From the
Lamina-associated polypeptides 1A-1C (LAPs1A-1C) are related
integral membrane proteins of the inner nuclear membrane that bind to
both A- and B-type lamins and have a putative role in the membrane
attachment and assembly of the nuclear lamina. In this study, we have
cloned a cDNA encoding LAP1C. The DNA sequence predicts a 506-amino
acid protein of largely hydrophilic character with a single
membrane-spanning region between residues 311-333. Mapping of the
epitope recognized by the anti-LAP1 monoclonal antibody RL13 indicates
that the hydrophilic domain containing residues 1-310 is exposed
to the nucleoplasm and thus that LAP1C is a type II integral membrane
protein. A second class of LAP1 cDNAs was isolated that contains two
protein-coding nucleotide insertions in the LAP1C sequence. These
probably encode parts of LAPs1A and/or -1B, suggesting that LAP1
isotypes arise from alternative splicing. Immunoblot analysis of mouse
P19 teratocarcinoma cells and the P19MES-differentiated derivative of
the latter suggest that LAP1 isotypes are differentially expressed
during development, similar to members of the nuclear lamin family.
Since the different LAP1 isotypes appear to bind lamins with different
affinities, these changes in expression could be important for
developmentally regulated alterations in nuclear structure.
The nuclear envelope (NE),
Mammalian lamins have been classified into A and B subtypes, based
on their sequence properties and state of membrane association during
mitosis. B type lamins (lamins B
Newly synthesized
lamins A, B
LAP2
is a
In attempts to isolate additional CO-related clones, CO was labeled
via the random-primed labeling method (Stratagene) and was used for
nucleic acid screening of this same unamplified library, yielding three
more independent clones. Six independent clones were found in a
Stratagene rat hepatoma library, and three more were isolated from a
rat macrophage library (generously provided by Dr. George Fey), all
using this initial clone as a nucleotide probe. Restriction digestion
and/or nucleotide sequencing indicated that seven of these clones
contained either one or both of the insertions characteristic of CO,
depending on whether they were sufficiently long enough to include both
insertion sites. However, none of these clones extended sufficiently in
the 5`-direction to yield cDNAs that would be predicted to encode the
amino-terminal ends of LAP1A or 1B. The longest clone containing these
two insertions had a 5`-end coinciding with nt 103 of LAP1C (predicted
to begin at residue 17 of LAP1C).
The original CL clone lacked a
guanine at nt 1264, compared with all other LAP1-related cDNAs that
were isolated (with or without the CO-like insertions). This would
result in a stop after amino acid 412 of LAP1C (Fig. 1). Since
all three LAP1 isotypes contain the sequence predicted at residues
466-479 of LAP1C as shown by reaction with anti-peptide
antibodies against this region, this nucleotide loss is very likely a
cloning artifact, and a CL clone reconstructed to contain this missing
nucleotide (Fig. 1) was used for this study.
We screened a cDNA expression library prepared from rat liver
poly(A)
To validate that clone CL
encoded a LAP1 isotype, we prepared polyclonal antibodies against a
trpE fusion protein containing residues 120-334 of the open
reading frame, as well as against a synthetic peptide encoding a second
region (residues 466-479) of the ORF (see ``Experimental
Procedures''). These antibodies were used to analyze various
mammalian cell and recombinant protein samples by immunoblotting (Fig.
2). RL13, the monoclonal antibody that originally defined the LAP1
antigens
(14) , recognized the
To further validate the identity of clone CL, we carried out
immunofluorescence staining of cultured rat cells with
affinity-purified antibodies against residues 120-334 of the ORF.
These antibodies stained the nuclear periphery in an approximately
continuous rim-like pattern (Fig. 3 c) that was identical to
the labeling obtained with RL13 (Fig. 3 a). In mitotic
cells where the NE was disassembled, the antibodies against residues
120-334 labeled the cytoplasm in a diffuse fashion (data not
shown), similar to the labeling of mitotic cells obtained with RL13
(14) . Thus, not only do the antibodies against the ORF of clone
CL react with LAP1 on immunoblots, they also specifically stain NEs in
a LAP1-like fashion. Considered together, these results indicate that
clone CL is a cDNA for a LAP1 isotype.
Consistent
with this view, Northern blot analysis with a radiolabeled fragment of
clone CL under high stringency conditions (see ``Experimental
Procedures'') detected three hybridizing species in
poly(A)
Kyte/Doolittle hydrophobicity analysis
revealed that most of LAP1C is hydrophilic (Fig. 1, bottom panel). It contains only a single hydrophobic stretch
sufficiently long to be a membrane-spanning segment, which is between
residues 311-333. This region is likely to be membrane-integrated
in vivo since LAPs1A-1C all fractionate as integral membrane
proteins resistant to extraction with harsh chaotrophs
(14) .
The LAP1C sequence contains 7 copies of the sequence (S/T)P XX,
which can be a recognition site for cdc2 kinase
(3) . All of
these sites occur in the large hydrophilic region on the amino-terminal
side of the putative transmembrane segment. We found no significant
similarity between the protein sequence of LAP1C and other translated
protein sequences from a data base search.
Assuming the sequence
between residues 311-333 is a membrane-spanning domain, two
topologies are possible for LAP1C in the inner nuclear membrane. In one
case, the sequence between residues 1-311 would be on the
nucleoplasmic surface of the inner nuclear membrane, and residues
334-506 would be on the lumenal surface. In a second scenario,
the orientation would be reversed. We previously localized the epitope
recognized by RL13 in isolated rat liver NEs and showed it to reside on
the nucleoplasmic surface of the inner nuclear membrane. To map the
epitope in the primary structure of LAP1C, we prepared several deletion
constructs of LAP1C, expressed these by in vitro transcription/translation, and determined whether they were
immunoadsorbed by RL13 (Fig. 7). Similar to the full-length LAP1C, a
fragment containing residues 1-325 was immunoadsorbed by RL13,
confirming the results of Fig. 2. Moreover, a construct from
which residues 129-303 were deleted was unreactive with RL13 and
appeared in the unbound supernatant (Fig. 7, lane 3,
asterisk). Together, these data demonstrate that the RL13
epitope, which is localized on the nucleoplasmic surface of the inner
nuclear membrane, occurs between residues 129-303 of LAP1C. Since
the RL13 epitope was shown previously to be localized to the
nucleoplasm
(14) , LAP1C appears to be a type II integral
membrane protein, containing a nucleoplasmic domain at residues
1-310, a transmembrane segment at residues 311-333, and a
lumenal segment at residues 334-506.
We examined the
expression patterns of the LAP1A-1C isotypes in several mouse cell
types reflecting different states of differentiation, using
affinity-purified polyclonal antibodies raised against residues
120-334 of rat LAP1C (Fig. 8). In mouse liver, three different
LAP1 species were detected by immunoblotting at
In this study, we have characterized rat LAP1C by cDNA
cloning and topological mapping and have begun an analysis of
developmental expression of LAP1 isotypes. LAP1C is predicted to
consist of 506 amino acids and is hydrophilic throughout most of its
length. It contains only one predicted membrane-spanning region, which
is found between residues 311-333. The epitope recognized by the
LAP1-specific monoclonal antibody RL13 was found to occur on the
amino-terminal side of this predicted transmembrane sequence. Since the
RL13 epitope is localized to the nucleoplasm
(14) , LAP1C
appears to be a type II integral membrane protein, with an
amino-terminal hydrophilic domain of
Up to now, we have been
unable to isolate full-length cDNAs encoding LAPs1A and -1B. However,
we have isolated cDNAs that appear to be partial length clones for one
or both of these. This class of cDNAs is identical to the LAP1C
sequence with the addition of two insertions. The first insertion,
which is found in the nucleoplasmic domain, places 27 novel amino acids
after amino acid 219 of LAP1C. The second insertion, which is found
immediately on the lumenal side of the predicted transmembrane
sequence, adds 51 novel amino acids after residue 334 of LAP1C and
results in loss of residue 335. These data, together with our results
that antibodies against two different LAP1 regions bind to all three
LAP1 isotypes, and our data that three different LAP1-like mRNAs are
detected on Northern blots by high stringency hybridization indicate
that different LAP1 isotypes are likely to arise from alternative
splicing. It should be noted that a hypothetical polypeptide containing
the LAP1C sequence with these two insertions would have a mass of 65.6
kDa, which is close to the mass of LAP1B estimated from SDS-gel
electrophoresis (68 kDa). However, we have not directly determined
whether LAP1B is represented by the LAP1C sequence with these two
insertions.
By comparing the LAP1 protein profiles in
undifferentiated mouse P19 teratocarcinoma cells, the differentiated
P19MES line, and mouse liver and 3T3 cells, we obtained evidence for
developmentally regulated expression of different LAP1 isotypes.
Assuming that the two larger M
Interestingly, a stretch of 27
residues encoded by the second sequence insertion into LAP1C that is
likely to be present in LAPs1A and/or -1B (the first 27 residues of the
insertion in Fig. 5 D) has a strong (>93%) probability
of forming a coiled-coil
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) U19614 and U20286.
We are grateful to Joanne Westendorf and other members
of our laboratory for helpful discussions throughout the course of this
work, to Dave Byrd for providing samples of isolated nuclear envelopes,
and to Christian Fritze for helpful comments on the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
the membrane
boundary of the nucleus in eukaryotic cells, consists of inner and
outer nuclear membranes, nuclear pore complexes, and nuclear lamina
(1) . The lamina is a filamentous protein meshwork lining the
inner nuclear membrane, which is thought to provide a structural
framework for the NE and an anchoring site at the nuclear periphery for
interphase chromosomes
(2, 3, 4) . The lamina
contains mainly a polymer of nuclear lamins, members of the
intermediate filament protein superfamily
(2, 3) . A
number of less abundant polypeptides are associated with the lamina as
well, including certain integral membrane proteins (see below).
and B
) are
present in somatic cells throughout development and remain membrane
attached during M-phase, while A type lamins (lamins A and C) are
expressed only during or following terminal differentiation in most
cells and are not membrane-associated during mitosis
(1, 3) . Recent studies have shown that both A- and
B-type lamins bind to chromatin
(5, 6, 7) or
DNA
(8, 9, 10) . Thus, it is likely that lamins
play a direct role in the attachment of chromatin to the NE, although
integral membrane proteins of the inner nuclear membrane also may
contribute to this interaction
(11) .
, and B
are isoprenylated at
carboxyl-terminal Caa X motifs, and this modification is
important for their targeting to the inner nuclear membrane
(2) . While the isoprenylated tail of lamin A is proteolytically
removed after the latter is assembled in the NE, B-type lamins remain
stably isoprenylated. Lamin-attached lipid is likely to promote the
association of the lamina with the inner nuclear membrane, possibly by
direct interaction with lipid bilayer. However, by analogy to other
systems involving protein prenylation
(12) , it is likely that
integral membrane proteins also are important for the lamina-inner
membrane interaction
(13) . Recent studies have identified
several integral membrane proteins localized to the inner nuclear
membrane that bind to lamins in vitro, which could play a role
in this lamina attachment. These include lamina-associated polypeptides
(LAPs)1 and -2 of mammalian cells
(14, 15) and a
54/58-kDa polypeptide found in avian and human cells
(16, 17, 18, 19) that is homologous to
sterol C14 reductase of Saccharomyces cerevisiae (4, 11) . Also, a 53-kDa protein (otefin) of the
inner nuclear membrane of Drosphila cells had been described
(20) , although its lamin binding has not been examined.
51-kDa polypeptide that specifically binds to both lamin B
and chromatin in a mitotic phosphorylation-regulated manner
(15) . cDNA cloning has shown that LAP2 contains a large
hydrophilic amino-terminal domain exposed to the nucleoplasm and a
single predicted membrane-spanning segment near its carboxyl
terminus.
(
)
Three different isotypes of LAP1
ranging in size from 55-75 kDa (LAPs1A, -1B, and -1C) have been
described in mammalian cells
(14, 15) . In this study,
we carried out cDNA cloning of LAP1C. We have found it to consist of
506 amino acids and have a substantially hydrophilic character, with
only a single putative transmembrane segment. Monoclonal antibody
epitope mapping combined with previous immunogold electron microscopy
demonstrated that LAP1C is a type II integral membrane protein with
approximately 60% of its mass exposed to the nucleoplasm. Analysis of
LAP1 expression in liver and cultured cells suggested that substantial
changes in the expression of LAP1 isotypes occur during development.
Since different LAP1 isotypes appear to bind lamins with different
affinities, this has important implications for understanding
developmentally regulated changes in the organization of the nucleus.
Isolation of cDNA Clones for LAP1
RNA
was isolated from rat livers (Sprague-Dawley males, 6-8 weeks)
using the guanidinium isothiocyanate method
(21) , and
polyadenylated RNA was prepared by chromatography on an oligo(dT)
cellulose column. The poly(A)RNA was used for
preparation of a cDNA library in the
LAPII expression vector (by
Marjorie Snead, Stratagene) using a mixture of random and oligo(dT)
primers. The cDNA library contained 3.3
10
primary
clones, with a background of 5% non-recombinants. Approximately 4
10
primary clones were screened with RL13
(
-LAP1) monoclonal antibody
(14) that had been preadsorbed
on an Escherichia coli/phage lysate. One positive clone with
an insert size of approximately 2 kb (clone CO) was isolated.
Oligonucleotides corresponding to the 5`-end of the clone CO were end
labeled with T4 polynucleotide kinase (Promega) and used to probe a rat
liver
gt11 cDNA library from Clontech, yielding a full-length
clone for LAP1C (clone CL). Clone CO differs from clone CL in that it
contains two additional short insertions of coding sequence. The first
is found after nucleotide 713 of clone CL and extends 81 nucleotides,
encoding 27 novel amino acids. The second is found after nucleotide
1059 of clone CL and extends 151 nucleotides, resulting in loss of
residue 335 of the CL sequence and addition of 51 novel amino acids.
Figure 1:
DNA sequence and deduced amino acid
sequence of LAP1C. The boxed amino acids comprise a predicted membrane-spanning region. The underlined amino acids were used to raise anti-peptide
antibodies in guinea pigs. Arrowheads indicate the two sites
where sequence insertions were found in multiple LAP1C-related clones
(see Fig. 5). The Kyte and Doolittle hydrophobicity plot (31) shown at
the bottom was generated using the GCG software package with an
11-amino acid window.
Plasmids were
rescued from ZAPII clones according to the manufacturer's
instructions; inserts were subcloned into pBluescript SK (Stratagene)
from
gt11 clones. All plasmids were propagated in XL-1 Blue cells
(Stratagene). DNA was sequenced using the dideoxy chain termination
method. Sequencing primers were synthesized as needed to obtain
sequence information from both DNA strands. Sequences were analyzed
using IntelliGenetics and GCG software.
Northern Blot Analysis
Total and
poly(A)RNA from HTC cells and poly(A)
RNA from rat liver were subjected to denaturing agarose gel
electrophoresis
(22) . After electroblotting onto HybondN
transfer membrane (Amersham Corp.) in 25 m
M sodium phosphate
buffer at 600 mA for 1.5 h, the RNA was cross-linked to the membrane
using ultraviolet light. The 5`- EcoRI fragment of clone CO
(containing base pairs 417-1059 of LAP1C plus additional insertions;
see above) was used to probe the blot. Hybridization was done in 5
SSPE
(22) , 50% formamide, 0.1% SDS, 0.5% nonfat dry
milk, and 100 µg/ml denatured, sonicated salmon sperm DNA at 37
°C. Filters were washed in 0.1
SSC
(22) , 0.1% SDS
at 55 °C.
Cell Culture
HTC, NIH 3T3, and P19 cells
were from ATCC, and P19MES cells
(23) were kindly provided by
Brian Burke. HTC, NIH 3T3, and P19MES cells were grown in
Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% fetal bovine serum, and P19 cells were
grown in -minimal essential Eagle's medium (Life
Technologies, Inc.) containing 7.5% bovine serum and 2.5% fetal bovine
serum.
In Vitro Translation and in Vivo Cell
Labeling
In vitro transcription and translation
was performed on both intact and linearized plasmid DNA using the
Promega TNT-coupled transcription translation kit according to the
manufacturer's instructions. For pulse labeling of cells in
vivo, HTC cells grown to 75% confluency were rinsed in
L-methionine- and
L-cysteine-deficient
Dulbecco's modified Eagle's medium (Sigma) supplemented
with 10% dialyzed fetal calf serum (starvation medium). Cells were then
incubated in starvation medium for 30 min at 37 °C. Starvation
medium containing 0.5 mCi/ml Tran
S label (ICN Biomedicals)
was added to the cells for 30 min, and cells were immediately harvested
as described for immunoprecipitation. For overnight labeling, cells
were maintained in labeling medium supplemented with cysteine and 20%
of the normal level of methionine with 50 µCi/ml Tran
S
label.
Preparation of NEs
NEs were isolated from
rat liver and were salt washed in a buffer containing 0.5
M NaCl as described
(24) . To prepare a NE-enriched fraction
from cultured cells, cell monolayers were scraped into cold
phosphate-buffered saline (PBS) (10 m
M sodium phosphate (pH
7.4), 140 m
M NaCl) and were pelleted at 100 g for 5 min. Cells were then washed by resuspension and pelleting in
cold homogenization buffer (HB) (10 m
M Tris-HCl (pH 8.0), 10
m
M KCl, 1.5 m
M MgCl
, 1 m
M dithiothreitol, and protease inhibitors (1 m
M phenylmethylsulfonyl fluoride and 1 µg/ml each of aprotinin,
leupeptin, and pepstatin)) and were swelled in one packed cell volume
of HB on ice for 20 min. Cells were then disrupted with 20-30
strokes in a tight fitting glass Dounce homogenizer. Nuclei were
pelleted at 500
g for 5 min, resuspended in five
packed cell volumes HB, given two more strokes in the Dounce
homogenizer, and repelleted. Crude nuclear pellets were then brought to
one cell volume of HB containing 50 µg/ml each of DNaseI and RNase
A. After 20 min in a room temperature water bath, the digested nuclei
were cooled to 0 °C. Next, an equal volume of 2
M NaCl, 10
m
M Tris-HCl (pH 8.0), 1 m
M dithiothreitol, and
protease inhibitors was added, and NEs were immediately pelleted out of
this solution at 2300
g for 10 min and frozen in
liquid N
.
Antibodies
Antibodies were raised in
rabbits against a trpE fusion protein containing a portion of LAP1. The
expression construct was made by ligating an EcoRI fragment
from clone CO into the PATH1 vector
(25) . The trpE fusion
protein contained the LAP1C region extending from residues
120-334 plus two short additional sequences: the 27 amino acids
of the first CO insertion (see above) and the first 5 amino acids of
the second. The fusion protein was expressed in E. coli,
purified by electrophoresis on an SDS gel, and used to immunize
rabbits. To prepare an affinity matrix for purifying anti-LAP1
antibodies from the rabbit antiserum, the EcoRI fragment from
clone CO (above) was subcloned into pGEX2T (Pharmacia Biotech Inc.).
This second fusion protein was expressed, purified on
glutathione-Sepharose, and coupled to CnBr-activated Sepharose
(Pharmacia) at 0.5-1.0 mg/ml according to the
manufacturer's instructions. Antibodies were adsorbed to the
affinity column for 1 h at room temperature and, after washing of the
column, were eluted with 3
M potassium thiocyanate, 0.5
M ammonium acetate, and 5 mg/ml bovine serum albumin
(26) .
Antibodies to a synthetic peptide corresponding to amino acids
466-479 of LAP1C were produced by coupling the peptide to keyhole
limpet hemocyanin and injecting the conjugate into guinea pigs.
Monoclonal RL13 IgG
(14) was purified on protein G-Sepharose
and coupled to CnBr-activated Sepharose at 3-5 mg/ml for use as
an immunoadsorbent.
Immunoadsorption
Extracts of cultured
cells for immunoadsorption were obtained by rinsing cells in PBS and
scraping into 1% Triton X-100, 20 m
M Tris-HCl (8.8), 500
m
M NaCl, 2 m
M EDTA, and protease inhibitors
(solubilization buffer). The cell lysate was sonicated briefly,
incubated on ice for 30 min, and spun at 265,000 g for
20 min to obtain a supernatant. Isolated rat liver NEs were solubilized
in a similar manner, while in vitro translation products were
simply diluted with 4 volumes of solubilization buffer. For
immunoadsorption of LAP1 antigens from these various extracts,
solubilized samples were incubated with RL13-Sepharose immunobeads
overnight at 4 °C. Antibody beads were then washed four times with
solubilization buffer and once with solubilization buffer lacking
Triton X-100. Bound proteins were eluted in SDS sample buffer lacking
dithiothreitol.
Gel Electrophoresis, Immunoblotting, and
Immunofluorescence Microscopy
SDS-polyacrylamide gel
electrophoresis was carried out on 10% polyacrylamide gels as described
(27) , and immunoblotting was performed essentially as
previously described
(24) . RL13 was used at 2.5 µg/ml and
was detected with peroxidase-conjugated donkey anti-mouse IgG (Jackson
Immunoresearch Laboratories Inc.) diluted 1:1000. Affinity-purified
antifusion protein antibody was detected with peroxidase-conjugated
donkey anti-rabbit IgG 1:1000 (Jackson). Guinea pig anti-peptide serum
against residues 466-479 of LAP1C was diluted 1:100. In
competition experiments, the diluted antibody was preincubated with 0.5
m
M immunizing peptide. Guinea pig antisera against lamins A/C
and lamin B were used at 1:500 dilution. Peroxidase-conjugated donkey
anti-guinea pig IgG 1:5000 (Jackson) was used to detect guinea pig
antibodies. ECL reagents (Amersham Corp.) were used to detect all the
secondary antibodies. For immunofluorescence microscopy, cells grown on
coverslips were fixed for 4 min in 4% formaldehyde in PBS and were
permeabilized for 4 min in 0.2% Triton X-100 in PBS. All antibodies
were diluted in 0.2% gelatin-containing PBS. RL13 was used at 20
µg/ml, and Texas red anti-mouse IgG (Molecular Probes Inc.) and
rhodamine anti-rabbit (Jackson) were diluted 1:200. Primary antibody
incubations were for 1 h at room temperature; secondary antibody
incubations were for 20 min.
mRNA with the LAP1-specific monoclonal
antibody RL13 to isolate clones encoding LAP1. A single
1.9-kb
positive clone was isolated from an initial screen. An oligonucleotide
corresponding to the 5`-end of this clone was used to screen a second
rat liver cDNA expression library, resulting in isolation of additional
cDNAs. DNA sequencing indicated that one of these clones (clone CL,
2307 nucleotides long) contained a large open reading frame (ORF) with
well defined start and stop sites that could encode a protein having
the approximate size of LAP1C. The nucleotide sequence of clone CL and
the deduced amino acid sequence of the large ORF are shown in Fig. 1.
The cDNA is predicted to encode a protein of 506 amino acids with a
calculated molecular weight of 56,696 and a pI of 6.42. It should be
noted that an in-frame stop codon is located nine nucleotides upstream
of the predicted start site, indicating that this clone encodes the
amino terminus of a protein-coding region.
75-,
68-, and
55-kDa bands corresponding to LAPs1A, -1B, and -1C, respectively,
in rat liver NEs (Fig. 2, RL13 panel, lane a) and in a sample of LAP1 antigens immunopurified from
NEs (Fig. 2, RL13 panel, lane b). In immunopurified LAP1 antigens from cultured rat
hepatoma (HTC) cells, RL13 recognized mainly LAP1C (Fig. 2,
RL13 panel, lane c), the only major
LAP1 isotype expressed in this cell type
(14) . However, a weak
LAP1A band also was seen in some experiments ( e.g. Fig. 2
,
peptide panel). RL13 reacted with a
number of weak bands in both rat liver and HTC cell samples, but these
varied in intensity between different preparations ( e.g. compare with Fig. 7) and most likely are proteolytic
products. Finally, RL13 reacted with a glutathione
S-transferase fusion protein containing residues 120-334
of the ORF, validating that the ORF we have isolated contains an RL13
epitope (Fig. 2, RL13 panel, lane d).
Figure 2:
Characterization of antibodies against
LAP1 by immunoblotting. Blots from 10% SDS-polyacrylamide gels were
incubated with the indicated antibodies (see ``Experimental
Procedures'' and text). Lanes a, salt-washed rat
liver NEs; lanes b, material immunoadsorbed from
salt-washed rat liver NEs with RL13; lanes c,
material immunoadsorbed from cultured HTC cells; lanes d, purified fusion protein containing amino acids
120-334 of LAP1C fused to glutathione S-transferase. The
positions of LAPs1A-1C and the fusion protein ( FP) are
indicated. comp., competition.
Figure 7:
Mapping the RL13 epitope in the LAP1C
sequence. Three LAP1C-derived constructs were used for in vitro transcription/translation. Products were immunoadsorbed with the
monoclonal antibody RL13 and analyzed by electrophoresis on a 10%
SDS-polyacrylamide gel and fluorography. Lane 1,
full-length LAP1C; lane 2, NcoI-truncated
LAP1C; lane 3, LAP1C from which the BbsI
fragment was removed. Shown are immunoadsorbed fractions (pellets) from
the three constructs and the unbound supernatant from BbsI
deletion only. The asterisk marks the specific in vitro translation product specific for construct 3, which is not found
in a control translation ( C). A schematic representation of
the DNA constructs is presented at the bottom of the figure.
The open box denotes the putative transmembrane
sequence, B marks the position of the BbsI
restriction sites at nt 430 and nt 962, and N shows the
NcoI site at nt 1019.
Affinity-purified antibodies against residues
120-334 of the ORF of clone CL reacted with a glutathione
S-transferase fusion protein containing this sequence, as
expected (Fig. 2, fusion panel). The antibodies
also reacted with three bands comigrating with LAPs1A-1C in isolated
rat liver NEs, as well as with immunopurified LAPs1A-1C of NEs and
immunopurified LAP1C of HTC cells (Fig. 2,
fusion
panel). Similarly, the anti-peptide antiserum against residues
466-479 reacted with three bands having the mobility of LAPs1A-1C
in isolated NEs, as well as with immunoadsorbed LAPs1A-1C of rat liver
NEs and immunopurified LAP1C of HTC cells (Fig. 2,
peptide
panel). The reaction of the anti-peptide antibodies with the LAP1
bands was specific, as antibody binding to these bands was selectively
abolished by including an excess of immunizing peptide in immunoblot
incubations (Fig. 2,
peptide + compet.
panel). Thus, polyclonal antibodies raised against two separate
regions of the ORF encoded by clone CL reacted specifically with all
three rat LAP1 isotypes, arguing that clone CL encodes a LAP1 species.
The data also support the possibility that the three LAP1 isotypes are
closely related, as previously suggested by their cross-reaction with
RL13.
Figure 3:
Immunofluorescence staining of cultured
HTC cells with antibodies against LAP1. HTC cells were grown on
coverslips, fixed with 4% formaldehyde, permeabilized with Triton
X-100, and labeled for immunofluorescence microscopy as follows:
panel a, RL13; panel b,
corresponding phase image of panel a; panel c, affinity-purified antibodies against residues
120-334 of the putative LAP1C. The anti-peptide antibodies raised
to residues 466-479 of putative LAP1C (Fig. 2) did not give a
signal in immunofluorescence microscopy (data not
shown).
The ORF encoded by clone CL
was expressed by coupled in vitro transcription/translation
and analyzed by SDS-gel electrophoresis (Fig. 4). The translation
product was found to have a very similar mobility to pulse-labeled
LAP1C that was immunoadsorbed from HTC cells (Fig. 4). Close
examination showed that the pulse-labeled LAP1C from the cultured
cells, which comigrated with LAP1C of rat liver NEs (data not shown),
migrated slightly (1 kDa) faster than the in vitro translation product (Fig. 4, compare lanes 1 and 2). This difference was confirmed by
coelectrophoresis of the two samples (Fig. 4, lane 3). Moreover, LAP1C immunoadsorbed from HTC cells that
had been continuously labeled for 16 h also migrated faster than the
in vitro translation product (Fig. 4, lanes 4 and 5). Hence, there is a small but
reproducible difference between the gel mobility of the in vitro translation product and LAP1C from cultured hepatoma cells. It is
conceivable that this is due to a posttranslational modification
associated with LAP1C of HTC cells that is absent from the in vitro translation product. In any case, because the in vitro translation product migrates very close to LAP1C of HTC cells, we
conclude that the ORF of clone CL very likely encodes LAP1C.
Figure 4:
Analysis of a LAP1C cDNA by in vitro transcription/translation of cloned LAP1C. Lane 1, protein immunoadsorbed by RL13 from an in vitro transcription/translation reaction containing LAP1C cDNA (Fig. 1);
lane 2, LAP1C immunoadsorbed from S
pulse-labeled HTC cells; lane 3, mixture of the
samples from lanes 1 and 2; lane 4, LAP1C immunoadsorbed from HTC cells that were labeled
with
S overnight; lane 5, mixture of the
samples from lanes 1-4. The open arrowhead marks the RL13-reactive product from in
vitro transcription/translation of cloned LAP1C; the closed arrowhead points to LAP1C immunoadsorbed from labeled
cells in culture.
We have
attempted to isolate cDNAs encoding LAP1A and LAP1B by nucleotide
screening of several cDNA libraries but have been unable to isolate
clones sufficiently large enough to encode these proteins.
Nevertheless, we have isolated eight independent LAP1C-like cDNAs that
are likely to contain partial length clones derived from either or both
LAPs1A and -1B (see ``Experimental Procedures''). This cDNA
class is identical in sequence to corresponding portions of the LAP1C
clone except that it contains insertions after nucleotides 713 and 1059
of LAP1C (indicated by arrowheads in the LAP1C sequence of
Fig. 1). The three clones of this group that are sufficiently long
enough to include both insertion sites contained both insertions. The
first insertion extends 81 base pairs and encodes 27 novel amino acids.
The second insertion extends 151 base pairs, resulting in loss of
residue 335 of LAP1C and addition of 51 novel amino acids (Fig. 5).
Considered together, these data suggest that the different LAP1
isotypes are likely to arise from alternative splicing.
RNA from HTC cells and rat liver at
2.25,
2.8, and
3.75 kb (Fig. 6). In rat liver, where the LAP1C
protein is relatively minor compared to LAP1A, the
2.25-kb RNA
species is substantially less abundant than the larger two species,
while in HTC cells where the LAP1C protein is very major compared to
LAPs1A and -1B, the
2.25-kb species is more abundant than the
larger two species. We conclude that the
2.25-kb
poly(A)
RNA probably represents the LAP1C mRNA, based
on the similarity of its size to clone CL and the correlation between
the relative abundance of the
2.25-kb species and the LAP1C
protein in different cell types. The two larger RNAs probably encode
LAPs1A and -1B, which are likely to be alternative splice variants of a
LAP1 transcript (see above).
82,
80, and
60 kDa (Fig. 8,
LAP1 panel). A similar
triplet of immunoreactive LAP1 species was expressed in mouse 3T3 cells
(Fig. 8,
LAP1 panel). The three mouse LAP1 bands
migrate significantly slower than comparable members of the LAP1
triplet from rat liver (Fig. 8,
LAP1 panel,
compare rat and mouse lanes). By contrast,
both lamin B
and lamins A/C of mouse essentially comigrate
with the homologous rat liver proteins (Fig. 8,
lamin
panels).
Figure 8:
Analysis of LAP1 isotype expression in
various cells and tissues. Shown are from salt-washed NEs from rat
liver and mouse liver (labeled rat and mouse) and
NE-enriched fractions isolated from mouse NIH 3T3 cells, mouse P19
teratocarcinoma cells, and mouse P19MES cells (a differentiated
derivative of P19 cells). Two times the amount of P19 protein is loaded
in the following lane ( 2 P19). Immunoblots
were probed with affinity-purified polyclonal anti-LAP1 antibody or
with guinea pig polyclonal antibodies against rat lamin B
or rat lamins A/C.
The most major LAP1 band of mouse liver was the
80-kDa species, while in 3T3 cells the overwhelming species was
the fastest migrating band of the triplet (the
60-kDa band;
Fig. 8
). This is similar to rat cells, where LAP1A (the slowest
migrating species) is the most prevalent band in liver, while LAP1C
(the fastest migrating species) is the only highly expressed band in
cultured cells
(14) . Additional evidence for developmentally
regulated expression of LAP1 isotypes came from comparing the LAP1
polypeptides of P19 cells, an undifferentiated teratocarcinoma cell
line, with those of P19MES cells, a differentiated derivative of P19
cells
(23) . As previously shown, P19 cells express lamin
B
but not lamins A/C (Ref. 28 and Fig. 8).
Interestingly, P19 cells express almost exclusively the
60-kDa
LAP1, while the differentiated derivative expressed a substantial
amount of the
80- and
82-kDa LAP1 species as well. Considered
together, these data indicate that different LAP1 isotypes, similar to
different lamin isotypes, are expressed in a developmentally regulated
fashion.
310 residues and a
carboxyl-terminal lumenal domain of
173 amino acids (Fig. 9).
Seven copies of the sequence (S/T)P XX, which is a potential
recognition site by cdc2 kinase
(29) , are found in LAP1C, and
all are localized to the nucleoplasmic domain. Thus, the
mitosis-specific phosphorylation described for LAP1
(15) could
involve these cdc2 sites. The function of mitotic LAP1 phosphorylation
is presently unknown. However, at least by itself, mitotic
phosphorylation of LAP1 does not detectably alter the binding of LAP1
to lamins in vitro (15) .
LAP1-related
polypeptides of mouse correspond to LAPs1A and -1B and the smallest
M
species represents LAP1C, our data showed that
LAPs1A and -1B are expressed strongly only in the differentiated cells,
similar to lamins A/C, while LAP1C is expressed in all the samples.
LAPs1A and -1B appear to interact more strongly with nuclear lamins
than LAP1C, based on in vitro binding of solubilized LAP1 to
nuclear lamins and relative susceptibility to extraction from the
nuclear lamina by nonionic detergent/salt treatment
(14, 15) . Thus, expression of LAPs1A and -1B in
differentiated cells could be important for promoting enhanced
stability of nuclear structure.
-helix when analyzed by the program of
Lupas et al. (32) . Coiled-coiled regions of a similar
size have the capacity to induce dimerization
(30) . If this
sequence specifies dimerization of LAP1 polypeptides, then its presence
in LAP1A and 1B but not in LAP1C could explain why LAPs1A and -1B
(hypothetical dimers) bind to polymeric lamins with higher avidity than
LAP1C (a hypothetical monomer).
Figure 5:
Insertions found in a class of LAP1 cDNAs
at two positions of LAP1C sequence. A, amino sequence of LAP1C
around the first insertion site at nt 713; B, deduced amino
acid sequence at the latter site containing the insertion; C,
amino acid sequence of LAP1C around the second insertion site at nt
1059; D, deduced amino acid sequence at the latter site
containing the insertion. In B and D, the residues
derived from the original LAP1C sequence are indicated in bold typeface, and the inserted sequences are indicated in
normal typeface. The sequence inserted at nt 1059
occurs at a mid-codon position, resulting in the loss of residue 335 of
LAP1C ( normal typeface in
C).
A number of potential functions can
be envisaged for LAP1. Because of its ability to specifically bind both
A- and B-type lamins, it could be involved in attaching the lamina to
the inner nuclear membrane and in targeting lamins to the NE during
interphase, thereby contributing to NE architecture
(15) . LAP1
(similar to LAP2) becomes concentrated at the surfaces of anaphase
chromosomes at early periods of nuclear membrane reassembly
(15) . Therefore, it could also play a role in NE reassembly by
helping to target lamins and membrane vesicles to chromosome surfaces
at the end of mitosis
(11) . The membrane topology of LAP1
revealed by our analysis, consisting of a single transmembrane sequence
and nucleoplasmic domain of substantial size, reflects an organization
that might be expected for a protein dedicated to attaching other
structures to the inner nuclear membrane. Moreover, LAP1 is
sufficiently abundant to carry out this function, since it is present
at an estimated 10% of the molar level of individual lamins in rat
liver NEs
(14) . The information reported in this study is
expected to provide the basis for a detailed investigation of the role
of LAP1 in NE structure and function.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.