 2. Further support for these structural subclasses
comes from two sources. First, with two exceptions
(RabH1d and RabB1b), intron/exon boundaries are conserved within each subclass, whereas 12 of the subclasses
have a unique structure (Figure 3). Second, a clear homologue for many of the subclasses can be identified in the
sequences available from other plants, suggesting that
these subclasses predate angiosperm diversification and
have been conserved during angiosperm evolution.
the best estimate is ‘at least eight but possibly about 18’.
In the remainder of this review, we discuss the available
information on and possible functions of each of the 18
Arabidopsis subclasses.
RabA1 to RabA6 (related to mammalian Rab11
and Rab25)
What is clear from these analyses is that 33 of the 41
mammalian subclasses have no clear orthologue in
Arabidopsis. This suggests that the diversification of Rab
GTPase functions and associated trafficking is characteristic
of mammalian cells has not occurred in the higher plant
lineage. In some cases, such as that of Rab3, which
functions in regulated secretion at the PM of neuronal
and neuroendocrine cells, the missing Rab subclasses may
perform functions that are specific to the animal lineage.
So, what clues might the quite distinct complement of
Rab GTPases and subclasses in Arabidopsis (and other
angiosperms) give us about the organisation of membrane
traffic in this species?
This branch of the Arabidopsis family is the most numerous,
accounting for almost half the total Rab complement, and
has been divided into six structural subclasses. The closest
mammalian homologues, Rab11a, Rab11b, and Rab25,
have all been localised to apical recycling endosomes in
polarised epithelial cells. They have been studied most
extensively in these cells, although they are also present
in other cell types [8,17]. Despite localising to the same
organelle, it has been suggested that the Rab11 and Rab25
subclasses may control distinct transport routes between
the recycling endosome and the Golgi or PM [17,18]. The
homologous subclass in S. cerevisiae, Ypt31/Ypt32, has been
implicated in export from a late Golgi compartment to a
pre-vacuolar/endosomal compartment and to the PM
[8,9,19]. As the diversification of subclasses RabA1–RabA6
has occurred only in the plant lineage, however, it is not
clear that any of the RabA subclasses is truly orthologous
to the yeast and/or mammalian subclasses.
It may be that each of the 18 structural subclasses discussed
above represents a distinct Rab GTPase function as do,
apparently, most mammalian subclasses. In yeast, however,
members of Ypt51 and Ypt52, two subclasses that were
defined by the same criteria as the mammalian subclasses,
have overlapping functions [9], but it is possible that this
reflects a redundancy of trafficking pathways rather than
redundancy of Rab function within a pathway. Conversely,
in mammals, two splice variants of Rab6a that differ in
only three residues in a relatively non-conserved region
of the protein perform distinct functions in the cell [16•].
Consequently, substantially more functional data will be
required before we can determine with any confidence
how many Rab functions Arabidopsis encodes. At present,
Green fluorescent protein (GFP) fusions have been used
to show that pea RabA3 (Pra2) and RabA4 (Pra3) proteins
each localise on distinct populations of punctate structures
that are likely to include Golgi and prevacuolar or putative
endosomal compartments [20••]. This is consistent with
the proposal that RabA proteins function in prevacuolar
or endocytic sorting events and that RabA3 and RabA4
proteins perform distinct functions. Monoclonal antibodies
raised against AtRabA5c (ARA4) labelled both the Golgi
and structures that were identified as trans-Golgi network
and Golgi-derived vesicles in pollen [21]. This is also
consistent with a role for plant RabA proteins in postGolgi/endosomal transport. Interestingly, the branch of the
syntaxin family of SNAREs that has undergone the
522
Cell biology
Figure 3
RabA
A1.a
A1.b
A1.c
A1.d
A1.e
A1.f
A1.g
A1.h
A1.i
A2.a
A2.b
A2.c
A2.d
A3
A4.a
A4.b
A4.c
A4.d
A4.e
A5.a
A5.b
A5.c
A5.d
A5.e
A6.a
A6.b
RabB
B1.a
B1.b
B1.c
.
.
....
.
..
.
.
....
.
..
.
.
....
.
..
.
.
....
.
..
.
.
....
.
..
.
.
....
.
..
.
.
....
.
..
.
....
.
..
.
.
....
.
..
.
.
....
.
.
....
.
..
....
.
..
.
.
....
.
.
.
....
.
.
....
RabE
E1.a
E1.b
E1.c
E1.d
E1.e
....
.
..
....
.
..
.
.
....
.
..
.
.
....
.
..
.
.
....
.
..
.
.
....
.
..
.
.
....
.
..
.
.
....
.
..
.
.
....
.
..
.
.
....
.
..
.
.
....
.
..
.
.
....
...
.
.
....
...
.
.
....
...
...
...
...
...
...
....
.......
.
.
....
..
.
.
....
..
.
.
.
....
..
.
.
.
....
..
.
.
.
....
..
.
.
....
..
.
.
....
..
.
.
....
..
.
.
....
..
.
....
...
.
.
....
...
.
.
....
...
...
...
RabG
G1
.
.
G2
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
RabH
H1.a
H1.b
H1.c
H1.d
H1.e
..
.
.
RabF
F1
F2.a
F2.b
G3.a
G3.b
G3.c
G3.d
G3.e
G3.f
.
.
C2.a
C2.b
D2.a
D2.b
D2.c
..
..
.
RabC
C1
RabD
D1
..
.
.
...
...
...
...
.
...
...
.
...
...
Current Opinion in Plant Biology
Intron/exon boundaries in the Arabidopsis Rab gene family. The 57
Arabidopsis Rab proteins are depicted schematically as coloured bars
that are proportional to the length of each sequence. Consecutive exons
are indicated by alternating yellow and blue bars. Green bars indicate the
positions of amino-acid residues where the codon has been bisected by
the intron. The positions of conserved residues that were used to align the
sequences are indicated in red. Gaps in the alignment are indicated by
dots. Genes that are known to be expressed are indicated in bold type.
greatest diversification in Arabidopsis is related to the late
Golgi/endosomal yeast SNARE, Tlg1 [6•].
distributions of PM proteins by actin-dependent membrane
trafficking to and from an internal compartment [22,23•–25•].
It may be that RabA sequences have diversified in the
plant lineage to facilitate such trafficking events. Rab11
and possibly Ypt32 facilitate an interaction between
membranes and the actin cytoskeleton [19,26]. Given that
It is tempting to speculate that the RabA subclass has
diversified to fulfil plant-specific functions. What could
these be? Plant cells can maintain and shift distinct polar
The Arabidopsis Rab GTPase family Rutherford and Moore
most organelle motility in plant cells is actin based, whereas
microtubules play a greater role in animal cells, it may be
that the diversity of plant RabA sequences reflects the
complexity of actin-based organelle motility in plants.
The toxicity of AtRabA5c (ARA4) in yeast ypt mutants
has been exploited to screen for interactions between this
protein and regulatory factors [27•]. The overexpression
and antisense expression of various RabA sequences have
resulted in various developmental and morphological
phenotypes (most recently [28•]), but have revealed little
about the trafficking functions of these proteins. One such
study of the pea RabA3 protein Pra2 in transgenic tobacco
concluded that RabA3 homologues act to integrate light
and brassinosteroid signalling pathways in the etiolation
response [29•]. Pra2 was proposed to reside on the ER
membrane and to stimulate DDWF1, a cytochrome P450
that can catalyse a step in brassinolide biosynthesis. A
primary role for a Rab GTPase in signalling and biosynthetic pathways of this sort would be unique. Given the
discrepancy between the ER localisation reported in this
study and that reported more recently [20••], together with
the limited stimulation of DDWF1 activity by Pra2 and
the modest alterations in the brassinosteroid content of
Pra2 antisense plants, it is probably premature to conclude
that RabA3 sequences are directly involved in brassinosteroid signalling or metabolism. Furthermore, the
proposed target of the Pra2 transgene in tobacco,
NtRab11d, is not in fact a member of the RabA3 subclass
([20••]; Figure 2). NtRab11d is clearly a member of the
RabA4 subclass along with the pea protein Pra3, which
acted as the negative control in all of the experiments with
Pra2. Finally, if the RabA3 and RabA4 subclasses act on
transport pathways to the vacuoles, as suggested by recent
localisation data, their influence on etiolation and
brassinosteroid physiology may have arisen indirectly from
a defect in vacuole function [30].
RabB (related to Rab2)
There is no RabB homologue in yeast, but in mammals,
Rab2 is localised on cis-Golgi membranes and interacts
with Golgi matrix proteins [31,32]. Rab2 is also implicated
in the maturation of vesicular tubular clusters (VTCs),
which are microtubule-associated intermediates in transport
between the ER and Golgi apparatus [33]. In plants, there
is no equivalent of the VTC and the Golgi is organised
quite differently from that in mammals [34]. The role of
Rab2 in plants is therefore of interest. Dominant inhibitory
mutants of a tobacco RabB have recently been shown to
slow pollen-tube growth and to inhibit the transport of
GFP markers between the ER and Golgi apparatus [35••].
These observations suggest that RabB function is required
to sustain normal membrane traffic from the ER to the
pollen tip, although the precise site of action of the
dominant inhibitory mutant is unclear. GFP fusions to the
tobacco RabB homologue labelled the Golgi stacks [35••],
consistent with a role for RabB in anterograde or retrograde
transport between the ER and Golgi.
523
RabC (related to Rab18)
The RabC branch also lacks a yeast orthologue. The
nearest mammalian homologue, Rab18, is implicated in
endocytic transport and is expressed most highly in
polarised epithelial cells [7,8,36]. However, the angiosperm
and animal sequences differ substantially in the conserved
domains that define subclass specificity, so it is not clear
that RabC and Rab18 will perform similar functions.
Furthermore, it is also unclear whether the endocytic
transport pathways of mammalian epithelial cells have
direct counterparts in plants.
RabD (related to Rab1)
The division of this branch into two subclasses, RabD1
and RabD2, is specific to plants. These subclasses are
closely related to the yeast and mammalian Ypt1/Rab1
proteins, which are involved in ER-to-Golgi transport and
the initial stages of intra-Golgi transport. The minor
RabD1 branch has a single representative in Arabidopsis
and is conserved in both monocots and dicots. Using a
dominant inhibitory mutant form of AtRabD2a and a
GFP-based membrane trafficking assay, Batoko et al. [37••]
showed that the RabD subclass is required for normal
ER-to-Golgi transport in tobacco. The AtRabD2a mutant
did not inhibit the Brefeldin A (BFA)-induced fusion of
the Golgi apparatus with the ER, but it did inhibit the
recovery of the Golgi after BFA washout [38]. This is
consistent with a primary role for RabD in anterograde
traffic between ER and Golgi.
RabE (related to post-Golgi Rab subclasses)
This subclass has been described as homologous to Rab8
and Rab10 in mammals [14] and to Sec4 in S. cerevisiae,
although its closest homologue is Ypt2 of S. pombe. All of
these proteins are known or suspected to be involved in
post-Golgi transport to the PM. It is misleading, however,
to single out Rab8 and Rab10 as possible mammalian orthologues of the RabE subclass. Figure 2 shows that Rab8 and
Rab10 reside in a large and complex group of mammalian
Rab sequences that have acquired specific functions, usually
in post-Golgi traffic, in cell types and in trafficking events
that have no clear counterpart in either yeast or plants. It is
likely that these Rabs have functions that are specific to the
mammalian lineage and have no orthologues in plants.
The implication is that post-Golgi transport to the PM has
not undergone the same diversification and specialisation
in angiosperms as it has in mammals.
RabF (related to mammalian Rab5 and Rab22)
The RabF branch contains three sequences divided into
two putative subclasses, RabF1 and RabF2. These proteins
are most similar to Rab5 and Rab22 of mammals and to
Ypt51/Ypt52/Ypt53 of yeast, all of which are involved in
endocytosis and endocytic-sorting pathways [7–9]. As with
post-Golgi traffic (discussed above), it appears that endocytic
trafficking has evolved rather differently in mammals and
yeast [39], and so perhaps also in plants. Most of the
mammalian subclasses that are involved in transport
524
Cell biology
Figure 4
1
2
3
4
5
CAI
RabD2.a
mi51
RabA3
RabA5.b
RabA1.a
RabA5.e
RabA2.b
GA1
RabE1.e
rga
RabC2.b
RNS1
MNSOD
PhyA
RabD1
RabE1.d
RabA4.d
RabC2.a
RabA2.a
g3715
RabA1.g
hy4
RabG3.c
SRP54A
DET1
mi421
RabA1.b
FLS
RabG3.a
RabH1.e
RabG3.f
RabA6.b
mi465
mi268
m235
RabG3.b
GLI
PhyB
RabB1.a
RabA1.e
RabB1.c
RabA1.d
RabG2
mi322
RabD2.c
RabF2.b
RabA1.i
UFO
AG
m433
nga139
RabH1.d
RabA4.e
RPS2
er
mi431
RabA5.d
RabB1.b
RabA1.h
cop1
RabE1.c
RabC1
AP2
RabA2.c
ASN1
RabH1.c
DHS1
mi441
RabG3.e
PHYC
RabG1
RabA4.b
LTP
RabE1.a
RabG3.d
RabA5.c
DFR
AP3
RabF1
RabF2.a
RabA1.c
RabH1.b
RabD2.b
RabA5.a
RabA4.c
ARR3
RabA
SBG9
RabB
RabE1.b
RabA2.d
RabC
RabD
RabA6.a
ADH
RabE
RabF
RabA1.f
LFY3
RabH1.a
RabA4.a
RabG
RabH
Current Opinion in Plant Biology
The Arabidopsis Rab GTPase family Rutherford and Moore
525
Figure 4 legend
Genomic positions of the 57 Rab GTPase genes. The five Arabidopsis
chromosomes are depicted as alternating black and grey bars, each of
which represents 1Mb of sequence, with circles representing the
centromere. The position of each Rab locus is indicated, with arrows
indicating the direction of transcription. Each Rab locus is colour coded
according to subclass (see key). A selection of classical loci are also
included. Major genome duplications involving Rab GTPases are indicated
by grey shading, which connects the duplicated regions. Dark grey shading
indicates duplications in which all of the Rab sequences have been
maintained on both copies of the duplication, whereas light grey shading
represents duplications in which one or more Rab sequence(s) has
apparently been lost from one copy of the duplication. The positional data,
direction of transcription and regions of genome duplication were obtained
from the Munich Information Centre for protein Sequences (MIPS)
Arabidopsis thaliana database (MatDB; http://mips.gsf.de/proj/thal/db) and
The Arabidopsis Information Resource (TAIR; http://www.Arabidopsis.org).
between endosomal, Golgi, and transcytotic compartments
are absent from both yeast and plants. These include
Rab4 and Rab9, which are required for important recycling
pathways in mammals [7,8]. One possibility is that in
plants, sequences in the RabA and RabC subclasses have
developed functions that are analogous to those of some of
the missing mammalian Rab subclasses.
YPT6 [45]. In mammals, Rab6A is implicated in retrograde
transport through the Golgi stack, and is also required for a
slow, COPI-independent, retrograde transport pathway
from Golgi to ER (reviewed in [46]). This pathway may
allow Golgi residents to be recycled through the ER for
scrutiny by ER quality-control systems. Remarkably, the
Rab6A′ splice variant, which differs from Rab6A by only
three residues, has a quite distinct function in retrograde
transport from the early-sorting endosomes to the transGolgi network [16•]. This activity appears to be similar to
that of the yeast RabH/Rab6 homologue Ypt6 [47].
These results underline the need for functional studies to
substantiate or falsify the classification proposed here.
The RabF1 subclass is one of the most intriguing in
higher plants. This subclass is currently unique among
the eukaryotic Rab families in that it associates with
membranes via amino-terminal N-myristoylation and
palmitoylation, rather than by carboxy-terminal geranylgeranylation [40••]. RabF1 was first identified in
L. japonicus [41] but is also known in rice and in the halophyte Mesembryanthemum crystallinum, in which its
expression is induced by salt stress [42]. Ueda and colleagues
[40••] showed that GFP fusions to Arabidopsis RabF1
(Ara6) and RabF2 (Ara7) co-localised principally with a
subset of punctate and spherical structures that could be
labelled with the styryl dye FM4-64, which is used as a
marker of the endocytic pathway. It will be interesting to
establish the relationship between the structures labelled
by RabF1, RabF2, RabA3 and RabA4 fusions, all of which
have been localised to putative endosomal or prevacuolar
structures [20••,40••].
RabG (related to mammalian Rab7)
It has proved more difficult to draw boundaries between
putative subclasses in the RabG group than in any other,
but we and others [11••] have divided the RabG group into
three provisional subclasses. The yeast and mammalian
homologues of RabG, Ypt7 and Rab7, are both involved
in transport to the vacuole/lysosome, whereas Ypt7 is also
required for homotypic vacuole fusion [7–9]. Cheon et al.
[43] showed that specific expression of a RabG3 antisense
transcript in soybean nodules prevented nodule development. The relatively large number of sequences in the
RabG and RabA branches in Arabidopsis may reflect the
diversity of vacuoles and associated trafficking pathways
that have evolved in higher plants (reviewed in [44]).
RabH (related to mammalian Rab6)
Of the five RabH sequences in the Arabidopsis genome
only two have been isolated as cDNA. AtRabH1b was able
to complement the temperature-sensitive growth defect
associated with deletion of the S. cerevisiae homologue
A multiplicity of genes within subclasses
Even if the 18 Rab subtypes that we recognise do represent
distinct subclasses with distinct trafficking functions,
many plant Rab subclasses are uncommonly large. Nine
of the 18 Arabidopsis subclasses contain three or more
members, compared to only five of the 111 Rab subclasses
in all of the other sequenced genomes. Inspection of
regions of genome duplication show that 44 of the 57 Rab
GTPases reside in these regions (Figure 4). Strikingly, in
14 cases, the Rab gene is not present on both copies of the
duplication. The simplest explanation is that one copy has
been lost, leaving only 15 of the 57 Arabidopsis Rab
GTPases that could be accounted for as passive participants
in large-scale duplication events. It is therefore plausible
that most of the 57 Arabidopsis Rab sequences have been
maintained by natural selection. This diversity may
represent the evolution of additional unrecognised subclasses (the most plausible example being the large and
diverse RabA1 subclass) or diversification within the
18 subclasses to generate isoforms that function in particular developmental, environmental or physiological
contexts. Some fairly specific expression patterns have
been identified for individual Rab genes [42,48–51], but
there is nothing as yet to suggest that they reflect significant functional specialisation. It may simply be that
the requirement for a particular Rab function in all cells of
the organism is met through the combined activities of
individual genes, each with a different but essentially
arbitrary expression pattern.
Conclusions
Genome sequencing has revealed that the Rab GTPase
family in Arabidopsis and other plants is quite unlike that
526
Cell biology
in yeast and mammals. Sequence analysis suggests the
existence of 18 or so functional subclasses, and the limited
functional and localisation data available to date are consistent with these groups. Much more work will be
necessary, however, to test the veracity of these predictions
and to establish the trafficking functions and redundancy
relationships of individual Rab genes.
Acknowledgements
We are indebted to Dr Robert Scotland, University of Oxford, for his
assistance with the phylogenetic analyses. We thank Dr Takashi Ueda for
sharing unpublished information about Arabidopsis Rab gene expression.
SR was supported by a grant (43/G15985) from the Biotechnology and
Biological Sciences Research Council (BBSRC).
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
The Arabidopsis Genome Initiative: Analysis of the genome
sequence of the flowering plant Arabidopsis thaliana. Nature
2000, 408:796-826.
2.
Shannon TM, Steer MW: The root cap as a test system for the
evaluation of Golgi inhibitors. I. Structure and dynamics of the
secretory system and response to solvents. J Exp Bot 1984,
35:1697-1707.
3.
Phillips DG, Preshaw C, Steer MW: Dictyosome vesicle production
and plasma membrane turnover in auxin-stimulated outer
epidermal cells of coleoptile segments from Avena sativa (L.).
Protoplasma 1988, 145:59-65.
4.
Thiel G, Kreft M, Zorec R: Unitary exocytotic and endocytotic
events in Zea mays coleoptile protoplasts. Plant J 1998,
13:101-104.
5.
•
Brandizzi F, Snapp EL, Roberts AL, Lippincott-Schwartz J, Hawes C:
Membrane protein transport between the endoplasmic reticulum
and the Golgi in tobacco leaves is energy dependent but
cytoskeleton independent: evidence from selective
photobleaching. Plant Cell 2002, 14:1293-1309.
Fluorescence-recovery after photobleaching (FRAP) of Golgi-targeted GFP
fusions is used to infer that the half-time for transfer of a membrane protein
to the Golgi apparatus is 2 min in tobacco epidermal cells. (This rapid rate
of transfer was possible even in the absence of Golgi motility and organised
actin or microtubules.) Despite this rapid rate of membrane exchange, Golgi
and ER membranes maintain distinct protein compositions. Similarly, two
independent estimates of the rate of secretory vesicle production by the
Golgi in rapidly growing coleoptile epidermis (using either capacitance
measurements or electron microscopy) [3,4] suggested that the quantity of
vesicle membrane that is delivered to (and probably recycled from) the PM
is sufficient to cause the entire surface area of the PM to be turned-over
once every 3–4 h. In hypersecretory maize root-cap cells, this turn-over time
may be as short as 10 min [2].
6.
•
Sanderfoot AA, Assaad FF, Raikhel NV: The Arabidopsis genome.
An abundance of soluble N-ethymaleimide-sensitive factor
adapter protein receptors. Plant Physiol 2000, 124:1558-1569.
This review summarises what is known about the SNARE protein families
and some of their interacting partners that are encoded within the
Arabidopsis genome. Like the Rab family, the syntaxin family in Arabidopsis
is more diverse than that in yeast and contains some branches that have no
clear orthologues in mammals or yeast. The authors propose a systematic,
phylogenetic nomenclature for Arabidopsis SNARE proteins.
7.
Zerial M, McBride H: Rab proteins as membrane organisers. Nat
Rev Mol Cell Biol 2001, 2:107-117.
8.
Segev N: Ypt and Rab GTPases: insight into functions
through novel interactions. Curr Opin Cell Biol 2001,
13:500-511.
9.
Lazar T, Gotte M, Gallwitz D: Vesicular transport: how many
Ypt/Rab-GTPases make a eukaryotic cell? Trends Biol Sci 1997,
22:468-472.
10. Jedd G, Richardson C, Litt R, Segev N: The Ypt1 GTPase is
essential for the first two steps of the yeast secretory pathway.
J Cell Biol 1995, 131:583-590.
11. Pereira-Leal JB, Seabra MC: Evolution of the Rab family
•• of small GTP-binding proteins. J Mol Biol 2001,
313:889-901.
Using rules defined in [12•], the authors of this article summarise the Rab
GTPase families in all of the eukaryotes that have sequenced genomes. Their
work standardises nomenclature and proposes the first systematic phylogenetic
classification of all Arabidopsis Rab sequences. Arabidopsis has more Rab
GTPase loci than yeast, Drosophila or Caenorhabditis.
12. Pereira-Leal JB, Seabra MC: The mammalian Rab family of small
•
GTPases: definition of family and subfamily sequence motifs
suggests a mechanism for functional specificity in the Ras
superfamily. J Mol Biol 2000, 301:1077-1087.
Extending the initial analysis described in [13], this paper defines and
applies criteria for identifying Rab GTPase sequences and for distinguishing
probable subclasses within Rab GTPase families in mammals.
13. Moore I, Schell J, Palme K: Subclass-specific sequence
motifs identified in Rab GTPases. Trends Biochem Sci
1995, 20:10-12.
14. Bischoff F, Molendijk A, Rajendrakumar CSV, Palme K:
GTP-binding proteins in plants. Cell Mol Life Sci 1999,
55:233-256.
15. Brennwald P, Novick P: Interactions of three domains
distinguishing the Ras-related GTP-binding proteins Ypt1 and
Sec4. Nature 1993, 362:560-563.
16. Mallard F, Luen-Tang B, Galli T, Tenza D, Saint-Pol A, Yue X, Antony C,
•
Hong W, Goud B, Johannes L: Early/recycling endosomes-to-TGN
transport involves two SNARE complexes and a Rab6 isoform.
J Cell Biol 2002, 156:653-664.
Two splice-variants of Rab6A are shown to have distinct membrane-trafficking
functions although they differ only in three amino acids. Two of these
substitutions, in consecutive amino acids in a relatively unconserved carboxyterminal region, are responsible for the differing behaviour. These two residues
reside close to one of the two ‘switch’ regions that alter conformation in
response to the phosphorylation state of the bound nucleotide. In this
position, they may influence the interaction between the two variants and
different regulatory or effector molecules. Rab6A is required for recycling
from the trans-Golgi network (TGN) and Golgi to ER, whereas the variant
Rab6A′ resembles its yeast counterpart Ypt6p in its role in trafficking from
endosome to TGN.
17.
Casanova JE, Wang X, Kumar R, Bhartur SG, Navarre J,
Woodrum JE, Altschuler Y, Ray GS, Goldenring JR: Association
of Rab25 and Rab11a with the apical recycling system of
polarised Madin–Darby canine kidney cells. Mol Biol Cell
1999, 10:47-61.
18. Rodman JS, Wandinger-Ness A: Rab GTPases coordinate
endocytosis. J Cell Sci 2000, 113:183-192.
19. Ortiz D, Medkova M, Walch-Solimena C, Nivick P: Ypt32 recruits the
Sec4p guanine nucleotide exchange factor, Sec2p, to secretory
vesicles; evidence for a Rab cascade in yeast. J Cell Biol 2002,
157:1005-1015.
20. Inaba T, Nagano Y, Nagasaki T, Sasaki Y: Distinct localization of two
•• closely related Ypt3/Rab11 proteins on the trafficking pathway in
higher plants. J Biol Chem 2002, 277:9183-9188.
In microsomal preparations of pea, Pra2 and Pra3, which are members of
the pea RabA3 and RabA4 families, respectively, distribute differently on
density gradients. In tobacco cells, GFP::Pra2 localises to a variety of punctate
structures. Some of these structures may be Golgi as GFP::Pra2 can be
found on an ER network (and other undefined structures) after BFA treatment.
Others may be endocytic/prevacuolar structures as they are labelled by the
dye FM4-64, which is thought to label endocytic and vacuolar membranes.
By contrast, GFP and red fluorescent protein (RFP) fusions to Pra3 label
dispersed punctate structures that are BFA-insensitive and distinct from
those labelled by GFP::Pra2. However, GFP/RFP::Pra3 largely co-localises
with an RFP fusion to the prevacuolar SNARE VTI11. These findings show
that RabA3 and RabA4 proteins can reside on different membrane systems
and are therefore likely to perform distinct roles in membrane traffic. In
contrast to [29•], these authors found that only a small portion of Pra2 is likely
to reside on the ER.
21. Ueda T, Anai T, Tsukaya H, Hirata A, Uchimiya H: Characterisation
and subcellular localisation of a small GTP-binding protein
(Ara-4) from Arabidopsis: conditional expression under control of
the promoter of the gene for heat-shock protein HSP81-1. Mol
Gen Genet 1996, 250:533-539.
22. Steinmann T, Geldner N, Grebe M, Mangold S, Jackson CL, Paris S,
Gälweiler L, Palme K, Jürgens G: Coordinated polar localisation of
auxin efflux carrier PIN1 by GNOM ARF GEF. Science 1999,
286:316-318.
The Arabidopsis Rab GTPase family Rutherford and Moore
23. Geldner N, Friml J, Stierhof Y-D, Jürgens G, Palme K: Auxin transport
•
inhibitors block PIN1 cycling and vesicle trafficking. Nature 2001,
413:425-428.
This work shows that although the steady-state polar localisation of PINFORMED1 (PIN1) protein in the PM is insensitive to actin-cytoskeleton
and auxin-transport inhibitors, both classes of molecule can inhibit the
BFA-induced redistribution of PIN1 and other proteins to and from the PM.
This confirms the involvement of the actin cytoskeleton in PIN1 cycling, and
suggests that some auxin-transport inhibitors may act more generally in
membrane traffic.
24. Swarup R, Friml J, Marchant A, Ljung K, Sandgerg G, Palme K,
•
Bennett M: Localization of the auxin permease AUX1 suggests
two functionally distinct pathways operate in the Arabidopsis root
apex. Genes Dev 2001, 15:2648-2653.
The authors show that the AUX1 auxin permease, in addition to the PIN family of auxin-efflux proteins, is polarly localised. Importantly, they also show
that PIN1 and AUX1 can adopt opposite polar distributions in protophloem
cells. These results show, for the first time, that a single plant cell can
maintain two proteins in distinct polar localisations. Given the implicit role of
membrane traffic in PIN1 localisation, distinct membrane trafficking of PIN1
and AUX1 on apical and basal pathways may be responsible for maintaining
distinct polar distributions of these proteins.
25. Friml J, Wistniewska J, Benkova E, Mendgen K, Palme K: Lateral
•
relocation of auxin efflux regulator PIN3 mediates tropism in
Arabidopsis. Nature 2002, 415:806-809.
The PIN3 protein is expressed in several specialised cells, including root cap
columella. In these cells, the distribution of PIN3 between internal punctate
structures and subdomains of the PM can be changed in an actin-dependent
manner in response to a change in the gravity stimulus. This work is of significance
here because selective actin-dependent endocytic recycling may be involved.
26. Lapierre LA, Kumar R, Hales C, Navarre J, Bhartur SG, Burnette JO,
Provance DW, Mercer JA, Bahler M, Goldenring JR: Myosin Vb is
associated with plasma membrane recycling systems. Mol Biol
Cell 2001, 12:1843-1857.
27.
•
Ueda T, Matsuda N, Uchimiya H, Nakano A: Modes of interaction
between the Arabidopsis Rab protein, Ara4, and its putative
regulator molecules revealed by a yeast expression system.
Plant J 2000, 21:341-349.
AtRabA5c (ARA4) and various mutations that cause defects in aspects of
GTP binding or hydrolysis are toxic to yeast ypt mutants. A screen for
interactions with factors that can suppress this toxicity resulted in the
identification of an Arabidopsis homologue of the Rab regulatory protein
Rab GDP dissociation inhibitor (Rab GDI) and a novel potential interacting
protein, SAY1, from Arabidopsis. The role of SAY1 is unclear.
28. Lu C, Zainal Z, Tucker G, Lycett G: Developmental abnormalities
•
and reduced fruit softening in tomato plants expressing an
antisense Rab11 GTPase gene. Plant Cell 2001, 13:1819-1833.
A member of the tomato RabA family that is expressed in ripening fruit (and
elsewhere) was introduced into tomato as an antisense construct. The fruit
of transgenic plants developed normal pigmentation but failed to soften.
Reduced levels of two enzymes that modify the cell wall were measured in
these plants, although it is unclear whether this reduction resulted from
reduced rates of synthesis or from mis-sorting during transport. A range of
other developmental abnormalities were observed in the transgenic plants.
29. Kang J-G, Yun J, Kim DH, Chung KS, Fujioka S, Kim JI, Dae HW,
•
Yoshida S, Takatsuto S, Song PS, Park CM: Light and brassinosteroid
signals are integrated via a dark-induced small G protein in
etiolated seedling growth. Cell 2001, 105:625-636.
Pra2, a RabA3 protein from pea, is normally expressed in a light-repressible
manner in the elongation zone of etiolated pea epicotyls, suggesting that it
has a role in etiolated growth. In this work, Pra2 was used to induce homologydependent gene silencing in tobacco. Dark-grown transgenic tobacco
seedlings exhibited short hypocotyls that could be rescued by exogenous
brassinosteroid or its immediate biosynthetic precursor. The effect of these
heterologous transgenes was attributed to an observed reduction in the
steady-state transcript level of a member of the tobacco RabA family
(NtRab11d). The phenotype was specific to Pra2; sense and antisense
constructs derived from a member of the pea RabA4 subclass, Pra3, failed
to alter hypocotyl elongation. The authors found that Pra2 could stimulate
the activity of DDWF1 by up to 1.3 fold, and hypothesised that this might
affect endogenous brassinosteroid metabolism and effect the observed
changes in hypocotyl elongation.
30. Schumacher K, Vafeados D, McCarthy M, Sze H, Wilkins T, Chory J:
The Arabidopsis det3 mutant reveals a central role for the vacuolar
H+-ATPase in plant growth and development. Genes Dev 1999,
13:3259-3270.
31. Chavrier P, Gorvel J-P, Stelzer E, Simons K, Gruenberg J, Zerial M:
Hypervariable C-terminal domain of a Rab protein acts as a
targeting signal. Nature 1991, 353:769-772.
527
32. Short B, Preisunger C, Koerner R, Kopajtich R, Byron O, Barr FA:
A GRASP55-rab2 effector complex linking Golgi structure to
membrane traffic. J Cell Biol 2001, 155:877-883.
33. Tisdale E, Balch W: Rab2 is essential for the maturation of
pre-Golgi intermediates. J Biol Chem 1996, 271:29372-29379.
34. Nebenführ A, Staehelin LA: Mobile factories: Golgi dynamics in
plant cells. Trends Plant Sci 2001, 6:160-167.
35. Cheung AY, Chen CY-H, Glaven RH, de Graaff BHJ, Vidali L,
•• Hepler PK, Wu H-M: Rab2 GTPase regulates trafficking between
the endoplasmic reticulum and the Golgi bodies and is important
to pollen tube growth. Plant Cell 2002, 14:945-962.
The first functional study of the RabB branch in plants. When expressed in
tobacco pollen, a GFP fusion to the tobacco RabB homologue NtRab2 was
shown to label mobile punctate structures, which were identified as Golgi
by immunoelectron microscopy with anti-GFP antisera. GFP::Rab2 locates
preferentially at the stack periphery. Expression of dominant inhibitory
mutants reduced the extracellular accumulation of soluble and membranebound secretory GFP markers and could cause an AtERD2::GFP fusion to
shift from a predominantly Golgi localisation to the ER. If these dominant
inhibitory mutants act by specifically inhibiting the activity of the RabB subclass, a point yet to be demonstrated, a role for this subclass in sustaining
ER to Golgi traffic could be inferred. The GFP fusions did not associate with
the Golgi in leaf cells or protoplasts, which express lower levels of NtRab2
than do pollen cells. However, we suspect that this may be an overexpression
artefact of the transient expression methodology as we have found the
Golgi-specific localisation of an Arabidopsis RabB::GFP fusion in these
cells using Agrobacterium-mediated transient expression (Neumann U,
Hawes C, Moore I, unpublished data).
36. Hashim S, Mukherjee K, Raje M, Basu K, Mukhopadhyay A: Live
Salmonella modulate expression of Rab proteins to persist in a
specialised compartment and escape transport to lysosomes.
J Biol Chem 2000, 275:16281-16288.
37.
••
Batoko H, Zheng H, Hawes C, Moore I: A Rab1 GTPase is required
for transport between the endoplasmic reticulum and Golgi
apparatus and for normal Golgi movement in plants. Plant Cell
2000, 12:2201-2217.
This paper describes the first functional study of the plant RabD subclass. A
dominant inhibitory form of the Arabidopsis RabD2a protein caused the
accumulation of secreted and Golgi markers in the ER. An N-glycosylated
Golgi marker accumulated in an endoglycosidase-H resistant form. All aspects
of the dominant mutant phenotype could be rescued by co-expression with
the wild-type protein, so it was inferred that the phenotypes resulted from
loss of activity of the RabD subclass. Intriguingly, the movement of the Golgi
apparatus over the ER was also inhibited, suggesting that there may be a
mechanistic link between Golgi movement and membrane traffic between
ER and Golgi as previously hypothesised [34]. However, recent data suggest
that the observed effect on motility is not specific to the Golgi (Moore I,
Batoko H, unpublished data).
38. Saint-Jore C, Evins J, Batoko H, Brandizzi F, Moore I, Hawes C:
Redistribution of membrane proteins between the Golgi apparatus
and endoplasmic reticulum in plants is reversible and not
dependent on cytoskeletal networks. Plant J 2002, 29:661-678.
39. Pelham HRB: Insights from yeast endosomes. Curr Opin Cell Biol
2002, 14:454-462.
40. Ueda T, Yamaguchi M, Uchimaya H, Nakano A: Ara6, a plant-unique
•• novel type Rab GTPase, functions in the endocytic pathway of
Arabidopsis thaliana. EMBO J 2001, 20:4730-4741.
The authors used GFP fusions to mutant proteins to show that the amino
terminus of Ara6, the Arabidopsis RabF1 protein, must be both palmitoylated
and N-myristoylated for this protein to be localised to FM4-64 labelled
structures. In protoplasts expressing either GFP::Ara6 or GFP::Ara7, some
FM4-64 labelled structures were unlabelled, suggesting that there may be
distinct classes of endocytic structures. The morphology of FM4-64 labelled
structures was altered upon expression of GFP fusions to AtRabF1 and
AtRabF2b mutants, which are likely to cause defects in GTP hydrolysis,
suggesting a role for AtRabF1 in trafficking or organisation of FM4-64 labelled
membranes. The mutant RabF1 localised to the PM as well as to internal
structures, whereas the mutant RabF2b did not, suggesting that the two
proteins may normally associate with distinct membrane systems.
41. Borg S, Brandstrup B, Jensen TJ, Poulsen C: Identification of new
protein species among 33 different small GTP-binding proteins
encoded by cDNAs from Lotus japonicus, and expression of
corresponding mRNAs in developing root nodules. Plant J 1997,
11:237-250.
42. Bolte S, Scheine K, Deitz K: Characterisation of a small
GTP-binding protein of the Rab5 family in Mesembryanthemum
crystalinum with increased level of expression during early salt
stress. Plant Mol Biol 2000, 42:923-936.
528
Cell biology
43. Cheon C-I, Lee N-G, Siddique A-BM, Bal AK, Verma DPS: Roles of
plant homologues of Rab1p and Rab7p in the biogenesis of the
peribacteroid membrane in soybean root nodules. EMBO J 1993,
12:4125-4135.
48. Inaba T, Nagano Y, Sakakibara T, Sasaki Y: DE1, a 12-base-pair cis
regulatory element sufficient to confer dark-inducible and light
down-regulated expression to a minimal promoter in pea. J Biol
Chem 2000, 275:19723-19727.
44. Chrispeels MJ, Herman EM: Endoplasmic reticulum-derived
compartments function in storage and as mediators of vacuolar
remodeling via a new type of organelle, precursor protease
vesicles. Plant Physiol 2000, 123:1227-1233.
49. Moore I, Diefenthal T, Zarsky V, Schell J, Palme K: A homologue of
mammalian Rab2 is present in Arabidopsis and is expressed
predominantly in pollen grains and seedlings. Proc Natl Acad Sci
USA 1997, 94:762-767.
45. Bednarek SY, Reynolds TL, Schroeder M, Grabowski R, Hengst L,
Gallwitz D, Raikhel NV: A small GTP-binding protein from
Arabidopsis thaliana functionally complements the yeast YPT6i
null mutant. Plant Physiol 1994, 104:591-596.
50. O’Mahoney P, Oliver M: Characterisation of a desiccation-responsive
small GTP-binding protein (Rab2) from the desiccation tolerant
grass Sporobolus stapfianus. Plant Mol Biol 1999, 39:809-821.
46. Storrie B, Pepperkok R, Nilsson T: Breaking the COPI
monopoly on Golgi recycling. Trends Cell Biol 2000,
10:385-391.
47.
Siniossoglou S, Pelham HR: An effector of Ypt6p binds the SNARE
Tlg1p and mediates selective fusion of vesicles with late Golgi
membranes. EMBO J 2001, 20:5991-5998.
51. Terryn N, Arias MB, Engler G, Tire C, Villaroel R, Van Montague M,
Inze D: rha1, a gene encoding a small GTP-binding protein from
Arabidopsis, is expressed primarily in developing guard cells.
Plant Cell 1993, 5:1761-1769.
52. Ostermeyer C, Brunger AT: Structural basis of Rab effector specificity:
crystal structure of the small G proterin Rab3A complexed with
the effector domain of Rabphillin-3A. Cell 1999, 96:363-374.