Department of Molecular Biology and Genetics, Cornell University, Ithaca,
NY 14853-2703, USA
* Present address: Department of Ophthalmology, SUNY Upstate Medical University,
Syracuse, NY13210, USA
Present address: Skirball Institute of Biomolecular Medicine, New York
University, New York City, NY 10016, USA
Author for correspondence (e-mail:
mfw5{at}cornell.edu)
Accepted 30 January 2003
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Summary |
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Key words: Nuclear lamina, Targeting, Yeast two-hybrid, Drosophila cell transfection, GFP fusion
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Introduction |
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Lamins, the major constituents of the nuclear lamina, are intermediate
filament proteins whose fibers support the nuclear envelope and provide
attachment sites for interphase chromatin
(Stuurman et al., 1998;
Taniura et al., 1995
). Most
eukaryotes contain multiple species of these hydrophilic coiled-coil proteins,
which can usually be subdivided into two groups: lamin A/C proteins found
primarily in differentiated cells, and lamin B proteins found in all cell
types examined, except for sperm in at least some organisms
(Liu et al., 1997
;
Moir and Spann, 2001
;
Moir et al., 2000
;
Riemer et al., 1995
). Recent
evidence suggests that the nuclear lamina functions, in part, as a scaffold
onto which a number of macromolecules are hung. Examples include inner
membrane proteins on the cytoplasmic side of the nuclear envelope and
chromatin and chromatin-binding proteins on the nucleoplasmic side
(Holmer and Worman, 2001
).
This suggests that lamins can target proteins to the nuclear periphery, either
directly or by preferential retention.
Protein targeting to the nuclear periphery is thought to occur after the
protein has entered the nucleus (Nakielny
and Dreyfuss, 1999). Targeting to the nuclear envelope by lamins
and integral nuclear membrane proteins is due to lipid modification motifs or
transmembrane domains in these proteins
(Gruenbaum et al., 2000
;
Holaska et al., 2002
). Lamins
A and B possess a lipid modification motif (CAAX) at their C-terminus that can
be isoprenylated, allowing insertion into membranes and targeting to the
nuclear periphery (Hofemeister et al.,
2000
; Holtz et al.,
1989
; Kitten and Nigg,
1991
). Transmembrane domains are essential for nuclear periphery
targeting of integral proteins to the inner nuclear membrane, such as the
lamin B-receptor (Soullam and Worman,
1995
), MAN1 (Lin et al.,
2000
; Wu et al.,
2002
), emerin (Fairley et al.,
1999
), nurim (Rolls et al.,
1999
) and nesprins (Zhang et
al., 2001
). Localization of these proteins can be explained by a
`diffusion-retention' model (Ellenberg et
al., 1997
). These proteins are translated on ER (endoplasmic
reticulum)-bound ribosomes and are inserted into the ER membrane. They diffuse
within that membrane, entering the inner nuclear membrane compartment.
Presence of these proteins at the nuclear periphery is often further
stabilized by interaction with lamin (e.g.
Furukawa et al., 1998
;
Vaughan et al., 2001
), and
chromatin and/or additional DNA bridging proteins such as BAF
(Holmer and Worman, 2001
;
Vlcek et al., 2001
).
Very little is known, about how hydrophilic non-membrane-associated
proteins are targeted to, and retained on, the nucleoplasmic side of the
nuclear envelope. The Drosophila YA (Young Arrest) protein is an
excellent test case to investigate this question. YA is an essential,
maternally provided, nuclear lamina protein found in Drosophila
oocytes, eggs and early embryos (Lin and
Wolfner, 1991; Lopez et al.,
1994
). This entirely hydrophilic protein, with no discernible
lipid modification or membrane-targeting motifs, is required for fertilized
eggs to enter the mitotic cleavage phase of early development. YA binds to
chromatin (Lopez and Wolfner,
1997
; Yu and Wolfner,
2002
) and localizes to the nuclear laminas of cleavage-stage
embryos in a cell-cycle-dependent manner
(Lin et al., 1991
). YA and
lamin Dm0 [the B-type lamin of Drosophila and the only
lamin present in embryos of this stage
(Harel et al., 1989
;
Riemer et al., 1995
)]
dissociate from the nuclear periphery at metaphase. Lamin Dm0
reassembles at the nuclear periphery at telophase; YA first appears at the
nuclear periphery at the start of the next interphase
(Lin and Wolfner, 1991
).
Nuclear envelope localization of YA appears to be necessary for its function
since mutant YA proteins that enter nuclei, but do not localize to the nuclear
periphery, do not rescue the null Ya phenotype
(Liu and Wolfner, 1998
).
To address how YA attains its subnuclear location, we searched for
YA-interacting proteins using a yeast two-hybrid assay
(Goldberg et al., 1998). Lamin
Dm0 was identified as a partner of YA in this screen. Only
full-length lamin interacted with YA, suggesting that YA binds only to
polymerized lamin. The C-terminal 190 amino acids (residues 506-696) of YA
were sufficient for a two-hybrid interaction with lamin Dm0
(Goldberg et al., 1998
).
Deletion of this region from YA prevented nuclear peripheral targeting of YA
in Drosophila, although the deleted YA still entered nuclei
(Liu and Wolfner, 1998
).
These data, coupled with the cell cycle kinetics of YA relative to those of
lamin Dm0, led us to propose that the interaction of YA with the
polymerized lamin network that is assembled at the nuclear periphery targets
YA to the nuclear periphery (Goldberg et
al., 1998). We test this hypothesis by narrowing down the
lamin-interaction domain of YA using two-hybrid analysis and testing whether
this region of YA can target a heterologous, non-membrane protein to the
nuclear lamina. We identify a hydrophilic domain capable of interaction with
lamin that targets a heterologous soluble protein to the Drosophila
nuclear periphery. The lamin-interaction domain could be useful for targeting
proteins to the nucleoplasmic side of the nuclear envelope.
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Materials and Methods |
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Plasmid constructions
Constructs containing regions of the C-terminal domain of YA were generated
by PCR by using gene-specific primers containing engineered EcoRI
sites (primer sequences will be provided upon request). The amplified products
were cloned into EcoRI site of pGBT9 and their sequences verified
(Cornell Bioresource Center, Ithaca NY).
For S2 cell transfections, the GFP-coding sequence was amplified from
eGFP-N1 (Clontech) using gene-specific primers with KpnI and
PstI overhangs. The SV40 T-antigen nuclear localization signal (NLS)
sequence (Makkerh et al.,
1996) was synthesized as oligonucleotides with PstI and
EcoRI overhangs and annealed. The digested PCR product and
oligonucleotides were ligated into pBS KS(+) (Stratagene) and verified by
sequencing. GFP-NLS was then cloned into the pMT V5 B vector (Invitrogen) to
generate pMT GFP-NLS. The metallothionein promoter in this construct allows
inducible expression in cultured Drosophila cells
(Bunch et al., 1988
).
EcoRI fragments from the YA C-terminal deletions in pGBT9 (see above)
were cloned into the EcoRI site of pMT GFP-NLS to generate pMT
GFP-NLS-YA constructs.
Nuclear envelope targeting assay
18 µg of pMT GFP-NLS (control) or pMT GFP-NLS-YA constructs was
transfected into S2 cells using the calcium phosphate method
(Di Nocera and Dawid, 1983);
(Drosophila Expression System, Invitrogen). The transfected cells
were induced to express GFP-NLS or GFP-NLS-YA with 0.5 mM CuSO4 per
well. 24 hours later, the cells were harvested, fixed and processed as
described by Sangoram et al. (Sangoram et
al., 1998
). Typically, 30-40% of the transfected cells expressed
the construct, although the levels of expression varied between cells.
Representative fields of cells were imaged and analyzed. Endogenous lamin
Dm0 in the transfected S2 cells was detected by staining with
affinity-purified rabbit polyclonal lamin Dm0 antibody, followed by
rhodamine-conjugated goat
-rabbit IgG secondary antibody (Jackson
ImmunoResearch Laboratories, West Grove, PA). Actin was detected by staining
with AlexaFluor-594-coupled phalloidin (Molecular Probes, Eugene, OR)
following the manufacturer's protocol. DNA in fixed cells was stained using
either DAPI (0.1 µg/100 µl) or propidium iodide (1 µg/ml).
Immunofluorescence imaging and confocal microscopy
The fixed, stained and mounted S2 cells were imaged using an Olympus BX-50
fluorescence/DIC microscope fitted with a high resolution Pentamax cooled CCD
camera (Princeton Instruments) and equipped with digital microscopy image
analysis software (Metamorph, Universal Imaging Corporation, PA). For further
resolution, a Bio-Rad MRC-600 attached to a Zeiss Axiovert 10-inverted
microscope was used to image single optical sections of transfected and
antibody stained cells at 63x or 100x magnification. Signals from
GFP and red (propidium iodide, rhodamine or Alexa Fluor 594) channels were
collected using a Z-series to determine colocalization. The acquired images
were processed using Confocal Assistant software (BioRad) and images were
assembled using Photoshop (Adobe).
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Results and Discussion |
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Control GFP-NLS protein expressed in S2 cells is detected in both nuclei
and cytoplasm, but appears slightly enriched in the former compartment,
presumably owing to targeting by its NLS
(Fig. 1A-C). Its fluorescence
was noticeable throughout the nucleus, including the nuclear periphery, and
the cytoplasm (Fig. 1B,C). We
believe that the presence of the fusion protein in the cytoplasm, despite the
presence of an NLS, is due to passive diffusion through the nuclear pores, as
the predicted size of GFP-NLS, 27.4 kDa, is below the 30 kDa size limit for
passive diffusion (Keminer and Peters,
1999). Analogous observations of GFP distribution in transfected
CHO-K1 cells indicate that GFP on its own does not show preferential retention
in any intracellular compartment (Broers et
al., 1999
).
|
Fusion of amino acids 506-696 of YA to GFP-NLS targets the fusion protein
preferentially to the nuclear periphery
(Fig. 1D-G,
Fig. 2A). GFP fluorescence was
exclusively nuclear, and within that compartment primarily circumferential
around the DNA; this was particularly evident in cells expressing high levels
of the reporter (Fig. 1F).
These results show that the C-terminal region of YA (residues 506-696), the
region that interacts with lamin Dm0 in the yeast two-hybrid
system, is sufficient to target a heterologous protein to the
Drosophila nuclear periphery. The location of the GFP-NLS-YA fusion
in transfected S2 cells was nearly identical to that of wild-type (untagged)
YA expressed in transfected Kc cells
(Lopez et al., 1994).
Interestingly, preliminary experiments using mammalian HEK cells (S.S.M.,
unpublished) suggest that the same YA domain fused to GFP-NLS is not
sufficient to target the reporter protein to the mammalian nuclear periphery.
This observation suggests that the targeting ability of YA 506-696 is not
species-general but instead may require a conformation or sequence of the
lamina (or lamin) present in the Drosophila nuclear envelope.
|
Dissection of the lamin-binding domain of YA
To further define the lamin-interaction domain of YA, we tested deletions
of YA (506-696) for their ability to interact with lamin Dm0 in the
yeast two-hybrid system (Table
1). The interaction was assessed by using the ability of cells
carrying the YA deletion in pGBT9 and lamin in pGAD424 to grow in the absence
of histidine and by using ß-galactosidase reporter activity. Lamin
interaction was not impaired by deleting 50 amino acids from the N-terminus of
the 190-residue YA region (YA 556-696). Deletion of up to 50 additional amino
acids from the N-terminus of the lamin-binding domain (YA 581-696 and YA
607-696) still allowed significant interaction with lamin. These data indicate
that the C-terminal 140 amino acids (YA 556-696), and possibly the C-terminal
90 residues (YA 607-696), are sufficient to interact with lamin.
|
Deletion of amino acids 506-696 from the C-terminus of YA abolished the interaction with lamin in the two-hybrid system (YA 506-647, YA 506-671 and YA 581-671; Table 1). This suggests that residues 671-696 of YA are necessary for optimal lamin interaction and/or for proper folding (see below). In summary, residues 607-696 of YA are sufficient for interaction with lamin Dm0, although additional residues N-terminal to this region (residues 556-606) may strengthen the association.
Subcellular localization of GFP-NLS-YA deletions in transfected S2
cells
We next tested the same subregions of YA's C-terminus of YA for their
ability to target GFP-NLS fusions to the nuclear periphery in transfected
Drosophila S2 cells. Nuclear envelope targeting is retained by the
N-terminal deletions that interact with lamin Dm0 in the two-hybrid
system (YA 556-696, YA 581-696, and YA 607-696)
(Fig. 2B-D). Although these
GFP-NLS-YA fusions are targeted to the nuclear periphery, there is also
nucleoplasmic staining. Because this distribution is like that seen with
full-length YA expressed in Kc cells
(Lopez et al., 1994), we
believe that it reflects binding of YA sequences to a minor population of
lamin Dm0 found by P. Fisher and colleagues to reside in the
interior of the Drosophila nucleus (P. Fisher, personal
communication) or to chromatin (Yu and
Wolfner, 2002
) or saturation of YA binding sites owing to high
level expression of the transfected DNA. Lamin-GFP fusions have also been
reported to localize to the interior of the nucleus (in addition to the
nuclear periphery) in transfected mammalian cells
(Broers et al., 1999
).
To test whether lamin-interacting sequences at the C-terminus of the 190
amino acid domain are essential for targeting, we carried out similar
experiments on GFP-NLS-YA 506-647, 506-671, and 581-671. Although in each case
the protein was present in transfected cells, it was distributed in a punctate
fashion in the cytoplasm and failed to translocate into the nucleus (for
example, see Fig. 2E). A likely
explanation for this apparent lack of nuclear entry by these NLS-containing
fusion proteins is that the C-terminal truncations of the YA domain cause
misfolding of the fusion protein. This would also explain why these deletions
fail to interact with lamin in the two-hybrid system. Secondary structure
analysis of amino acids in the lamin-interaction domain (506-696) predicts a
strong propensity to form -helices and turns
(Chou and Fasman, 1978a
;
Chou and Fasman, 1978b
;
Garnier et al., 1978
).
Disrupting this region could cause a conformation unsuitable for proper
folding of the fusion protein. In particular, amino-acid residues from YA 647
to 696 include a number of charged residues (38%) that could form hydrogen
bonds or participate in electrostatic interactions. Such interactions are
believed to stabilize the intrahelical ion pairing and contribute to
higher-order multimolecular interactions in the case of intermediate filament
proteins (Letai and Fuchs,
1995
). Future studies using site-directed mutagenesis can shed
light on the role of this region, and of the charged amino acids within it, in
mediating the interaction of YA with polymerized lamin Dm0.
Our results, summarized in Fig.
3, favor a model in which YA is targeted to, and/or retained at,
the nuclear periphery by interaction with the polymerized lamin network that
has already assembled at this location. This targeting mechanism, for a
largely hydrophilic protein, differs from mechanisms reported for nuclear
periphery targeting of proteins that insert into the nuclear membranes in the
course of their targeting (Ashery-Padan et
al., 1997; Ellenberg et al.,
1997
; Foisner and Gerace,
1993
; Hofemeister et al.,
2000
; Soullam and Worman,
1995
; Worman et al.,
1990
; Wu et al.,
2002
). For one of those proteins, mammalian LAP2, and for a
heterologous transmembrane reporter protein, a lamin-binding domain was
suggested to stabilize targeting to the nuclear periphery by preventing the
protein's diffusion from the inner nuclear membrane compartment
(Furukawa et al., 1998
). Our
results indicate that binding to lamin Dm0 can also target proteins
to the Drosophila nuclear periphery from the nucleoplasmic side of
the envelope without requiring a membrane tether. The requirement of lamin A
for the presence of lamin C [a lamin variant that lacks the C-terminal CAAX
motif (Lin and Worman, 1993
)]
at the mammalian nuclear periphery is consistent with use of an analogous
mechanism to target hydrophilic proteins to the nuclear envelope of vertebrate
cells (Pugh et al., 1997
;
Vaughan et al., 2001
).
|
The cell cycle dynamics of YA are consistent with this model. Lamin
Dm0 is present in the nuclei of all cells in which YA is in the
nuclear envelope (Lin et al.,
1991; Lin and Wolfner,
1991
; Liu et al.,
1995
; Lopez et al.,
1994
). At metaphase, the two proteins disappear from the nuclear
periphery simultaneously, as one would expect if the presence of YA at the
nuclear periphery depended on the presence of polymerized lamin. After
anaphase, both proteins reappear at the nuclear periphery, but lamin
Dm0 is detected there before YA reappears
(Lin and Wolfner, 1991
),
consistent with the idea that lamin polymerizes to form a scaffold to which YA
becomes attached. Our model is further supported by the finding that the
YA-lamin interaction in yeast is abolished if either the head or tail domain
of lamin is deleted, suggesting that YA interacts with a multimeric form of
lamin Dm0 (Goldberg et al.,
1998
).
The 90 amino-acid region of YA that we have defined here also provides a
useful tool for further studies of nuclear structure and function in the
Drosophila model system. It could be used to bring to the nuclear
envelope hydrophilic proteins that are not normally found at the nuclear
periphery, for studies of nuclear assembly, replication or transcriptional
silencing of chromatin (Andrulis et al.,
1998). It would also be interesting to see if this domain can
compensate for loss of a nuclear targeting domain from nuclear envelope
proteins that normally use other mechanisms to target to this cellular
compartment.
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Acknowledgments |
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