Departments of Medicine and of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA
* Author for correspondence (e-mail: hjw14{at}columbia.edu )
Accepted 9 January 2002
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Summary |
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Key words: Nuclear envelope, Inner nuclear membrane, LEM domain, Fluorescence recovery after photobleaching, ng, Membrane proteins, Muscular dystrophy
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Introduction |
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MAN1 is an integral protein of the inner nuclear membrane encoded by a gene
on human chromosome 12q14 (Lin et al.,
2000). It is predicted to have a nucleoplasmic, N-terminal domain
followed by two hydrophobic segments and a nucleoplasmic, C-terminal tail.
Indirect evidence suggests that MAN1 is associated with the nuclear lamina
(Paulin-Levasseur et al.,
1996
). Protein sequence analysis reveals that MAN1 contains a
conserved globular module of approximately 40 amino acids, which has been
termed the LEM domain because it is found in LAP2, emerin and MAN1
(Lin et al., 2000
). The LEM
domain is composed mainly of two large parallel alpha helices in a fold
similar to some bacterial dehydrogenase multienzyme complexes
(Laguri et al., 2001
;
Wolff et al., 2001
).
Biochemical experiments have shown that the LEM domain binds to
barrier-to-autointegration factor
(Shumaker et al., 2001
).
It is not known how MAN1 is targeted to or retained in the inner nuclear membrane. To investigate the intracellular trafficking of MAN1, we have transfected cells with plasmids that express different domains and chimeric constructs and examined their intracellular locations. We have also used fluorescence recovery after photobleaching (FRAP) to measure the diffusion of a MAN1-green fluorescent protein (GFP) fusion protein in living cells. Our results are consistent with the `diffusion and retention' model for MAN1 targeting to the inner nuclear membrane, with retention being mediated by its N-terminal domain.
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Materials and Methods |
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Plasmids FL, MAN538 and CT were constructed to express full-length MAN1,
amino acids 1-538 (MAN1 nucleoplasmic, N-terminal domain followed the first
hydrophobic segment) and the C-terminal tail with two hydrophobic segments of
MAN1, respectively. The cDNAs for cloning were generated by polymerase chain
reaction (PCR) (Saiki et al.,
1987) using the Gene Amp PCR System 2400 (Applied Biosystems,
Foster City, CA), with restriction endonuclease sites engineered at the
5' ends of the oligonucleotide primers. The recombinant pSVK3 construct
that encoded full-length MAN1 with a FLAG epitope tag at its N-terminus
(plasmid FL) was used as a template to generate the other cDNAs. In most
instances, an EcoRI site was engineered in the sense primer and a
XhoI site in the antisense primer. PCR products were digested with
the appropriate restriction endonucleases and ligated into pBFT4, which was
digested with EcoRI and XhoI. pBFT4 is a pBluescript II KS-
(Stratagene, La Jolla, CA) based plasmid containing a Kozak consensus
sequence, ATG, and the FLAG coding sequence 5' to the multiple cloning
site. The resulting plasmids, which contained a SpeI site 5' to
the Kozak consensus sequence and a XhoI site 3' to the cloned
cDNA insert, were then digested with SpeI and XhoI and the
isolated cassette ligated into pSVK3, which was digested with XbaI
(an isoschizomer of SpeI) and XhoI.
Plasmids MAN476-CHL and MAN351-CHL were constructed to express FLAG-tagged
proteins containing the first 476 and first 351 amino acids of MAN1,
respectively, followed by the transmembrane domain and portion of the
C-terminal domain of chicken hepatic lectin (CHL). The protein-coding cassette
was isolated from the previously described plasmid LMBR-CHL
(Soullam and Worman, 1993) by
restriction endonuclease digestion with EcoRI and XhoI. This
isolated cassette was ligated into pBFT4, which was also digested with
EcoRI and XhoI. This plasmid was digested with
EcoRI and BspEI, which cuts in the cDNA codon for amino acid
24 of CHL, and then the PCR products encoding amino acids 1-476 and amino
acids 1-351 of MAN1, amplified using sense primers with an EcoRI site
and antisense primers with a BspEI site, were ligated into it. These
plasmids were then digested with SpeI and XhoI and the
excised DNA ligated into pSVK3. To delete the coding region for amino acids
85-336 of MAN1 from plasmid MAN476-CHL, it was digested with NotI and
the ends re-ligated to generate plasmid MAN
85-336-CHL. To delete the
nucleotides encoding amino acids 8-199 of MAN1 from plasmid MAN476-CHL, it was
digested with NarI and the ends religated to generate plasmid
MAN
8-199-CHL.
Plasmid FG-CHL was constructed to express FLAG-tagged protein containing the transmembrane domain and a portion of the C-terminal domain of CHL. The pBFT4 construct containing the cDNA encoding the first 476 amino acids of MAN1 fused to CHL was digested with XmaI and BspEI and the compatible cohesive ends ligated. This construct was then digested with SpeI and XhoI and the excised DNA ligated into pSVK3.
To express a transmembrane protein that had a nucleoplasmic domain with a
molecular mass of >60 kDa and the inner nuclear membrane targeting signal
of MAN1, plasmid CMPK-MAN538 was constructed. This plasmid encoded a
FLAG-tagged protein containing the first 538 amino acids of MAN1 fused to
amino acids 17-476 of chicken muscle pyruvate kinase (CMPK) at the MAN1
N-terminus. This truncated form of CMPK lacks its mitochondrial signal
sequence and is a soluble cytosolic protein
(Frangioni and Neel, 1993).
DNA encoding amino acids 17-476 of CMPK was amplified by PCR using plasmid
p3PK (Frangioni and Neel,
1993
) as template, using sense and antisense primers containing
EcoRI sites. The PCR product, encoding amino acids 1-538 of MAN1,
amplified using a sense primer with an EcoRI site and an antisense
primer with an XhoI site, was ligated into pBFT4. The amplified
product encoding amino acids 17-476 of CMPK was digested with EcoRI
and ligated in-frame into the construct encoding amino acids 1-538 of MAN1.
The resulting chimeric cDNA was excised by restriction endonuclease digestion
with SpeI and XhoI and subcloned into pSVK3.
For studies of MAN1 fused to GFP, a FLAG-tagged construct containing amino
acids 1-538 of MAN1 was fused, via its C-terminus, to the F64L, S65T, H231L
variant of GFP. A cDNA fragment encoding a FLAG-tagged protein of the first
538 amino acids of MAN1 was isolated from plasmid MAN538 (see above) by
restriction endonuclease digestion with SacI. This fragment was
ligated into pEGFP-N1 (CLONTECH Laboratories), which also was digested with
SacI. The resulting plasmid, MAN538-GFP, expressed the first 538
amino acids of MAN1 preceded by a FLAG epitope and followed by GFP. Plasmids
MAN5388-199-GFP and MAN538
85-336-GFP, which express GFP fusion
proteins, are similar to MAN-538-GFP, with amino acids 8-199 and 85-336 of
MAN1 deleted. To generate these plasmids, MAN538 was digested with
NotI and NarI, respectively, and the ends re-ligated to
generate plasmids MAN538-
8-199 and MAN538-
85-336, respectively.
The cDNA fragments were isolated from these two plasmids by restriction
endonuclease digestion with SacI and ligated into pEGFP-N1 that was
digested with SacI.
To confirm proper plasmid construction, cDNAs were sequenced using an ABI Prism 377 automated sequencer (Applied Biosystems).
Cell culture and transfection
COS-7 cells were grown in DME medium containing 10% fetal bovine serum and
2 mM L-glutamine. For transfection, cells were grown to 70-90% confluency on
six-well plates, and transfected using LIPOFECT AMINE PLUSTM
reagent (Life Technologies, Gaithersburg, MD), following the manufacturer's
instructions. The cells were overlaid with the lipid-DNA complexes for 10-22
hours and allowed to grow in fresh medium for approximately 24-36 hours
post-transfection. Cells were then washed with phosphate-buffered saline (PBS)
and split into Chamber Slides or Chamber Coverglass (Nalge Nunc International
Corp., Naperville, IL) and grown for an additional 12-24 hours before
preparation for immunofluorescence microscopy or photobleaching.
Immunofluorescence microscopy
Transfected cells were washed three times with PBS and then fixed with
methanol for 6 minutes at -20°C. The cells were permeabilized with 0.5%
Triton X-100 in PBS for 2 minutes at room temperature, washed three times with
0.1% Tween-20 in PBS (Solution A) and incubated with the primary antibodies
diluted in PBS containing 0.1% Tween-20 and 2% bovine serum albumin (Solution
B) for 40 minutes at 37°C. Primary antibodies were anti-FLAG M5 monoclonal
antibody (Sigma-Aldrich, St Louis, MO) used at a dilution of 1:200, anti-lamin
B1 polyclonal antibody (Cance et al.,
1992) used at a dilution of 1:1,000 and
anti-signal-sequence-receptor
(anti-SSR-
) polyclonal antibodies
(gift of Christopher Nicchitta, Duke University, Durham, NC) used at a
dilution of 1:500. After washing four times with Solution A, the cells were
incubated with secondary antibodies diluted 1:200 in Solution B. Secondary
antibodies used were rhodamine-conjugated goat anti-rabbit IgG (Biosource
International, Camarillo, CA), fluorescein isothiocyanate (FITC)-conjugated
goat anti-mouse IgG (Jackson ImmunoResearch Labs, Inc., West Grove, PA) and
rhodamine-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Labs, Inc.).
The cells were then washed four times with Solution A and three times with
PBS. The slides were dipped in methanol, air-dried and the coverslips mounted
using anti-fade mounting medium (Slowfade Light Antifade Kit, Molecular
Probes, Eugene, OR).
Digitonin-permeabilization of cells was carried out as described previously
(Adam et al., 1992).
Transfected cells were washed three times with PBS and fixed with 2%
paraformaldehyde in PBS for 30 minutes on ice. They were then washed three
times with PBS and incubated with pre-cooled 40 mg/ml digitonin (Calbiochem,
La Jolla, CA) in PBS for 10 minutes on ice. The cells were then washed and
incubated with antibodies as described above, except that Tween-20 was
excluded from the buffers and all steps were performed on ice.
Immunofluorescence microscopy was performed using a Zeiss LSM 410 confocal laser scanning system attached to a Zeiss Axiovert 100TV inverted microscope (Carl Zeiss, Inc., Thornwood, NY). Images were processed using PhotoShop software (Adobe Systems, Inc., San Jose, CA) on a Macintosh G3 computer (Apple Computer, Inc., Cupertino, CA).
Fluorescence photobleaching experiments
Fluorescence recovery after photobleaching was performed on the Zeiss LSM
410 confocal laser scanning system using a 488 nm line of a 15 mW Kr/Ar laser
in conjunction with a 100x objective for optimum resolution or a
40x objective to achieve sufficient depth for bleaching in the optical
axis (z). Command macros for programming the microscope for photobleaching
experiments were downloaded from the Internet at
http://dir.nichd.nih.gov/CBMB/pb2labob.htm
. For qualitative experiments, the outlined box was photobleached at full
laser power (100% power, 100% transmission) and recovery of fluorescence
monitored by scanning the whole cell at low power (25% power and 13%
transmission) in 10 second intervals. For quantitative experiments, the
photobleached stripe was 2 µm wide and extended across the cell and through
its entire depth. Fluorescence within the strip was measured at low laser
power before the bleach and then photobleached with full laser power. Recovery
was followed with low laser power at 2 second intervals until the intensity
reached a steady plateau. Images were processed using PhotoShop software and
average intensities were measured using NIH Image J software on a Macintosh G3
computer.
Other chemicals
Unless otherwise indicated, routine chemicals were obtained from either
Fisher Scientific Co. (Pittsburgh, PA) or Sigma-Aldrich. Enzymes and enzyme
buffers for DNA cloning were obtained from either Fisher Scientific Co. or New
England Biolabs (Beverly, MA).
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Results |
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To confirm the intracellular locations of full-length MAN1 and CHL, COS-7
cells were transfected with plasmids FL and FG-CHL
(Fig. 1A). Full-length MAN1 was
localized to the nuclear rim (Fig.
1B). CHL is a type II integral membrane protein localized to the
ER, endosomes and plasma membrane. It has an N-terminal domain of 23 amino
acids that faces the cytoplasm, a single transmembrane segment and a luminal,
C-terminal domain of 160 amino acids
(Chiacchia and Drickamer,
1984; Mellow et al.,
1988
). Strong labeling of the ER and other cytoplasmic membranes,
along with weaker labeling of the plasma membrane, was seen in the transfected
cells that expressed CHL (Fig.
1B). The fluorescence enhancement at the nuclear periphery in
cells that expressed CHL was consistent with labeling of the outer nuclear
membrane and ER, as the rough ER is concentrated around the nucleus and shares
proteins with the contiguous outer nuclear membrane.
|
To determine the domain of MAN1 responsible for its inner nuclear membrane localization, we transiently transfected COS-7 cells with plasmids expressing truncated and chimeric forms of the protein (Fig. 2A). COS-7 cells were transfected with plasmid MAN538, which encodes the nucleoplasmic, N-terminal domain and the first transmembrane segment of MAN1. This protein was detected at the nuclear rim by immunofluorescence microscopy, showing colocalization with lamin B1, a marker for the lamina and inner nuclear membrane, consistent with an inner nuclear membrane localization (Fig. 2B). MAN1 devoid of its N-terminal nucleoplasmic domain was localized to the ER and possibly endosomes (Fig. 2B, CT), showing that the N-terminal domain is necessary for inner nuclear membrane targeting. To determine if the N-terminal domain of MAN1 can function as a nuclear envelope targeting signal for another integral membrane protein, it was attached to the transmembrane segment of CHL. In cells expressing a FLAG-tagged chimeric protein containing the N-terminal domain of MAN1 fused to the N-terminal side of the transmembrane segment of the CHL, nuclear rim fluorescence without ER labeling was observed, consistent with inner nuclear membrane localization (Fig. 2B). Hence, the N-terminal domain of MAN1 can function as a nuclear envelope targeting signal sufficient to direct an integral membrane protein synthesized on the ER to the inner nuclear membrane.
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To confirm that the N-terminal domain of MAN1 attached to a transmembrane
segment localized to the inner as opposed to the outer nuclear membrane, the
plasma membranes of cells expressing the first 538 amino acids of MAN1
(N-terminal domain and first transmembrane segment) with a FLAG epitope were
selectively permeabilized with digitonin
(Adam et al., 1992). To confirm
further that the FLAG-tagged protein was detected at the intact nuclear rim
but inaccessible to anti-FLAG antibodies, cells expressing the FLAG-tagged
protein containing the first 538 amino acids of MAN1 fused to GFP
(Fig. 3A) were treated with
Triton X-100 or digitonin. The endogenous fluorescence from GFP was then
visualized simultaneously to antibody labeling with anti-FLAG antibodies
recognized by rhodamine-conjugated secondary antibodies
(Fig. 3B). Cells in which the
nuclear envelopes were permeabilized with Triton X-100 showed an overlap of
the signals from the rhodamine channel and the GFP channel. In cells
permeabilized with digitonin, green fluorescence was detected at the nuclear
rim but it was not labeled with anti-FLAG antibodies. This experiment clearly
showed that the protein containing the N-terminal domain of MAN1 and its first
transmembrane segment was located in the inner nuclear membrane.
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We next attempted to determine if a smaller portion of the N-terminal
domain of MAN1 could mediate inner nuclear membrane targeting and retention.
COS-7 cells were transfected with plasmids MAN8-199-CHL,
MAN
85-336-CHL and MAN351-CHL, from which amino acids 8-199, 85-336 and
352-476, respectively, were deleted from the protein containing the MAN1
N-terminal domain followed by CHL (Fig.
4A). In cells transfected with these plasmids, the expressed
proteins were detected in the ER and nuclear envelope of transfected cells
(Fig. 4B). However, unlike
full-length MAN1 (Figs 1,
2), fluorescence labeling with
these internally truncated constructs was never exclusive to the nuclear
envelope. Fluorescence labeling of the ER and nuclear envelope does not
exclude the possibility that some of the expressed protein is in the inner
nuclear membrane or nuclear pore membranes; however, a large portion of
protein is in the ER. Therefore, even if some portion of these chimeric
proteins reaches the inner nuclear membrane, their retention signals are
significantly weaker than those present in proteins containing the whole
N-terminal domain of MAN1. These results suggest that the entire
nucleoplasmic, N-terminal domain of MAN1 is essential for efficient inner
nuclear membrane retention.
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Enlargement of the nucleoplasmic, N-terminal domain of MAN1 prevents
its inner nuclear membrane targeting
The results presented so far suggest that N-terminal of MAN1 can target an
integral membrane protein synthesized on the ER to the inner nuclear membrane.
For this to occur, the protein would presumably have to diffuse through the
nuclear pore complexes. The nuclear pore complex contains a central channel,
through which soluble proteins are thought to be actively transported, as well
as lateral channels with diameters of 10 nm, through which proteins with a
molecular mass of <60 kDa can presumably diffuse
(Hinshaw et al., 1992). The
lateral channels are located adjacent to the pore membrane domain
(Hinshaw et al., 1992
), and
the cytoplasmically exposed domains of transmembrane proteins would have to
pass through them en route to the inner nuclear membrane. To test if a
transmembrane protein targeted by the N-terminal domain of MAN1 gained access
to the inside of the nucleus via the lateral channels of the nuclear pore
complexes, we enlarged the cytoplasmically synthesized domain of a MAN1
polypeptide that was normally localized in the inner nuclear membrane.
We fused the N-terminal domain of MAN1 with its first transmembrane segment
to truncated CMPK at MAN1's N-terminus
(Fig. 5A). This truncated CMPK
is a non-membrane protein localized to the cytosol
(Frangioni and Neel, 1993;
Soullam and Worman, 1995
). The
molecular mass of the cytoplasmically synthesized domain of this CMPK-MAN1
chimeric protein preceding the transmembrane segment was increased to
approximately 100 kDa. This chimeric protein did not concentrate in the inner
nuclear membrane but remained primarily in the ER
(Fig. 5B), despite containing
an inner nuclear membrane targeting domain. This implies that MAN1 diffuses
from the ER to the inner membrane through the lateral channels of the nuclear
pore complexes.
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Diffusional mobility of MAN1 in the inner nuclear and ER
membranes
To examine the diffusion of MAN1 in the inner nuclear and ER membranes, we
performed FRAP experiments using a protein with the first 538 amino acids of
MAN1 (nucleoplasmic, N-terminal domain plus first transmembrane segment) fused
at its C-terminus to GFP (Fig.
3A). In cells overexpressing this GFP fusion protein at a
relatively high level, it `backed up' in the ER, probably when its binding or
retention sites in the nuclear membrane were filled. This allowed us to
measure the protein's diffusion in the inner nuclear and ER membranes, as we
have done previously for LBR (Ellenberg et
al., 1997) and emerin
(Östlund et al., 1999
).
In cells expressing the MAN1-GFP fusion protein, the bleached nuclear envelope
area did not regain full fluorescence intensity 290 seconds after
photobleaching (Fig. 6A),
whereas in the bleached ER area, fluorescence recovered quickly, regaining
approximately 70% of its original fluorescence intensity after about 60
seconds (Fig. 6B).
Approximately 80% of the nuclear envelope fluorescence recovered 10 minutes
after photobleaching.
|
Quantitative FRAP experiments were performed to measure the diffusional
mobility of the MAN1-GFP fusion protein in the nuclear envelope and ER
membranes. Normalized mean fluorescence intensities in a 2 µm bleached
strip before and after the photobleach were plotted versus time to determine
the diffusion constants (D), using previously described methods
(Ellenberg et al., 1997), in
the two different membrane pools (Fig.
7). D was 0.28±0.04 µm2/second for the ER
pool and 0.12±0.02 µm2/second for the nuclear envelope
pool (mean±s.d.). These results showed that MAN1 was significantly less
mobile in the inner nuclear membrane compared with the ER
(Table 1).
|
|
We also examined the diffusional mobilities of two GFP fusion proteins of
the first 538 amino acid of MAN1 from which amino acids 8-199
(MAN5388-199-GFP) and 85-336 (MAN538
85-336-GFP) were deleted.
Deletion of either of these two stretches of amino acids prevents the
N-terminal domain of MAN1 from functioning as an efficient nuclear targeting
signal (Fig. 4). The measured
diffusion constant in the ER was 0.26±0.02 µm2/second for
MAN538
8-199-GFP and 0.29±0.02 µm2/second for
MAN538
85-336-GFP, not significantly different than for the GFP fusion
protein containing the entire N-terminal domain of MAN1. An immobile fraction
in the nuclear envelope could not be measured because of the predominant
ER/outer nuclear membrane localization that was always present. These findings
further indicate that MAN1 can diffuse rather freely in the ER and that the
entire N-terminal domain is necessary for efficient inner nuclear membrane
retention.
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Discussion |
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The results of our present studies on MAN1 are also consistent with its
targeting to the inner nuclear membrane by a `diffusion-retention' mechanism.
Its nucleoplasmic, N-terminal domain mediates accumulation in the inner
nuclear membrane after synthesis on the ER membrane. The N-terminal domain of
MAN1 with its first transmembrane segment diffuses rather freely in the ER
membrane, but its lateral diffusion constant is decreased by greater than
half, from D=0.28 µm2/second in the ER to D=0.12
µm2/second, in the inner nuclear membrane. These values are
similar to those reported for emerin of D=0.32 µm2/second in the
ER and D=0.10 µm2/second in the nuclear envelope
(Östlund et al., 1999).
By contrast, LBR appears to be more immobile in the nuclear envelope compared
to MAN1 and emerin (Ellenberg et al.,
1997
).
The N-terminal domain of LBR binds to B-type lamins
(Ye and Worman, 1994) and
human orthologues of Drosophila heterochromatin protein 1
(Ye and Worman, 1996
;
Ye et al., 1997
). The
N-terminal domain of LAP2ß similarly binds to lamins
(Furukawa et al., 1998
) and
chromatin (Foisner and Gerace,
1993
) and the inner nuclear membrane targeting domain localizes
with its lamin binding domain (Furukawa et
al., 1998
). The N-terminal domain of emerin binds to nuclear
lamins (Clements et al., 2000
)
and probably one or more chromatin components based on the colocalization of
different portions of emerin with different chromosomal regions during mitosis
(Haraguchi et al., 2000
).
Specific nuclear proteins that bind to MAN1 have not yet been identified;
however, MAN1 cofractionates with nuclear lamins, suggesting an interaction
with the lamina (Paulin-Levasseur et al.,
1996
). Like emerin, MAN1 also contains a LEM domain
(Lin et al., 2000
;
Wolff et al., 2001
), which
binds to the predominantly nuclear protein barrier-to-autointegration factor
(Shumaker et al., 2001
). Most
or all of the 476 amino acid N-terminal domain of MAN1 is necessary for
efficient inner nuclear membrane retention, suggesting that binding to more
than one nuclear component is required for concentration there. Alternatively,
a complex quaternary structure of the entire N-terminal domain may be required
for binding to one nuclear structure.
Transmembrane domains of some integral membrane proteins also contribute to
their localization in the inner and pore membrane domains of the nuclear
envelope. This may occur as a result of oligomerization in the plane of the
membrane or differential retention in membranes of varying thickness
(Bretscher and Munro, 1993;
Nilsson and Warren, 1994
).
Transmembrane segments contribute to the inner nuclear membrane localization
of LBR (Smith and Blobel,
1993
; Soullam and Worman,
1995
) and nurim (Rolls et al.,
1999
) and the pore membrane targeting of glycoprotein gp210, whose
cytoplasmic tail and transmembrane segment can mediate targeting to this
location (Wozniak and Blobel,
1992
). However, proteins very similar in structure to LBR in their
transmembrane segments but lacking hydrophilic N-terminal domains are
localized predominantly to the ER (Holmer
et al., 1998
), suggesting that the N-terminal domain of LBR is its
most important targeting signal. By contrast, the transmembrane segments of
nurim (Rolls et al., 1999
) and
gp210 (Wozniak and Blobel,
1992
) may be their major targeting determinants. For the inner
nuclear membrane proteins emerin
(Östlund et al., 1999
;
Tsuchiya et al., 1999
) and
LAP2ß (Furukawa et al.,
1995
) and the nuclear pore membrane protein POM121
(Söderqvist et al.,
1997
), the transmembrane segments do not mediate targeting to a
significant degree. This is also the case for MAN1.
Is the inner nuclear membrane a specialized domain of the ER?
The `diffusion-retention' model for inner nuclear membrane protein
targeting and the observed morphology of the nuclear envelope imply that the
inner nuclear membrane is actually a specialized domain of the ER. The outer
nuclear membrane, which contains ribosomes on its outer surface, is directly
continuous with the rough ER and similar or identical to it in composition
(Amar-Costesec et al., 1974;
Pathak et al., 1986
). Viral
integral membrane proteins synthesized on the ER are also found in the outer,
pore and inner membrane domains of the nuclear envelope
(Bergmann and Singer, 1983
;
Torrisi and Bonnatti, 1985
).
Studies using FRAP (Ellenberg et al.,
1997
; Östlund et al.,
1999
; Rolls et al.,
1999
), including the present study, have shown that the lateral
diffusion of integral proteins is significantly different in the ER/outer
nuclear membrane and inner nuclear membrane, with the diffusion of resident
proteins being significantly decreased in the inner nuclear membrane. The
inner nuclear membrane is separated from the remainder of the ER by the
nuclear pore complexes, which form an immobile network and have a very low
turnover in live mammalian cells (Daigle
et al., 2001
). The nuclear lamina, a meshwork of intermediate
filaments, is associated with the inner nuclear membrane, and FRAP experiments
have shown that the peripheral lamina is also relatively immobile in
interphase cells (Broers et al.,
1999
; Moir et al.,
2000
). The pore complexes and the lamina are also associated in
cells (Aaronson and Blobel,
1975
), further contributing to the establishment of a `rigid'
macromolecular structure supporting the inner nuclear membrane. The adjacent
chromatin, another immobile structure in the interphase nucleus
(Marshall et al., 1997
), may
also contribute to the different properties of the inner nuclear membrane
compared with the rest of the ER. Most integral proteins are probably
localized to the inner nuclear membrane primarily because they are immobilized
there by binding to the adjacent rigid lamina and chromatin. In mitosis,
integral proteins such as LBR and emerin, which are relatively immobile in the
inner nuclear membrane in interphase, diffuse more freely in the ER
(Ellenberg et al., 1997
;
Haraguchi et al., 2000
). This
suggests that the inner nuclear membrane loses its differentiation from the
remainder of the ER in mitosis when the nuclear lamina depolymerizes, the pore
complexes disassemble and the chromatin condenses into chromosomes.
Although the inner and outer nuclear membranes are directly continuous via
the pore membrane, the pore complexes present a topological barrier to the
movement of MAN1 and other integral proteins from their site of synthesis on
the ER membrane to the inner nuclear membrane. Structural studies have
indicated that aqueous channels of approximately 10 nm are present at the
periphery of the pore complex immediately adjacent to the pore membrane
(Hinshaw et al., 1992). A
globular protein of approximately 60 kDa can freely diffuse through an aqueous
channel of this size. When truncated CMPK was fused to the MAN1 N-terminal
domain, resulting in a domain of approximately 100 kDa preceding the
transmembrane segment, it was retained in the ER. Similar results have been
observed when the nucleoplasmic, N-terminal domain of LBR
(Soullam and Worman, 1995
) was
enlarged. These results show that the lateral channels of the nuclear pore
complexes provide barriers that prevent integral proteins with cytoplasmically
synthesized domains greater than
60 kDa from reaching the inner nuclear
membrane. Along these lines, all isoforms of the six integral membranes
proteins definitely localized to the inner nuclear membrane in interphase
LBR, LAP1, LAP2, emerin, nurim and MAN1 have nucleocytoplasmic
domains with molecular masses of <60 kDa. Hence, the nuclear pore complexes
play an important role in differentiating the inner nuclear membrane from the
rest of the ER.
Inner nuclear membrane protein trafficking in inherited diseases
Inherited mutations in inner nuclear membrane proteins cause human
diseases. Mutations in emerin were first shown to cause X-linked
Emery-Dreifuss muscular dystrophy (Bione et
al., 1994). Most of these mutations lead to a loss of emerin from
all cells, including skeletal and cardiac muscle
(Manilal et al., 1996
;
Nagano et al., 1996
). A wide
range of mostly missense and deletion mutations in nuclear lamins A and C,
which interact with emerin (Clements et
al., 2000
), causes an autosomal dominantly inherited form of
Emery-Dreifuss muscular dystrophy (Bonne et
al., 1999
; Bonne et al.,
2000
). In addition, missense and deletion mutations cause two
phenotypically overlapping disorders: dilated cardiomyopathy with conduction
deficit (Fatkin et al., 1999
)
and limb girdle muscular dystrophy type 1b
(Muchir et al., 2000
).
Localized mutations in a specific region of the C-terminal tail domain of
lamins A and C cause autosomal dominant Dunnigan-type familial partial
lipodystrophy (Cao and Hegele,
2000
; Shackleton et al.,
2000
; Speckman et al.,
2000
).
How do mutations in emerin and lamins A and C cause tissue-specific human
diseases? This question has been the subject of considerable recent
experimental attention and theoretical speculation but remains unanswered
(Emery, 2000;
Flier, 2000
;
Morris, 2000
;
Nagano and Arahata, 2000
;
Hutchison et al., 2001
;
Wilson et al., 2001
). Our
current results show that the LEM domain-containing protein MAN1, like emerin
(Östlund et al., 1999
),
is immobilized in the inner nuclear membrane in interphase. In cells from
Lmna knockout mice that do not express lamins A and C, some emerin is
lost from the nuclear envelope and mislocalized in the ER
(Sullivan et al., 1999
).
Expression of some mutant forms of lamin A from patients with autosomal
dominant Emery-Dreifuss in transfected cells causes a loss of some emerin from
the nuclear envelope (Raharjo et al.,
2001
; Östlund et al.,
2001
). Hence, mutations in an integral inner nuclear membrane
protein and associated lamins may lead to an overall disruption in the
structural integrity of the nuclear envelope, causing increased lateral
mobility of integral proteins in the inner nuclear membrane. Measurements of
the diffusional mobilities of integral proteins of the inner nuclear membrane
in cells from patients with Emery-Dreifuss muscular dystrophy and
Dunnigan-type partial lipodystrophy may provide further insights into how
mutations in emerin and lamins A and C lead to alterations in the inner
nuclear membrane in inherited human diseases. Increased lateral diffusion of
integral proteins in the inner nuclear membrane and possibly back into the
contiguous ER may lead to structural weakness in cells and cause
tissue-specific defects that result in skeletal muscular dystrophy and
cardiomyopathy.
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Acknowledgments |
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References |
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