The small GTPase Ran is highly conserved in eukaryotic cells
and has roles in the control of nucleocytoplasmic transport
(Melchior et al., 1993; Moore and Blobel, 1993), mitotic
spindle assembly (Carazo-Salas et al., 1999; Kalab et al., 1999;
Ohba et al., 1999; Wilde and Zheng, 1999; Zhang et al., 1999)
and nuclear envelope assembly (Hetzer et al., 2000; Zhang and
Clarke, 2000). Proper spatial coordination of these processes
relies upon the distribution of GTP- versus GDP-bound forms
of Ran, which is in turn controlled by the localisation of its
regulators (Clarke and Zhang, 2001; Dasso, 2002; Görlich and
Mattaj, 1996; Hetzer et al., 2002). The only known guanine
nucleotide exchange factor (GEF) for Ran, RCC1, is localised
to chromatin throughout the cell division cycle (Bischoff and
Ponstingl, 1991; Moore et al., 2002; Ohtsubo et al., 1989). By
contrast, RanGAP1, the sole GTPase-activating factor that
stimulates GTP hydrolysis by Ran (Bischoff et al., 1994), is
localised to cytoplasm in interphase, in particular the
cytoplasmic face of nuclear pore complexes, and to the mitotic
spindle and kinetochores in mitosis (Joseph et al., 2002;
Mahajan et al., 1997; Matunis et al., 1996). Together with the
concentration of Ran in the nucleus by its active import, the
distinct localisation of its regulators results in a high
concentration of Ran-GTP in the nucleoplasm and a low
concentration of Ran-GDP in the cytoplasm, whereas in
mitosis, Ran-GTP is concentrated around chromosomes
(Caudron et al., 2005; Kalab et al., 2006; Kalab et al., 2002).
Nucleocytoplasmic transport is carried out by the importin
? family of transport proteins, or karyopherins, which carry
cargo across the nuclear envelope via nuclear pores, with
directionality conferred by the high concentration of Ran-GTP
in the nucleoplasm (Görlich and Mattaj, 1996). As import
cargo complexes enter the nucleus through nuclear pore
complexes, Ran-GTP binding to importin ?
disassembly of importin-cargo complexes, releasing cargo into
the nucleoplasm (Stewart, 2007). Conversely, Ran-GTP is an
essential component of export complexes involving the export
receptor, Crm1 (Fornerod et al., 1997). These export
complexes disassemble on entry into the cytoplasm as
RanGAP1 catalyses hydrolysis of GTP by Ran, converting it
into Ran-GDP (Bischoff et al., 1994).
During mitosis, when the nuclear envelope breaks down in
animal cells, similar mechanisms are involved in the
orchestration of spindle assembly by Ran. Ran-GTP generated
around mitotic chromosomes binds to importin ? and causes
the release of spindle assembly factors such as TPX2 from
inhibitory complexes with importins, thereby promoting
microtubule nucleation and stabilisation in the vicinity of
chromosomes (Gruss et al., 2001; Nachury et al., 2001; Wiese
et al., 2001). It has been proposed that a gradient of Ran-GTP
concentration, declining at greater distances from mitotic
chromosomes, provides a spatial signal that is important for
orchestrating activity of different factors required for spindle
assembly (Hetzer et al., 2002).
RCC1 is the guanine nucleotide exchange factor for Ran
GTPase. Generation of Ran-GTP by RCC1 on chromatin
provides a spatial signal that directs nucleocytoplasmic
transport, mitotic spindle assembly and nuclear envelope
formation. We show that RCC1 is expressed in human cells
as at least three isoforms, named RCC1? ?, RCC1? ? and
RCC1? ?, which are expressed at different levels in specific
tissues. The ? ? and ? ? isoforms contain short inserts in their
N-terminal regions (NTRs) that are not present in RCC1? ?.
This region mediates interaction with chromatin, binds
importin ? ?3 and/or importin ? ?, and contains regulatory
phosphorylation sites. RCC1? ? is predominantly localised to
the nucleus and mitotic chromosomes like RCC1? ?.
However, compared to RCC1? ?, RCC1? ? has a greatly
reduced interaction with an importin ? ?3-? ? and a stronger
interaction with chromatin that is mediated by the
extended NTR. RCC1? ? is also the isoform that is most
highly phosphorylated at serine 11 in mitosis. Unlike
RCC1? ?, RCC1? ? supports cell proliferation in tsBN2 cells
more efficiently when serine 11 is mutated to non-
phosphorylatable alanine. Phosphorylation of RCC1? ?
therefore specifically controls its function during mitosis.
These results show that human RCC1 isoforms have
distinct chromatin binding properties, different molecular
interactions, and are
phosphorylation, as determined by their different NTRs.
selectively regulated by
Supplementary material available online at
Key words: RCC1, Ran, Nuclear transport, Mitosis
RCC1 isoforms differ in their affinity for chromatin,
molecular interactions and regulation by
Fiona E. Hood and Paul R. Clarke*
Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK
*Author for correspondence (e-mail: email@example.com)
Accepted 30 July 2007
Journal of Cell Science 120, 3436-3445 Published by The Company of Biologists 2007
Journal of Cell Science
JCS ePress online publication date 11 September 2007
Regulation of RCC1 isoforms
The localisation of RCC1 to chromatin is crucial for the
spatial organisation of Ran-GTP and is therefore important for
the functions of Ran throughout the cell division cycle. The
crystal structure of RCC1 reveals it to form a seven-bladed
propeller, from the seven characteristic RCC1 sequence repeats
(Renault et al., 1998). The association of RCC1 with chromatin
involves interactions between its core region and histones H2A
and H2B (Nemergut et al., 2001). The binding of RCC1 to
chromatin in vitro is promoted by nucleotide-free or GDP-
bound Ran, and inhibited by Ran-GTP (Li et al., 2003; Zhang
et al., 2002). Ran interacts separately with histones H3 and H4
(Bilbao-Cortes et al., 2002) and the affinity of RCC1 with
chromatin is reduced in a D182A mutant (Moore et al., 2002;
Hutchins et al., 2004), which disrupts the interaction with Ran
(Azuma et al., 1999). However, the localisation of RCC1 to
chromatin is critically dependent on a flexible N-terminal
region (NTR) (Moore et al., 2002) which is likely to extend
beyond the core structure (Renault et al., 1998). Deletion of the
NTR disperses RCC1 from mitotic chromosomes and causes a
variety of spindle abnormalities consistent with disruption of
the proper spatial organisation of Ran-GTP (Moore et al.,
2002). The NTR is required for binding of RCC1 directly to
DNA in vitro via residues 21-25 (Seino et al., 1992), although
it is not known if this binding occurs during the interaction with
The NTR of RCC1 also mediates interactions with other
proteins and is the target for post-translational modification.
There are two clusters of basic residues in the NTR that form
a bipartite nuclear localisation signal (NLS) that binds to
importin ? via the adaptor importin ?3 (Kohler et al., 1999;
Nemergut and Macara, 2000; Talcott and Moore, 2000).
Lysines 21 and 22 in the second cluster are essential for
importin ?3 binding whereas the first cluster strengthens the
interaction (Friedrich et al., 2006). The N-terminal methionine
of RCC1 is removed and the ?-amino group of serine 2 is
mono-, di- or tri-methylated, which is required for interaction
with chromatin, but does not appear to be regulated during the
cell cycle (Chen et al., 2007). By contrast, phosphorylation of
serines 2 and 11 by CDK1-cyclin B1 during mitosis disrupts
importin binding and regulates the dynamic interaction with
mitotic chromosomes (Hutchins et al., 2004; Li and Zheng,
Although animal cells contain only one RCC1 gene, human
and hamster cells express RCC1 proteins of different lengths
that are probably generated by alternative mRNA splicing.
However, no apparent difference in activity or cellular function
of these novel isoforms compared to the shorter, more abundant
isoform was found (Miyabashira et al., 1994). No further
consideration of the possible importance of different RCC1
isoforms has been made.
Here we show that human cells express at least three
isoforms of RCC1, which we name RCC1?, RCC1? and
RCC1?. Normal human tissues show different levels of
expression of these isoforms. We show that RCC1? has
increased affinity for chromatin in cells, reduced binding to
importins, and is the isoform that is most highly
phosphorylated at serine 11 during mitosis. In a cell
proliferation assay, RCC1? promotes cell proliferation more
efficiently when phosphorylation at serine 11 is abolished by
mutation. This work demonstrates that isoforms of RCC1 in
mammalian cells have different molecular interactions and
divergent mechanisms of regulation, suggesting that they have
distinct functions during the cell division cycle and in
Multiple isoforms of RCC1 are expressed in mammalian
Alignment of the amino acid sequences of mammalian RCC1
molecules derived from cDNA sequences (Fig. 1A) indicates
that three isoforms are expressed in human cells. These
correspond to the originally identified short isoform, a second
isoform with a 31 amino acid insert previously identified by
Miyabashira et al. [(Miyabashira et al., 1994) RCC1-I], and a
third isoform with a 17 amino acid insert that we also identified
by RT-PCR amplification of RCC1 cDNA from HeLa cells. We
named these three isoforms RCC1?, RCC1? and RCC1?,
respectively. The insert in the novel human isoform, RCC1?,
consists of the first 17 residues of the insert in RCC1?. RCC1?
is closely related to the hamster and mouse RCC1-I proteins
previously identified (Miyabashira et al., 1994), and is
predicted in rhesus monkey, whereas RCC1? is predicted in
chimpanzee. This suggests that the expression of all three
isoforms is conserved in primates.
Examination of the genomic sequence of human RCC1
indicates that the latter part of exon 6? that is present in RCC1?
but not RCC1? contains a GT-AG splice donor and acceptor
sites, and also a potential branch site, suggesting that the
differences between all three RCC1 transcripts could be due to
alternative splicing (Fig. 1B). Comparison of the genomic
sequence of mouse with human shows the sequence
conservation of the 6? exon of RCC1 (supplementary material
Fig. S1), including the alternative splice sites identified in
human, indicating that homologues of all three isoforms may
be expressed in non-primate mammals as well as in primates.
An isoform containing a four amino acid insert is found in
Xenopus laevis, indicating that multiple isoforms are also
present in other vertebrates.
To examine expression of RCC1 proteins in human cells,
two antibodies were generated (Fig. 1C). The first antibody,
anti-R-INS, was raised to a peptide corresponding to the insert
region of RCC1?, and so should detect both RCC1? and
RCC1?. The second antibody, anti-R-DIS, was raised to a
peptide corresponding to the region either side of the insert site.
This epitope was predicted to be disrupted when the insert is
present, and therefore should only detect RCC1?. A similar
strategy was used successfully by Miyabashira et al.
(Miyabashira et al., 1994). These antibodies were tested by
immunoblotting of proteins retrieved from HeLa cell extracts
using glutathione S-transferase (GST) expressed as a fusion
with RanT24N, a mutant that forms a stable complex with
RCC1 (Fig. 1D). Anti-R-DIS detected a single band
precipitated specifically by GST-RanT24N that corresponded
to the major polypeptide detected by an antibody raised against
the invariant C terminus of RCC1, consistent with it being
(45 kDa). Anti-R-INS detected two bands
corresponding to the two higher molecular mass bands detected
with the total RCC1 antibody. The migration of these larger
polypeptides is consistent with the predicted sizes of RCC1?
and RCC1? of 46.7 and 48.2 kDa, respectively, indicating that
both are expressed in HeLa cells, with RCC1? being expressed
at a higher level than RCC1?. In addition, a very minor band
Journal of Cell Science
with an apparent molecular mass less than that of RCC1? was
detected by the total RCC1 antibody in the GST-RanT24N
precipitate but not by either anti-R-DIS or anti-R-INS
antibodies. This may represent an additional shorter isoform
that lacks the anti-R-DIS epitope.
Immunoblotting of lysates from a range of human cell lines
showed expression of both RCC1? and insert-containing
isoforms in all of those tested (Fig. 1E). When the relative
levels of expression of RCC1? and RCC1? were analysed
during the cell cycle, no significant changes in the amount or
the ratio of the two isoforms was observed. However, the
relative expression levels of RCC1 isoforms differed between
HeLa and U2OS cells (supplementary material Fig. S2).
Analysis of the expression of RCC1 isoforms in normal human
tissues showed remarkable differences (Fig. 1F). RCC1? was
the major isoform expressed in most tissues, but the level of
Journal of Cell Science 120 (19)
expression varied widely. RCC1? was not detected or only
weakly expressed in small intestine, lung and stomach. The
expression levels of insert-containing isoforms also varied
between tissues, but the pattern differed from that of RCC1?.
RCC1? and ? were expressed in all tissues tested, but were the
predominant isoforms in stomach and were also strongly
expressed in lung. The ratio of ? and ? isoforms also varied
between tissues, but ? was the more abundant isoform except
in skeletal muscle. In addition, a low molecular mass band was
detected in skeletal muscle and testis which might correspond
to the low molecular mass form weakly expressed in HeLa
cells (Fig. 1D).
Localisation of RCC1? and RCC1? in cells
When RCC1? was expressed in cells as a fusion with green
fluorescent protein (GFP) it was found to be mainly nuclear in
Fig. 1. Three RCC1 transcript variants
are expressed in humans. (A) Linear
representation of RCC1 protein
domains (not to scale), showing
sequence alignment of the NTR
(residues 1-27) of human RCC1?
protein with insert-containing RCC1
isoforms from: human (hs); chimpanzee
(pt); rhesus monkey (macm); golden
hamster (ma); mouse (mm); and
African clawed frog (xl). RCC1?
homologues in species other than
human are not shown. Insert sequences
are shown in bold. Phosphorylation
sites (serines 2 and 11) are indicated by
circles labelled P. (B) Schematic
showing how alternative mRNA
splicing around and within exon 6?
could generate the known RCC1
transcript variants. (C) Isoform-specific
antibodies were generated to peptides
corresponding to RCC1 sequences.
Anti-R-INS should detect insert-
containing isoforms, whereas the anti-
R-DIS epitope should be disrupted by
them. (D) Precipitation of RCC1
proteins from asynchronous HeLa cell
extracts by GST-RanT24N or GST as a
control, with analysis by
immunoblotting with RCC1 isoform-
specific antibodies. (E) Immunoblotting
of human cultured cell lysates.
(F) Immunoblotting of normal human
tissue lysates on an Instablot
(IMGENEX) membrane (Cambridge
Bioscience), which is pre-loaded with
SDS-PAGE-separated normal tissue
lysates (20 ?g protein/lane). The blot
was probed using anti-R-INS (upper
panel), then re-probed using anti-RCC1
(lower panel). RCC1 isoforms are
indicated, with the question mark
signifying a possible RCC1 band of
unknown identity. Molecular mass (in
kDa) is indicated on the left of the
Journal of Cell Science
Regulation of RCC1 isoforms
interphase and localised predominantly to chromosomes
during mitosis, similar to GFP-RCC1? (Fig. 2A). However,
when the localisation of the two isoforms was quantified in live
U2OS cells, more cells expressing GFP-RCC1? than GFP-
RCC1? showed a fluorescent signal in the cytoplasm, albeit at
much lower levels than in the nucleus (Fig. 2B). The proportion
of cells expressing GFP constructs that were in mitosis was
reduced when a higher concentration of DNA was used for
transfection, and this effect was greater for GFP-RCC1? (2.1%
and 0.7% at 0.4 and 0.8 ?g DNA/well, respectively) than for
GFP-RCC1? (3.4% and 1.4% at 0.4 and 0.8 ?g DNA/well,
respectively). This suggests that overexpression of RCC1 is
detrimental to cell cycle progression, particularly with the ?
RCC1? has a stronger interaction with chromatin
The interaction of RCC1 with chromatin in cells is dynamic
(Cushman et al., 2004; Hutchins et al., 2004; Li et al., 2003;
Li and Zheng, 2004). To examine whether the dynamics of this
interaction varied among the isoforms, we used fluorescence
recovery after photobleaching (FRAP). A laser was targeted at
the nuclei of live interphase U2OS cells expressing GFP-
RCC1? or GFP-RCC1? in order to bleach a spot of about 1
?m in diameter. The recovery of fluorescence within the spot
was monitored as a measure of the mobility of GFP-RCC1
proteins, from which the stability of their interaction with
chromatin can be inferred (Fig. 3A). These experiments
showed that the half-time of signal recovery for GFP-RCC1?
(1.62±0.36 seconds) was approximately twofold greater than
that for GFP-RCC1? (0.71±0.19 seconds), indicating that
RCC1? has a more stable interaction with chromatin than
RCC1?. Consistent with this finding, when subcellular
fractionation of U2OS cells was used to separate the cytoplasm
and soluble nuclear material (supernatant) from insoluble
nuclear material including chromatin (pellet), a greater
proportion of RCC1? or ? than RCC1? was present in the
pellet (Fig. 3B).
Since the localisation of RCC1? to mitotic chromosomes is
strongly dependent on the NTR (Moore et al., 2002), we
compared the localisation of the isolated NTRs of RCC1? and
RCC1? attached to a GFP-GST (GG) fusion. Only a small
proportion (14%) of mitotic cells expressing GG-NTR?
showed concentration of the fusion protein on chromosomes,
similar to previous results (Moore et al., 2002), whereas in the
majority of mitotic cells expressing GG-NTR?, the fusion was
localised to chromosomes (Fig. 3C). Thus, the extended NTR
of RCC1? is sufficient to target a fusion protein to mitotic
chromosomes. Together, these results show that RCC1? has a
stronger interaction with chromatin in cells than RCC1? and
that this difference is due to its extended NTR.
RCC1? has greatly reduced binding to importins
The inserts of RCC1? and ? are located adjacent to the NLS
within the NTR, and introduces an acidic aspartate residue next
to the second cluster of basic residues of the NLS that are
critical for importin-?3 binding (Friedrich et al., 2006). We
therefore tested whether the presence of the insert could affect
binding of importin ?3-? to RCC1?. We used GST-RCC1? or
GST-RCC1? coupled to beads to precipitate endogenous
importin ? from asynchronous HeLa extracts, with or without
addition of His6-importin ?3. In contrast to the strong importin
?3-dependent association of importin ? with GST-RCC1?,
little or no importin ? was associated with GST-RCC1? (Fig.
4A), indicating a greatly reduced interaction. When GST-
importin ?-coated beads were used to precipitate endogenous
RCC1 from HeLa cell extracts, much less RCC1? was
precipitated than RCC1?, again showing a strongly reduced
interaction of RCC1? with importins compared to RCC1?
Fig. 2. Localisation of GFP-
RCC1? and GFP-RCC1? in
U2OS cells. (A) Representative
deconvolved images of different
cell cycle stages of live cells
transiently co-transfected with
GFP-RCC1? or GFP-RCC1? and
RFP-histone H2B. Bars, 12 ?m.
(B) Proportion of live cells
transiently transfected with each
GFP-RCC1 isoform with each
pattern of localisation (left),
shown as mean percentages from
three experiments ± s.d., with
over 1200 cells for each
parameter. Examples are shown
(right). Bar, 20 ?m.
Journal of Cell Science
(Fig. 4B). These results provide an explanation for the greater
proportion of cells with cytoplasmic GFP-RCC1? than GFP-
RCC1? (Fig. 2B), since the nuclear localisation of RCC1? is
in part dependent on its active import mediated by importin ?3-
? which is disrupted in RCC1?.
The interaction of RCC1 with importins does not affect
exchange activity (supplementary material Fig. S3); however,
the interaction of the NTR with an importin ?3-? dimer might
influence the dynamic interaction of RCC1 with chromatin
(Hutchins et al., 2004; Li and Zheng, 2004). To test the possible
effect of importin ?3-? on the interaction of RCC1 isoforms
with chromatin, we incubated recombinant RCC1 proteins with
the resuspended chromatin
fractionation of HeLa cells with or without GST-importin ?
and His6-importin ?3. We found that importin ?3-? efficiently
competed with the association of RCC1? but not RCC1? with
chromatin in vitro (Fig. 4C).
The NTR of RCC1 is also required for DNA binding in vitro
(Seino et al., 1992), although it not yet clear if this binding is
important in the interaction with chromatin. To test the ability
of importin ?3-? to compete with DNA for binding of RCC1,
we incubated HeLa extract with DNA-cellulose with or
without GST-importin ? and His6-importin ?3. Consistent with
its ability to bind RCC1? selectively, the importin ?3-? dimer
strongly inhibited the binding of RCC1? but not RCC1? to
DNA (Fig. 4D).
These results indicate that the more stable interaction of
RCC1? with chromatin compared to RCC1? (Fig. 3) is in part
due to reduced competition from importin ?3-? for the NTR.
It remains possible that RCC1? also has an intrinsically higher
affinity for chromatin than RCC1?.
pellet from subcellular
Journal of Cell Science 120 (19)
RCC1? is the isoform most highly phosphorylated at
serine 11 in mitosis
Human RCC1 is regulated by phosphorylation in mitosis at
sites in the NTR close to the insert site (Hutchins et al., 2004;
Li and Zheng, 2004). Previous studies have shown that RCC1?
can be phosphorylated at these sites by CDK1-cyclin B1,
although the identity of the endogenous phosphorylated RCC1
isoform(s) is unknown.
When we compared the separation of RCC1 isoforms
between the chromatin pellet and supernatant in lysates of
mitotic and interphase (asynchronous) HeLa cells, we found
that most of RCC1? was released from the pellet in mitosis
whereas a greater proportion of RCC1? was retained (Fig. 5A).
A major RCC1 band was detected by a phospho-specific
antibody against the serine 11 site in lysates of mitotic, but not
asynchronous HeLa cells. In addition, a less abundant
phosphorylated RCC1 band of lower molecular mass was
detected. A further very weak band of higher molecular mass
was also detected, but this latter band was not depleted by a
total RCC1 antibody and is therefore unlikely to be an RCC1
isoform (data not shown). We found that the major
phosphorylated form of RCC1 was strongly enriched in the
chromatin pellet of mitotic cells (Fig. 5A). This major band
exactly comigrated with RCC1? (Fig. 5B) and is therefore very
likely to represent this isoform. This indicates that
phosphorylated RCC1? is predominantly associated with
chromatin, suggesting that phosphorylation of this isoform
promotes this interaction.
The less abundant phosphorylated RCC1 band is most likely
to be RCC1?. Interestingly the phosphorylated form of RCC1?
was not associated with chromatin but was released into the
Fig. 3. RCC1? has a more stable
interaction with chromatin in U2OS cells
than RCC1?. (A) FRAP of live
interphase cells expressing GFP-RCC1
isoforms. Example images from one
photobleach are shown, with the bleach
spot indicated by an arrow. Mean half-
times of recovery for each isoform in
seconds are shown, ± s.d., calculated
from 90 cells for GFP-RCC1? and 63
cells for GFP-RCC1? over three separate
experiments. (B) Subcellular
fractionation. Soluble nuclear markers are
Ran, Crm1 and importin ?; cytoplasmic
marker is GAPDH; and insoluble nuclear
marker is lamin B. (C) Localisation of
GFP-GST-NTR? (GG-NTR?), GFP-
GST-NTR? (GG-NTR?) and GFP-GST
constructs in fixed cells. Values shown
are from >22 mitotic cells for each
construct counted over four experiments.
Bar, 16.3 ?m.
Journal of Cell Science
Regulation of RCC1 isoforms
supernatant (Fig. 5A). This indicates that phosphorylation of
RCC1? does not promote its interaction with chromatin, in
contrast to phosphorylation of RCC1?.
These results indicate that RCC1? is the endogenous
isoform of RCC1 that is most highly phosphorylated at serine
11 during mitosis, although both RCC1? and RCC1? can be
phosphorylated in cells when over-expressed (supplementary
material Fig. S4) (Hutchins et al., 2004). The differences in
phosphorylation between RCC1? and RCC1? in cells could be
due to differential regulation of the isoforms, rather than
intrinsic differences in the effectiveness of the molecules as a
CDK1-cyclin B substrate. However, when recombinant RCC1
isoforms were incubated in mitotic HeLa extracts, RCC1? was
Fig. 4. RCC1? interacts less well with importins than RCC1?.
(A) Endogenous importin ? precipitated from asynchronous HeLa
extracts using beads coupled to GST-RCC1?, GST-RCC1? or GST, with
or without His6-importin ?3 (Imp ?3). (B) Endogenous RCC1
precipitated from asynchronous HeLa extracts using beads coupled to
GST-importin ? (Imp ?) or GST, with or without His6-importin ?3.
(C) Binding of untagged RCC1?, RCC1? or GST to the chromatin pellet
from subcellular fractionation of HeLa cells, with His6-importin ?3 and
GST-importin ?, or with GST as a control. (D) Endogenous RCC1
isoforms from asynchronous HeLa extracts bound by DNA-cellulose, with
or without His6-importin ?3 and GST-importin ?. Molecular mass (in
kDa) is indicated on the left.
Fig. 5. Phosphorylation of RCC1 isoforms. (A) Subcellular
fractionation of mitotic (M) and asynchronous (A) HeLa cells.
Soluble nuclear markers are Ran, Crm1 and importin ?;
cytoplasmic marker is GAPDH; insoluble nuclear marker is
lamin B; and mitotic chromatin marker is phospho-S10 histone
H3. (B) Alignment of the total RCC1 and pS11 RCC1 from
SDS-PAGE and immunoblotting of fractionated chromatin
pellet run in triplicate. (C) Phosphorylation of untagged
RCC1? and RCC1? by p13Suc1precipitates of CDKs from
mitotic HeLa extract, or GS4B precipitates as a control.
Molecular mass (in kDa) is indicated on the left.
Journal of Cell Science
more highly phosphorylated at serine 11 than RCC1?
(supplementary material Fig. S5). Furthermore, RCC1? was
more highly phosphorylated than RCC1? at serine 11 by
CDK1-cyclin B semi-purified from mitotic HeLa cell extracts
(Fig. 5C). Therefore the RCC1? molecule is an intrinsically
better substrate for the kinase than RCC1?.
Journal of Cell Science 120 (19)
The function of RCC1? in cells is regulated by
phosphorylation at serine 11
To determine whether RCC1? is functional like RCC1? in
cells, we compared the ability of the isoforms to replace
endogenous RCC1 in tsBN2 cells (Fig. 6). This temperature-
sensitive cell line carries a single point mutation in RCC1
(S256F), which causes the protein to be rapidly degraded when
cells are shifted from the permissive temperature (32°C) to the
restrictive temperature (39.7°C) (Uchida et al., 1990). We
transiently expressed GFP-RCC1? and GFP-RCC1? in tsBN2
cells, along with the non-phosphorylatable mutants GFP-
RCC1? S11A and GFP-RCC1? S11A in order to investigate
the contribution of phosphorylation
(supplementary material Fig. S6). All four constructs
complemented tsBN2 cell proliferation at the restrictive
temperature, as assessed by numbers of GFP-positive cells
(Fig. 6A) or total numbers of cells (Fig. 6B). However, GFP-
RCC1? supported tsBN2 cell proliferation less well than GFP-
RCC1?, whereas GFP-RCC1? S11A worked as well as either
GFP-RCC1? or GFP-RCC1? S11A (Fig. 6). Because the
levels of GFP expression per cell declined during the time
course of the experiment (supplementary material Fig. S6), we
confirmed that GFP-RCC1? was less efficient at supporting
cell proliferation by analysing the number of cells on each day
as a percentage of those transfected at day 0 (Fig. 6C). These
results demonstrate that phosphorylation of RCC1? at serine
11 regulates its cellular function and its ability to support cell
at serine 11
RCC1 is the only known GEF for Ran GTPase, and as such
plays a central role in cellular organisation and trafficking. We
have identified and characterised a novel isoform of human
RCC1 which we named RCC1?. RCC1? and a generally less
abundant isoform, RCC1?, differ from RCC1? in the length
of their N-terminal regions (NTR) and are likely to be
generated by alternative splicing during pre-mRNA processing.
The NTR has been previously identified in the context of the
? isoform as the domain that is critical for interaction of RCC1
with chromatin in cells, and that binds importin ?3-? in an
interaction controlled by phosphorylation during mitosis. Our
results show that the extended NTR of RCC1? confers specific
properties compared with RCC1?, namely that it has a much
weaker interaction with importin ?3-? and a stronger
interaction with chromatin. It is also a better substrate for
phosphorylation by CDK1-cyclin B and we show that
phosphorylated RCC1? preferentially associates with mitotic
chromatin. Additionally, our cell proliferation assay shows that
regulation of RCC1? by phosphorylation plays an important
role in its cellular function. By contrast, RCC1? is only weakly
phosphorylated at serine 11 during mitosis and this
phosphorylation does not promote chromatin enrichment of
RCC1?. Thus, we show that RCC1 isoforms have divergent
mechanisms of regulation and may play specialised cellular
Sequence analysis of the human RCC1 gene suggests that
RCC1? is produced using a previously unidentified splice
donor site within exon 6?. This results in expression of a protein
that contains an insert of 17 amino acids after residue 24
compared to RCC1?. Alternative splicing around and within
the 6? exon of the RCC1 gene appears to be an evolutionarily
GFP positive cells (%)
DAPI positive cells (%)
DAPI positive cells
/ transfected cells
Days at restrictive temp. (39.7ºC)
Days at restrictive temp. (39.7ºC)
Days at restrictive temp. (39.7ºC)
Fig. 6. Proliferation of tsBN2 cells expressing RCC1 isoforms. GFP-
RCC1? and GFP-RCC1?, and their respective S11A non-
phosphorylatable mutants, were transiently expressed in tsBN2 cells.
Following transfection, cells were split equally between dishes and
kept at the permissive temperature (32°C) for 24 hours (day 0), then
shifted to the restrictive temperature (39.7°C) for the time periods
indicated. The number of GFP-positive cells (A) and DAPI-positive
cells (B) is expressed as a percentage of the number of each present
at day 0. Mean of four values for each data point ± s.d. is shown.
About 60 GFP-positive cells and more than 150 DAPI-positive cells
were counted for each data point at day 0. (C) Number of DAPI-
positive cells expressed as a multiple of those initially transfected at
day 0. In order to account for the few cells that survive at the
restrictive temperature in the absence of transfected RCC1, the mean
percentage of surviving DAPI-positive cells from an untransfected
control were first subtracted from the mean percentage of total
DAPI-positive cells in each transfected sample. The resultant value
was then divided by the mean percentage of transfected cells
determined at day 0.
Journal of Cell Science
Regulation of RCC1 isoforms
conserved mechanism. The expression of RCC1?, RCC1? and
RCC1? in mammals, and the likely presence of multiple
isoforms in other vertebrates, strongly suggests that they have
distinct and conserved functions. In the cultured human cells
that we analysed, all three isoforms are expressed in order of
decreasing abundance ?>?>?. However, in normal human
tissues, we find clear differences in the relative expression
levels of the isoforms, with some tissues apparently lacking
RCC1?. This strongly suggests specific roles for the different
isoforms in differentiated cells.
We show that the NTR of RCC1? is sufficient to localise a
fusion protein to chromosomes, strongly supporting the
proposal that differences in chromatin association between the
full-length isoforms are due solely to their different NTRs and
is not dependent on differences in Ran- or histone-binding
ability. This difference is likely to be due, in part, to the
intrinsically higher affinity of the NTR of RCC1? for
chromatin, which possibly involves a direct interaction with
DNA. In addition, the binding of RCC1? to chromatin or DNA
is not competed by the binding of importin ?3-?, in contrast
to RCC1?, and this may promote the relatively more stable
association of RCC1? with chromatin in cells. The reduced
binding of importin ?3-? to RCC1? also provides an
explanation for the slightly greater proportion of cells with
cytoplasmic GFP-RCC1? than GFP-RCC1? (Fig. 2B), since
the nuclear import of RCC1 is partly dependent on this
interaction, although it is clear that RCC1? can also be
localised to the nucleus in an NLS-independent manner
(Nemergut and Macara, 2000; Moore et al., 2002), and this
may account for the relatively small effect of the insert on
In mitosis, we find that RCC1? also differs from RCC1? in
its phosphorylation status at serine 11. The major
phosphorylated endogenous isoform is RCC1?, most likely
because this isoform is an intrinsically better substrate for
CDK1-cyclin B. Previous work on RCC1 phosphorylation
indicated that mitotic phosphorylation disrupts the interaction
of RCC1 with importin ?3-? and also affects the dynamics of
its interaction with chromatin. Phosphorylation of RCC1 was
proposed to be required for the generation of a Ran-GTP
gradient in mitosis (Hutchins et al., 2004; Li and Zheng, 2004).
In the case of our previous experiments conducted using
transfected RCC1? (which does become phosphorylated;
supplementary material Fig. S4), the data indicated that
phosphorylation of this isoform releases the protein from
importin ?3-? but also destabilises its dynamic interaction with
mitotic chromatin in cells (Hutchins et al., 2004). This is
supported by our new observation that endogenous RCC1?
phosphorylated at serine 11 is released from mitotic chromatin
during cell fractionation (Fig. 5A). However, we now show that
the major phosphorylated isoform in mitotic cells is RCC1?,
and phosphorylation of this isoform at serine 11 is associated
with stable interaction with chromatin, in contrast to RCC1?.
Thus, phosphorylation of serine 11 differentially regulates
RCC1 isoforms in mitosis.
Surprisingly, our experiments using non-phosphorylatable
alanine mutants indicate that phosphorylation of serine 11 in
the context of RCC1? restrains tsBN2 cell proliferation, which
is otherwise prevented at the restrictive temperature due to the
loss of endogenous RCC1 isoforms. This demonstrates that
mitotic phosphorylation at this site strongly regulates the
function of RCC1?. One explanation for RCC1? being less
efficient at supporting tsBN2 cell proliferation compared to its
S11A mutant is that phosphorylation of this isoform at serine
11 enhances its activity to the extent that it is detrimental when
the protein is expressed in the absence of other isoforms or at
a higher level than normal. If this is the case, then
phosphorylation would enhance the function of RCC1?
specifically during mitosis by stabilising its interaction with
chromatin, where it would generate Ran-GTP in a highly
localised manner. This would suggest that RCC1? has a
particularly important role during mitosis, whereas RCC1?
may be the most important isoform during interphase. It
remains conceivable, however, that phosphorylation of RCC1?
rather inhibits its function during mitosis, although the
mechanism would be unclear.
To summarise, we have identified a novel isoform of
mammalian RCC1, RCC1?, which contains an insert in the
NTR adjacent to the NLS. This insert reduces importin
binding, increases the stability of its interaction with chromatin
and makes it a better substrate for CDK1-cyclin B1 in mitosis.
Phosphorylation of RCC1? at serine 11 regulates its function
in mitotic cells. The distinct biochemical properties of RCC1
isoforms, their different expression patterns in normal human
tissues and the conservation of their expression in other
mammals suggest that they have distinct roles in vivo.
Materials and Methods
Amplification of RCC1
RCC1? and RCC1? cDNAs were amplified from HeLa cells by RT-PCR and cloned
into the pENTR/D-TOPO entry vector of the Gateway system (Invitrogen) and
transformed into E. coli DH5?. Colony PCR using primers designed to amplify a
small section including the spliced region (5?-GCA TAG CTA AAA GAA GGT
CCC-3? and 5?-CCA ATC ACA CCG TTA TTG TCC-3?) was used to screen for
different isoforms, as insert-containing isoforms were predicted to be of lower
abundance. The LR Clonase reaction was used to transfer open reading frames
(ORFs) into pDEST15 for bacterial expression with a GST tag. ORFs were also
subcloned into pEGFP.C3, pEGFP.N1 and pGEX6p1. The linker region between
GFP and the RCC1 coding sequences differs from that in a previously used GFP-
RCC1? construct; the current constructs contain sequence encoding SGRTQISR
between GFP and RCC1, whereas in the previously published construct this encoded
YSDLE (Moore et al., 2002). The first 27 codons of RCC1? and codons 1-44 of
RCC1? (the NTRs) were amplified from plasmid template and subcloned into a
modified pEGFP.C1 vector containing an ORF for GST in the multiple cloning site,
to give GFP-GST-tagged constructs. Phosphorylation site mutants were generated
with the QuikChange site-directed mutagenesis kit (Stratagene). RFP-Histone H2B
was a gift from J. Swedlow, University of Dundee.
Recombinant proteins were expressed in E. coli BLR (DE3) cells as described
previously (Moore et al., 2002). GST-RCC1 and GST-importin ? proteins were
purified using glutathione Sepharose 4B (GS4B, GE Healthcare) as described
previously (Hutchins et al., 2004). For GST-tagged proteins with a cleavable tag,
the tag was removed using PreScission protease (GE Healthcare) while protein was
bound to GS4B. His6-importin ?3 was purified on Ni-NTA agarose (Qiagen) as
described previously (Hutchins et al., 2004).
Sequence alignment was performed using the ClustalW method. Accession numbers
are as follows: Homo sapiens RCC1?, NM_001269; H. sapiens RCC1?,
NM_001048194; H. sapiens RCC1?, NM_001048195; Pan troglodytes RCC1-I,
XP_513256; Macaca mulatta RCC1-I, XR_013696.1; Mesocricetus auratus RCC1-
I (insert sequence only), P23800; Mus musculus, AAH57645; and Xenopus laevis
RCC1-I, P25183. All sequences are available through NCBI.
Antibodies against the C terminus of RCC1 (Santa Cruz Biotechnology, C20),
Importin ? (Transduction Labs), Ran (Transduction Labs), actin (Sigma), GST
(Molecular Probes), lamin B (Calbiochem), Crm1 (Transduction Labs), phospho-
S10 histone H3 (Upstate), anti-caspase 3 (Santa Cruz Biotechnology, N-19) and
GAPDH (Ambion) are all commercially available. Anti-phospho-serine 11 RCC1
antibody was purified from rabbit serum as before (Hutchins et al., 2004). For
isoform specific antibodies, peptides derived from RCC1 sequences were
Journal of Cell Science
synthesised: ‘R-INS’, DTRAAASRRVPGARS and ‘R-DIS’, CPKSKKVKVS -
HRSHST (Cancer Research UK, London Institute). Peptides were conjugated to
KLH and used to raise polyclonal antibody sera in rabbits (Moravian Biotechnology,
Brno, Czech Republic). Sera were negatively selected against the other isoform
(RCC1? used for anti-R-INS and RCC1? for anti-R-DIS) coupled to CNBr-
activated Sepharose (GE Healthcare), then affinity purified against the appropriate
peptide immobilised on Reacti-gel beads (Pierce Biotechnology).
U2OS and HeLa cells were obtained from Cancer Research UK London Research
Institute. tsBN2 cells were a generous gift from Prof. T. Nishimoto (Fukuoka
University, Japan) via Prof. H. Ponstingl (DKFZ, Heidelberg, Germany). Cell lines
were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen),
supplemented with 10% foetal calf serum (Biosera), 2 mM L-glutamine
(Invitrogen), 50 IU/ml Penicillin G (Invitrogen) and 50 ?g/ml streptomycin
(Invitrogen). Cells were grown at 37°C with 5% CO2, apart from tsBN2 cells which
were grown at 32°C and shifted to 39.7°C to remove endogenous RCC1 protein.
U2OS cells were transfected with 0.8 ?g DNA/2 cm well (unless otherwise stated)
using Superfect (Qiagen), whereas tsBN2 cells were transfected with 2 ?g DNA/2
cm well using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s
For live cell microscopy, cells were transfected and grown in glass-bottomed dishes
(Willco) in phenol red-free DMEM (Invitrogen). Cells were imaged on a Zeiss
Axiovert 200M microscope, in a 37°C Incubator XL-3 with 5% CO2, and
photographed through a 63? or 100? lens using a cooled CCD camera
(Hamamatsu) under the control of Volocity software (Improvision). Images for z
stacks were taken at 1 ?m intervals and deconvolved using Volocity. FRAP
experiments were performed and analysed at the Light Microscopy Facility, College
of Life Sciences, University of Dundee, as described previously (Hutchins et al.,
2004). For microscopy using fixed cells, cells were fixed in 1:1 methanol acetone
at –20°C as described previously (Hutchins et al., 2004) and counterstained with
DAPI. Cells were visualised on a Zeiss Axioplan 2 microscope. Figures were
assembled using Photoshop (Adobe).
For mitotic samples, cells were arrested in prometaphase using 100 ng/ml
nocodazole for 17 hours, mitotic cells were collected by wash-off, and when
required the remaining G2-arrested cells were collected by trypsinisation. For
asynchronous samples, adherent cells were collected by trypsinisation. To arrest
cells in G1-S phase, 3 mM hydroxyurea was added to the medium and cells
incubated for 16 hours before harvesting by trypsinisation.
Mitotic or asynchronous cells were harvested, then washed three times with cold
PBS and once with cold STM buffer [50 mM Tris-HCl pH 7.5, 250 mM sucrose, 5
mM MgCl2, 10 mM iodoacetamide, 0.1 mM phenylmethanesulphonyl fluoride
(PMSF), 0.1 mM benzamidine, 1 ?g/ml each of aprotinin, leupeptin and pepstatin
A, and 1 ?M okadaic acid (Biomol)] (Batchelor et al., 2004). Cells were
resuspended in STM-N buffer (STM plus 0.5% NP40), then incubated for 5 minutes
on ice. Half of the suspension was removed into sample buffer; the remaining
suspension was centrifuged at 1000 g for 15 minutes to pellet nuclear material. The
supernatant was removed into sample buffer, representing the soluble fraction. The
pellet was resuspended in 1 ml STM buffer (STM without protease inhibitor or
okadaic acid) and layered onto a 200 ?l sucrose cushion (STM plus 40% sucrose),
then centrifuged at 16,000 g for 15 minutes to re-pellet nuclear material. The pellet
was resuspended in STM-N buffer to the same volume as the other samples, then
added to SDS sample buffer. Equal volumes of each fraction were analysed by SDS-
PAGE and immunoblotting.
HeLa cell extracts were made in EBS buffer (80 mM ?-glycerophosphate, 20 mM
EGTA, 15 mM MgCl2, 100 mM sucrose, 1 mM dithiothreitol (DTT), and 1 mM
PMSF) as described previously (Hutchins et al., 2004).
HeLa extracts were supplemented with an ATP regenerating system (10 mM
creatine phosphate, 40 ?g/ml creatine kinase, and 1 mM ATP) and diluted in EBS
to 7.5 mg protein/ml extract. For experiments in which recombinant importins were
added, extracts were pre-incubated for 30 minutes at 30°C with 5 ?M GST-importin
? and/or 5 ?M His6-importin ?3. For GST-RCC1 precipitations, GS4B was loaded
with GST-RCC1 proteins or GST in HBS (20 mM Hepes, pH 7.5, 150 mM NaCl)
plus 2 mM DTT. GST-RCC1 beads were incubated with HeLa extract for 30 minutes
at 30°C with shaking. Beads were washed in 20 mM Tris-HCl at pH 7.5, 150 mM
NaCl, 10% glycerol, 0.1% Triton X-100 and 2 mM EDTA. For GST-importin ?
precipitations, GS4B beads were loaded with GST-importin ? or GST in IPB (50
mM Tris-HCl pH 7.5, 200 mM NaCl, 2 mM MgCl2, 5 mM ?-mercaptoethanol) then
Journal of Cell Science 120 (19)
incubated with HeLa extract for 45 minutes at 30°C with shaking. Beads were
washed in IPB. For GST-RanT24N precipitations, GS4B beads were loaded with
GST-RanT24N or GST in HBS plus 2 mM DTT, then incubated with HeLa extracts
diluted 2.5 fold in HBS plus 2 mM DTT overnight at 4°C with rotation. Beads were
washed in HBS plus 2 mM DTT. For DNA precipitations, DNA-cellulose beads
(Sigma; at 0.5 ?g/?l) in DCB (50 mM Tris pH 7.5, 50 mM NaCl, 10% v/v glycerol)
were incubated with HeLa extract for 45 minutes at 30°C with shaking. Beads were
washed in DCB. In each case, proteins were eluted in SDS sample buffer and
analysed by SDS-PAGE and immunoblotting.
Chromatin-binding competition assays
The pellet fraction from subcellular fractionation of typically four 15 cm dishes of
mitotic HeLa cells was resuspended in 200 ?l HBS + 5 mM DTT and distributed
in 20 ?l aliquots. 10 ?l of premix containing RCC1?, RCC1? or GST; and His6-
importin ?3, GST-importin ? or GST, as required was added. This gave final
concentrations of 5 ?M of each importin or 10 ?M GST plus 2.2 ?M RCC1 or
2.2 ?M GST. The reaction was incubated for 15 minutes at 30°C. The reaction was
resuspended in 100 ?l HBS + DTT and spun through a 50 ?l 40% sucrose cushion.
The resulting pellet was resuspended in SDS sample buffer and analysed by SDS-
PAGE and immunoblotting.
tsBN2 cell proliferation assay
This assay was similar to that used previously (Li and Zheng, 2004; Miyabashira
et al., 1994). Cells (3?105) were seeded in 2 cm wells for transfection the next day.
Cells were transfected and 1/8 of each well seeded onto coverslips. Cells were
grown at 32°C for 24 hours to allow expression of constructs, then shifted to 39.7°C
for the specified period, and the number of GFP-and DAPI-positive cells in 21 fields
of view under 63? magnification were counted at each timepoint. Experiments were
repeated to give four values for each data point, from which mean values were
determined as a percentage of the number of cells at day 0 for that transfection.
Guanine nucleotide exchange assays
Guanine nucleotide exchange assays were performed as described previously
(Nicolas et al., 2001) using 1.8 pmole RCC1 (3.6 nM) and 36 nM each of GST-
importin ? and His6-importin ?3 where indicated, in an exchange reaction with 50
pmoles GST-Ran loaded with [8-3H]GDP.
FRAP was carried out at the University of Dundee Light
Microscopy Facility with help from Sam Swift. Thanks to Sonia Lain
and Oliver Staples for use of the microscope for live cell imaging.
Thanks also to Helen Sanderson and Lindsey Allan. This work was
supported by the Biotechnology and Biological Sciences Research
Council, a Medical Research Council Studentship (F.E.H.) and a
Royal Society-Wolfson Research Merit Award (P.R.C.).
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