(Received for publication, December 26, 1995; and in revised form, February 14, 1996)
From the
Employing avian erythrocytes, we have previously isolated a
multimeric complex consisting of the lamin B receptor (LBR, or p58),
the nuclear lamins, an LBR-specific kinase, a 34-kDa protein, and an
18-kDa polypeptide termed p18. As the LBR kinase and the 34-kDa
component have been recently characterized, we now proceed in the
characterization of p18. We show here that p18 is an integral membrane
protein specific to the erythrocyte nuclear envelope which binds to LBR
and B-type lamins. NH-terminal sequencing indicates that
p18 is distinct from other nuclear envelope components, but has
similarity to the mitochondrial isoquinoline-binding protein. In
situ analysis by immunoelectron microscopy and examination of
digitonin-permeabilized cells by indirect immunofluorescence show that
p18, unlike LBR and other lamin-binding proteins, is equally
distributed between the inner and outer nuclear membrane. Furthermore,
cycloheximide inhibition experiments reveal that the fraction of p18
that resides in the outer nuclear membrane does not represent nascent
chains en route to the inner nuclear membrane, but rather
material in equilibrium with the p18 that partitions with the inner
nuclear membrane. The paradigm of p18 suggests that transmembrane
complexes formed by the nuclear lamins and LBR provide potential
docking sites for integral membrane proteins of the nuclear envelope
that equilibrate between the rough endoplasmic reticulum and the inner
nuclear membrane.
Relatively few integral membrane proteins of the nuclear
envelope have been characterized so far (for reviews, see Gerace and
Foisner (1994) and Georgatos et al.(1994)). These include the
``lamin B receptor'' (LBR ()or p58) (Worman et
al., 1988; Bailer et al., 1991), the lamina-associated
polypeptides (LAPs) (Senior and Gerace, 1988; Foisner and Gerace,
1993), and a protein termed otefin (Padan et al., 1990).
Otefin has been identified in Drosophila cells, but its
properties are not yet known. However, some molecular information
concerning the LAPs and the LBR is available.
The LAP group of
proteins includes the so-called LAP1 A, LAP1 B, LAP1 C, and LAP2. LAP1
C and LAP2 are typical intrinsic membrane proteins with a single
(predicted) transmembrane domain and two hydrophilic end-regions
(Martin et al., 1995; Furukawa et al., 1995). LAP1 A
and B represent splicing variants of LAP1 C, but their exact amino acid
sequence has not been determined yet (Martin et al., 1995).
Data base searches show that LAP2 is identical to a previously cloned
protein, thymopoietin (Harris et al., 1994). Thymopoietin is
known to have important immunological functions (Goldstein, 1974;
Goldstein et al., 1979; Ranges et al., 1982) and is
expressed in many tissues, most abundantly in the thymus. It comprises
three distinct variants (,
, and
) generated by
differential splicing.
thymopoietin (which is identical to LAP2)
and
thymopoietin possess potential membrane-spanning domains and
are located in the nuclear envelope; however,
thymopoietin lacks
a hydrophobic region and seems to be nucleoplasmic (Harris et
al., 1994). The LAPs bind directly to lamin paracrystals under in vitro conditions; LAP1 A and 1B interact with all lamin
types, while LAP2 associates exclusively with B-type lamins (Foisner
and Gerace, 1993). LAP1 C does not show detectable binding to purified
lamins in vitro, but clearly associates with A-type lamins
under in vivo conditions (Powell and Burke, 1990).
Interestingly, LAP2 also interacts with isolated chromosomes in
vitro (Foisner and Gerace, 1993).
LBR possesses a long, charged
NH-terminal domain, eight potential membrane-spanning
segments, and a hydrophilic COOH-terminal region (Worman et
al., 1990; Ye and Worman, 1994; Schuler et al., 1994).
Its NH
-terminal domain, which is exposed to the
nucleoplasm, contains multiple phosphorylation sites (Simos and
Georgatos, 1992; Courvalin et al., 1992), DNA-binding motifs
(Worman et al., 1990; Ye and Worman 1994), as well as a
stretch rich in serine/arginine motifs (Simos and Georgatos, 1994). LBR
is widely expressed in human and avian cells (Bailer et al.,
1991; Chaudhary and Courvalin, 1993). In addition, three yeast proteins
that exhibit significant similarity to the vertebrate LBR have been
molecularly cloned (Chen et al., 1991; Lorenz and Parks, 1992;
Shimanuki et al., 1992). All three yeast proteins lack the
NH
-terminal domain of vertebrate LBR, and one of them is an
enzyme involved in ergosterol metabolism (Lorenz and Parks, 1992).
(Ergosterol is a fungal sterol not found in higher eukaryotes.)
Finally, a putative yeast homologue of vertebrate LBR has been
identified by immunochemical and biochemical means (Georgatos et
al., 1989), but it is not clear whether this polypeptide
corresponds to one of the already cloned proteins. LBR associates with
B-type lamins both in vitro and in vivo (Worman et al., 1988; Simos and Georgatos, 1992; Ye and Worman, 1994;
Smith and Blobel, 1994), consistent with its presumed function as a
``lamin receptor.''
In the terminally differentiated avian erythrocyte, LBR is known to form a multimeric complex, which includes the nuclear lamins, a specific kinase (LBR kinase), and two other polypeptides with molecular masses of 18 kDa (p18) and 34 kDa (p34) (Simos and Georgatos, 1992). Considering that the LBR complex might constitute a ``junctional'' assembly responsible for the coupling of the nuclear lamina to the inner nuclear membrane, we have undertaken a systematic effort to characterize the non-lamin nearest neighbors of LBR. As it turns out, p34 is the avian equivalent of a human nuclear protein known as p32 (Simos and Georgatos, 1994), which co-isolates with splicing factor 2 (SF2) (Krainer et al., 1991) and interacts with the human immunodeficiency virus 1 product Rev in vivo (Luo et al., 1994). Other data show that the LBR kinase is a resident protein of the nuclear envelope (Simos and Georgatos, 1992), which phosphorylates specifically serine/arginine dipeptide motifs present in LBR and in splicing factors (Nikolokaki et al., 1996). Finally, observations described below indicate that p18, the last component of the LBR complex to be characterized, is itself an integral membrane protein of the erythrocyte nuclear envelope that interacts specifically with LBR and B-type lamins. The implications of these observations in nuclear envelope structure and dynamics are discussed below.
Figure 1:
Partitioning of p18 upon cell
fractionation and determination of its NH-terminal
sequence. Subcellular fractions and extracts of turkey erythrocytes
were prepared as specified under ``Experimental Procedures.''
Samples of these fractions were then analyzed by SDS-PAGE and either
stained by Coomassie blue (A) or immunoblotted with a
polyclonal antibody against p18 (B). Panels show total
erythrocyte ghosts (G, lane 1), plasma membranes (M, lane 2), salt-washed nuclear envelopes (E, lane 3), urea extract of nuclear envelopes (S, lane 4), and urea-insoluble residue of nuclear
envelopes (P, lane 5). C,
NH
-terminal sequencing of p18. ``Authentic'' p18 (1, p18) isolated by immunoprecipitation from whole
cell lysates (Simos and Georgatos, 1992) and an 18-kDa protein purified
from electrophoresed urea-extracted nuclear envelopes (2, 18 kDa) were microsequenced in parallel. Comparison of the two
sequences indicates identity. Data base searches also indicate that
turkey p18 is similar to bovine IBP (3, IBP), an
18-kDa integral membrane protein known as the isoquinoline-binding
protein subunit of the PBRs. Identical residues (one-letter
code) between bovine IBP and turkey p18 are included in boxes. Conservative substitutions are indicated by double
dots. X indicates unknown amino
acid.
To find out whether the 18-kDa polypeptide
corresponded to p18, urea-extracted nuclear envelopes were resolved by
preparative SDS-PAGE and material was isolated either by electroelution
of gel pieces, or by electrotransfer and excision of the corresponding
band from blots (for technical details, see ``Experimental
Procedures''). The isolated 18-kDa protein was then microsequenced
in parallel to ``authentic'' p18 co-immunoprecipitated with
LBR from whole cell lysates (Simos and Georgatos, 1992) and purified in
the same way from SDS gels. The NH-terminal sequences of
the two proteins were identical (Fig. 1C).
Data base
searches using the NH-terminal sequence of p18 (34 residues
in the order
MWAYTVGFTVPHVGGFLGXFINRRETPVXYEXL; X = unknown amino acid) confirmed that p18 was neither a
degradation product of a known protein, nor a histone. The only
sequenced protein that exhibited similarity to p18 was a
17-18-kDa polypeptide, the isoquinoline-binding protein (IBP) (Fig. 1C), which had been characterized
previously as a component of the mitochondrial peripheral-type
benzodiazepine receptors (Sprengel et al., 1989; Riond et
al., 1989; Parola et al., 1991; Riond et al.,
1991; for comments on this, see ``Discussion'').
To
confirm the nuclear envelope localization of p18 in a more direct way,
we immunized rabbits and mice with electrophoretically purified protein
and raised one polyclonal and five monoclonal antibodies against it.
The antibodies precipitated an 18-kDa, detergent-soluble polypeptide,
which had the same NH-terminal sequence with p18. Using
Western blotting (Fig. 1B), we could readily detect p18
in fractions of whole erythrocyte ghosts (lane 1), salt-washed
nuclear envelopes (lane 3), and urea-extracted nuclear
envelopes (lane 5). However, p18 could not be detected in the
plasma membrane fraction (lane 2) or in urea extracts of
nuclear envelopes (lane 4). The same results were obtained
when turkey erythrocyte fractions were probed by the polyclonal or the
monoclonal antibodies. However, whereas the latter reacted equally well
with turkey and chicken red blood cells, the former reacted strongly
with turkey erythrocyte p18 and less strongly with chicken erythrocyte
p18. None of the antibodies reacted with material obtained from
mammalian cells (e.g. mouse erythroleukemia (MEL) cells,
Chinese hamster ovary (CHO) cells, and normal rat kidney (NRK) cells;
data not shown).
The NH-terminal sequence of p18
indicated the existence of a 20-amino acid stretch
(-WAYTVGFTVPHVGGFLGXFI-), which had features (i.e. size and hydrophobicity) of a membrane-spanning region. This,
combined with the fact that p18 could not be extracted from the nuclear
envelopes by 8 M urea or alkali, suggested that it represents
an intrinsic membrane protein. To substantiate this interpretation, we
extracted the nuclear envelopes with the detergent Triton X-114 at low
temperature and induced phase separation by warming up the solubilized
material at 37 °C. Immunoblotting of the various fractions showed
that the bulk of the p18 is solubilized by Triton X-114 and that the
extracted material partitions exclusively with the detergent phase (Fig. 2).
Figure 2: Extraction of p18 by Triton X-114. Turkey erythrocyte nuclear envelopes were extracted by Triton X-114 at 4 °C, and the extract was warmed up to 37 °C to induce phase separation between the detergent phase and the aqueous phase. The resulting fractions were analyzed by SDS-PAGE and either stained by Coomassie Blue (A) or immunoblotted with an anti-p18 polyclonal antibody (B). Panels show salt-washed nuclear envelopes (E, lane 1), insoluble fraction after extraction of the nuclear envelopes with 1% Triton X-114 (P, lane 2), total soluble fraction after extraction with 1% Triton X-114 (S, lane 3), aqueous phase of soluble fraction (Aq, lane 4), and detergent phase of soluble fraction (D, lane 5).
Figure 3: Subcellular distribution of p18 as detected by immunoelectron microscopy. a, turkey erythrocytes were fixed with formaldehyde, permeabilized by Triton X-100, and incubated with a polyclonal anti-p18 antibody and protein A-gold. Notice the heavy decoration of the nuclear surface and the lack of labeling in the area of the nuclear pores (arrows). Similar results were obtained with the monoclonal antibodies against p18. ``Unit membranes'' do not appear in this image due to removal of the lipids by the detergent; however, the nuclear lamina is clearly discernible as a thick electron dense layer and so is the fibrillar membrane-skeleton, which underlies the plasma membrane. Also visible in a are long 10-nm filaments, which seem to extend from the nuclear pores to the plasma membrane. These filaments show the same level of background staining with anti-p18 antibodies and preimmune sera. Inset shows a low power view of the cell depicted at high magnification in a. b, low power view of an immunodecorated ultrathin frozen section of a non-lysed erythrocyte that has been exposed to hypotonic media (10 mM sodium phosphate). Observe the dilated perinuclear cisterna and the heavy staining with anti-p18 antibodies along the inner and outer nuclear membrane. c, immunodecorated ultrathin frozen section of an turkey erythrocyte ghost depicting at high magnification the region of the nuclear envelope. Membrane profiles are clearly visible. Heavy staining with the anti-p18 antibody is observed along the inner (open arrowheads) and the outer (closed arrowheads) nuclear membrane, whereas the plasma membranes (PM) are not decorated. N indicates the cell nucleus. Bars correspond to 100 nm.
To differentiate between an inner or outer nuclear membrane localization, ultrathin frozen sections were prepared from non-extracted erythrocyte ghosts and processed for immunogold staining. Consistent with the previous data, the nuclear envelope was specifically decorated, whereas the plasma membrane was not. Upon closer examination, we noticed that both the inner and the outer nuclear membrane were stained by the anti-p18 antibodies (Fig. 3c). The partitioning of p18 with the inner and outer nuclear membrane could be better appreciated by examining intact (i.e. unlysed) erythrocytes in which the perinuclear cisternae were dilated by prior exposure to hypotonic media (Fig. 3b). Morphometric analysis of specimens such as the ones shown in Fig. 3(b and c) confirmed that equal numbers of immunogold particles were present in the outer and the inner nuclear membrane.
The localization of p18 on both sides of the nuclear envelope was unanticipated, because all other integral membrane proteins of the nuclear envelope so far characterized (LAPs and LBR) reside exclusively in the inner nuclear membrane. To ensure that p18 was indeed present in the outer nuclear membrane by a different method, we examined digitonin-permeabilized cells. Digitonin is known to permeabilize the cholesterol-rich plasma membrane without affecting the integrity of the nuclear envelope. Thus, in digitonin-treated cells, antigens exposed on the outer nuclear membrane are accessible to exogenously added antibodies, whereas antigens located in the inner nuclear membrane are not. Digitonin-treated or Triton X-100-permeabilized erythrocytes (control) were doubly-stained with anti-p18 and anti-lamin B antibodies and examined by indirect immunofluorescence microscopy. Data depicted in Fig. 4c show that the anti-p18 antibodies labeled readily the surface of the nucleus, yielding a ``patchy'' staining pattern. In contrast, the nuclear lamina of digitonin-permeabilized cells was not decorated (Fig. 4d). In good agreement with previous studies (Soullam and Worman, 1995), the nuclei of digitonin-permeabilized erythrocytes were not stained by anti-LBR antibodies confirming the exclusively inner nuclear membrane localization of this protein (data not shown). Upon Triton X-100 permeabilization, staining of erythrocytes with anti-p18 or anti-lamin B antibodies yielded the same smooth rim fluorescence pattern typically observed with nuclear envelope antigens (Fig. 4, a and b). From these experiments, it seems reasonable to conclude that p18 is exposed on both sides of the nuclear envelope. In retrospect, the partitioning of p18 with the inner and outer nuclear membrane explains why nuclear envelope fractions, which during isolation lose parts of the outer nuclear membrane, contain variable amounts of p18.
Figure 4: Localization of p18 in digitonin-permeabilized erythrocytes and in cells treated with cycloheximide. Turkey red blood cells were incubated in the absence (a-d) or presence (e and f) of 100 µg/ml cycloheximide (10 min to 2 h, 37 °C; only the 30-min sample is shown here). After this incubation, the cells were permeabilized with Triton X-100 (a and b), or digitonin (c-f). Panels on the left show indirect immunofluorescence patterns after staining with a polyclonal antibody against p18. Panels on the right show the same specimens decorated with a monoclonal anti-lamin B2 antibody. (For further explanations see text.) Bars correspond to 1 µm.
Unlike their mammalian counterparts, avian
erythrocytes are biosynthetically active. We could confirm that by
incubating mature turkey erythrocytes with
[S]methionine/cysteine in the presence and
absence of cycloheximide and performing immune precipitation
experiments with the anti-p18 antibodies (Fig. 5). To examine
whether the pool of p18 in the outer nuclear membrane represents
nascent chains en route to the inner nuclear membrane and
whether the protein can be ``chased'' from one membrane
compartment to the other (for pertinent information, see Bergmann and
Singer(1981), Torrisi and Bonatti(1985), and Torrisi et
al.(1987)), we performed the following experiment. Turkey red
blood cells were incubated at 37 °C for 10-120 min in the
presence or absence of cycloheximide, a protein synthesis inhibitor
that does not interfere with intracellular transport (Green et
al., 1981; Jamieson and Palade, 1968). At the end of these
incubations, the cells were permeabilized with digitonin and stained
with anti-p18 and anti-lamin B antibodies.
Figure 5:
Cycloheximide inhibition experiment.
Washed erythrocytes were incubated in minimal essential medium
containing [S]methionine and cysteine (10
mCi/ml) in the presence (+CX) or absence
(-CX) of cycloheximide. The cells were extracted with
PBS, 2 mM MgCl
, 1% Triton X-100, 1 mM PMSF, and the lysates were either analyzed directly by SDS-PAGE (a) or used for immune precipitation experiments (b).
Shown here is an autoradiogram of an SDS-polyacrylamide gel loaded with
the following samples. Lane 1, sample of molecular weight
markers; lane 2, total cell lysate of metabolically labeled
turkey erythrocytes (G); lane 3, Triton
X-100-insoluble pellet of metabolically labeled turkey erythrocytes (P); lanes 4 and 5, samples corresponding to 2 and 3 using erythrocytes that have been incubated
in the presence of cycloheximide; lanes 6 and 7,
Triton X-100-solubilized material from metabolically labeled
erythrocytes in the absence or presence of cycloheximide, respectively (S); lanes 8 and 10, immune precipitation
from the Triton X-soluble fraction using the specific anti-p18
polyclonal antibody; lanes 9 and 11, material
precipitated from the same lysates by preimmune
serum.
As shown in Fig. 4(e and f), cycloheximide treatment did not alter the immunostaining pattern observed before; p18 was still detectable on the outer nuclear membrane in digitonin-permeabilized cells. Validating this observation, cell counting showed that equal numbers of digitonin-permeabilized cells were decorated by the anti-p18 antibodies in untreated and cycloheximide-treated specimens. From these experiments, it can be inferred that p18 residing at the outer nuclear membrane does not represent nascent chains in transit.
Figure 6: Tissue distribution of p18. Frozen sections of chicken intestine (a-a"), chicken heart (b-b"), chicken liver (c-c" and e-e"), and turkey liver (d-d") were probed with a mixture of monoclonal antibodies recognizing p18 (a`, b`, c`, and e`), a polyclonal antibody against p18 (d`), or a polyclonal antibody against histone H5 (e"). Panels (a-e) on the left show DAPI staining of the DNA, while panels (a"-d") on the right show phase contrast images. Note that only erythrocytes are labeled. Apparent in a", b", and c" are blood vessels that contain packed red blood cells. Bars, 3 µm.
The appearance of the positively stained cells in the various tissue sections suggested that they were red blood cells. To substantiate this interpretation, liver sections were doubly stained with anti-p18 monoclonal antibodies and a polyclonal antibody against the erythrocyte-specific histone H5. Indeed, all anti-p18-positive cells were readily decorated with anti-H5 antibodies (Fig. 6, row e).
Figure 7:
Binding of radiolabeled p18 to LBR and
B-type lamins. Turkey erythrocyte fractions (PM, plasma
membranes; S, urea extract of nuclear envelopes; P,
urea-insoluble residue of nuclear envelopes) and a sample of molecular
weight markers (Mr; for molecular weights see Fig. 1)
were run on SDS-polyacrylamide gels, transferred to nitrocellulose
filters, and probed by I-p18 as specified under
``Experimental Procedures.'' After drying, the nitrocellulose
filters were exposed to x-ray film and autoradiograms prepared. A shows Coomassie Blue-stained gel; B shows an
autoradiogram of the nitrocellulose filter after ligand blotting. The
positive signals in the area of lamin B (LmB) and LBR (LBR) are indicated. In another experiment, identical samples
of I
-labeled p18 were incubated with 1 µg (lanes
1 and 2) or 2 µg (lanes 3 and 4) of
purified LBR and LBR was precipitated with affinity-purified aR1
antibodies in the absence (lanes 1 and 3) or the
presence (lanes 2 and 4) of the antigenic peptide R1.
The immunoprecipitates were analyzed by SDS-PAGE and either stained by
Coomassie Blue (C) or autoradiographed (D).
To
confirm these results by another method, we performed binding
experiments in solution using I-p18 and purified LBR. As
illustrated in Fig. 7B (lanes 1 and 3),
I-p18 bound to LBR and the binary complex of
the two proteins was readily precipitated by affinity-purified anti-LBR
antibodies (aR1). The specificity of this interaction was demonstrated
by performing the same experiment in the presence of the antigenic
peptide R1, against which the anti-LBR antibodies were raised. Under
these conditions, neither LBR nor
I-p18 were precipitated
with aR1 IgG (Fig. 7B, lanes 2 and 4). Consistent with a concentration-dependent binding, about
twice as much
I-p18 was co-precipitated with LBR when the
input LBR was doubled (Fig. 7B, compare lanes 1 and 3).
To rule out the possibility that binding to
LBR and lamin B was due to a minor I-labeled contaminant (e.g. a core histone), we repeated the binding experiments
employing a different approach. In this version of the binding assay,
purified nuclear envelope proteins (for SDS-PAGE profiles of the
preparations used, see Fig. 8A) were mixed in various
combinations and the complexes formed were subsequently precipitated
with affinity-purified anti-LBR or anti-lamin antibodies. The immune
pellets were run on SDS gels, blotted, and probed with anti-p18,
anti-LBR, or anti-lamin antibodies. As shown in Fig. 8B (lanes 1 and 5), unlabeled p18 and LBR were
co-precipitated by aR1. However, p18 could not be detected in the
immune pellet when LBR was omitted from the reaction (lanes 2 and 6). Co-incubation of p18 with lamin B and
precipitation with anti-lamin antibodies (aLI) yielded a small but
detectable amount of p18 in the immune pellet (lanes 3 and 7). This weak binding appeared to be specific, as no p18 was
seen in the corresponding control (lanes 4 and 8).
The specificity of the p18-lamin B interaction could be further
demonstrated by repeating the binding assay with equivalent quantities
of lamin B and lamin A (Fig. 8C). No binding of p18 to
nuclear lamin A was seen (lanes 3 and 6), whereas
binding of p18 to lamin B was readily detectable (lanes 2 and 5). Taken together, these data show that p18 binds
specifically to LBR and B-type lamins. Apparently, binding of p18 to
LBR is stronger than binding to the B-type lamins.
Figure 8: Binding of unlabeled p18 to LBR and nuclear lamin B. A, Coomassie Blue-stained gel showing the purified proteins used in the binding experiments. Designations are as in previous figures. M is a sample of molecular weight markers with values as specified in Fig. 1. The band at 36 kDa in lane 5 (asterisk) is a dimer of p18, which forms during electrophoresis and reacts with anti-p18 antibodies (G. Simos and S. D. Georgatos, unpublished results). B, binding assay in which p18 was incubated with LBR (lanes 1 and 5), lamin B (lanes 3 and 7), or buffer alone (lanes 2, 4, 6, and 8). The incubation mixtures were used for immune precipitation with affinity-purified aR1 or aLI antibodies, which recognize LBR and A/B lamins, respectively. The supernatants (after trichloroacetic acid precipitation, lanes 1-4) and the immune pellets (lanes 5-8) were analyzed by SDS-PAGE. LBR, lamin B, and p18 were identified by cutting the blots in half and performing Western blotting with the corresponding antibodies. C, binding assay in which p18 was incubated with buffer alone (lanes 1 and 4), with lamin B (lanes 2 and 5) or with lamin A (lanes 3 and 6). The reaction mixtures were immunoprecipitated with affinity-purified aLI antibodies. The supernatants (after trichloroacetic acid precipitation, lanes 1-3) and the immune pellets (lanes 4-6) were analyzed by immunoblotting using anti-p18 antibodies. Only the relevant parts of the blots are shown.
That about half of the p18 complement does not
co-localize (and thus does not interact) with LBR and the lamins
appears somewhat paradoxical. However, precedent for this exists in the
case of the carbonate/chloride exchanger (band 3 protein), an abundant
integral membrane protein of the erythrocyte plasma membrane. At any
one instance, only 10% of band 3 is associated with the
spectrin-actin membrane skeleton (via ankyrin), whereas
90% of it
is uncoupled (for a discussion, see Pinder et al.(1995)). At
this point we do not know whether the two subpopulations of p18 are
structurally identical, or whether this protein is post-translationally
modified in a compartment-specific manner.
As indicated by the cycloheximide inhibition experiments, the fraction of p18 that resides in the outer nuclear membrane does not represent nascent chains en route to the inner nuclear membrane. Instead, p18 equilibrates between the inner and the outer nuclear membrane. One explanation for this may be that, at some point, the abundance of p18 exceeds the binding sites provided by the the LBR complex. However, an alternative interpretation that need to be further investigated could be that resident proteins of the rough endoplasmic reticulum provide alternative binding sites for p18 and actively anchor this protein at the outer nuclear membrane.
This work is dedicated to Stavros and Adamantia Politis.