From the Departamento de Biología Molecular,
Centro de Biología Molecular "Severo Ochoa" (Consejo
Superior de Investigaciones Científicas/Universidad
Autónoma de Madrid), Universidad Autónoma de Madrid,
E-28049 Madrid, Spain and the
European Molecular Biology
Laboratory, Gene Expression Programme,
D-69117 Heidelberg, Germany
Received for publication, February 14, 2002, and in revised form, November 7, 2002
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In red blood cells, protein 4.1 (4.1R) is an
80-kDa protein that stabilizes the spectrin-actin network and anchors
it to the plasma membrane. The picture is more complex in nucleated
cells, in which many 4.1R isoforms, varying in size and intracellular location, have been identified. To contribute to the characterization of signals involved in differential intracellular localization of 4.1R,
we have analyzed the role the exon 5-encoded sequence plays in 4.1R
distribution. We show that exon 5 encodes a leucine-rich sequence that
shares key features with nuclear export signals (NESs). This sequence
adopts the topology employed for NESs of other proteins and conserves
two hydrophobic residues that are shown to be critical for NES
function. A 4.1R isoform expressing the leucine-rich sequence binds to
the export receptor CRM1 in a RanGTP-dependent fashion,
whereas this does not occur in a mutant whose two conserved hydrophobic
residues are substituted. These two residues are also essential for
4.1R intracellular distribution, because the 4.1R protein containing
the leucine-rich sequence localizes in the cytoplasm, whereas the
mutant protein predominantly accumulates in the nucleus. We hypothesize
that the leucine-rich sequence in 4.1R controls distribution and
concomitantly function of a specific set of 4.1R isoforms.
Red blood cell protein 4.1, 4.1R, or 4.1R80 was
identified in human erythrocytes as an 80-kDa multifunctional protein
that stabilizes the spectrin-actin network. Protein 4.1R anchors this
network to the overlying lipid bilayer through interactions with
cytoplasmic domains of transmembrane proteins (1). For the maintenance of normal erythrocyte morphology and the mechanical strength of the
membrane, the formation of the spectrin-actin-4.1R ternary complex is
essential; alterations in the spectrin-actin-binding site of 4.1R,
located at the C-terminal region of the molecule (2-5) are associated
with congenital hemolytic anemias (6).
In nucleated cells, multiple isoforms of 4.1R are expressed as a result
of extensive alternative splicing of the 4.1R pre-mRNA (7-10).
This event is cell- and tissue-specific and also dependent on growth
and differentiation stages of the cell (10-13). Immunological studies
have detected 4.1R epitopes at different intracellular sites (14-20).
Concomitantly, the association of 4.1R with proteins localized at
different intracellular sites have been reported (21-27), thus
suggesting that 4.1R may be involved in many processes in nucleated
cells. A possible role for 4.1R in organizing the nuclear and
microtubule architecture and the mitotic spindle poles has been
suggested. 4.1R is known to interact with nuclear components of the
splicing machinery (19, 22), pICln (28), a regulator of a chloride
channel recently shown also to associate with spliceosomal proteins
(29), interphase microtubules (30), a novel centrosomal protein termed
CPAP (31), and the nuclear mitotic apparatus protein (32). Transfection
studies using 4.1R cDNAs isolated from different sources have
allowed the identification of specific nuclear isoforms of 4.1R
(32-35) and the signals involved in 4.1R nuclear targeting (33, 34,
36) and the abrogation of its nuclear accumulation (34, 37).
Nuclear transport in eukaryotic cells is triggered by specific
transport signals that are recognized by soluble receptors that
interact with the nuclear pore complexes. Nuclear import is mediated by
nuclear localization signals present in nuclear proteins. In the
case of classical basic nuclear localization signals, this process
involves the importin In our previous studies, which focused on the identification of signals
involved in differential intracellular localization of proteins 4.1R
(33, 34, 37), we showed that a constitutive region of the 4.1R
molecule, one that is therefore present in all 4.1R isoforms and is
thus designated as the "core region," was responsible for nuclear
targeting of 4.1R (34). Because 4.1R isoforms expressing exon 5 are
predominantly localized in the cytoplasm (34), in this study we have
aimed to identify the amino acid sequence that is responsible for this
effect. We show here that exon 5 encodes a leucine-rich sequence
resembling a NES. This sequence and, more specifically, two leucine
residues that are also conserved in NESs are necessary for 4.1R
cytoplasmic localization and for 4.1R binding to the export receptor CRM1.
Cell Culture and Transfection--
COS-7 cells were used for
transient cDNA expression, immunofluorescence, and biochemical
analyses. The cells were grown as described (33). Transient
transfections were performed by electroporation using the Electro Cell
Manipulator 600 (BTX, San Diego, CA). The cells were always processed
48 h after transfection. For each cDNA construct tested, more
than 300 cells from at least five independent replicates were counted.
cDNA Cloning, Composite cDNA Constructs, and
Mutagenesis--
4.1R80
The mutated constructs 4.1R80 Antibodies--
Anti-c-Myc (9E10) monoclonal antibody (46) was
obtained from the American Type Culture Collection. Goat anti-mouse IgG
secondary antibodies conjugated with horseradish peroxidase or
fluorescein isothiocyanate were obtained from Southern Biotechnology
Associates, Inc. (Birmingham, AL). Anti-GST monoclonal antibody was
purchased from GeneTex (San Antonio, TX).
Immunofluorescence Microscopy--
Cells grown on glass
coverslips were fixed, permeabilized, and blocked as described (33).
The cells were incubated with the appropriate antibodies and processed
as reported (16). The preparations were examined using a Zeiss
epifluorescence microscope. Controls to assess the specificity and lack
of cross-labeling included incubations with nonimmune rabbit serum and
control monoclonal antibodies or omission of either of the primary antibodies.
Protein Expression and Western Blot Analysis--
For binding
assays, GST fusion proteins were expressed and purified using standard
procedures (47). To verify their size, as well as those of the proteins
expressed in transfection experiments using COS-7 cells, total protein
extracts were obtained, and the protein fractions were analyzed by
SDS-PAGE (48) and Western blot. The membranes were processed and
developed as described elsewhere (16).
Solution Binding Assays--
For binding assays with zz-tagged
CRM1 (45, 49), 10 µg of zz-CRM1/binding reaction were incubated with
IgG-Sepharose beads for 30 min at 4 °C in 100 µl of binding buffer
(100 mM potassium acetate, 30 mM HEPES-KOH, pH
7.5, 2 mM magnesium acetate, and 0.001% Triton X-100). The
beads were then recovered by gentle centrifugation and washed with
binding buffer. The beads were subsequently incubated with candidate
proteins in a final volume of 100 µl for 60 min at 4 °C. The beads
were recovered by gentle centrifugation and washed four times with 1 ml
of binding buffer. The bound proteins were eluted by the addition of 1 ml of 1 M MgCl2, and the eluted proteins were
precipitated with isopropanol. The protein pellets were resuspended in
Laemmli (48) sample buffer and analyzed by SDS-PAGE and Western blot.
Exon 5-encoded Sequence Alters the Subcellular Localization of
Nuclear GFP--
To study the role that the exon 5-encoded sequence
plays in 4.1R intracellular distribution, we prepared different
cDNA fusion constructs in which the sequence coding for GFP was
appended at the 3' end of 4.1R coding sequences (Fig.
1). Transfection experiments were
performed in COS-7 cells and the distribution patterns of the expressed
proteins were analyzed 48 h post-transfection by fluorescence
microscopy. Cells expressing only GFP had intense nuclear staining with
some fluorescence also detected in the cytoplasm (Fig.
2A and Table
I). Cells expressing a fusion protein
containing the complete amino acid sequence of a nuclear 4.1R isoform
that lacked expression of exon 5 (4.1R60 A Leucine-rich Sequence Resembling a NES in 4.1R Isoforms
Expressing the Alternative Exon 5--
NES sequences are short
sequence motifs that are necessary and sufficient to mediate the
nuclear export of large proteins (38, 39, 50). Important for their
function is a characteristic spacing of hydrophobic residues, mainly
leucine or isoleucine (38, 39). Analysis of the amino acid sequence
coded by exon 5 allowed the identification of a hydrophobic region,
L26LKRVCEHLNLL, which is significantly similar to
leucine-rich NES sequences (Fig.
3A). The key hydrophobic
residues shown to be important for NES function in other proteins (51,
52) are also found in the putative NES in 4.1R and correspond to
Leu34 and Leu36 (numbered as in the erythroid
4.1R sequence reported in Ref. 10). The crystal structure of the
N-terminal 30-kDa domain of erythroid 4.1R comprising exon-5-encoded
sequence has been determined (53). From the tertiary structure it may
be inferred that the 4.1R hydrophobic sequence,
L26LKRVCEHLNLL, adopts an Mutation of Two Essential Residues within the Leucine-rich Sequence
Alters the Cytoplasmic Localization of Protein 4.1R--
Mutation of
two critical hydrophobic amino acids within NES sequences has been
shown to affect the function of NES in many proteins (51, 52). We next
investigated whether mutations of the two conserved and presumably
critical hydrophobic amino acids within the 4.1R leucine-rich sequence
also affect 4.1R subcellular distribution. COS-7 cells were transfected
with either a cDNA coding the wild-type protein for the expression
of the leucine-rich sequence (4.1R80
Wild-type 4.1R80
The binding site of band 3 to 4.1R consists of the L37EEDY
sequence that is adjacent to the NES (57). Mutation of
L37EEDY to S37RAGN (protein
4.1R80 Protein 4.1R80 In recent years, our view of strict compartmentalization of
nuclear and non-nuclear components has been challenged as plasma membrane (59), tight junction (60), endocytic (61), and cytoskeletal
(50) proteins have been described in the nucleus. Moreover, proteins
interacting with 4.1R and with predominant extranuclear localization
and function have recently been detected in the nucleus. One of these
proteins is actin, which is a ubiquitous, essential cytoskeletal
protein and is therefore like 4.1R80 Our previous studies on signals involved in 4.1R differential
intracellular localization showed that all 4.1R molecules have a common
region, designated the core region, involved in their nuclear targeting
(34). However, in this study we show that 4.1R isoforms expressing exon
5 contain a leucine-rich sequence that predominantly excludes them from
the cell nuclei. A third signal comprising basic amino acids coded by
the alternative exon 16 have also been involved in 4.1R nuclear
targeting (33, 36). Finally, a fourth region mediating 4.1R
intracellular localization is the 209-amino acid head piece (HP)
of high molecular weight 4.1R135 isoforms that inhibits
nuclear targeting of 4.1R (37). The complexity of signals and regions
identified to date as being involved in 4.1R nuclear and cytoplasmic
distribution and the hierarchical fashion in which they regulate the
intracellular localization of 4.1R are summarized in Fig.
6A. Fig. 6B
represents schematically possible models for the intracellular traffic
experienced by different 4.1R isoforms. A first group of
4.1R80 isoforms containing the constitutive core region but
lacking the alternative leucine-rich sequence is directed to the
nucleus by a mechanism that has yet to be elucidated. This group would be comprised of the nuclear set of 4.1R isoforms (Fig. 6B,
panel a). A second group of 4.1R80 isoforms
contains the leucine-rich sequence, and these enter the nucleus via the
core region and might bind to CRM1-RanGTP to be exported to the
cytoplasm (Fig. 6B, panel b). A third group of
4.1R80 isoforms expresses exon 16, and they enter the
nucleus by binding to human importin
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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and importin
proteins, whose function is
regulated by Ran GTPase (38, 39). Like import, nuclear export is
mediated by specific signals known as nuclear export signals
(NESs).1 There are two
relatively well defined exportins that transport proteins: CAS and
CRM1. These receptors associate in the nucleus with their export
substrates in the presence of Ran-GTP forming trimeric export complexes
that are then transferred to the cytoplasm (40). Human CRM1 binds
leucine-rich NESs found in different proteins such as Rev,
mitogen-activated protein kinase kinase 1, cAMP-dependent protein kinase inhibitor, and cyclin B
(41-44).
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16 and 4.1R80
5,16
cDNAs were cloned and tagged as described (34).
4.1R80
16-GFP and 4.1R60
16,18-GFP were
constructed by PCR using pSR
4.1R80
16 and pSR
4.1R60
16,18 as templates (34), respectively, and
appropriate sense and antisense primers complementary to both ends of
their respective open reading frames. E5-GFP was constructed by PCR
using pSR
4.1R80
16 as a template and sense and
antisense primers complementary to both ends of 4.1R exon 5 coding
sequence. The PCR-amplified sequences were cloned into the vector
pcDNA3.1/CT-GFP-TOPO® (Invitrogen) following the manufacturer's
instructions. GST-4.1R80
16 was prepared as described
(30). The constructs zz-CRM1, GST-RevNES, GST-M10, and RanGTP were
prepared as reported (45).
16NESmut,
4.1R80
16LEEDYmut, and
GST-4.1R80
16NESmut were obtained using the
QuikChange site-directed mutagenesis kit (Stratagene). The reactions
were performed following the manufacturer's instructions, using
pSR
4.1R80
16 or pGEX 4.1R80
16 (30) as a
template and the appropriate pair of oligonucleotides covering both
strands of the region of interest. In
4.1R80
16NESmut and
GST-4.1R80
16NESmut, L34NL
was mutated to ANQ. In 4.1R80
16LEEDYmut,
L37EEDY was mutated to SRAGN. The amino acids were numbered
according to the erythroid 4.1R sequence (10), GenBankTM
accession number M61733.
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DISCUSSION
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16,18-GFP) also
showed predominant nuclear staining (Fig. 2B and Table I).
By contrast, cells expressing a 4.1R isoform containing the sequence
encoded by exon 5 (4.1R80
16-GFP) presented predominantly
cytoplasmic staining (Fig. 2C and Table I). Consistently,
appending the 35 amino acids encoded by exon 5 to GFP (E5-GFP) also
resulted in an increase in the number of cells showing cytoplasmic
fluorescence (Fig. 2D and Table I).
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Fig. 1.
Scheme of the exon map for the 4.1R protein
and the cDNA constructs used in this study. A,
schematic representation for the protein 4.1R. The exons are coded as
follows: striped, alternative; white,
constitutive; black, noncoding. The number of each
individual exon is represented. Three translation initiation sites at
exons 2' (ATG-1), 4 (ATG-2), and 8 (ATG-3) are indicated, as well as
the stop codon (TGA) at exon 21. These data have been taken from Refs.
10, 12, 13, 34, 35, and 65. B, exon composition of the 4.1R
cDNAs and GFP and GST fusion proteins used in this study. The
nucleotide sequence encoding the c-Myc-epitope tag (myc) was
added at the 3' end of cloned cDNAs. GFP protein is expressed at
the C terminus, whereas GST is at the N terminus of the expressed 4.1R
chimeras. Mutations introduced in different 4.1R cDNAs and
constructs are indicated. In 4.1R80 16NESmut
and GST-4.1R80
16NESmut, residues
Leu34 and Leu36 within the leucine-rich
sequence were replaced by Ala and Gln, respectively. In
4.1R80
16LEEDYmut, the sequence
L37EEDY was replaced by SRAGN (see "Experimental
Procedures" for details).
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Fig. 2.
Effect of the exon 5-encoded sequence of 4.1R
on the subcellular localization of GFP. COS-7 cells were
transfected with GFP (A), 4.1R60 16,18-GFP
(B), 4.1R80
16-GFP (C), or E5-GFP
(D) cDNAs and examined by fluorescence microscopy
48 h after transfection. Green represents fluorescence
of GFP; blue represents nuclear staining with
4'-6'-diamidino-2-phenylindole.
Subcellular distribution of various proteins used in this study and
expressed in COS-7 cells
300 cells from five independent transfection
experiments. The percentages of transfected cells showing
immunofluorescence staining only in the cytoplasm (c), predominantly in
the cytoplasm (c > n), at similar levels in the cytoplasm and the
nucleus (c = n), predominantly in the nucleus (n > c), and
only in the nucleus (n) were estimated 48 h after transfection.
-helix
conformation (Fig. 3D). Leu36 is exposed on one
side of the
-helix, whereas Leu34 and the hydrophobic
residues Leu26 and Val30 are exposed on the
opposite side (Fig. 3B). A similar topology has been
described for other NES (50, 54, 55), such as that of protein p53, in
which Leu350 is located on one side of the
-helix,
whereas Leu348 and the hydrophobic residues
Met340 and Leu344 appear on the opposite side
(Fig. 3C).
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Fig. 3.
The leucine-rich sequence encoded by exon 5 of 4.1R resembles NESs characterized in a variety of proteins.
A, alignment of the leucine-rich sequence of 4.1R with NES
sequences of mitogen-activated protein kinase kinase
(MAPKK), zyxin, RevHIV, p53, actin, and
cAMP-dependent protein kinase inhibitor, form
(PKI
). The conserved hydrophobic amino acids are
blue; the two most highly conserved residues crucial to the
proper function of NES are marked with two dots.
B, three-dimensional structure of the leucine-rich sequence
present in protein 4.1R. Amino acids are color-coded as follows:
yellow for hydrophobic, red for acidic,
blue for basic, and green for polar residues.
C, three-dimensional structure of the NES present in p53
protein. D, three-dimensional structure of the FERM domain
of 4.1R (53) showing the position of the leucine-rich sequence in an
exposed
-helix. The two conserved residues, Leu34 and
Leu36, are shown. E, predicted three-dimensional
structure of the mutated FERM domain of
4.1R80
16NESmut showing the position of the
two mutated residues, Ala34 and Gln36.
A was prepared using the ClustalX alignment program (66).
B-E were modeled using the Swiss PDB Viewer program (67),
based on the crystallographic coordinates of the 4.1R80
FERM (53) and p53 tetramerization (55) domains deposited in the Protein
Data Bank with accession numbers 1GG3 and 1AE1, respectively. In
E, the FOLD-X program (56), available at
fold-x.embl-heidelberg.de, was used to predict the coordinates of the
mutated FERM domain. Note that the overall structure of the domain is
not altered and that the
-helix comprising the mutated leucine-rich
sequence remains exposed to the solvent.
16) or a cDNA
encoding a protein with Leu34 and Leu36
replaced by Ala34 and Gln36 within the
leucine-rich sequence (4.1R80
16NESmut). The
subcellular distribution of the expressed proteins, tagged with c-Myc
sequences at the C terminus, was determined by staining with antibody 9E10.
16 protein had a predominantly
cytoplasmic distribution (Fig.
4A and Table I) with a small
percentage of the transfected cells containing the expressed protein in
the nucleus and cytoplasm (Table I). By contrast, protein
4.1R80
16NESmut was predominantly distributed
in the nucleus (Fig. 4B and Table I). An explanation for the
results obtained for the mutated 4.1R protein could be that it is first
directed to the nucleus via its core region (34) and that once there it
cannot be exported to the cytoplasm because the two mutated hydrophobic
amino acids, Leu34 and Leu36, are essential for
nuclear export. The predicted folding of the mutant was determined
using as template the crystallographic coordinates of the
4.1R80 FERM domain (53) deposited in the Protein Data Bank
(accession number 1GG3) and the FOLD-X computer algorithm (56) (Fig. 3E). Substitution of Leu34 and Leu36
by Ala34 and Gln36 is not predicted to cause
perturbation of folding and is slightly favorable to protein stability
(the free energy of folding is 52.32 kcal·mol
1 for the
wild type versus 52.14 kcal·mol
1 for the
mutant). It is very unlikely that the results described above for the
mutant are due to perturbation of folding. Isoform 4.1R80
5,16, which is similar to 4.1R80
16
except for lacking exon 5-encoded sequences, also accumulates in the
nucleus (Fig. 4C and Table I). All of these results indicate that although wild-type 4.1R80
16 is found predominantly
in the cytoplasm, either the mutation in the leucine-rich sequence or
the deletion of exon 5 clearly resulted in the accumulation of the
protein in the nucleus. Thus, the leucine-rich sequence and, more
specifically, amino acids Leu34 and Leu36 play
a pivotal role in 4.1R cytoplasmic distribution in COS-7 cells.
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Fig. 4.
Two conserved leucine residues within the
sequence L26LKRVCEHLNLL are involved in 4.1R cytoplasmic
distribution. COS-7 cells were transfected with
4.1R80 16 (A),
4.1R80
16NESmut (B),
4.1R80
5,16 (C), or
4.1R80
16LEEDYmut (D) cDNAs,
labeled with anti-c-Myc (9E10) antibody (green) and examined
by epifluorescence microscopy 48 h after transfection.
Blue represents nuclear staining with
4'-6'-diamidino-2-phenylindole.
16LEEDYmut), unlike
4.1R80
16NESmut, did give rise to predominant
cytoplasmic staining of 4.1R80
16LEEDYmut
(Fig. 4D and Table I). The capacity of protein
4.1R80
16 to be exported from the nucleus was analyzed by
injecting recombinant protein GST-4.1R80
16 into
Xenopus laevis oocyte nuclei and processing the
samples as described (58). At 3 h post-injection, most of the
protein GST-4.1R80
16 was detected in the cytoplasmic
fraction supporting nuclear export of 4.1R80
16 (data not
shown). The mutant protein
GST-4.1R80
16NESmut was also detected in the
cytoplasmic fraction. This unexpected result suggests that 4.1R
contains additional nuclear export signal(s) that appear(s) to be
functional in the X. laevis oocyte system.
16, but not
4.1R80
16NESmut, Associates with the Nuclear
Export Protein CRM1 in a RanGTP-dependent
Manner--
Proteins containing a leucine-rich NES are recognized in
the nucleus by the exportin CRM1 and form a trimeric complex with RanGTP, which is exported from the nucleus to the cytoplasm (40). We
investigated whether 4.1R80
16 associates with CRM1 in
the RanGTP-dependent manner characteristic of export
substrates. zz-tagged CRM1 was bound to IgG-Sepharose and incubated
with the different substrates used in the binding assays. Bead-bound
material was eluted from the column and processed as indicated under
"Experimental Procedures." A functional export substrate containing
the NES of Rev fused to the C terminus of GST (GST-RevNES) (45, 49) and
an export-deficient control substrate containing a disrupted NES of Rev
(GST-M10) were used as positive and negative controls, respectively.
All of the binding assays were performed in the absence and presence of
RanGTP. GST-RevNES bound efficiently to zz-CRM1, in a
RanGTP-dependent manner (Fig. 5, compare lanes 1 and
2). Similarly, GST-4.1R80
16 bound to CRM1 in
a RanGTP-dependent manner (Fig. 5, compare lanes
5 and 6). By contrast, the export deficient control
(GST-M10) and the 4.1R mutant in the leucine-rich sequence
(GST-4.1R80
16NESmut) did not bind to CRM1
even in the presence of RanGTP (Fig. 5, lanes 3 and
4 and lanes 7 and 8, respectively).
The RanGTP-dependent binding of CRM1 to
GST-4.1R80
16 but not to
GST-4.1R80
16NESmut was more clearly revealed
by Western blot analysis (Fig. 5, lanes 9-12).
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Fig. 5.
Protein 4.1R binds to CRM1 in a
RanGTP-dependent manner. Proteins bound to CRM1 were
isolated as indicated under "Experimental Procedures," analyzed by
SDS-PAGE and Coomassie staining (lanes 1-8), or blotted to
PVDF and detected by incubation with anti-GST antibody (lanes
9-12). GST-RevNES (RevNES) and GST-M10 (M10) were used as
positive and negative controls, respectively. Note that
GST-4.1R80 16 protein (4.1R80
16) binds
zz-CRM1 in the presence, but not in the absence, of RanGTP, whereas the
mutated GST-4.1R80
16NESmut protein
(4.1R80
16NESmut) does not.
Arrowheads mark the positions of GST-RevNES, RanGTP, and
GST-4.1R80
16.
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16 with a
predominant cytoplasmic localization at steady state. Actin contains
two functional NES that mediate its nuclear export in a CRM1-mediated
manner. However, the physiological relevance of the shuttling of actin
between the nucleus and cytoplasm is not known (50). ZO-1 interacts
with 4.1R in cell tight junctions (24), and ZO-1 has been shown to
accumulate in the nucleus in a cell density-dependent
fashion (60). hCASK interacts with 4.1R, establishing a link between
the extracellular matrix and the cortical actin cytoskeleton (21). It
has been reported that hCASK directly enters the nucleus and thereby
regulates gene expression (62). Conversely, a 4.1R-interacting protein,
the nuclear splicing factor U2AF35 (22), has recently been shown to
shuttle from the nucleus to the cytoplasm, but its role in the
cytoplasm remains to be elucidated (63). This study shows that 4.1R
isoforms expressing exon 5 contain a leucine-rich sequence that shares
key features with NESs known to trigger the rapid, active delivery of
proteins and RNA-protein complexes from the nucleus to the cytoplasm
(39, 40, 64). Thus, the key hydrophobic residues important for NES
function in other proteins are also found to be essential in 4.1R, and
the topology adopted for the NES of proteins such as p53 (55) or actin
(50) is similar to that adopted for 4.1R (53). Moreover, the
leucine-rich sequence is required for 4.1R binding to CRM1 in a
RanGTP-dependent fashion.
2, Rch1 (36) (Fig.
6B, panel c). Finally, a fourth group of
4.1R135 isoforms contains the HP domain that inhibits
nuclear targeting of 4.1R (37). The HP domain might adopt a
conformation masking the regions involved in 4.1R nuclear targeting.
However, an association between the HP domain and a cytoplasmic partner
could also account for this effect (Fig. 6B, panel
d). Thus, there must be orchestrated regulatory mechanisms
controlling the traffic of specific 4.1R isoforms to play distinct
roles in different compartments.
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Fig. 6.
Hierarchical organization of 4.1R targeting
signals. A, the 4.1R cDNA has been divided into
four regions represented by white boxes. Region
1, the 209-amino acid sequence, or HP, specific for 4.1R isoforms
translated from ATG-1, which spans from ATG-1 to ATG-2; region
2, the sequence including the alternative exon 5 encoding the
leucine-rich sequence (NES) and spanning from ATG-2 to
ATG-3; region 3, the core region encompassing the sequence
encoded from ATG-3 to the in-frame-ATG located at exon 17 (ATG-E17);
region 4, the C-terminal region encoded from ATG-E17 to the
4.1R stop codon at exon 21 (TGA). The striped
boxes indicate the alternative exons 5 and 16. Arrows
show the sequences involved in 4.1R differential targeting. The
directions of the arrows represent the predominant
localization determined by the expression of every block of sequence:
nuclear for the core region and for exon 16 (in the presence of exon 5)
and cytoplasmic for exon 5 (in the absence of exon 16) and for the HP
domain (33, 34, 36, 37). The hierarchical effect of these regions on
4.1R intracellular localization is as follows: the constitutive core
region determines nuclear localization, which is blocked by expression
of exon 5 (the leucine-rich sequence) and restored by the simultaneous
expression of exons 5 and 16. Finally, the HP domain specific to 4.1R
isoforms translated from ATG-1 is dominant over all the signals, being
sufficient to abrogate nuclear entry (37). B, schematic
representation of possible models indicating the intracellular traffic
experimented by different sets of 4.1R isoforms (for explanation, see
"Discussion").
We do not know the physiological role that 4.1R isoforms containing the
leucine-rich sequence play in nucleated cells, and it is rather
speculative, therefore, to propose possible cytoplasmic or nuclear
roles. If the presence of 4.1R8016 in the cell nucleus
has deleterious consequences for the cell, it is possible that the
function of the leucine-rich sequence of 4.1R is to control the access
of 4.1R to nuclear components. Alternatively, the sequence may be
important for maintaining the cytoplasmic localization of a putative
binding partner that would otherwise gain access to the nucleus to
carry out specific functions. Lastly, 4.1R80
16 may
transit to the nucleus to deliver a nuclear component, either protein
or RNA, to the cytoplasm. Protein 4.1R80
16, and other
4.1R isoforms expressing exon 5, may be candidates for participation in
the relay of information between the cytoplasm/plasma membrane and the
nucleus. Investigation of the functions of 4.1R isoforms containing the
leucine-rich sequence will provide new insights into this
multifunctional protein.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Iain Mattaj for invaluable help and critical reading of the manuscript and Drs. Gareth Griffiths and Joost Schymkowitz for helpful suggestions (European Molecular Biology Laboratory, Heidelberg, Germany). We also acknowledge Dr. Scott Kuersten for generous help and Drs. Rainer Saffrich and Wilhelm Ansorge for hosting Dr. Luque in a short term visit to the European Molecular Biology Laboratory (Heidelberg, Germany). We are indebted to Dr. Miguel A. Alonso (Centro de Biología Molecular Severo Ochoa, Madrid, Spain) for helpful discussions.
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FOOTNOTES |
---|
* This work was supported by Grant BMC202-00978 from the Ministerio de Ciencia y Tecnología (Spain) and Grant 08.3/0004.1/99 from the Comunidad de Madrid (Spain). Institutional financial support was received from the Fundación Ramón Areces, Spain.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a fellowship from the European Advanced Light Microscopy Facility at European Molecular Biology Laboratory in Heidelberg, Germany. Postdoctoral fellow of the Comunidad Autónoma de Madrid. Present address: European Molecular Biology Laboratory, Developmental Biology Programme, D-69117 Heidelberg, Germany.
¶ Postgraduate fellow of the Ministerio de Educación, Ciencia y Cultura, Spain.
** Present address: Bardehle et al., Patent Attorneys, Possartstrasse 18, D-81679 Munich, Germany.
To whom correspondence should be addressed. Fax:
34-91-397-8087; E-mail: icorreas@cbm.uam.es.
Published, JBC Papers in Press, November 9, 2002, DOI 10.1074/jbc.M201521200
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ABBREVIATIONS |
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The abbreviations used are: NES, nuclear export signal; GST, glutathione S-transferase; HP, head piece domain of 4.1R; GFP, green fluorescent protein.
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