Download Arabidopsis RHD3 mediates the generation of the tubular ER

2241
Research Article
Arabidopsis RHD3 mediates the generation of the
tubular ER network and is required for Golgi
distribution and motility in plant cells
Jun Chen1, Giovanni Stefano2, Federica Brandizzi2 and Huanquan Zheng1,*
1
Developmental Biology Research Initiatives, Department of Biology, McGill University, 1205 Dr Penfield Avenue, Montreal, QC H3A 1B1, Canada
Michigan State University-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
2
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 14 March 2011
Journal of Cell Science 124, 2241-2252
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.084624
Summary
In plant cells, the endoplasmic reticulum (ER) and Golgi apparatus form a unique system in which single Golgi stacks are motile and
in close association with the underlying ER tubules. Arabidopsis has three RHD3 (ROOT HAIR DEFECTIVE 3) isoforms that are
analogous to the mammalian atlastin GTPases involved in shaping ER tubules. We used live-cell imaging, genetic complementation,
split ubiquitin assays and western blot analyses in Arabidopsis and tobacco to show that RHD3 mediates the generation of the tubular
ER network and is required for the distribution and motility of Golgi stacks in root and leaf epidermal cells. We established that RHD3
forms homotypic interactions at ER punctae. In addition, the activity of RHD3 on the tubular ER is specifically correlated with the
cellular distribution and motility of Golgi stacks because ER to Golgi as well as Golgi to plasma membrane transport was not affected
by RHD3 mutations in the conserved GDP/GTP motifs. We found a possible partial redundancy within the RHD3 isoforms in
Arabidopsis. However, yeast Sey1p, a functional atlastin homologue, and RHD3 are not interchangeable in complementing the
respective loss-of-function mutants, suggesting that the molecular mechanisms controlling ER tubular morphology might not be
entirely conserved among eukaryotic lineages.
Key words: RHD3, Tubular ER, Golgi distribution, GTP/GDP, Protein secretion
Introduction
The endoplasmic reticulum (ER) appears as an extended network
of interconnected tubules and cisternae stretching throughout the
cytoplasm. The shape of the ER often undergoes drastic changes,
but the mechanisms underlying the ER rearrangements are not
fully elucidated (Federovitch et al., 2005; English et al., 2009).
Recent data have indicated that continuous fusion and fission
reactions of ER tubules are necessary for the dynamic restructuring
of the ER network (Dreier and Rapoport, 2000; Voeltz et al., 2006;
Shibata et al., 2009). It has also been generally accepted that the
cytoskeleton plays an important role, possibly as a facilitator in the
generation and distribution of the ER network in living cells
(Runions et al., 2006; Vedrenne and Hauri, 2006; Shibata et al.,
2009).
Two families of ER membrane proteins, the reticulons and
DP1/Yop1, have been shown to be structural components that
shape the ER tubules in mammalian and yeast cells (Voeltz et al.,
2006) as well as in proteoliposomes (Hu et al., 2008). The reticulons
and DP1/Yop1 proteins have a wedge-like transmembrane topology
and they can oligomerize in the tubular ER, serving as arc-like
scaffolds around ER tubules (Shibata et al., 2008). It is assumed
that they can deform the lipid bilayer into curved tubules through
their wedge-like hydrophobic insertion and/or scaffolding
mechanisms (Shibata et al., 2009; Sparkes et al., 2010). Recent
work also indicated that a class of ER-membrane-bound, dynaminlike GTP binding proteins, which includes the atlastins in mammals
and Sey1p in yeast (Saccharomyces cerevisiae), plays an important
role in the generation of the interconnected ER tubules (Hu et al.,
2009; Orso et al., 2009). Atlastins and Sey1p do not share sequence
homology but the membrane topology of the proteins is similar,
with four conserved GTP motifs at the N-terminus, and two
transmembrane domains, spaced by a few amino acids, located in
the lumen of the ER at the C-terminus (Hu et al., 2009). It has been
hypothesized that the atlastins and Sey1p might be required in the
generation of local ER membrane curvatures that are necessary for
either fission or fusion of ER tubules (Hu et al., 2009). In humans
there are three atlastin isoforms (Rismanchi et al., 2008). Mutations
in atlastin-1 frequently cause hereditary spastic paraplegia (HSP),
a disease primarily affecting the development of the long axons of
corticospinal neurons (Rismanchi et al., 2008).
Similar to animal and yeast cells, in growing cells of most plant
tissues, the cortical ER is organized into a tubular reticulate network
interconnected by three-way junctions (Ridge et al., 1999; Zheng
et al., 2004). The ER of plant cells undergoes drastic rearrangements
in response to both developmental cues and outside influences
(Ridge et al., 1999; Hawes et al., 2001; Runions et al., 2006). In
mammalian cells, ER tubules move mainly along microtubules
(Vedrenne and Hauri, 2006); in plants, although the motility and
orientation of the ER can be microtubule dependent (Mathur et al.,
2003; Foissner et al., 2009), actin–myosin-based motility is believed
to be the primary means of ER movement (Boevink et al., 1998;
Sparkes et al., 2009; Ueda et al., 2010). Compared with other
eukaryotic cells, plant cells have developed a unique ER–Golgi
interface (Moreau et al., 2007; Staehelin and Kang, 2008). The
plant Golgi apparatus is made of functionally independent Golgi
stacks, each consisting of several polarized cisternae (Boevink et
Journal of Cell Science
2242
Journal of Cell Science 124 (13)
al., 1998; Nebenfuhr et al., 1999). The Golgi stacks in plant cells
are closely associated with the ER tubules and travel along the
actin tracks throughout the cytoplasm (Boevink et al., 1998;
Nebenfuhr et al., 1999). However, the function and molecular
mechanism of Golgi motility on the actin tracks remain unknown.
From observations of Golgi stacks alternating motile and stationary
phases, a ‘stop-and-go’ model was proposed to correlate Golgi
movement with ER-to-Golgi transport (Nebenfuhr et al., 1999;
Staehelin and Kang, 2008). However, although disruption of the
actin track could arrest Golgi movement, both ER-to-Golgi and
Golgi-to-ER transport appear not to be dependent on the actin
filaments (Brandizzi et al., 2002b; Saint-Jore et al., 2002). In
plants, ER exit sites are found closely associated with and tracked
with Golgi stacks (daSilva et al., 2004; Stefano et al., 2006). It was
therefore suggested that ER exit sites and Golgi stacks might
constitute entire mobile functional units and that the stack
movement might not be required to reach ER export sites for ERto-Golgi transport to occur (daSilva et al., 2004; Stefano et al.,
2006).
Arabidopsis thaliana ROOT HAIR DEFECTIVE 3 (RHD3) is
a large GTP-binding protein initially isolated in a genetic screen
for mutants defective in root hair development (Wang et al., 1997).
In rhd3 mutants, the root hairs are short and wavy (Wang et al.,
1997), a defect reminiscent of axon growth in HSP. RHD3 is
ubiquitously expressed in plants, so the cell growth defect is
prominent but not restricted to root hairs in rhd3 mutants (Wang et
al., 1997). Arabidopsis RHD3 has been classified as a member of
atlastin GTPases (Hu et al., 2009). In Arabidopsis, there are three
RHD3 isoforms (Hu et al., 2003) but the cellular function of each
and their relationship with other members of atlastin GTPases
remain to be determined. A study on the steady-state distribution
of the soluble ER marker, GFP-HDEL, and of the bulk flow
marker, secGFP in rhd3-1 (a line with an Ala575 to Val mutation
in RHD3) suggested that RHD3 is required for both ER morphology
and perhaps ER-to-Golgi protein transport (Zheng et al., 2004).
Here, we provide evidence that RHD3 is primarily involved in the
generation of the interconnected tubular ER network in plant cells.
The expression of mutant forms of RHD3 alters the cellular
distribution and motility of Golgi stacks but does not affect ER
membrane export, indicating a specific role of RHD3 in the
morphological integrity of the ER rather than in export functions.
Results of complementation experiments suggest that RHD3
proteins might be functionally redundant in planta, but that they do
not complement a yeast sey1 yop1 mutant (Hu et al., 2009), and
that Sey1p cannot complement an rhd3 loss-of-function mutant.
These results underscore that the detailed mechanisms underlying
ER tubular integrity might not well be conserved among eukaryotes.
Results
RHD3 is localized to the ER and it concentrates in punctae
along the ER tubules
As the first step to define the cellular role of Arabidopsis RHD3,
we tagged RHD3 with GFP at its N-terminus to determine its
subcellular location. GFP–RHD3 is functional because it fully
Fig. 1. GFP–RHD3 complements rhd3-1 and localizes
to the ER. (A–C) Root and root hairs of Col-0 (A), rhd31 (B) and rhd3-1::GFP–RHD3 (C) plants. The short and
wavy root hair phenotype in rhd3-1 plants (B) was fully
rescued in rhd3-1::GFP–RHD3 plants (C). (D)Mature
Col-0, rhd3-1 and rhd3-1::GFP–RHD3 plants. The dwarf
phenotype of rhd3-1 was rescued by GFP–RHD3.
(E)GFP–RHD3 in rhd3-1::GFP–RHD3 root epidermis
cells is localized to an ER-like network but frequently
concentrates on punctae along the ER tubules (arrows),
often at the junction of three-way connections (inset).
Scale bars: 5m. (F)The ER in rhd3-1 root epidermal
cells stained with Rhodamine B hexyl ester. Scale bar:
5m. (G–I) Confocal images of Arabidopsis leaf
epidermal cells expressing GFP–RHD3 (G) and showing
Rhodamine hexyl B staining (H). (I)Merged images of G
and H. Scale bar: 5m (G–I).
Journal of Cell Science
RHD3 in the generation of the tubular ER network
complements the Arabidopsis rhd3-1 mutant throughout the
development of root, root hairs and adult plants (Fig. 1A–D; see
also Fig. 3A and Fig. 8A for rhd3-1 phenotype). Confocal imaging
of rhd3-1::GFP–RHD3 root epidermal cells revealed that the fusion
protein marked a fine meshwork reminiscent of the cortical ER
(Fig. 1E; supplementary material Movies 1, 2). In rhd3-1 cells, the
fine polygonal tubular ER appeared severely compromised, as
shown in rhd3-1 root epidermal cells stained with Rhodamine B
hexyl ester, a dye used to stain the ER (Fig. 1F) (Zheng et al.,
2005a). Staining of rhd3-1::GFP–RHD3 cells with Rhodamine B
hexyl ester suggested that the cortical network marked by GFP–
RHD3 is indeed ER (Fig. 1G–I).
We frequently observed that GFP–RHD3 was also concentrated
in punctae along the ER tubules (Fig. 1E, arrows; supplementary
material Movie 2), often at the three-way junctions of tubules (Fig.
1E, inset). This distribution of the punctae was very dynamic
(supplementary material Movies 1, 2). We also noted that, similar
to Sey1p–GFP in yeast cells, the punctate labeling of RHD3
appeared weak in some cells with low levels of fluorescence (Fig.
1G), probably due to the low abundance of RHD3. Simultaneous
visualization of GFP–RHD3 and Golgi stacks labeled by either the
trans-Golgi marker ST–YFP or the ER and cis-Golgi marker
ERD2–YFP (Boevink et al., 1998; Brandizzi et al., 2002a) indicated
that the RHD3 punctae do not correspond to Golgi stacks (Fig.
2A,B, arrows in the merged image). The punctae appeared much
more enriched in GFP–RHD3 than in ER soluble [YFP–HDEL
(Irons et al., 2003)] or membrane [P241d–YFP (Langhans et al.,
2008)] protein markers, or could even be devoid of the latter two
proteins (Fig. 2C,D, arrows in the merged images). The expression
of all GFP and YFP ER and Golgi markers are known to not affect
plant cell development (Boevink et al., 1998; Brandizzi et al.,
2002b; Zheng et al., 2004; Chen et al., 2009). We have not noticed
any impact of the expression of GFP–RHD3 and P241d–YFP
constructs on cell and plant development (supplementary material
Fig. S1).
Atlastins and Sey1p physically interact with the reticulons and
DP1/Yop1 in generating tubular ER network (Hu et al., 2009). In
Arabidopsis, a Yop1 homolog has been identified as HVA22d,
which has been suggested to function as a negative regulator of
autophagy (Chen et al., 2009). We found that HVA22d–YFP was
also localized to the tubular ER network and was concentrated in
punctae along the ER tubules (Fig. 2E, arrows in the merged
image). Interestingly, GFP–RHD3 and YFP–HVA22d were
colocalized to the ER tubules and at the punctae (Fig. 2E;
supplementary material Movie 3). Together with the evidence that
the COPII coat proteins, which are responsible for cargo export,
are located at the Golgi-associated ER exit sites (daSilva et al.,
2004; Stefano et al., 2006), the data presented here suggest that
RHD3 acts at the level of the ER, and that the RHD3 punctae are
separate entities from the COPII-mediated Golgi-associated ER
export sites, but might contain the protein machinery that is
important for the ER morphology.
RHD3 S51N and T75A mutants phenocopy rhd3-1 and
affect the generation of the tubular ER network
RHD3 is classified as a member of the class of dynamin-like
atlastin GTPases (Hu et al., 2009). The GTPases of this class
contain the signature motifs (G1, G2 and G3) characteristic of
dynamin GTPases but they also possess a distinct G4 motif
comprising three hydrophobic residues preceding an Arg-Asp (RD)
sequence (Hu et al., 2009). Transgenic wild-type Col-0 plants
2243
Fig. 2. GFP–RHD3 colocalizes with HVA22d, but not with Golgi markers.
(A)Confocal images of a tobacco epidermal cell coexpressing GFP–RHD3
and ST–YFP. ST–YFP marks Golgi bodies but does not colocalize with the
RHD3 punctae (arrows). (B)As in A but coexpressing GFP–RHD3 and
ERD2–YFP. (C)As in A but coexpressing GFP–RHD3 and YFP–HDEL. The
YFP–HDEL does not colocalize with the RHD3 punctae (arrows). (D)As in A
but coexpressing GFP–RHD3 and P241d–YFP. The P241d–YFP does not
colocalize with the RHD3 punctae (arrows). E as in A but coexpressing GFP–
RHD3 and HVA22d–YFP. Note that the HVA22d–YFP is colocalized with
RHD3 at both ER tubules and RHD3 punctae (arrows). Scale bar: 5m (A–E).
expressing either YFP–RHD3(S51N) (a single S51N substitution
in G1) or YFP–RHD3(T75A) (a single T75A substitution in G2)
phenocopied rhd3-1 (Fig. 3A), whereas expression of YFP–RHD3
had no effect on plant development (data not shown). We noted
that both mutant forms of RHD3 were predominantly localized to
some condensed tubules in root epidermal cells that did not appear
well interconnected (Fig. 3B,C, inset, arrows). YFP–RHD3 in wild
type Col-0 root epidermal cells marked a polygonal tubular network
and punctae along the tubules (Fig. 3D, arrows). Interestingly,
RHD3(T75A) tended to be concentrated at punctae along the
tubules, specially at the tubular junctions (Fig. 3C, arrowheads;
supplementary material Movie 4), whereas RHD3(S51N) mainly
marked condensed tubules (Fig. 3B, arrows). Rhodamine B hexyl
ester staining suggested that, similar to rhd3-1 plants (Fig. 1F)
(Zheng et al., 2004), the tubular ER network in these transgenic
plants was altered, in that it lacked the fine meshwork of
interconnected ER tubules (Fig. 3E,F). We also found that, similar
to transgenic Arabidopsis expressing RHD3(S51N) and
Journal of Cell Science
2244
Journal of Cell Science 124 (13)
Fig. 3. Transgenic Arabidopsis expressing YFP–RHD3(S51N) and
YFP–RHD3(T75A) phenocopy rhd3-1 mutants. (A)Root hairs of
Col-0, rhd3-1 and transgenic plants expressing YFP–RHD3(S51N)
and YFP–RHD3(T75A). Note the short wavy root hairs in transgenic
plants expressing YFP–RHD3(S51N) and YFP–RHD3(T75A).
(B–F) Confocal images of root epidermal cells. (B)Cell expressing
YFP–RHD3(S51N) shows condensed less branched tubules (arrows).
(C)Cell expressing YFP–RHD3(T75A) shows condensed less
branched tubules (arrows) and punctae (arrowheads) in the junctions
of condensed tubules. (D)Cell expressing YFP–RHD3 shows a typical
polygonal ER network and RHD3 punctae (arrowheads). (E)Cell
expressing YFP–RHD3(S51N) stained with Rhodamine B hexyl ester.
Note the colocalization of less branched ER tubules (revealed by
Rhodamine B hexyl ester) with the condensed tubules marked by
YFP–RHD3(S51N). (F)Cell expressing YFP–RHD3(T75A). Scale
bars: 5m (B–E).
RHD3(T75A), transient expression of both RHD3 mutants in
tobacco (Nicotiana benthamiana) leaf epidermal cells led to
unbranched ER tubules (supplementary material Fig. S2A,B;
supplementary material Movies 5, 6). Together, these data imply
that RHD3 is involved in shaping the fine tubular ER and this
function requires amino acid residues in the GTP motifs that are
conserved among dynamin GTPases.
RHD3(S51N) alters ER morphology but not ER export of
Golgi membrane cargo
In order to test whether morphological changes of the ER could be
correlated with a perturbation of ER-to-Golgi protein trafficking,
we tested the effects of RHD3(S51N) in a transient expression
system. In Agrobacterium-mediated transient protein expression in
tobacco leaf epidermal cells, expression of a protein starts roughly
24 hours post-infiltration and gradually reaches a peak 67 hours
post-infiltration (Zheng et al., 2005b). Leaves were infiltrated with
the Agrobacterium strains transformed with either the ER lumenal
marker GFP-HDEL or the Golgi marker ST–GFP (Batoko et al.,
2000), and 24 hours later, the same sectors were re-infiltrated with
the Agrobacterium strain containing RHD3(S51N), as well as the
following controls: RHD3, RAB-D2a and RAB-D2a(N121I)
(Zheng et al., 2005b). Infiltration of the leaf areas with the ER and
Golgi markers followed by a re-infiltration with the mutant protein
ensured that the markers are expressed before the mutant protein
has any effect on their traffic. We expected that if ST–GFP export
to the Golgi was inhibited by the RHD3 mutant, then the marker
would be partially distributed to the ER (Zheng et al., 2005b). We
found a loss of fine tubular ER network roughly 38 hours after
infiltration with RHD3(S51N) (Fig. 4A); however, targeting of
ST–GFP to Golgi was largely unaffected, even after 67 hours (Fig.
4C). By contrast, an inhibition of ST–GFP trafficking by RABD2a(N121I) was clearly visible as early as 38 hours post-infiltration
(Fig. 4D), as expected (Zheng et al., 2005b); in the presence of this
mutant, however, no morphological changes in the ER could be
detected, even after 67 hours (Fig. 4B). Expression of wild-type
RHD3 and RAB-D2a had no effect on either ER and ER-to-Golgi
trafficking (Fig. 4E–H). Together, these results indicate RHD3 is
primarily involved in the generation of interconnected ER tubules
and that alteration of the ER meshwork by the RHD3 mutant does
not affect ER-to-Golgi protein trafficking.
RHD3(S51N) specifically alters the distribution and
motility of Golgi stacks rather than affecting protein
secretion to the cell surface
Because it has been proposed that the ER and the Golgi are
attached in highly vacuolated plant cells (Sparkes et al., 2009), we
questioned whether RHD3-induced alteration of the ER
morphology could also affect Golgi distribution and motility. To
test this we compared the Golgi distribution in cells expressing
either RHD3 or the RHD3(S51N) mutant. In cells expressing wildtype RHD3, we found that Golgi stacks were dispersed along the
tubular ER network throughout the cytoplasm (Fig. 4G; Fig. 5E–
G; supplementary material Fig. S3B) (Boevink et al., 1998;
Nebenfuhr et al., 1999) with occasional aggregation of a few
stacks. In the presence of RHD3(S51N), however, 8–15 Golgi
stacks were aggregated around condensed ER tubules and especially
at the junctions of the tubules (Fig. 4C; Fig. 5A–C, arrows;
supplementary material Fig. S3A). We noted that these Golgi
stacks often underwent slow wiggling-type motions around the
condensed ER tubules for a prolonged time (Fig. 5B; supplementary
material Fig. S3A, arrows in different colors). We tracked and
Journal of Cell Science
RHD3 in the generation of the tubular ER network
monitored the motility of randomly selected Golgi stacks in eight
different cells expressing wild-type RHD3 and RHD3(S51N). In
wild-type cells, Golgi stacks often traveled with velocities between
0.5 and 4 m/second in the cytoplasm (Fig. 5H), consistent with
previous reports (Boevink et al., 1998; Nebenfuhr et al., 1999).
Although it is possible to quantify the instantaneous velocity of
individual Golgi stacks at a given instance of time, because of the
long-range movement and the variability of the stack velocity, we
found it was difficult to average the velocity of Golgi stacks over
a long period of time. However, we noted that the majority of
Golgi stacks (89.1%, n55; Fig. 5H) in the presence of wild-type
RHD3 moved quickly (>1 m/second), at least at one instance of
time over a period of 50 seconds. In the presence of RHD3(S51N),
the number of Golgi stacks that moved quickly (>1 m/second)
was greatly reduced (32.8%, n64). Most Golgi stacks (67.2%,
n64) only moved slowly (<1 m/second) along the ER (Fig. 5D).
The Golgi is a sorting station for proteins destined to post-Golgi
compartments. Because expression of dominant-negative versions
of RHD3 alters cellular distribution and motility of Golgi stacks
but not ER-to-Golgi trafficking, we asked whether expression of
dominant-negative forms of RHD3 could inhibit protein trafficking
to the cell surface. In the presence of RAB-D2a(WT), the apoplast
markers secGFP (Zheng et al., 2004) and AtCTL1–GFP [a cellwall-localized chitinase-like1 involved in biosynthesis of cell walls
(Zhong et al., 2002)] were secreted, producing dim GFP
fluorescence (Fig. 5A,D) owing to sub-optimal pH in the apoplast
for GFP fluorescence and protein truncation (Zheng et al., 2004;
Zheng et al., 2005b). In the presence of dominant-negative RABD2a(NI), as expected (Zheng et al., 2005b), secretion of the these
proteins was inhibited, as shown by an accumulation of the GFP
signal in the ER (compare Fig. 6B with A, 6E with D;
supplementary material Fig. S4B with A, S4E with D, quantification
in G). By contrast, transport of these proteins to the cell wall was
not impaired in the presence of the mutant RHD3 as no increase
in intracellular GFP intensity was observed (compare Fig. 6C with
A, 6F with D; supplementary material Fig. S4C with A, S4F with
D, quantification in G). The presence of RHD3(S51N) is indicated
in Fig. 6C,F (YFP channel). This was further confirmed by a
western blot analysis of tobacco leaves expressing secGFP in the
presence of either wild-type and mutated RAB-D2a or wild-type
and mutated RHD3 (Fig. 6G). SecGFP is known to be partially
cleaved in the apoplast (Zheng et al., 2004). On western blots with
anti-GFP serum this gives two bands with the top band
corresponding to full-length intracellular GFP, and a stronger lower
band corresponding to the cleaved apoplastic product (Zheng et al.,
2004). We found that the intensity ratio of the two bands was
comparable in extracts of leaves expressing either wild-type RABD2a, wild-type or mutant RHD3 (Fig. 6G). In the presence of
mutant RAB-D2a(NI), the intensity of the signal of the lower band
was much reduced compared with the upper band, indicating that
GFP is not secreted efficiently to the apoplast, where it would be
cleaved. These data indicate that secretion of secGFP is not
compromised in the presence of the RHD3 forms. Similarly,
targeting of AHA2–GFP, a plasma membrane localized H+-ATPase
(Kim et al., 2001), to the plasma membrane was also not prevented
by RHD3(S51N) (compare Fig. 6H with I, arrows). These data
show that the aberrant functioning of RHD3 affects specifically
ER morphology and Golgi distribution. This, in turn, does not
compromise ER-to-Golgi protein transport or Golgi-to-plasmamembrane protein traffic.
2245
Fig. 4. ER morphology but not trafficking at the ER–Golgi interface is
altered in the presence of YFP–RHD3(S51N). (A–H) Confocal images of
tobacco epidermal cells. (A)Cell expressing GFP–HDEL in the presence of
YFP–RHD3(S51N). (B)Cell expressing GFP–HDEL in the presence of RABD2a(NI). (C)Cell expressing ST–GFP in the presence of YFP–RHD3(S51N).
Note the aggregated Golgi stacks (arrow) along the ER tubules. (D)Cell
expressing ST–GFP in the presence of RAB-D2a(NI). (E)Cell expressing
GFP–HDEL in the presence of YFP–RHD3(WT). (F)As in E, but in the
presence of untagged RAB-D2a(WT). (G)Cell expressing ST–GFP in the
presence of YFP–RHD3(WT). (H)As in G but in the presence of RABD2a(WT).
RHD3 forms a homotypic interaction
In animal cells, atlastins homo-oligomerize and GTP binding is
crucial for this self-interaction (Rismanchi et al., 2008; Orso et al.,
2009). Using a mating-based split ubiquitin system in yeast, we
found that RHD3 homotypically interacts with another RHD3
protein, but not with controls that included the plasma membrane
protein KAT1 (Obrdlik et al., 2004) and the ER membrane protein
2246
Journal of Cell Science 124 (13)
Journal of Cell Science
Fig. 5. The Golgi movement is altered in the presence of YFP–RHD3(S51N). (A–C) Confocal images of a tobacco epidermal cell coexpressing ST–GFP with
YFP–RHD3S51N at 5 seconds (5⬙; A) and 50 seconds (50⬙; C). B is an average projection of 20 frames between A and C. Aggregated Golgi stacks (arrow) are
present along the ER tubules. (D)Percentage of Golgi stacks that underwent fast (>1m/second;m/s) and slow (<1m/second) movement in the presence of
YFP–RHD3(S51N) over 50 seconds. (E–G) Confocal images of a tobacco epidermal cell coexpressing ST–GFP with YFP–RHD3(WT) at 5 seconds (E) and 50
seconds (G). F is an average projection of 20 frames between E and G. (H)Percentage of Golgi stacks that underwent fast movement (>1m/second) and slow
movement (<1m/second) in the presence of YFP–RHD3 over 50 seconds. Scale bars: 5m.
P241d (Langhans et al., 2008) (Fig. 7A). The homotypic
interaction of RHD3 was further confirmed with bimolecular YFP
fluorescence complementation in plant cells. When the N-terminal
half of YFP (YFPn) fused with RHD3 (YFPn–RHD3) was
coexpressed with the C-terminal half YFP (YFPc) fused RHD3
(YFPc–RHD3), YFP fluorescence was re-established (Fig. 7B,
arrows; 32% of infiltrated cells, n247). No YFP fluorescence was
discerned when YFPn–RHD3 was coexpressed with YFPc fused to
P241d (P241d–YFPc) (Fig. 6C; 0% of infiltrated cells, n234).
Interestingly, coexpression of the two split-YFP fusions to RHD3
used for bimolecular fluorescence complementation with GFP–
RHD3 revealed that the self interaction of RHD3 occurred largely
at the RHD3 punctae (Fig. 7D,E, arrows).
Overexpressed RHD3-like2 can functionally replace RHD3
in the generation of the tubular ER network
In Arabidopsis, in addition to RHD3, there are two RHD3-like
GTP binding proteins encoded by the genetic loci At1g72960 and
At5g45160 (Hu et al., 2003). In this report, we refer to them as
RHD3-like1 (RL1) and RHD3-like2 (RL2), respectively. RL2 is
ubiquitously expressed at very low levels throughout plant tissues,
RL1 is pollen specific (Hu et al., 2003; Schmid et al., 2005).
Fig. 6. Transient expression of YFP–RHD3(S51N)
does not prevent cell wall secretion and protein
targeting to the plasma membrane.
(A–C) Confocal images of tobacco epidermal cells
coexpressing secGFP with RAB-D2a(WT) (A),
RAB-D2a(NI) (B) and YFP–RHD3(S51N) (C).
secGFP is retained in the ER in the presence of
RAB-D2a(NI) (B) but not in the presence of RABD2a(WT) (A) or YFP–RHD3(S51N) (C).
(D–F) Confocal images of tobacco epidermal cells
coexpressing AtCTL1–GFP with RAB-D2a(WT)
(D), RAB-D2a(NI) (E) and YFP–RHD3(S51N) (F).
AtCTL1–GFP is retained in the ER in the presence
of RAB-D2a(NI) (E) but with RAB-D2a(WT) (D)
or YFP–RHD3(S51N) (F). (G)Western blot
analyses with anti-GFP of total extracts of tobacco
leaf epidermal cells expressing secGFP with wildtype or mutant forms of either RAB-D2a or RHD3.
The upper band corresponds to non-cleaved GFP.
The lower band corresponds to cleaved apoplastic
GFP. Note the secGFP+RAB-D2a(NI) sample
loading was one third that of the loading in the other
lanes. (H,I)Confocal images of tobacco epidermal
cells coexpressing AHA2–GFP with RAB-D2a(WT)
(H) and YFP–RHD3(S51N) (I). Targeting of AHA2–
GFP to the plasma membrane (arrows) was not
prevented in both cases. Scale bars: 10m.
RHD3 in the generation of the tubular ER network
2247
(Fig. 8C), indicating that the S54N substitution in RL2 also has a
dominant-negative effect on the generation of the tubular ER
network. Coexpression of wild-type GFP–RHD3 rescued this
negative effect of RL2(S54N) (Fig. 8D). Furthermore, RL2 and
RHD3 were found to form homotypic and heterotypic interactions
when expressed in yeast cells (Fig. 8E). These data indicate that
members of the RHD3 family, at least in conditions of
overexpression, can replace the function of other members in the
maintenance of the ER tubular morphology, suggesting that they
might act on similar targets in the cells.
Journal of Cell Science
RHD3 and Sey1p are not interchangeable
Fig. 7. RHD3 is capable of homotypic interactions at ER punctae. (A)A
yeast two-hybrid system showing the interaction within RHD3 (right plate, 2)
on synthetic complete (SC) medium without histidine SC-Leu-Trp-His, but not
between RHD3 and P241d (right plate, 3) or KAT1 (right plate, 4). The
interaction was confirmed by a -galactosidase activity assay (blue). The left
plate shows cells growing on SC-Leu-Trp medium, as mating controls.
(B,C)Confocal images of a tobacco epidermal cell coexpressing YFPn–RHD3
and YFPc–RHD3 (B) or P241d–YFPc (C). B shows punctate YFP signal
(arrows) but it is absent in C. (D,E)Confocal images of a tobacco epidermal
cell coexpressing GFP–RHD3 with YFPn–RHD3 and YFPc–RHD3 (D) or
with YFPn-RHD3 and P241d–YFPc (E). Note the colocalization of GFP–
RHD3 and reconstituted YFP signals in the RHD3 punctae (arrows). Scale bar:
5m (B–E).
Knockout mutants of RL1 and RL2 produce no obvious
developmental defect (supplementary material Fig. S5). To
understand the functional relationship within the RHD3 GTPases
in plants, we expressed GFP fused RL2 in rhd3-1 under the control
of the constitutive promoter CaMV 35S. RL1 was not analyzed
because of expression specificity at a tissue level. We found that
GFP–RL2 fully complemented rhd3-1 (Fig. 8A). Confocal
microscopy of GFP–RL2 expressed in rhd3-1 cells revealed that,
similar to RHD3, RL2 was also localized to the ER tubules and
often concentrated on punctae along the ER tubules (Fig. 8B).
Simultaneous visualization of GFP–RL2 and YFP–RHD3 indicated
that RL2 was colocalized with RHD3 on the ER tubules and the
RHD3 punctae (Fig. 8B, arrows). Transient expression of YFP–
RL2(S54N), a mutated version of RHD3L2 with a S54N
substitution in the G1 motif, revealed that RL2(S54N) was also
predominantly localized to some condensed tubules (Fig. 8C,
arrowheads). The morphology of the tubular ER network indicated
by GFP–HDEL was also altered in the presence of this mutant RL2
In the family of dynamin-like atlastin GTPases, Sey1p shares
sequence homology with RHD3 but not with the atlastins (Hu et
al., 2009). It was proposed that Sey1p and RHD3 might work in
protozoans and plants, whereas the atlastins might function in
metazoan (Hu et al., 2009). Having established the primary role for
RHD3 in the generation of the tubular ER network in plants, we
expressed Sey1p and RHD3 in plants and yeasts, reciprocally. We
found that unlike RHD3 (Fig. 1A–D), Sey1p was unable to rescue
the developmental defect of the rhd3-1 mutant (Fig. 8A;
supplementary material Fig. S6). When YFP–Sey1 was expressed
in tobacco leaf epidermal cells, it was targeted to the ER
(supplementary material Fig. S6B). At the same time, when
Arabidopsis RHD3 was expressed in sey1 yop1 yeast cells with
Sec63p fused with GFP as an ER marker (Hu et al., 2009), RHD3
was unable to rescue the altered ER morphology in the sey1
yop1 cells (compare Fig. 9C with A), whereas Sey1p did (compare
Fig. 9B with A). When GFP–RHD3 was expressed in yeast cells,
it was localized to the ER (supplementary material Fig. S6C–E).
These results indicate that although Sey1p and RHD3 have a
similar function, in the formation of the tubular ER network in the
corresponding organisms, unique functional features of Sey1p and
RHD3 probably arose during evolution to suit different cell systems.
Discussion
Role of RHD3 in the generation of the tubular ER network
RHD3 is a large GTP-binding protein originally identified in a
screen for root hair defective mutants (Wang et al., 1997) whose
primary cellular function remains elusive (Hu et al., 2003; Zheng
et al., 2004). We show here that RHD3 is primarily involved in the
generation of the tubular ER network rather than in secretory
traffic. RHD3 resides in ER tubules; however, it also concentrates
as punctae on the ER tubules (we termed them RHD3 punctae).
RHD3 is also colocalized with HVA22d, an Arabidopsis homolog
of DP1/Yop1p that is involved in shaping ER tubules (Voeltz et al.,
2006) at the RHD3 punctae. This result suggests that the RHD3
punctae define subdomains that control the tubular morphology of
the ER.
A recent structural study on recombinant atlastin-1 has indicated
that atlastin-1 undergoes a GTP-dependent oligomerization in
solution (Byrnes and Sondermann, 2011). Here we show that
RHD3 is capable of homotypic interaction, which appears to occur
largely within the RHD3 punctae on the ER tubules. Generally in
the large dynamin-like GTPases, the amino acid stretch GxxxxGKS
(G1) is involved in coordinating phosphate binding and the tyrosine
(T) in the G2 motif is required for GTP hydrolysis (Marks et al.,
2001). The expression of both RHD3(S51N), a mutant form of
RHD3 with a single amino acid change in the G1 motif, and
RHD3(T75A), with a single amino acid change in the G2 motif,
causes the ER to be less branched. Interestingly, our data suggest
2248
Journal of Cell Science 124 (13)
Journal of Cell Science
Fig. 8. Overexpression of RL2 but not Sey1p rescues rhd3-1.
(A)5-day-old seedlings of Col-0, rhd3-1, rhd3-1::GFP–RL2 and
rhd3-1::Sey1p. Scale bar: 1 cm. (B)Confocal images of the
colocalization of GFP–RL2 and YFP–RHD3 in both ER tubules
and RHD3 punctae (arrows). (C)Confocal images of less
branched ER tubules marked by GFP–HDEL in the presence of
YFP–RL2(S54N). Note YFP–RL2(S54N) also marks condensed
tubules (arrowheads) as well as the unconnected ER tubules
marked by GFP–HDEL (arrows). (D)Confocal images of the fine
ER tubular network seen with YFP–RL2(S54N) when
coexpressed with GFP–RHD3. Scale bar: 5m (B–D). (E)A yeast
two-hybrid system showing the interaction between RHD3 and
RL2 (4) and within RL2 (6) identified on SC-Leu-Trp-His
medium, confirmed by -galactosidase activity assay (blue).
that the mode of action of the two RHD3 mutants in cells is
different. We found that RHD3(S51N) is largely associated with
less branched ER tubules, whereas RHD3(T75A) is seen frequently
on large punctae in the junction of the less branched ER tubules.
It is known that, in the presence of GTP, dynamins are capable of
deforming membranes by transiently polymerizing on them. Upon
GTP hydrolysis, triggered by polymerization, the dynamin scaffold
disassembles, resulting in a membrane release for spontaneous
membrane tubulation and fission (Bashkirov et al., 2008). Although
it remains to be demonstrated how RHD3(S51N), as well as
RHD3(T75A), could affect homotypic interaction of RHD3 in
plant cells, it is tempting to speculate that RHD3(S51N) might
have a reduced ability in homotypic interaction and therefore a
reduced ability to form RHD3 punctae, but RHD3(T75A) might
have an enhanced ability in homotypic interaction and therefore
form large RHD3 punctae in the junction of the less branched ER
tubules.
It has been proposed that the GTP-dependent conformational
changes of atlastin at discrete points along the tubules is crucial in
the formation of the tubular ER network with three-way junctions,
by either fission of the ER tubules or fusion of two different ER
tubules (Hu et al., 2009). Atlastin in Drosophila has been shown
to mediate the fusion of different ER tubules (Orso et al., 2009).
At the moment, we do not know whether RHD3 is involved in
fission or fusion of ER tubules in the generation of tubule ER
network. Given the structural similarity between RHD3 and atlastin,
it is possible that RHD3 also mediates the fusion of different ER
tubules.
Proteins involved in RHD3 function in plants
The atlastins and Sey1p interact with the previously identified ER
tubule-shaping proteins of the reticulon and DP1/Yop1 families
(Hu et al., 2009). Here we revealed that RHD3 is colocalized with
HVA22d at the RHD3 punctae. HVA22d has been implicated as a
negative regulator of autophagy (Chen et al., 2009) although its
homologue, DP1/Yop1, is known to play a role in ER tubule
shaping (Voeltz et al., 2006). It is possible that autophagy and ER
tubular shape maintenance are linked processes (Chen et al., 2009).
A role for Arabidopsis reticulons in remodeling of ER tubules has
also been proposed (Tolley et al., 2008; Sparkes et al., 2010). It
will be interesting to test whether RHD3 could interact with
reticulons and HVA22 proteins to coordinate the generation of
tubular ER network in plant cells.
We revealed in this study that Sey1p, a yeast functional
homologue of atlastin, and RHD3 are not interchangeable in
rescuing the respective loss-of-function mutants. Although it is
Journal of Cell Science
RHD3 in the generation of the tubular ER network
Fig. 9. RHD3 does not rescue the altered ER morphology in sey1 yop1
yeast cells. (A)Altered ER morphology revealed by Sec63–GFP in sey1
yop1 double mutant cells. (B)Sey1p rescues altered ER morphology in sey1
yop1 cells. (C)RHD3 does not rescues altered ER morphology in sey1
yop1 cells. Scale bar: 1m (A–C). (D)The percentage of cells with abnormal
ER.
possible that RHD3 does not fold properly in yeast and that Sey1
might not fold properly in Arabidopsis, the evidence that fluorescent
protein fusions of RHD3 and Sey1 are targeted to the ER in the
alternative system suggests that this possibility is unlikely.
Therefore, it seems that although the function of the atlastin GTPase
members in the generation of the tubular ER network is conserved
among eukaryotes (Hu et al., 2009) (this study), their molecular
mechanisms might have diversified in the different organisms. A
possible reason for the diversified mechanisms could be the
different relationship between the ER and the cytoskeleton in
animal, plant and yeast cells (Bola and Allan, 2009).
In mammalian cells, ER tubules are moved mainly along
microtubules (Vedrenne and Hauri, 2006). Atlastin-1 interacts
with spastin, a katanin-like ATPase microtubule severing factor
to disassemble microtubules (Lee et al., 2009) in order to remodel
the ER network. In plants, actin–myosin-based motility is the
primary means of moving the ER (Boevink et al., 1998; Sparkes
et al., 2009; Ueda et al., 2010), although the motility and
orientation of the ER can be microtubule dependent (Mathur et
al., 2003; Foissner et al., 2009). In rhd3-1 cells, the organization
of cortical actin filaments is affected (Hu et al., 2003) and the
transition of cortical microtubules in response to low doses of
propyzamide, a -tubulin-binding pronamide that affect
microtubule dynamics, is also impaired (Yuen et al., 2005). It
will be interesting to test whether and how RHD3 could physically
and genetically interact with actin and microtubule elements to
control the stability of the cytoskeleton in the generation of
tubular ER network and cell growth in plants. One obvious
2249
candidate to be tested is the Arabidopsis homologue of katanin.
Consistently, Arabidopsis plants with mutations in the katanin
gene are dwarf (Burk and Ye, 2002).
In Arabidopsis, there are three isoforms of RHD3 (Hu et al.,
2003). RL2 is ubiquitously expressed but at very low level, whereas
RL1 is pollen specific (Hu et al., 2003; Schmid et al., 2005). In our
functional analysis of RL1 and RL2, we revealed no obvious
developmental defect in knockout mutants of RL1 and RL2.
However, rhd3-1 can also be rescued by the overexpression of
RL2. Expression of an RL2 mutant [RL2(S54N)] leads to ER with
less branched tubules. Coexpression of wild-type RHD3 with
RL2(S54N) rescues the defect. Furthermore, RHD3 and RL2 can
form a heterotypic interaction when expressed in yeast cells.
Together, these results indicate that there is functional overlap
between members of the RHD3 protein class in the generation of
the tubular ER network. It seems that RL2 is functional but at a
low level copy number so that the loss of RL2 in the knockout line
is compensated by RHD3. rhd3-4, a T-DNA insertional knockout
mutant results in slightly longer root hairs than rhd3-1 (Wang et
al., 1997), an allele with an A575V mutation in RHD3. It is
possible that the loss of RHD3 in rhd3-4 is also partially
compensated by low level RL2. In humans, three atlastins are
differentially localized in ER domains, and atlastin-1, but not
atlastin-2 or -3, interacts with spastin (Rismanchi et al., 2008). It
remains to be determined whether RHD3 proteins function
differently in the generation of tubular ER network in different
regions of the ER.
The role of RHD3 in Golgi distribution and protein
transport
Compared with other eukaryotic cells, a unique feature of the plant
ER–Golgi interface is that individual plant Golgi stacks are closely
associated with ER tubules and are motile along the actin tracks
with variable velocities (Boevink et al., 1998; Nebenfuhr et al.,
1999). In the presence of dominant-negative RHD3 mutants, Golgi
stacks tend to aggregate; many of them undergo slow wiggling
motion along the condensed ER tubules for a prolonged time. This
suggests that, in addition to the actin tracks, the tubular ER network
could also be a molecular highway for fast linear movements of
the Golgi stacks. Interestingly, although the distribution and motility
of Golgi stacks is altered in the presence of mutant RHD3, ER-toGolgi transport is not prevented. This result is consistent with the
observation that, when actin filaments are disrupted the traffic of
cargo from the ER to Golgi stacks is not affected (Brandizzi et al.,
2002b). We think this can be at least partially explained by the fact
that in plants Golgi stacks are closely associated with ER exit sites
where they act as a functional unit (daSilva et al., 2004; Stefano et
al., 2006).
Here we show that in the presence of mutant RHD3, ER-toGolgi protein transport is not affected, and we also noted that
general secretion of cell wall proteins and targeting of plasma
membrane proteins are not prevented. This is consistent with the
findings in mammalian cells that in atlastin-depleted HeLa cells
the plasma membrane targeting of VSVG, a vesicular stomatitis
virus glycoprotein, is not impaired (Rismanchi et al., 2008). It
seems, therefore, that the minor transport defect detected in some
cells of rhd3-1 (Zheng et al., 2004) might be a secondary effect of
the ER disorganization, perhaps resulting from stress during the
period of plant growth. In particular, we cannot exclude that the
fluorescent signal found in some rhd3-1 cells (Zheng et al., 2004)
is due to accumulation of autofluorescent molecules, as might
2250
Journal of Cell Science 124 (13)
happen in conditions of stress (Chalker-Scott et al., 1999). It is also
possible that reduced vacuolar expansion in rhd3-1 mutants
(Galway et al., 1997) is linked to altered pH of the lumen of
secretory organelles of some rhd3-1 cells. The secGFP reporter
used by Zheng et al. is pH sensitive (Zheng et al., 2004). Therefore,
accumulation of GFP signal in some rhd3-1 cells might also be
linked to pH changes during growth.
Journal of Cell Science
Conclusions
Our analyses indicate that the Arabidopsis rhd3 mutants could be
a useful tool for investigating the regulation and functional roles
of ER remodeling in directional cell elongation. Approximately
50% the known dominant forms of HSP have been linked to
mutations in atlastin-1, REEP1(DP1-like) and spastin, three proteins
that interact in the generation of tubular ER (Park and Blackstone,
2010). However, the actual functional role of ER restructuring in
the development of the long axons of corticospinal neurons has not
been established. Searches for genes that suppress rhd3-1 could
provide valuable therapeutic insights into HSP, and identification
of rhd3-1 enhancers could shed light on how the generation of the
tubular ER network is controlled and how it is involved in cell
elongation. For example, synergistic genetic interaction of rhd3
with scn1-1 (RhoGDI), tip1-2 (S-acyl-transferase) and shv2-1 (a
plasma-membrane-localized COBRA-like protein) (Parker et al.,
2000) certainly warrants further investigation.
Materials and Methods
Molecular cloning and site-directed mutagenesis
RHD3, p241d, HVA22d, AtCTL1 and RHD3L2 were amplified by RT-PCR using a
Superscript III first-strand synthesis system kit (Invitrogen) with RNAs from wildtype Col-0 Arabidopsis. RHD3-1(A575V) was amplified from rhd3-1. RHD3 was
first cloned in pBluescript-II(KS) as an XbaI–SalI–BamHI fragment and sequenced.
The sequenced RHD3 was then subcloned as a SalI–BamHI fragment into pVKH16–
GFPN (Zheng et al., 2005b) to generate pVKH18–GFP–RHD3. RHD3 and RHD3L2
was also cloned into the entry vector pDONR222 and sequenced. p241d, HVA22d,
AtCTL1 were cloned into the entry vector pCR8/GW/TOPO and sequenced. The
corresponding genes were then subcloned into destination vector pEarleyGate104
[Arabidopsis Biological Resource Center (ABRC) stock DB3-686] to generate YFP–
RHD3; into pEarleyGate101 (ABRC stock DB3-683) to produce P241d–YFP and
HVA22d–YFP; into pEarleyGate103 (ABRC stock DB3-685) to generate AtCTL1–
GFP; and into pMDC43 (Curtis and Grossniklaus, 2003) to create GFP–RHD3L2.
The gene encoding Sey1p from Saccharomyces cerevisiae was amplified from
pRS315 (Hu et al., 2009), sequenced and subcloned into the binary vector pFGC5941.
YFP–Sey1 was made in pEarleyGate104. All ER, Golgi and transport markers were
described by Zheng et al. (Zheng et al., 2005b).
Site-directed mutagenesis of RHD3 and RHD3L2 was performed in pDONR222
using Quick Change Lighting Site-Directed Mutagenesis Kit (Stratagene).
RHD3(S51N) was generated using primers 5⬘-CCACAAAGTAGTGGGAAGAATACGCTCTTGAATCATTTG-3⬘ and 5⬘-CAAATGATTCAAGAGCGTATTCTTCCCACTACTTTGTGG-3⬘. RHD3(T75A) was created using primers 5⬘-GAGGAAGGTCTCAGACGGCTAAGGGAATTTGGATT-3⬘ and 5⬘-AATCCAATTCCCTTAGCCGTCTGAGACCTTCCTC-3⬘. RHD3L2(S54N) was produced using primers
5⬘-GGTCCTCAAAGTAGTGGGAAGAATACACTTTTGAATCATCTTTTT-3⬘ and
5⬘-AAAAAGATGATTCAAAAGTGTATTCTTCCCACTACTTTGAGGACC-3⬘.
The mutated genes were sequenced and then cloned into destination vector
pEarleyGate104 to produce YFP–RHD3(S51N), YFP–RHD3(T75A) and YFP–
RHD3L2(S54N).
Plant materials and growth conditions
rhd3-1 expressing GFP–RHD3, YFP–RHD3, GFP–RHD3L2 and Sey1p, wild-type
Col-0 expressing YFP–RHD3(S51N) and YFP–RHD3(T75A) were generated by
Agrobacterium-mediated transformation of corresponding plasmids into rhd3-1 and
wild-type Col-0 plants, respectively (Clough and Bent, 1998). Transformed
Arabidopsis seeds were selected on AT (Somerville and Ogren, 1982) or MS
(Murashige and Skoog, 1962) growth medium supplied with either kanamycin (50
g/l; Wisent, Montreal, Canada) or hygromycin (20 g/l; Wisent) or BASTA (20
g/ml; Calbiochem). To confirm the expression of Sey1 in transgenic plants, RNA
was isolated from 2-week-old seedlings (RNeasy Plant Mini Kit; Qiagen). The firststrand cDNA was then synthesized using the SuperScript III first-strand synthesis
system (Invitrogen). PCR was performed using the High-fidelity DNA Polymerase
(Finnzymes, Espoo, Finland) for 35 cycles. For an internal control, the constitutive
gene Ubiquitin10 was used. Seedlings on AT or MS plates or plants in soil were
grown at 20–22°C under constant light.
Transient expression in Nicotiana benthamiana
Transient expression of YFP–RH3D, RAB-D2a and other GFP-based markers in
Nicotiana benthamiana leaf lower epidermal cells was performed using
Agrobacterium transformation as described by Batoko et al. with slight modifications
(Batoko et al., 2000). Plants were cultivated in short-day (8 hours light) conditions.
After the Agrobacterium culture reached the stationary growth phase at 28°C with
agitation, cells were pelleted and resuspended in the infiltration buffer (100 M
acetosyringone in 10 mM MgCl2). The final OD600 for Agrobacterium cultures was
0.01 except for RAB-D2a(WT) and RAB-D2a(NI) (OD6000.03) as used by Zheng
et al. (Zheng et al., 2005b).
Western blot analyses
Total proteins were extracted with 1⫻ SDS loading buffer [50 mM Tris-HCl, pH 7.5,
100 mM dithiothreitol, 2% SDS (w/v), 0.1% Bromophenol Blue (w/v) and 10%
glycerol] from tobacco leaves expressing secGFP with RabD2a(NI), RabD2a(WT),
RHD3(WT) and RHD3(SN). Proteins were boiled for 5 minutes and loaded on a
12% SDS-PAGE gel. SDS-PAGE was performed on a Protean III apparatus (BioRad) and separated proteins were transferred onto a PVDF membrane. Western
blotting was carried out with a rabbit anti-GFP antibody (Sigma-Aldrich) at 1:2000
dilution and the secondary anti-Rabbit IgG-peroxidase (Sigma-Aldrich) at 1:10,000
dilution. Signals were detected using Invitrogen Novex ECL (HRP Chemiluminescent
Substrate Reagent Kit) according to the manufacturer’s recommendations.
ER staining and confocal laser scanning fluorescence imaging
For ER staining with Rhodamine B hexyl ester, seedlings of rhd3-1::GFP–RHD3,
Col-0::YFP–RHD3(S51N) and Col-0::YFP–RHD3(T75A) were immersed in
Rhodamine B hexyl ester solution (1.6 M) for 10 minutes before imaging. For
fluorescence imaging of GFP, YFP and Rhodamine B hexyl ester, samples were
analyzed with an inverted Zeiss LSM510meta laser scanning microscope with
either a C-apochromat 40⫻/1.2W numerical aperture oil-immersion lens, or a Capochromat63⫻/1.2W numerical aperture oil-immersion lens as described by
Zheng et al. (Zheng et al., 2005b). Golgi motility was analyzed using volocity
quantification (PerkinElmer). Zeiss LSM software (Carl Zeiss), Photoshop (Adobe)
and NIH ImageJ (http://rsbweb.nih.gov/ij/) were used for post-acquisition image
processing.
Yeast sey1 yop1 double mutant complementation
To rescue the sey1 yop1 yeast double mutant and locate RHD3 in yeast cells,
RHD3 was subcloned into a yeast expression vector pGREG505 and pGREG574,
respectively (Jansen et al., 2005). sey1 yop1 cells expressing Sec63–GFP (Hu et
al., 2009) were transformed with Sey1p in pRS315 (Hu et al., 2009) and RHD3 in
pGREG505. Sec63–GFP in yeast cells were imaged with a Leica DMI 6000B
microscope (Leica), 100⫻ oil lens and Volocity Acquisition software. GFP–RHD3
expressed in yeast cells was examined with a spinning disc confocal microscope
equipped with an EM-CCD camera (Hamamatsu C9100-B).
Protein–protein interaction analysis
For the yeast two-hybrid analyses, the corresponding wild-type and mutated genes
in the entry vectors were subcloned into the destination vector pNCW-GWRFC.1 to
generate all the N-terminal Cub fusions used in this paper, and pNX32 or pXN22
for N- or C-terminal NubG fusion used. KAT1-Cub (ABRC stock CD3-815) and
KAT1-NubG (ABRC stock CD3-816) (Obrdlik et al., 2004) were obtained from
ABRC. The yeast two-hybrid analysis was carried out according to the method of
Obrdlik et al. (Obrdlik et al., 2004).
For bimolecular fluorescence complementation analysis, YFPn–RHD3, YFPc–
RHD3 and P241d–YFPc were made by fusion of either residues 1–174 of YFP
(termed YFPn) or residues 175–239 (termed YFPc) in corresponding Gateway
destination vectors pMDC32–YFPn-C, pMDC32–YFPc-C and pMDC32–N-YFPc.
These Gateway compatible destination vectors were created by inserting HindIII–
SstI fragments of pUGW2–nEYFP, pUGW2–cEYFP and pUGW0–cEYFP
(Nakagawa et al., 2007) into the same cutting site of binary vector pMDC32 (Curtis
and Grossniklaus, 2003). YFPn–RHD3 was coexpressed with YFPc–RHD3 or
P241d–YFPc at OD6000.03 in tobacco leaf lower epidermal cells. Fluorescence of
YFP was then assessed 68 hours post-infiltration with a Zeiss LSM510meta system
(Zeiss).
We thank Sylvie Lalonde (Carnegie Institution, Stanford, CA, USA)
for pNX32 and pXN22 mbSUS Gateway vectors; David Bird
(University of Manitoba, Winnipeg, Canada) for pNCW-GWRFC.1;
Tsuyoshi Nakagawa (Shimane University, Matsue, Japan) for pUGW2nEYFP, pUGW2-cEYFP and pUGW0-cEYFP BiFC gateway vectors;
Mark Curtis (University of Zurich, Zurich, Switzerland) for pMDC43;
William Prinz (National Institute of Diabetes and Digestive and Kidney
Diseases, NIH, Bethesda, MD, USA) for the Sey1p clone and sey1
RHD3 in the generation of the tubular ER network
yop1::Sec63–GFP yeast double mutant; Inhwan Hwang (Pohang
University of Science and Technology, Pohang, Korea) for AHA2–
GFP; and Tamara Western for critical reading of manuscript. This
work was supported by a grant from The National Science and
Engineering Research Council of Canada and a startup grant from
McGill University (Montreal, Canada) to H.Z. and a grant from
National Science Foundation (MCB-0948584) and from the
Department of Energy Great Lakes Bioenergy Research Center and
the Chemical Sciences, Geosciences and Biosciences Division, Office
of Basic Energy Sciences, Office of Science, US Department of Energy
(award number DE-FG02-91ER20021) to F.B.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/124/13/2241/DC1
Journal of Cell Science
References
Bashkirov, P. V., Akimov, S. A., Evseev, A. I., Schmid, S. L., Zimmerberg, J. and
Frolov, V. A. (2008). GTPase cycle of dynamin is coupled to membrane squeeze and
release, leading to spontaneous fission. Cell 135, 1276-1286.
Batoko, H., Zheng, H. Q., Hawes, C. and Moore, I. (2000). A rab1 GTPase is required
for transport between the endoplasmic reticulum and golgi apparatus and for normal
golgi movement in plants. Plant Cell 12, 2201-2218.
Boevink, P., Oparka, K., Santa Cruz, S., Martin, B., Betteridge, A. and Hawes, C.
(1998). Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network.
Plant J. 15, 441-447.
Bola, B. and Allan, V. (2009). How and why does the endoplasmic reticulum move?
Biochem. Soc. Trans. 37, 961-965.
Brandizzi, F., Frangne, N., Marc-Martin, S., Hawes, C., Neuhaus, J. M. and Paris, N.
(2002a). The destination for single-pass membrane proteins is influenced markedly by
the length of the hydrophobic domain. Plant Cell 14, 1077-1092.
Brandizzi, F., Snapp, E. L., Roberts, A. G., Lippincott-Schwartz, J. and Hawes, C.
(2002b). 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 14, 1293-1309.
Burk, D. H. and Ye, Z. H. (2002). Alteration of oriented deposition of cellulose microfibrils
by mutation of a katanin-like microtubule-severing protein. Plant Cell 14, 2145-2160.
Byrnes, L. J. and Sondermann, H. (2011). From the cover: structural basis for the
nucleotide-dependent dimerization of the large G protein atlastin-1/SPG3A. Proc. Natl.
Acad. Sci. USA 108, 2216-2221.
Chalker-Scott, L. (1999). Environmental significance of anthocyanins in plant stress
responses. Photochem. Photobiol. 70, 1-9.
Chen, C. N., Chen, H. R., Yeh, S. Y., Vittore, G. and Ho, T. H. (2009). Autophagy is
enhanced and floral development is impaired in AtHVA22d RNA interference
Arabidopsis. Plant Physiol. 149, 1679-1689.
Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743.
Curtis, M. D. and Grossniklaus, U. (2003). A gateway cloning vector set for highthroughput functional analysis of genes in planta. Plant Physiol. 133, 462-469.
daSilva, L. L., Snapp, E. L., Denecke, J., Lippincott-Schwartz, J., Hawes, C. and
Brandizzi, F. (2004). Endoplasmic reticulum export sites and Golgi bodies behave as
single mobile secretory units in plant cells. Plant Cell 16, 1753-1771.
Dreier, L. and Rapoport, T. A. (2000). In vitro formation of the endoplasmic reticulum
occurs independently of microtubules by a controlled fusion reaction. J. Cell Biol. 148,
883-898.
English, A. R., Zurek, N. and Voeltz, G. K. (2009). Peripheral ER structure and function.
Curr. Opin. Cell Biol. 21, 596-602.
Federovitch, C. M., Ron, D. and Hampton, R. Y. (2005). The dynamic ER: experimental
approaches and current questions. Curr. Opin. Cell Biol. 17, 409-414.
Foissner, I., Menzel, D. and Wasteneys, G. O. (2009). Microtubule-dependent motility
and orientation of the cortical endoplasmic reticulum in elongating characean internodal
cells. Cell Motil. Cytoskeleton 66, 142-155.
Galway, M. E., Heckman, J. W., Jr and Schiefelbein, J. W. (1997). Growth and
ultrastructure of Arabidopsis root hairs: the rhd3 mutation alters vacuole enlargement
and tip growth. Planta 201, 209-218.
Hawes, C., Saint-Jore, C., Martin, B. and Zheng, H. Q. (2001). ER confirmed as the
location of mystery organelles in Arabidopsis plants expressing GFP! Trends Plant Sci.
6, 245-246.
Hu, J., Shibata, Y., Voss, C., Shemesh, T., Li, Z., Coughlin, M., Kozlov, M. M.,
Rapoport, T. A. and Prinz, W. A. (2008). Membrane proteins of the endoplasmic
reticulum induce high-curvature tubules. Science 319, 1247-1250.
Hu, J., Shibata, Y., Zhu, P. P., Voss, C., Rismanchi, N., Prinz, W. A., Rapoport, T. A.
and Blackstone, C. (2009). A class of dynamin-like GTPases involved in the generation
of the tubular ER network. Cell 138, 549-561.
Hu, Y., Zhong, R., Morrison, W. H., 3rd and Ye, Z. H. (2003). The Arabidopsis RHD3
gene is required for cell wall biosynthesis and actin organization. Planta 217, 912-921.
Irons, S. L., Evans, D. E. and Brandizzi, F. (2003). The first 238 amino acids of the
human lamin B receptor are targeted to the nuclear envelope in plants. J. Exp. Bot. 54,
943-950.
Jansen, G., Wu, C., Schade, B., Thomas, D. Y. and Whiteway, M. (2005). Drag&Drop
cloning in yeast. Gene 344, 43-51.
2251
Kim, D. H., Eu, Y. J., Yoo, C. M., Kim, Y. W., Pih, K. T., Jin, J. B., Kim, S. J.,
Stenmark, H. and Hwang, I. (2001). Trafficking of phosphatidylinositol 3-phosphate
from the trans-Golgi network to the lumen of the central vacuole in plant cells. Plant
Cell 13, 287-301.
Langhans, M., Marcote, M. J., Pimpl, P., Virgili-Lopez, G., Robinson, D. G. and
Aniento, F. (2008). In vivo trafficking and localization of p24 proteins in plant cells.
Traffic 9, 770-785.
Lee, M., Paik, S. K., Lee, M. J., Kim, Y. J., Kim, S., Nahm, M., Oh, S. J., Kim, H. M.,
Yim, J., Lee, C. J. et al. (2009). Drosophila Atlastin regulates the stability of muscle
microtubules and is required for synapse development. Dev. Biol. 330, 250-262.
Marks, B., Stowell, M. H. B., Vallis, Y., Mills, I. G., Gibson, A., Hopkins, C. R. and
McMahon, H. T. (2001). GTPase activity of dynamin and resulting conformation
change are essential for endocytosis. Nature 410, 231-235.
Mathur, J., Mathur, N., Kernebeck, B., Srinivas, B. P. and Hulskamp, M. (2003). A
novel localization pattern for an EB1-like protein links microtubule dynamics to
endomembrane organization. Curr. Biol. 13, 1991-1997.
Moreau, P., Brandizzi, F., Hanton, S., Chatre, L., Melser, S., Hawes, C. and SatiatJeunemaitre, B. (2007). The plant ER-Golgi interface: a highly structured and dynamic
membrane complex. J. Exp. Bot. 58, 49-64.
Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays
with tobacco tissue culture. Physiol. Plant 15, 473-497.
Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka,
K., Matsuoka, K., Jinbo, T. and Kimura, T. (2007). Development of series of gateway
binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant
transformation. J. Biosci. Bioeng. 104, 34-41.
Nebenfuhr, A., Gallagher, L. A., Dunahay, T. G., Frohlick, J. A., Mazurkiewicz, A. M.,
Meehl, J. B. and Staehelin, L. A. (1999). Stop-and-go movements of plant Golgi
stacks are mediated by the acto-myosin system. Plant Physiol. 121, 1127-1142.
Obrdlik, P., El-Bakkoury, M., Hamacher, T., Cappellaro, C., Vilarino, C., Fleischer,
C., Ellerbrok, H., Kamuzinzi, R., Ledent, V., Blaudez, D. et al. (2004). K+ channel
interactions detected by a genetic system optimized for systematic studies of membrane
protein interactions. Proc. Natl. Acad. Sci. USA 101, 12242-12247.
Orso, G., Pendin, D., Liu, S., Tosetto, J., Moss, T. J., Faust, J. E., Micaroni, M.,
Egorova, A., Martinuzzi, A., McNew, J. A. et al. (2009). Homotypic fusion of ER
membranes requires the dynamin-like GTPase atlastin. Nature 460, 978-983.
Park, S. H. and Blackstone, C. (2010). Further assembly required: construction and
dynamics of the endoplasmic reticulum network. EMBO Rep. 11, 515-521.
Parker, J. S., Cavell, A. C., Dolan, L., Roberts, K. and Grierson, C. S. (2000). Genetic
interactions during root hair morphogenesis in Arabidopsis. Plant Cell 12, 1961-1974.
Ridge, R. W., Uozumi, Y., Plazinski, J., Hurley, U. A. and Williamson, R. E. (1999).
Developmental transitions and dynamics of the cortical ER of Arabidopsis cells seen
with green fluorescent protein. Plant Cell Physiol. 40, 1253-1261.
Rismanchi, N., Soderblom, C., Stadler, J., Zhu, P. P. and Blackstone, C. (2008).
Atlastin GTPases are required for Golgi apparatus and ER morphogenesis. Hum. Mol.
Genet. 17, 1591-1604.
Runions, J., Brach, T., Kuhner, S. and Hawes, C. (2006). Photoactivation of GFP
reveals protein dynamics within the endoplasmic reticulum membrane. J. Exp. Bot. 57,
43-50.
Saint-Jore, C. M., Evins, J., Batoko, H., Brandizzi, F., Moore, I. and Hawes, C. (2002).
Redistribution of membrane proteins between the Golgi apparatus and endoplasmic
reticulum in plants is reversible and not dependent on cytoskeletal networks. Plant J.
29, 661-678.
Schmid, M., Davison, T. S., Henz, S. R., Pape, U. J., Demar, M., Vingron, M.,
Scholkopf, B., Weigel, D. and Lohmann, J. U. (2005). A gene expression map of
Arabidopsis thaliana development. Nature Genet. 37, 501-506.
Shibata, Y., Voss, C., Rist, J. M., Hu, J., Rapoport, T. A., Prinz, W. A. and Voeltz, G.
K. (2008). The reticulon and DP1/Yop1p proteins form immobile oligomers in the
tubular endoplasmic reticulum. J. Biol. Chem. 283, 18892-18904.
Shibata, Y., Hu, J., Kozlov, M. M. and Rapoport, T. A. (2009). Mechanisms shaping
the membranes of cellular organelles. Annu. Rev. Cell Dev. Biol. 25, 329-354.
Somerville, C. R. and Ogren, W. L. (1982). Isolation of photorespiratory mutants of
Arabidopsis. In Methods in: Chloroplast Molecular Biology (ed. R. B. Hallick and N.
H. Chua), pp. 129-139. New York: Elsevier.
Sparkes, I., Runions, J., Hawes, C. and Griffing, L. (2009). Movement and remodeling
of the endoplasmic reticulum in nondividing cells of tobacco leaves. Plant Cell 21,
3937-3949.
Sparkes, I., Tolley, N., Aller, I., Svozil, J., Osterrieder, A., Botchway, S., Mueller, C.,
Frigerio, L. and Hawes, C. (2010). Five Arabidopsis reticulon isoforms share
endoplasmic reticulum location, topology, and membrane-shaping properties. Plant
Cell 22, 1333-1343.
Staehelin, L. A. and Kang, B. H. (2008). Nanoscale architecture of endoplasmic reticulum
export sites and of Golgi membranes as determined by electron tomography. Plant
Physiol. 147, 1454-1468.
Stefano, G., Renna, L., Chatre, L., Hanton, S. L., Moreau, P., Hawes, C. and Brandizzi,
F. (2006). In tobacco leaf epidermal cells, the integrity of protein export from the
endoplasmic reticulum and of ER export sites depends on active COPI machinery. Plant
J. 46, 95-110.
Tolley, N., Sparkes, I. A., Hunter, P. R., Craddock, C. P., Nuttall, J., Roberts, L. M.,
Hawes, C., Pedrazzini, E. and Frigerio, L. (2008). Overexpression of a plant reticulon
remodels the lumen of the cortical endoplasmic reticulum but does not perturb protein
transport. Traffic 9, 94-102.
Ueda, H., Yokota, E., Kutsuna, N., Shimada, T., Tamura, K., Shimmen, T., Hasezawa,
S., Dolja, V. V. and Hara-Nishimura, I. (2010). Myosin-dependent endoplasmic
2252
Journal of Cell Science 124 (13)
Journal of Cell Science
reticulum motility and F-actin organization in plant cells. Proc. Natl. Acad. Sci. USA
107, 6894-6899.
Vedrenne, C. and Hauri, H. P. (2006). Morphogenesis of the endoplasmic reticulum:
beyond active membrane expansion. Traffic 7, 639-646.
Voeltz, G. K., Prinz, W. A., Shibata, Y., Rist, J. M. and Rapoport, T. A. (2006). A class
of membrane proteins shaping the tubular endoplasmic reticulum. Cell 124, 573-586.
Wang, H., Lockwood, S. K., Hoeltzel, M. F. and Schiefelbein, J. W. (1997). The ROOT
HAIR DEFECTIVE3 gene encodes an evolutionarily conserved protein with GTPbinding motifs and is required for regulated cell enlargement in Arabidopsis. Genes
Dev. 11, 799-811.
Yuen, C. Y. L., Sedbrook, J. C., Perrin, R. M., Carroll, K. L. and Masson, P. H. (2005).
Loss-of-function mutations of ROOT HAIR DEFECTIVE3 suppress root waving,
skewing, and epidermal cell file rotation in Arabidopsis. Plant Physiol. 138, 701-714.
Zheng, H., Kunst, L., Hawes, C. and Moore, I. (2004). A GFP-based assay reveals a role
for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus. Plant
J. 37, 398-414.
Zheng, H., Rowland, O. and Kunst, L. (2005a). Disruptions of the Arabidopsis EnoylCoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in
cell expansion during plant morphogenesis. Plant Cell 17, 1467-1481.
Zheng, H., Camacho, L., Wee, E., Batoko, H., Legen, J., Leaver, C. J., Malho, R.,
Hussey, P. J. and Moore, I. (2005b). A Rab-E GTPase mutant acts downstream of the
Rab-D subclass in biosynthetic membrane traffic to the plasma membrane in tobacco
leaf epidermis. Plant Cell 17, 2020-2036.
Zhong, R., Kays, S. J., Schroeder, B. P. and Ye, Z. H. (2002). Mutation of a chitinaselike gene causes ectopic deposition of lignin, aberrant cell shapes, and overproduction
of ethylene. Plant Cell 14, 165-179.