Download ACTIN-RELATED PROTEIN8 Encodes an F-Box

Plant Cell Physiol. 49(5): 858–863 (2008)
doi:10.1093/pcp/pcn053, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Short Communication
ACTIN-RELATED PROTEIN8 Encodes an F-Box Protein Localized to the
Nucleolus in Arabidopsis
Muthugapatti K. Kandasamy, Elizabeth C. McKinney and Richard B. Meagher *
Genetics Department, Davison Life Sciences Building, University of Georgia, Athens, GA 30602, USA
far (Kandasamy et al. 2003, Deal et al. 2005). Indeed, the
nuclear ARPs are present in yeast and vertebrates as
components of various chromatin-modifying complexes and
are only known to function as part of such complexes (Chen
and Shen 2007). Interestingly, conventional actin is also
found in many of these complexes together with ARPs.
Based on their activities, the nuclear ARP-containing
complexes can be classified as belonging to one of three
functional categories: ATP-dependent nucleosome movement and remodeling (NR) complexes; histone variant
exchange (HVE) complexes; and histone acetyltransferase
(HAT) complexes. The NR and HVE complexes all contain
a DNA-dependent ATPase subunit and use the energy of
ATP hydrolysis to drive the repositioning of nucleosomes
on DNA or to exchange one histone type for the other
within nucleosomes. The HAT complexes, on the other
hand, acetylate the N-terminal tails of histone proteins and
thereby alter the chromatin structure indirectly (Meagher
et al. 2007).
No single specific role has been defined for nuclear
ARPs within chromatin remodeling complexes (Olave et al.
2002, Meagher et al. 2007). Although the nuclear ARPs are
highly divergent from actin, they are still predicted to
maintain the actin fold, which is comprised of two protein
domains held together by a hinge region, creating a flexible
nucleotide-binding pocket. In actin, this region undergoes a
relatively large conformational change upon nucleotide
binding, ATP hydrolysis or ATP/ADP exchange, which
can stimulate the polymerization and depolymerization of
F-actin. Hence, based on this potential ‘lock and key’ shift
in structure, it has been proposed that the nuclear ARPs
may aid in the assembly or modulation of chromatin
remodeling complexes (Sunada et al. 2005). Moreover, yeast
ARP4 and ARP8 have been shown to bind directly to core
histones (Harata et al. 1999, Shen et al. 2003), thus ARPs
might be required for targeting the ARP-containing complexes to the chromatin. ARPs may also play a role in
stimulating core ATPase activity in NR and HVE complexes (Meagher et al. 2007).
Arabidopsis ARP8, which is the subject of the present
study, shows 30 and 29% amino acid identity to yeast actin
Arabidopsis encodes six nuclear actin-related proteins
(ARPs), among them ARP8 is unique in having an F-box
domain and an actin homology domain. Analysis of the ARP8
promoter–b-glucuronidase (GUS) fusion suggests that ARP8
is ubiquitously expressed in all organs and cell types.
Immunocytochemical analysis with ARP8-specific monoclonal antibodies revealed that ARP8 protein is localized to the
nucleolus in interphase cells and dispersed in the cytoplasm in
mitotic cells. The cell cycle-dependent subcellular patterns of
distribution of ARP8 are conserved in other members of
Brassicaceae. Our findings provide the first insight into the
possible contributions of plant ARP8 to nucleolar functions.
Keywords: ARP8 — Chromatin remodeling — Nuclear
actin-related proteins — Nucleolar functions — Ribosome
biogenesis.
Abbreviations: ARP, actin-related protein; DAPI, 40 ,6diamidino-2-phenylindole; GUS, b-glucuronidase; HAT, histone
acetyltransferase; HVE, histone variant exchange; NOR, nucleolar
organizing region; NR, nucleosome movement and remodeling;
PEPC, phosphoenolpyruvate carboxylase.
The actin family consists of conventional actin and
various actin-related proteins (ARPs), which share moderate sequence homology and their basal structure with actin.
ARPs are found in all eukaryotes and, based on their degree
of similarity to actin, they are grouped into several classes.
For example, the 10 ARPs of budding yeast are classified
into classes 1–10, where ARP1 is the most and ARP10 is the
least similar to actin (Poch and Winsor 1997). Some of the
ARPs (e.g. ARP1–ARP3) that are well conserved relative
to actin are cytoplasmic and participate in cytoskeletal
functions (Schafer and Schroer 1999, Kandasamy et al.
2004). However, several of the more divergent ARPs
accumulate in the nucleus, and a few of them are known
to be involved in important nuclear functions. In yeast,
ARPs 4–9 are localized to the nucleus (Weber et al. 1995,
Harata et al. 2000), whereas in plants only ARP4, APR6
and ARP7 have been demonstrated to be in the nucleus so
*Corresponding author: E-mail, [email protected]; Fax, þ1-706-542-1387.
858
Plant nucleolar ARP8 protein
and Arabidopsis ACT2 in the regions of alignment,
respectively (Supplementary Table S1). The presence of
large regions of poor conservation in the Arabidopsis ARP8
sequence obscures its phylogenetic relationships to the other
known ARPs. Because it is not closely related to yeast or
human ARP8 and shows similar weak homology to yeast
ARP8 and ARP9, the Arabidopsis ARP8 is considered a
plant-specific orphan ARP. Moreover, Arabidopsis ARP8
has a complex gene structure encoding a novel protein with
distinct F-box and actin homology domains (McKinney
et al. 2002). To understand the function of this unique
protein, herein we have characterized its pattern of
expression using an ARP8 promoter–b-glucuronidase
(GUS) reporter fusion, and examined the subcellular
localization using ARP8-specific antibodies. We have
shown that unlike other known eukaryotic nuclear ARPs,
Arabidopsis ARP8 is localized to the interphase nucleolus,
suggesting a role for ARP8 in nucleolar functions.
The ARP8 gene sequence in Arabidopsis comprises 12
exons and encodes a protein of 471 amino acids (Fig. 1A).
In addition to the requisite actin-related (A) domain of 381
amino acids, the Arabidopsis ARP8 protein has unique
additional domains, including an N-terminal 40 amino acid
hydrophobic leader (L) and a 50 amino acid F-box (F)
homology domain (Fig. 1B), neither of which are found in
fungal or animal ARP8 or other nuclear ARPs. The
genomes of the evolutionarily distant dicot grape (Vitis
vinifera) and monocot rice (Oryza sativa) also encode a
similarly organized ARP8 homolog with 65 and 63% amino
acid identity to the Arabidopsis sequence, respectively (see
Supplementary Fig. S1 and Table S1). Thus, this unusual
gene structure is ancient, pre-dating the split between
monocots and dicots almost 200 million years ago.
However, an ARP8 homolog is absent from the genomes
of the moss Physcomitrella and green alga Chlamydomonas,
suggesting that it may be specific to angiosperms.
Transcripts resulting from an alternative shorter splice
variant (representing exons 1–6 and exon 12) encoding a 242
amino acid protein are found in Arabidopsis flowers, but
not leaves or other cDNA libraries (McKinney et al. 2002).
However, this truncated ARP8 protein is not detectable on
Western blots of flower or inflorescence samples, which
suggests it may not be functional or may be only very
weakly expressed.
Previous analysis of mRNA levels suggested that ARP8
was expressed at low levels in most plant organs (McKinney
et al. 2002, Zimmermann et al. 2004). To refine our
knowledge about the organ- and tissue-specific expression
of ARP8, we analyzed the activity of ARP8 regulatory
sequences and ARP8 protein. When transgenic plants
expressing an ARP8pt::GUS fusion were incubated in
X-glucuronide substrate, blue staining from GUS activity
was observed in all organs and tissues at various stages
859
A ARP8 Gene
ATG
1
2 3 4 5 6
7
8
9
10
11
TGA
12
ARP8-N
ARP8-C
B ARP8 Protein
N- L
C
F
C
A
1–471 aa,
52 kDa
N- and C-terminal antibodies
APR8r
A. th. APR8r
A. th. APR8r
-52 kDa
MAbARP8-N
D
MAbARP8-C Coomassie
MAbARP8-N
MAbARP8-C
Control
ARP8
DNA
Merge
Fig. 1 ARP8 gene structure and antibody specificity. (A) ARP8
gene with exons and flanking sequence and the location of ARP8N and ARP8-C peptides. Introns are not drawn to scale. (B) ARP8
protein with leader [L], F-box [F] and actin-homology [A] domains.
(C) Western blots showing the reaction of MAbARP-N and
MAbARP8-C antibodies to recombinant ARP8 (ARP8r) and ARP8
in Arabidopsis (A. th.) plant extracts. A Coomassie-stained gel of
ARP8r is shown to the right. (D) Immunoreactivity of the MAbARPN and MAbARP8-C antibodies with ARP8 in the nucleolus of root
cells. The pre-immune serum control is shown on the right panel.
Antibody and DAPI staining are shown in green and red,
respectively. Merged images are at the bottom.
of development, as shown for representative plant samples
in Fig. 2. In particular, strong expression was observed in
the cotyledons and hypocotyls of young seedlings (Fig. 2A)
as well as in developing and mature rosette leaves and roots
of relatively older seedlings (Fig. 2B, C). The reporter gene
expression in the root was strongest in vascular tissues
and weakest in root tips (Fig. 2A). Moreover, root hairs
and trichomes showed positive staining (not shown). GUS
expression was also observed in most floral organs and
pollen (Fig. 2D–F). Staining was relatively weak in ovules,
ovary and petals (Fig. 2D, F, G). Soon after fertilization,
the developing seeds revealed strong GUS staining and the
mature embryos were also positive for ARP8 expression
(Fig. 2G, H).
860
Plant nucleolar ARP8 protein
A
C
B
D
E
G
F
H
Fig. 2 Constitutive expression of the ARP8pt::GUS reporter gene.
(A) Four-day-old seedlings. The inset shows a close up of a root tip.
(B) and (C) Eleven- and 18-day-old seedlings, respectively. (D)
Open flower. (E) Flower bud 1 d before opening. (F) Pistil and
stamens. The anther insert shows pollen staining. (G) Developing
seeds. Arrowheads point to fertilized ovules. (H) Isolated mature
embryos.
To examine ARP8 protein expression, we prepared
monoclonal antibodies to ARP8 N-terminal and C-terminal
peptides (Fig. 1A). These two antibodies, MAbARP8-N
and MAbARP8-C, each reacted with the full-length 52 kDa
recombinant ARP8 protein expressed in Escherichia coli
and endogenous ARP8 of identical molecular weight in
Arabidopsis plant extracts (Fig. 1C). Although MAbARP8C reacted strongly with recombinant ARP8, it reacted
relatively weakly with ARP8 protein in plant samples. Thus,
MAbARP8-N was used for further analysis of tissuespecific ARP8 expression. MAbARP8-N detected ARP8
protein in all vegetative and reproductive organs examined
including seedlings, roots and siliques, although higher
concentrations were observed in developing flower buds
and flowers within the inflorescence (Fig. 3Q, upper panel).
Equal loading of total protein was confirmed by re-probing
the blot with a polyclonal antibody specific for the
constitutively expressed 110 kDa phosphoenolpyruvate carboxylase (PEPC; Fig. 3Q, lower panel). To confirm the
ubiquitous but differential levels of ARP8 expression in
vegetative and reproductive tissues, we examined the
expression of its homolog in a relatively distant crucifer,
Brassica. As in Arabidopsis, MAbARP8-N detected higher
levels of the Brassica ARP8 homolog of 52 kDa in the
inflorescence, compared with relatively low levels in root
samples (Fig. 4A). Thus, the differential expression of
ARP8 is conserved in Arabidopsis and Brassica, and its
ubiquitous presence in all organs is identical to the other
nuclear ARPs (ARP4, ARP6 and ARP7) characterized in
plants (Kandasamy et al. 2003, Deal et al. 2005).
Plant ARP8 was classified as a potential nuclear
protein based on its phylogenetic affinity for other known
nuclear ARPs and its significant divergence from cytoplasmic ARPs and actin (McKinney et al. 2002, Kandasamy
et al. 2004). Moreover, most of the previously characterized
divergent ARP proteins of other eukaryotes, including yeast
and humans, have been localized to the nucleoplasm of
interphase cells. Because Arabidopsis ARP8 is unique in
having an F-box domain, we wanted to examine whether it
shows a similar subcellular distribution to other diverse
nuclear ARPs or whether it may have a different subcellular
localization and possibly have different function. We
therefore immunolabeled fixed and dissociated Arabidopsis
root cells with MAbARP8-N antibody. Interestingly, our
immunolabeling revealed intense staining of the root cell
nucleolar region within nuclei as revealed by the merged
images of antibody labeling and 40 ,6-diamidino-2-phenylindole (DAPI) staining (Fig. 1D, left panel). The nucleolar
localization of ARP8 was confirmed by labeling cells with
MAbARP8-C monoclonal antibody (Fig. 1D, middle
panel), which also showed a strong staining of the
nucleolus. Occasionally a few cells showed faint staining
in the nucleoplasm surrounding a rather densely stained
nucleolus (see the upper middle panel in Fig. 1D).
Apparently, ARP8 labeling with both the antibodies did
not reveal any substructure in the nucleolus. To determine
whether the nucleolar accumulation of ARP8 is conserved
in Brassica, we immunolabeled root cells from B. napus
(Fig. 4B–E) and B. campestris (not shown) with
MAbARP8-N, which showed the same restriction of
ARP8 staining to the nucleolar compartment.
A
A
B
C
B. campestris
root
Merge
B. napus root
DNA
861
B. napus
inflorescence
ARP8
Arabidopsis
inflorescence
Plant nucleolar ARP8 protein
ARP8
ARP8
PEPC
D
ARP8
DNA
B
DNA
E
i
Merge
i
m
E
F
C
Merged
i
F
ARP8
i
m
t
i
DNA
G
H
vn
sn
Flower
N
Inflorescence
Root
Q
Seedling
M
K
L
O
P
Silique
J
Flower
buds
I
ARP8
PEPC
Fig. 3 ARP8 expression and localization in different Arabidopsis
tissues. ARP8 labeling with MAbARP8-N is shown in green and
DAPI staining is shown in red. Separate and merged images are
presented. (A–C) Root cortical cell. (D–F) A file of root apical cells.
The arrow points to cells towards the apical meristem. (G) Root
cells at metaphase (m), telophase (t) and interphase (i) stages. (H)
DAPI image of cells shown in G. (I–P) ARP8 labeling and merged
images of microspore (I, J), pollen (K, L) and tapetal cells (M–P). sn,
sperm nucleus; vn, vegetative nucleus. Bars ¼ 10 mm. (Q) Western
blot showing ARP8 expression in different organs (upper panel).
The lower control panel shows PEPC levels in different samples.
D
p
t
G
H
Fig. 4 ARP8 expression and localization in Brassica. (A) Western
blots probed with MAbARP8-N (top) or PEPC (bottom) antibodies.
(B–D) Root cells at interphase. (B) ARP8; (C) DAPI; (D) merged
image. (E) Merged image of a root cortical cell. (F–H) Root cells
at different stages of the cell cycle. m, metaphase; t, telophase;
p, prophase; i, interphase. Bars ¼ 10 mm.
A more thorough analysis of the nucleolar expression
patterns was undertaken with MAbARP8-N to examine all
major organs and tissues in Arabidopsis, shown in Fig. 3.
Mature cortical root cells (Fig. 3A–C) and a file of young
root apical cells (Fig. 3D–F) showed strong nucleolar
staining in all interphase cells, except for the few apical
initials adjacent to the quiescent center that contain very
small nucleoli. In mitotic cells at metaphase, when the
nuclear membrane is broken down, ARP8 staining was
dispersed into the cytoplasm and not associated with the
chromosomes (Fig. 3G). This cytoplasmic diffusion is very
similar to the Arabidopsis nuclear ARP4 and ARP7
localization during metaphase and anaphase (Kandasamy
et al. 2003), but contrasts sharply with human ARP8, which
has recently been shown to be associated with mitotic
chromosomes (Aoyama et al. 2008). However, during early
telophase stage, ARP8 appeared associated with the
chromatin, although there was still no distinct demarcation
of a nucleolar region (Fig. 3G, H). A similar cell cycledependent subcellular distribution of plant ARP8 protein
was also observed in Brassica (Fig. 4F–H). In developing
Arabidopsis mono-nucleate microspores, ARP8 was clearly
observed in the nucleolus (Fig. 3I, J). However, in
trinucleate pollen, ARP8 was associated with one or more
nucleolar regions of the vegetative nucleus, but no staining
862
Plant nucleolar ARP8 protein
was visible in the two sperm cell nuclei (Fig. 3K, L). In the
polyploid and sometimes multinucleate tapetal cells,
MAbARP8-N reacted with several small nucleoli in each
cell (Fig. 3M–P). Thus, plant ARP8 is localized to the
nucleolar compartment within the interphase nuclei in
essentially all cell types.
The nucleolus is the ribosomal factory where rRNAs
are synthesized, processed and assembled with ribosomal
proteins to form the ribosome subunits that are then
exported to the cytoplasm (Olson et al. 2000, HernandezVerdun 2006). Specific complexes participate in and
accomplish these different steps by interacting with the
rDNA in the nucloeolar organizing regions (NORs) or with
rRNA. In Arabidopsis there are two active NORs located on
chromosomes 2 and 4 with several hundred rDNA genes
each (Copenhaver and Pikaard 1996). Considering that
the nuclear ARPs function as components of various
chromatin-modifying complexes and Arabidopsis ARP8 is
localized to the nucleolus, it is reasonable to speculate about
possible roles for ARP8 in nucleolar chromatin remodeling
complexes involved in the epigenetic activation and/or
silencing of the rDNA genes. This assumption is particularly appealing, because of the tight regulation of both
ARP8 and rDNA activities. Although chromatin remodeling closely controls the replication timing and transcription
of the rDNA genes (Grummt and Pikaard 2003, Li et al.
2005), no ARP has been identified to date as part of any
nucleolar chromatin remodeling complex (e.g. NoRC).
Moreover, proteomic analyses of nucleoli from humans
and Arabidopsis have failed to identify any nuclear ARP
component (Scherl et al. 2002, Brown et al. 2005). Thus,
plant ARP8 is the first candidate ARP with the potential to
participate in the epigenetic control of rDNA activity.
Moreover, the angiosperm ARP8 protein sequences
contain an F-box motif upstream of the actin homology
domain. The F-box motifs generally function as a site of
protein–protein interaction and thus may link the resident
proteins (e.g. ARP8) to the target proteins such as a
ubiquitin–ligase complex for ubiquitin tagging and subsequent proteolytic degradation. F-box-mediated protein
degradation has been linked to cell growth, aging and the
cell cycle in diverse organisms (Itoh et al. 2003, Dong et al.
2006). In addition, F-box proteins also participate in
ubiquitin tagging of histones in the nucleus, which is
required for gene silencing (de Napoles et al. 2004, Baarends
et al. 2005). Thus, ARP8 may have important nucleolar
functions, with the F-box domain contributing to its tight
regulation at the level of protein turnover and/or to
ubiquination of histones, which may control gene regulation at rDNA loci.
In conclusion, we have shown that the novel plant
ARP8 protein is strongly and ubiquitously expressed in all
actively growing and differentiating organs and tissues.
We have clearly demonstrated with two distinct monoclonal
antibodies that ARP8 protein is localized to the nucleolar
compartment(s) within the nucleus. Based on its unique
structure and subcellular distribution, we infer that plant
ARP8 has important roles in the epigenetic regulation of
nucleolar genes.
Materials and Methods
To generate ARP8pt::GUS construct, we replaced the ADF8
promoter region in the previously characterized ADF8pt::GUS
construct with a 1,104 bp ARP8 promoter sequence (Ruzicka et al.
2007). For recombinant ARP8 protein expression, the full-length
ARP8 coding region was amplified from a flower cDNA library
(Invitrogen, Carlsbad, CA, USA), cloned into the pET-15b
vector (Novagen, Madison, WI, USA) and then the protein was
expressed from this vector in E. coli as described earlier (McKinney
et al. 2002).
Arabidopsis ecotype Columbia plants were grown at 228C
with 16 h light and 8 h dark periods. Plants were transformed
with the ARP8pt::GUS construct by vacuum infiltration, and
the transgenic plants were selected on MS medium containing
35 mg ml–1 kanamycin.
ARP8-specific monoclonal antibodies were prepared to 25
amino acid N-terminal (ARP8-N, amino acids 2–26, ILKKVWG
SVWNRSNSGKDLVNHQRA) and C-terminal (ARP8-C, amino
acids 447–471, SNLSIFPGPWCITRKQFRRKSRLMW) multiple
antigenic peptides, as described earlier (Li et al. 2001). For
preparation of protein samples, various plant organs were ground
in a high salt extraction buffer [20 mM Tris–HCl (pH 7.4), 100 mM
NaCl, 10% glycerol, 1 mM b-mercaptoethanol and protease
inhibitor cocktail (Roche, Mannheim, Germany)]. After centrifugation, the pellet proteins were extracted with sample buffer
(Laemmli 1970) and assayed by Western blotting as described
previously (Kandasamy et al. 2003). The recombinant ARP8
protein was prepared by boiling the bacterial pellets directly in
sample buffer.
Immunocytochemistry was performed on fixed and dissociated cells as explained previously (Kandasamy et al. 2003). GUS
histochemical staining and microscopic analysis of ARP8pt::GUS
expression was performed as described earlier (Deal et al. 2005).
Supplementary material
Supplementary material mentioned in the article is available
to online subscribers at the journal website www.pcp.
oxfordjournals.org.
Funding
The National Institutes of Health (GM 36397-21).
Acknowledgments
We are grateful to Gay Gragson and Dan Ruzicka for critical
reading of the manuscript.
Plant nucleolar ARP8 protein
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(Received February 7, 2008; Accepted March 27, 2008)