(Received for publication, May 30, 1996, and in revised form, November 6, 1996)
From the Otefin is a 45-kDa nuclear envelope protein with
no apparent homology to other known proteins. It includes a large
hydrophilic domain, a single carboxyl-terminal hydrophobic sequence of
17 amino acids, and a high content of serine and threonine residues. Cytological labeling located otefin on the nucleoplasmic side of the
nuclear envelope. Chemical extraction of nuclei from
Drosophila embryos revealed that otefin is a peripheral
protein whose association with the nuclear envelope is stronger than
that of lamin. Deletion mutants of otefin were expressed in order to
identify regions that direct otefin to the nuclear envelope. These
experiments revealed that the hydrophobic sequence at the carboxyl
terminus is essential for correct targeting to the nuclear envelope,
whereas additional regions in the hydrophilic domain of otefin are
required for its efficient targeting and stabilization in the nuclear
envelope.
The nuclear envelope separates the nucleoplasm from the cytoplasm.
It is a complex structure composed of outer and inner membranes that
are separated by the perinuclear space. The two membranes are joined at
the nuclear pore complexes. Underneath the inner membrane, there is a
proteinaceous meshwork of intermediate filaments termed the nuclear
lamina (for reviews, see Refs. 1-3). The inner and outer membranes of
the nuclear envelope differ in their morphology and composition.
Nevertheless, the two membranes are joined at the nuclear pore
complexes. The way by which inner membrane proteins reach their target
membrane and are retained there has been the subject of several recent
studies. Isoprenylation of the CAAX motif in conjunction
with a nuclear localization signal has been shown to be necessary in
order to direct newly synthesized nuclear lamins to the inner nuclear
envelope (4). The CAAX motif-mediated modifications,
although necessary, are not sufficient for stable association of lamins
with membranes (5). Therefore, additional factors such as inner
membrane proteins must be involved in lamin-membrane association
(6-8). At least two independent nucleoplasmic regions of
LAP2 1 are responsible for its nuclear
envelope targeting, while the transmembrane domain of LAP2 mediates
nonspecific membrane binding at the endoplasmic reticulum (9). Both the
first transmembrane domain (10) and the amino-terminal domain of LBR
(11, 12) mediate the targeting of LBR to the inner nuclear membrane.
The highly charged amino-terminal domain of LBR can also direct
cytosolic proteins to the nucleus and type II integral membrane
proteins to the inner nuclear membrane (10). It was proposed that the mechanism for inner membrane targeting and retention of LBR and LAP2
involves lateral diffusion in the interconnected membranes of the
endoplasmic reticulum and nuclear envelope and interaction with
components of the nuclear lamina and chromatin (9, 12).
The Drosophila nuclear envelope protein otefin has a
mobility on SDS-polyacrylamide gel electrophoresis consistent with a molecular mass of 53 kDa (13). The otefin gene encodes a putative primary translation product of 45 kDa with no apparent homology to
known proteins. It is highly hydrophilic with a relatively high content
of serine and threonine residues and a putative site for
phosphorylation by Cdc2 kinase. The 17 carboxyl-terminal residues of
the otefin protein are hydrophobic and resemble membrane-spanning domains of integral membrane proteins (14). Polyclonal antibodies raised against Drosophila otefin revealed conservation of
mammalian nuclear envelope epitope(s) (14). Immunoelectron microscopy showed that otefin is associated with the nuclear envelope, yet it is
excluded from the nuclear pores (13).
In this study, we have determined by immunogold electron microscopy
that otefin is present underneath the inner nuclear membrane, facing
the nucleoplasm. Extraction studies have demonstrated that in contrast
to LAP1, LAP2, and LBR, otefin behaves as a peripheral protein.
Expression in COS-7 cells of deletion mutants of otefin and of fusion
constructs between otefin truncations and the lacZ gene
coupled to a nuclear localization signal (NLS-lacZ) revealed that the COOH-terminal 17-amino acid hydrophobic sequence of otefin is
essential for the targeting of otefin to the nuclear periphery. This
sequence alone can target NLS-lacZ to the nuclear rim,
albeit with low efficiency. Other sequences that are located between amino acids 173 and 372 of otefin are required for efficient targeting to the nuclear envelope, while sequences between amino acids 35 and 172 are required for stabilizing the interaction of otefin with the nuclear
envelope.
Anti-Drosophila lamin Dm mAb 611A3A6
and anti-Drosophila otefin mAb 618A2O7 have been described
(13, 15). Polyclonal anti-Drosophila otefin antibodies were
prepared by injecting 500 µg of otefin cDNA translation product
into rabbits. Anti- Vector pSP64-497-524.Z (16) is a
eukaryotic expression vector carrying an SV40 enhancer, an pCA1038-otefin was prepared by subjecting the EcoRI fragment
containing the complete otefin cDNA (14) to end filling of the
recessed 3 The construction of fusions between
otefin cDNA and the lacZ gene was performed in a pT7-7
vector in which the NdeI and EcoRI restriction
sites were destroyed. The NLS-lacZ region was then cloned
into the BamHI site of pT7-7. The NLS-lacZ-otefin
cDNA fusions (see below) were subcloned into the BamHI
restriction site, replacing the normal lacZ gene in
pSP64-497-524.Z with the NLS-lacZ-otefin fusions.
To prepare NLS-lacZ-otefin, the AAAATG translation start
sequence in otefin cDNA was polymerase chain reaction-mutated to the NdeI site CATATG. The NdeI-EcoRI
fragment of otefin cDNA, which contains the complete open reading
frame, was cloned into the NdeI and EcoRI sites
at the 3 COS-7 cells (American Type
Culture Collection CRL1651, SV40-transformed CV-1 cells) were grown in
Dulbecco's modified Eagle's medium containing 10% FCS, 40 mM glutamine, 200 units/ml penicillin, and 0.2 mg/ml
streptomycin (Beit Haemek, Israel). For transfection, 5 × 105 cells were plated on 10 × 10-mm coverslips
(prewashed with 70% ethanol and treated with poly-L-lysine
(Sigma)) in a six-well plate and grown overnight.
Lipofection mixtures were prepared by vortexing 1 ml of Opti-MEM (Life
Technologies, Inc.) and 15 µl of Lipofectin transfection reagent
(Boehringer Mannheim catalog No. 1202375) in polystyrene tubes. 5 µg
of supercoiled plasmid DNA (prepared with a Qiagen plasmid kit, QIAGEN
GmbH, Hilden, Germany) were then added and mixed gently for 10 min with
the lipofection mixture. Following overnight incubation, the cells were
washed once with Opti-MEM, and the lipofection mixture was then added
to the cells. After a 5-h incubation, 1 ml of Dulbecco's modified
Eagle's medium supplemented with 20% FCS was added to the culture.
After 24 h, the transfection medium was replaced with fresh
Dulbecco's modified Eagle's medium containing 10% FCS. Following an
additional 24 h, the cells were fixed and stained for
immunofluorescence analysis.
Drosophila embryos (0-7 h old) were
dechorionated and homogenized in buffer NM (250 mM sucrose,
2.5 mM MgCl2, 50 mM KCl, 1 mM dithiothreitol, 10 µM leupeptin, 10 µg/ml aprotinin). Nuclei were separated by centrifugation of the
homogenate at 10,000 × g for 10 min at 4 °C. The
isolated nuclei were washed at 4 °C with PBS, fixed for 30 min at
room temperature in PBS containing 4.0% paraformaldehyde and 0.1%
glutaraldehyde, and washed with PBS containing 1% bovine serum albumin
and 0.1% Triton X-100 (PBSBT). The nuclei were then incubated for
16 h at 4 °C with polyclonal anti-Drosophila otefin
antibody diluted in PBSBT, washed three times for 1 h each with
PBSBT, and then incubated for 2 h at room temperature with 12-nm
colloidal gold-conjugated goat anti-rabbit IgG diluted 1:25 in PBSBT.
Nuclei were washed for 2 h with PBSBT and then post-fixed with PBS
containing 2% formaldehyde and 2% glutaraldehyde for 30 min at room
temperature. Following a wash with PBS, nuclei were washed for 16 h at 4 °C with 100 mM sodium cacodylate (pH 7.5) and
then fixed for 90 min at room temperature with 1% OsO4 in
100 mM sodium cacodylate. Samples were dehydrated, embedded, sectioned, stained, and viewed as described previously (13).
Transfected cells were grown to ~80% confluence on
10 × 10-mm coverslips, washed once with PBS, fixed with 100%
methanol for 10 min at 4 °C, washed again with PBS, and fixed again
for 30 min at 20 °C with 4% formaldehyde in PBS containing 0.1%
Triton X-100 (PBSTR). Cells were then washed three times for 30 min: first with PBS, then with PBSTR, and then with PBSTR containing 2%
FCS. These washes were followed by a single 10-min wash with PBSTR
containing 5% low fat milk. Cells were then incubated overnight at
4 °C with mAb 618A2O7 or mAb Z387A diluted in PBS. Following incubation with the antibody, the cells were washed three times for 30 min each with PBSTR and were stained for 2 h with
Cy3A-conjugated goat anti-mouse IgG in PBS containing 1%
FCS. The cells were then washed three times for 30 min with PBSTR and
stained with 1 µg/ml 4 Triton X-100 extraction of cells was performed by incubation of the
cells prior to fixation with 1% Triton X-100 for 10 min at 4 °C,
followed by a brief wash with PBS, after which the cells were fixed and
treated as described above.
A Bio-Rad MRC-1024 confocal scanhead
coupled to a Zeiss Axiovert 135M inverted microscope was used to
acquire images of the stained cells. A 63×/NA = 1.4 objective was
used. Excitation light was provided by a 100-microwatt air-cooled argon
ion laser run in the multiline mode. The 514-nm line of the laser was
used for excitation and was selected with an interference filter. The
emission filter was a D580/32 (32 band pass centered about 580 nm). The confocal iris diameter was between 1.3 and 3.5 mm, with the larger opening used for weaker signals. Vertical resolution was between 0.5 and 1 µm, depending on the iris setting. Two to four images were
averaged in order to reduce point noise. Images of 512 × 512 pixels were acquired using a hardware zoom of 3-10 (0.106-0.032 µm/pixel). When needed, a 3 × 3 median filter was used to
remove point noise from the digital images.
Labeling was with horseradish
peroxidase-conjugated antibodies, and visualization was with the
chromogen 3,3 Nuclei were prepared according to Adra
et al. (17) by thawing frozen 0-15-h-old
Drosophila (Canton S) embryos on ice and homogenizing them
in 10 volumes of buffer D (10 mM Tris-HCl (pH 8), 5 mM MgCl2, 1.3 M sucrose). The
homogenate was filtered through a 125-µm nylon mesh overlaid above a
cushion of buffer D and centrifuged at 10,000 × g for
10 min at 4 °C. The supernatant was removed, and the nuclear pellet
was washed twice with buffer B (20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 100 µM
leupeptin, 100 mM TPCK) containing 40% glycerol. The
nuclei were resuspended to a concentration equivalent to 2 µl of
packed embryos/µl of buffer and frozen in liquid N2. For
chemical extraction, nuclei were thawed on ice, washed once in buffer
B, and digested for 15 min with 1 µg/ml DNase I and 1 µg/ml
RNase A at 23 °C. The nuclear pellet was further extracted for 15 min at 4 °C in 4 volumes of buffer B supplemented with the
extraction reagent. Extraction with high pH was performed in buffer B
devoid of Tris-HCl, the pH of which was adjusted with NaOH.
After extraction, the residual nuclear pellet was separated from the
supernatant by centrifugation at 14,000 × g. The
supernatant was centrifuged once more for 10 min at 150,000 × g. From each fraction, amounts equivalent to 5 µl of
packed embryos were subjected to protein blot analysis using specific
mAbs. Colorimetric detection of alkaline phosphatase activity was
according to McGrady (19).
To reveal the exact orientation of otefin within the
nuclear envelope, polyclonal anti-otefin antibodies were raised against the otefin cDNA translation product. These were used to refine the
localization of otefin in Drosophila embryonic nuclei by
immunogold electron microscopy. As shown in Fig. 1,
otefin was localized on the nucleoplasmic side of the inner nuclear
membrane. Although the labeling of otefin in the current experiments
was highly specific, the immunolabeling was weaker than in a previous
study in which, due to the sensitivity of the available epitope to
fixatives such as glutaraldehyde, lighter fixation conditions and 5-nm
gold particles were used (13).
Extraction with detergents, salt, and chaotropic
reagents was employed in order to study the mode of the association of
otefin with the nuclear envelope (20). Drosophila embryonic
nuclei were isolated, digested with DNase I and RNase A, and extracted with buffer B containing different reagents. The supernatant and pellet
were then separated and analyzed by protein blot analysis using mAb
618A2O7 (anti-Drosophila otefin) and mAb 611A3A6
(anti-Drosophila lamin Dm) (12, 14). Extraction with 8 M urea, with 4 M guanidine HCl, or with buffer
B at pH 13 resulted in extraction of both otefin and lamin Dm (Fig.
2A). These data show that the association of
otefin with the inner nuclear membrane is peripheral and different from
that of other known integral membrane proteins (20). Otefin remained
associated mostly with the nuclear envelope after washing with buffer B
containing 1 M NaCl, 1 M LiCl, or 3 M NaBr (data not shown) or after washing with buffer B at
pH 11. Under the same conditions, a fraction of the lamin Dm molecules
were extracted (Fig. 2A). These data reveal that association
of otefin with the nuclear membrane is stronger than that of lamin Dm.
It was interesting to note that both otefin and lamin remained
associated with the nuclear envelope after a 1% Triton X-100
extraction (Fig. 2B), which suggests that both otefin and
lamin are present in a protein complex that is stable to extraction
with Triton. Both lamin and otefin were solubilized with 1% Triton
X-100 combined with 1 M NaCl (Fig. 2A).
Wild-type and
truncated otefin forms were visualized following transient transfection
into COS-7 cells in order to analyze sequences in otefin that
participate in its targeting to the nuclear periphery (Fig.
3A). Expression of the otefin constructs was
detected in ~1% (pCA1038) or 10% (pSP64-497-524.Z) of the
transfected cells. COS-7 transfection with wild-type otefin labeled the
nuclear rim in all otefin-expressing cells. In some transfected cells,
otefin was also localized to the cytoplasm (Fig.
4C and Table I). In Drosophila Schneider cells, otefin was resistant to
extraction with 1% Triton X-100 as judged by protein blot (Fig.
2B) and immunofluorescence (Fig. 4, A and
B) analysis. Similarly, otefin remained associated with the
nuclear rim in ~60% of the otefin-expressing COS-7 cells following
extraction with Triton X-100, while most of the cytoplasmic staining
disappeared (Fig. 4D and Table I). The remaining cytoplasmic staining appeared punctated probably due to aggregation of the protein
at these sites.
Summary of the results obtained following transfection of COS-7 cells
with the different wild-type and truncated otefin constructs
Department of Genetics,
Antibodies
-galactosidase (Z378A) and anti-tubulin (T9026)
mAbs were purchased from Promega and Sigma, respectively.
Cy3A-conjugated goat anti-rabbit IgG (H + L),
Cy3A-conjugated goat anti-mouse IgG, alkaline
phosphatase-conjugated goat anti-rabbit IgG (H + L), and anti-rabbit
IgG conjugated to 12-nm gold particles (catalog No. 111-205-144) were
purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove,
PA).
-globin
promoter, NLS sequence of the glucocorticoid receptor (amino acids
497-524) fused 5
to the lacZ sequences encoding the
-galactosidase protein, and polyadenylation sequences. pCA1038 (a
kind gift from Dr. Nissim Benvenisty) is an expression vector
containing the mouse phosphoglycerate kinase I promoter (17), a
multiple cloning site, and polyadenylation sequences.
-ends with DNA polymerase I Klenow fragment and cloning it
into the SmaI site of pCA1038.
388-406 was prepared by
inserting a stop codon after amino acid 387 to pCA1038-otefin.
35-172 was prepared by digesting pCA1038-otefin with
EagI and HincII. Following end filling with a DNA
polymerase I Klenow fragment, the construct was self-ligated to create
a deletion between amino acids 34 and 173.
35-172,
388-406 was
prepared by replacing the carboxyl terminus of
35-172 at the
BglII site with the carboxyl terminus of
388-406.
-end of the lacZ coding sequences. This created an
in-frame fusion between lacZ and otefin.
NLS-lacZ-
1-33 was prepared by inserting the
EagI-EcoRI fragment of otefin cDNA, encoding
amino acids 34-406, into the EcoRI site of the
lacZ gene. Both the EagI-EcoRI
fragment of otefin and the EcoRI site in lacZ were subjected to end filling of the recessed 3
-ends prior to the
ligation. NLS-lacZ-
372-406 was prepared by digesting
NLS-lacZ-otefin with StuI and EcoRI.
The digested construct was subjected to end filling of the recessed
3
-ends and self-ligated. NLS-lacZ-
1-371 was prepared by
digesting otefin cDNA with StuI and EcoRI.
The StuI-EcoRI fragment, encoding amino acids
1-371, was cloned into the NdeI site of the lacZ
gene. Both the StuI-EcoRI fragment of otefin and
the NdeI site in lacZ were subjected to end
filling of the recessed 3
-ends prior to the ligation.
,6-diamidino-2-phenylindole in PBS. Coverslips
were mounted with 2% (w/v) n-propyl gallate in 50%
glycerol/PBS (1:1) and viewed with a Leitz epifluoresence microscope
using a 63×/NA = 1.4 Planapo objective lens. To better localize
the immunofluorescence signal, the same cells were also viewed with a
Bio-Rad confocal microscope (see below).
-diaminobenzidine tetrahydrochloride and
H2O2 since they allowed the detection of the
few cells that expressed the construct with a light microscope before
visualization with an electron microscope. COS-7 cells were transfected
with NLS-lacZ-otefin. After 48 h, the cells were collected; washed with PBS; and fixed for 30 min at room temperature in
PBS (pH 7.4) containing 4% paraformaldehyde, 0.2% picric acid, and
0.05% glutaraldehyde. Subsequently, the cells were washed five
times with PBS and twice with TBS. The fixed and washed cells were
incubated for 30 min with 0.25% sodium borohydride in water; washed
eight times with TBS; and then incubated with blocking solution
containing 0.05% Triton X-100, 2% egg albumin, 0.5% glycine, 0.5%
lysine, and 0.9% NaCl in 0.5 M Tris-HCl (pH 7.4). Cells
were then incubated for 72 h at 4 °C in 4 µg/ml mAb Z378A
(anti-
-galactosidase) in TBS containing 0.05% Triton X-100 and 1%
egg albumin. The cells were washed eight times with TBS and incubated
for 3 h at room temperature with biotinylated secondary anti-mouse
antibody. The cells were then incubated for 2 h in
avidin-biotinylated horseradish peroxidase (Vectastain, Elite ABC kit,
Vector Laboratories, Inc.) and washed eight times with TBS. The
horseradish peroxidase was visualized using the chromogen
3,3
-diaminobenzidine tetrahydrochloride and 0.02%
H2O2. The cells were washed eight times with
TBS and embedded in 2% low melting point agarose. The cells were then post-fixed for 1 h at room temperature in 0.1 M
cacodylate buffer containing 1% OsO4 and 1.5%
K3Fe(CN)6. After brief washes with 0.1 M cacodylate, the cells were dehydrated in ascending
concentrations of ethanol and infiltrated with epoxy resin. Embedding,
sectioning, staining, and viewing were as described (13).
Otefin Is Localized to the Nucleoplasmic Side of the Nuclear
Envelope
Fig. 1.
Immunoelectron microscopy localization of
otefin in Drosophila embryonic nuclei.
Drosophila embryonic nuclei were incubated with polyclonal
anti-otefin antibodies, followed by an incubation with goat anti-rabbit
antibody conjugated to 12-nm gold particles. Arrowheads mark
selected areas in the inner nuclear membrane where gold particles are
seen. Bar = 30 nm.
[View Larger Version of this Image (120K GIF file)]
Fig. 2.
Solubility properties identify otefin as a
peripheral protein. Drosophila embryonic nuclei (0-15 h
old) were digested for 15 min in buffer B containing 1 µg/ml DNase I
and 1 µg/ml RNase A at 23 °C. A, digested nuclei were
extracted in buffer B containing 1 M NaCl; buffer B
containing 1% Triton X-100 and 1 M NaCl; buffer B at pH 13 (without Tris); buffer B at pH 11 (without Tris); buffer B containing 1 M LiCl; 8 M urea; or 4 M guanidine
HCl (GuHCl). B, digested nuclei (control) were
extracted in buffer B containing 1% Triton X-100. Following
extraction, the residual pellets (P) and the supernatants
(S) were separated by centrifugation, and proteins were
separated by 10% SDS-polyacrylamide gel electrophoresis and analyzed
by immunoblotting using mAb 618A2O7 (anti-otefin) and mAb 611A3A6
(anti-lamin Dm). The positions of otefin and lamin Dm are
indicated.
[View Larger Version of this Image (24K GIF file)]
Fig. 3.
Schematic diagrams of proteins expressed in
the transfection experiments. Shown are schematic diagrams of the
constructs. The different sequences are presented as a broken
line (NLS--galactosidase (NLS-
gal)), a
dotted bar (the hydrophilic sequences in otefin), and a
solid bar (the hydrophobic sequence in otefin).
A, native and truncated otefin mutants: full-length otefin
(wild type), truncation of the hydrophobic sequence (
388-406),
truncation of amino acids 35-172 (
35-172), and truncation of both
the hydrophobic sequence and amino acids 35-172
(
35-172,
388-406). Constructs for these proteins were prepared
for transfection in the pCA1038 vector. B, fusion proteins
between NLS-
-galactosidase and native or truncated forms of otefin:
full-length otefin (NLS-lacZ-otefin), otefin truncated at
amino acids 1-33 (NLS-lacZ-
1-33), otefin truncated at
amino acids 372-406 (NLS-lacZ-
372-406), and otefin truncated at amino acids 1-371 (NLS-lacZ-
1-371).
Constructs for these proteins were prepared for transfection in the
pSP64-497-524.Z vector. The amino terminus of each protein is shown on
the left.
[View Larger Version of this Image (22K GIF file)]
Fig. 4.
Cellular localization of the otefin protein
in Schneider and COS-7 cells by immunofluorescence. Cells were
viewed using a Leitz fluorescence microscope for
4,6-diamidino-2-phenylindole (DAPI) and Cy3A
(FM) staining. Cy3A staining was also viewed
with a Bio-Rad confocal microscope (CONFOCAL). A,
Schneider cells were fixed with methanol and 4% formaldehyde and
permeabilized with 0.1% Triton X-100. The cells were than incubated
with mAb 618A2O7 (anti-otefin) and with Cy3A-conjugated
goat anti-mouse IgG, followed by staining with
4
,6-diamidino-2-phenylindole. Antibody staining was localized to the
nuclear rim. B, Schneider cells were extracted with 1%
Triton X-100 for 10 min at 4 °C and then fixed and stained as
described for A. Following the extraction with Triton X-100, staining remained in the nuclear rim. C, COS-7 cells were
Lipofectin-transfected with full-length otefin in the pCA1038 vector.
After 24 h, the transfection medium was replaced with fresh
Dulbecco's modified Eagle's medium containing 10% FCS. Following an
additional 24 h, the cells were fixed and stained as described for
A. Staining was localized to the nuclear rim and the
cytoplasm (please note the cytoplasmic staining in the left cell in the
FM image). D, the transfected COS-7 cells were
washed with 1% Triton X-100 for 10 min at 4 °C, followed by
fixation and staining as describe for C. Staining remained
in the nuclear rim. In some cells, some aggregates remained in the
cytoplasm as shown in the FM images. Cells with relatively
high levels of cytoplasmic staining are presented in D.
Bars = 5 µm (A and B) and 10 µm (C and D). ~1% of the transfected cells
expressed otefin. mAb 618A2O7 (anti-otefin) did not recognize a native
otefin in mammalian cells, as indicated by cells that do not express
Drosophila otefin (C and D).
[View Larger Version of this Image (78K GIF file)]
, low intensity; and +/
, intensities close to background
levels. The percent resistance to Triton X-100 was measured as the
fraction of expressing cells in the culture following treatment with
Triton X-100 divided by the fraction prior to treatment with Triton
X-100. Low immunofluorescence intensity (+/
) was probably not
detected following extraction with Triton X-100. The fractions were
calculated from three different experiments. Please note the 13%
background level for the NLS-lacZ constructs.
Construct
Location
Resistance to
Triton X-100
After transfection
After
Triton X-100
N
NE
Cyt
N
NE
Cyt
%
Otefin
++
+
++
+/
60
35-172
++
+
++
+/
20
388-406
++
+/
0
35-172,
388-406
++
+/
0
NLS-lacZ
++
+/
+/
13
NLS-lacZ-otefin
++
+/
++
+/
77
NLS-lacZ-
1-33
++
+/
++
+/
NDa
NLS-lacZ-
374-406
++
+/
16
NLS-lacZ-
1-373
+
+
+
+
35
a
ND, not determined.
In contrast to wild-type otefin, transfection with the 388-406
construct, which lacks the hydrophobic sequence, or with the
35-172,
388-406 construct, which lacks amino acids 35-172 and 388-406, resulted in the exclusive localization of the proteins to the
nucleoplasm (Fig. 5, A and C). The
lack of nuclear rim localization of the
388-406 and
35-172,
388-406 proteins shows that the last 17 amino acids of
otefin are essential for directing otefin to the nuclear envelope. The
388-406 and
35-172,
388-406 proteins were completely soluble
following a Triton X-100 extraction (Table I).
The localization of the 35-172 protein was similar to that of the
normal protein (Fig. 5B), but was significantly less stable to extraction with Triton X-100. Only ~20% of the cells that
expressed the
35-172 protein remained stained following extraction
with Triton X-100 (Table I), and the level of labeling in these cells was significantly reduced (data not shown). The extraction of
35-172 with Triton X-100 demonstrated that although the
amino-terminal portion of the hydrophilic domain has little or no
effect on the cellular localization of otefin, it does play a role,
when present together with the hydrophobic sequence, in stabilizing the
interaction of otefin at the nuclear periphery.
To further analyze the role of
specific sequences in targeting otefin to the nuclear periphery, a
series of constructs was prepared in which the otefin sequences were
fused in frame to the 3-end of the open reading frame of the
lacZ gene in the pSP64-497-524.Z vector (Fig.
3B). These constructs were transiently transfected into
COS-7 cells. A comparison of the results that were obtained with the
different constructs is presented in Table I.
When the pSP64-497-524.Z vector encoding an NLS--galactosidase
protein was transiently transfected into COS-7 cells, the expressed
protein was localized in the nucleoplasm as judged by indirect
immunofluorescence (Fig. 6D). In ~70% of
the expressing cells, low levels of cytoplasmic staining were also
observed, perhaps due to the high expression levels in these cells. In
contrast, the NLS-
-galactosidase protein fused to amino acids 1-406
of otefin was detected mainly in the nuclear periphery (Fig.
6A). In some cells, reticular cytoplasmic staining was also
observed. Both anti-
-galactosidase (Fig. 6A) and
anti-otefin (618A2O7; data not shown) mAbs demonstrated similar
behavior. Extraction of NLS-lacZ-otefin with Triton X-100
resulted in punctated perinuclear staining, similar to the pattern
observed in COS-7 cells transfected with otefin (compare Fig.
4D with Fig. 6A).
The location of the NLS--galactosidase-otefin fusion protein was
further determined with electron microscopy immunolocalization using
anti-
-galactosidase mAb and horseradish peroxidase-conjugated anti-rabbit antibodies. Horseradish peroxidase staining appeared under
the inner membrane of the nuclear envelope of the lac
Z-expressing cells (Fig. 7, A-C), but
not in cells that do not express the fusion protein (Fig.
7D). In addition to the inner nuclear membrane staining,
horseradish peroxidase staining was also observed in the outer nuclear
membrane and in the cytoplasm of many cells.
The NLS--galactosidase-
372-406 protein, which lacks the
hydrophobic sequence, showed a nucleoplasmic localization, similar to
that of the NLS-
-galactosidase protein alone (Fig. 6E).
While confirming the importance of the hydrophobic sequence in
directing otefin to the nuclear periphery, these findings imply that
the hydrophilic sequences of otefin are not sufficient for promoting localization to the nuclear envelope.
To examine whether the hydrophobic sequence is sufficient for nuclear
envelope localization, COS-7 cells were transfected with
NLS-lacZ-1-371, encoding the NLS-
-galactosidase
protein fused to amino acids 372-406. This fusion protein had a
complex localization pattern. In ~60% of the expressing cells,
staining of the nuclear rim was observed (Fig. 6C). In
addition, in all of the expressing cells, nonhomogeneous cytoplasmic
labeling of unidentified blobs was detected. In ~40% of the cells,
nucleoplasmic staining was also observed. Triton X-100 extracted most
of the cytoplasmic and nuclear background, while the nuclear rim
staining remained in ~20% of the expressing cells (Table I),
apparently reflecting the fraction of this fusion protein that stably
interacts with component(s) of the nuclear envelope (Fig.
6C). It should be noted, however, that the estimation of the
percent of extractability by Triton X-100 was qualitative; following
extraction with Triton X-100, cells that expressed low levels of the
truncated protein could have been missed. Together, these data indicate
that while low efficiency targeting to the nuclear periphery of
NLS-containing proteins is obtained with the hydrophobic sequence of
otefin alone, high efficiency targeting requires additional sequences
in otefin.
Removal of the first 33 amino acids in otefin
(NLS-lacZ-1-33) resulted in a localization pattern
similar to that of the complete otefin (Fig. 6B). Extraction
of these cells with Triton X-100 resulted in punctated nuclear rim
staining, similar to NLS-lacZ-otefin (Table I). These data,
together with the transfection experiments, allow the identification of
three domains in otefin that are important for directing otefin to the
nuclear envelope. The hydrophobic sequence at the carboxyl terminus is
essential for the sorting of otefin to the nuclear envelope, while the
domain containing amino acids 35-172 is required to confer a stable
protein complex in the nuclear envelope. Additional sequences at the
hydrophilic domain play a role in the efficient targeting of otefin to
the nuclear periphery.
In this study, we used polyclonal anti-otefin antibody and the immunogold electron microscopy technique to show that in the nuclear envelope, otefin is facing the nucleoplasm. These findings add to our previous study (13), in which otefin was localized by mAb to the nuclear envelope, but its topology within the nuclear envelope could not be determined.
The solubility properties identify otefin as a peripheral protein of the nuclear envelope. A major operational criterion for this classification was its extraction with 8 M urea, with 4 M guanidine HCl, or with buffer B at pH 13. Therefore, the topology of the association of otefin is different from that of LAP1, LAP2, and LBR, which are all type II integral membrane proteins (21-23). The only other known peripheral proteins of the inner nuclear membrane are the lamins. The association of otefin with the nuclear membrane was found to be stronger than that of lamins since it remained resistant to extractions with high salt or high pH (pH 11), while lamin was solubilized. Otefin also remained associated with the nuclear envelope following extraction with Triton X-100. Hence, otefin maintained its association with the insoluble lamin fraction, similar to LAP1, LAP2, and LBR (23).
There are several indications that otefin may also interact in vivo with the nuclear lamina and/or chromatin. These indications include the peripheral nucleoplasmic localization of both otefin and lamin; the resistance of otefin to extraction with Triton X-100, similar to lamin; the finding that in early embryos, otefin remains associated with the spindle envelope during mitosis, like lamin Dmmit (13); and the finding that in the maternal pool, otefin is associated with the same membrane vesicle fractions as lamin Dmmit. These results motivate further studies to determine if such interactions exist.
Sequences That Are Required for the Targeting of Otefin to the Nuclear EnvelopeThe data presented here indicate that as in Drosophila nuclei, Drosophila otefin interacts with factor(s) that are present only at the nuclear periphery of the COS-7 cells to form a Triton-stable complex. In a fraction of the otefin-expressing cells, some of the label was found associated with the cytoplasm. This cytoplasmic association was largely solubilized with Triton X-100 and therefore did not involve the formation of Triton-stable complexes.
The hydrophilic domain of otefin was not sufficient for the targeting
of proteins to the nuclear envelope. The localization of otefin to the
nuclear envelope depended absolutely on the COOH-terminal hydrophobic
sequence. Removal of the short hydrophobic sequence of otefin resulted
in the nuclear localization of the 388-406 and
NLS-lacZ-
372-406 proteins, which is in line with our
previous suggestion that otefin has putative NLS sequences (14). The hydrophobic sequence of otefin and its neighboring 11 amino acids had,
by itself, only limited capability for directing the
NLS-
-galactosidase protein to the nuclear envelope. Other sequences
in otefin were required for the efficient localization of otefin to the
nuclear envelope and for its interaction with other nuclear envelope
proteins. Since the
35-172 and NLS-lacZ-
1-33
proteins had mainly a nuclear rim localization, similar to wild-type
otefin, efficient targeting to the inner membranes requires the
hydrophobic sequence together with hydrophilic sequences between amino
acids 173 and 389.
Removal of amino acids 35-172 from otefin did not affect the localization of otefin. However, the expressed construct became sensitive to extraction with Triton X-100. Although this region in otefin may be involved in the interaction of otefin with other factors that are present at the nuclear envelope, we cannot exclude the possibility that the sensitivity to Triton X-100 is due to misfolding of the mutant protein. It is interesting to note that this region in otefin contains a conserved site for Cdc2 kinase phosphorylation (SPKK) that may be involved in the regulation of the formation of a complex in vivo.
Based on these observations, an attractive model for the topology of otefin is that it is attached to the inner nuclear membrane by its hydrophobic sequence. This attachment is stabilized by integral membrane proteins. Treatment with guanidine HCl, urea, or pH 13 disrupts these protein-protein interactions and allows the extraction of otefin from the protein complex. Further study is required to determine whether the known integral membrane proteins of the nuclear envelope are involved in the formation of an otefin-containing complex.
Mechanisms for Targeting Proteins to the Nuclear PeripheryIt is possible that otefin is directed to the nuclear envelope by entering the nucleus with the aid of its NLS(s), followed by a hydrophobic sequence-dependent sorting to the nuclear envelope and retention by other nuclear envelope proteins. This mechanism of targeting resembles the mechanism by which lamins are localized to the nuclear envelope. However, in the case of lamins, this association with the nuclear envelope requires a post-translational modification resulting in the addition of an isoprenyl group to the CAAX motif at the carboxyl terminus (24). In a previous report, it was noted that the stretch of amino acids 74-81 in otefin has homology to known NLSs (14). In the present study, we show that deletion of this region and the carboxyl-terminal domain does not abolish nuclear targeting. The ability of the deleted otefin to reach the nucleus can be explained either by the presence of a different functional NLS or by a passive diffusion of this ~30-kDa protein into the nucleus and retention by binding to nuclear ligands. The location of NLSs in otefin is currently under investigation.
Since otefin was found to be associated with membrane vesicles in the maternal pool, sorting and retention at the nuclear envelope could also occur at the end of mitosis, when vesicles attach to chromatin and fuse to form the nuclear envelope. In that case, the precise localization of otefin within the inner nuclear membrane would require direct or indirect interactions of otefin with other nuclear envelope proteins or with proteins that are present on the surface of the chromatin.
Another mechanism suggested for the transport of type II integral
membrane proteins to the inner nuclear membrane includes insertion of
the protein into endoplasmic reticulum membranes, followed by lateral
diffusion through the endoplasmic reticulum network and accumulation at
the inner membrane by binding interactions (9, 12). Like otefin, which
is a peripheral protein, the targeting of these integral membrane
proteins involves more than one region in the protein. However, the
ability of the large-size fusions of NLS--galactosidase-otefin
(~170 kDa) to reach the inner nuclear membrane suggests that in the
COS-7 cells, they did not enter the nuclear envelope by lateral
diffusion. Nevertheless, since otefin is probably small enough for
lateral diffusion, we cannot rule out the possibility that in
vivo, a fraction of otefin is targeted to the inner membrane by
association with other inner membrane proteins at the endoplasmic
reticulum.
We thank Drs. D. Picard, K. R. Yamamoto, and N. Benvenisty for the pSP64-497-524.Z and pCA1038 constructs. We also thank Hermona Soreq, Pierre Goloubinoff, and Amnon Harel for valuable discussions.