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INTRODUCTION |
Bidirectional transport between the nucleus and the cytoplasm is
mediated by nuclear pore complexes
(NPCs)1 (1, 2). These massive
structures extend across the nuclear envelope (NE) and are bound to the
pore membrane domain (see Refs. 1-3). Integral membrane proteins
associated with the NPC are speculated to play an important role in the
molecular organization of the NPC and its biogenesis. In metazoan
cells, two pore membrane proteins have been identified and
characterized: gp210 and Pom121p (4-7). An important step in
evaluating the function of these proteins and their interactions with
other components of the NPC has been the determination of their
topology within the pore membrane domain. Both gp210 and Pom121p have
been proposed to be type I integral membrane proteins, i.e.
they contain a single transmembrane segment with their N terminus
positioned in the lumen of the NE and their C terminus located within
the nuclear pore in a position to directly interact with other NPC
proteins (termed nucleoporins; Refs. 5, 8, and 9). A marked difference
between these two proteins is the size of their lumenal and pore-side
domains. Whereas most of the mass of gp210 is positioned in the lumen
of the NE (5, 8), the majority of Pom121p is on the pore side of the
membrane, including a region containing repetitive sequences of the
type FXFG also found in a subset of nucleoporins (6, 9).
Regions of gp210 and Pom121p responsible for their localization to the
pore membrane domain have been identified. It has been proposed that
after their integration into the endoplasmic reticulum membrane, these
proteins laterally diffuse through the lipid bilayer to their
destination within the pore membrane (10). For gp210, the transmembrane
segment is the dominant sorting determinant for its targeting to the
pore membrane (10). Its short cytoplasmic tail, however, also appears
to contain sorting information. Although directing its integration into
the endoplasmic reticulum membrane system, the transmembrane segment of
Pom121p does not act as a pore membrane sorting determinant. Instead,
the sorting signal is present in the pore-exposed portion of Pom121p
(11).
Whereas several nucleoporins found in vertebrates have homologues in
yeast, neither gp210 nor Pom121p shares any significant sequence
similarity with open reading frames present in the yeast genome.
However, we have recently identified an integral pore membrane protein
in yeast termed Pom152p (12). It represents one of the most abundant
proteins in the highly enriched yeast NPC fraction. Surprisingly, a
yeast strain lacking the POM152 gene is viable, suggesting
that Pom152p might be a member of a group of functionally overlapping
proteins. This phenotype was utilized in genetic screens to identify a
number of genetically interacting nucleoporins including Nup170p (13),
Nup188p (14, 15), Nup59p (16), and the previously identified
nucleoporin Nic96p (13, 17). Like Pom152p, each of these proteins is an abundant constituent of the yeast NPC. Their abundance has led us to
hypothesize that they are components of the repetitive substructures that form the 8-fold symmetrical core of the NPC (15). However, the
functional basis for the observed genetic interactions between Pom152p
and these nucleoporins remains unclear.
In a continuing effort to understand the function of Pom152p, we have
conducted a series of experiments aimed at defining its topology in the
pore membrane and examining functional interactions between its
different domains and several of the abundant nucleoporins. Our data
demonstrate that Pom152p is a type II integral membrane protein with
its N-terminal domain positioned on the pore side of the membrane and
its C terminus located within the lumen of the NE. With this
information, we tested a series of deletion mutations to identify
regions of Pom152p that are capable of rescuing the viability of
strains containing mutations allelic to NUP170, NIC96, NUP188, and NUP59. Although the
N terminus in each case is necessary to rescue the mutants examined,
differing requirements exist for the transmembrane segment and the
lumenal domain.
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Media--
The yeast strains used in this
study are listed in Table I. All strains
were grown in either YPD (1% yeast extract, 2% bactopeptone, and 2%
glucose) or synthetic minimal media supplemented with the appropriate
amino acids, bases, and 2% glucose. 5-FOA (Toronto Research
Chemicals)-containing plates were made as described previously (18).
All strains were grown at 30 °C. Procedures for yeast manipulation
were conducted as described previously (18).
Plasmids--
The plasmids used in this study are as follows:
(a) pRS315, CEN/LEU2 (19); (b)
pPOM152, pRS315 containing the POM152 gene (13); and
(c) pPOM152-HA, pRS315 containing HA-tagged
POM152 (termed pPM1-HA in Ref. 12). Several deletion
variants were constructed from pPOM152 or pPOM152-HA by restriction
enzyme digestions or by using PCR techniques and are listed in Fig. 1.
In the case of the deletion constructs p170-301-HA and p170-1337-HA,
the POM152 promoter and the first two codons of
POM152 are followed in frame by the codon for amino acid
residue 170.
Preparation of Crude Nuclei, Nuclei, and NEs--
Yeast nuclei
and NEs were isolated using the procedure of Kilmartin and Fogg (20)
with more recent modifications (21). For the isolation of crude nuclei,
we used the following mini-scale procedure. Cells were grown in the
appropriate media to mid-log phase. All of the following steps were
performed at 4 °C, unless otherwise indicated. Cells were harvested
from a 100-ml culture (~1-2 × 109 cells) and
washed two times with water and two times with 1.1 M
sorbitol. The cells were then resuspended in 1 ml of 1.1 M
sorbitol. To digest the cell wall, 40 µl of glusulase (DuPont) and 10 µl of 1% zymolyase (ICN Biomedicals Inc.) were added, and the cells were incubated for 2-3 h at 30 °C with occasional shaking. After digestion, spheroplasts were collected by centrifugation (5,000 × g for 20 min), washed one time with 1.1 M
sorbitol, and resuspended in 800 µl of 1.1 M sorbitol.
This cell suspension was then layered over a cushion of 7.5% Ficoll
400 in 1.1 M sorbitol and then sedimented at 5,000 × g for 20 min. Spheroplasts were then resuspended in 1 ml of
8% polyvinylpyrrolidone in 0.5 mM MgCl2 and 20 mM KH2PO4, pH 6.5, (PVP medium)
containing 1% of solution P (0.1 M phenylmethylsulfonyl fluoride and 0.4 mg/ml pepstatin A (Boehringer Mannheim)) and lysed
with a Polytron (4 × 30 s). 1 ml of 0.6 M
sucrose in PVP medium containing solution P was then added, and nuclei
were sedimented at 10,000 × g for 10 min. The pellet
containing crude nuclei was resuspended in 500 µl of 1.5 M sucrose, 0.1 mM MgCl2, and 10 mM bis-Tris, pH 6.5. This procedure typically yielded ~1
mg of crude nuclei.
Alkaline Extraction--
100 µl of yeast crude nuclei
(~100-200 µg of protein) were diluted with 400 µl of BTM buffer
(10 mM bis-Tris, pH 6.5, and 0.1 mM
MgCl2) containing 1 mM phenylmethylsulfonyl
fluoride. 500 µl of 40 mM NaOH, pH 12.0, were added and
incubated on ice for 20 min. Extracted proteins were separated from the
membrane fraction by centrifugation at 100,000 × g for
30 min. Proteins in the supernatant were precipitated by the addition
of trichloroacetic acid to a final concentration of 10% (v/v). This
precipitate and the membrane pellet were solubilized in SDS sample buffer.
Trypsin Digestion--
Yeast NE (or nuclei) containing
Pom152p-HA or the p170-1337-HA deletion variant were diluted in BTM
buffer and sedimented through a sucrose cushion (1 M
sucrose in BTM buffer) at 100,000 × g for 15 min. The
pellet was then resuspended in BTM buffer and divided into equal
aliquots. These aliquots were incubated with different concentrations
of trypsin (from 0 to 500 µg/ml) in the presence or absence of 1% of
Triton X-100 for 20 min at 0 °C. The reaction was stopped by the
addition of trichloroacetic acid (to 10%) or solution P (to 1%) and
soybean trypsin inhibitor (Boehringer Mannheim). To examine the
membrane association of protease protected fragments of Pom152p, the
membranes were sedimented through a sucrose cushion (1 M
sucrose in BTM buffer) at 100,000 × g for 15 min after
trypsin digestion and the addition of trypsin inhibitors. The pellet
was then washed with BTM buffer, and alkaline was extracted as
described above.
Endoglycosidase H Treatment--
Crude yeast nuclei (~100 µg
of total protein) were washed several times with BTM buffer and then
resuspended in endoglycosidase H (Endo H) buffer (50 mM
sodium citrate, pH 5.5, 0.1% SDS, and 1 mM
dithiothreitol). Samples were incubated at 75 °C for 20 min to
denature proteins and solubilize membranes. 5 milliunits of Endo H
(Boehringer Mannheim) or a mock solution as a control and phenylmethylsulfonyl fluoride to a final concentration of 1 mM were added. Samples were then incubated overnight at
37 °C. Proteins in the reaction mixtures were then recovered by
trichloroacetic acid precipitation and solubilized in SDS sample buffer.
Western Blot Analysis--
Proteins were separated by
SDS-polyacrylamide gel electrophoresis and then electrophoretically
transferred to nitrocellulose membranes (Hybond-C; Amersham). Antibody
incubations using the monoclonal antibodies mAb 12CA5 and mAb118C3 and
subsequent washes were performed in phosphate-buffered saline
containing 0.1% Tween-20. Binding was detected using enhanced
chemiluminescence with horseradish peroxidase-conjugated sheep
anti-mouse IgG or rabbit anti-mouse IgG followed by horseradish
peroxidase-labeled donkey anti-rabbit IgG (Amersham). For the Western
blot analysis of total cell lysates, cells from liquid cultures were
collected by centrifugation. Pellets were resuspended in 2 M NaOH containing 8%
-mercaptoethanol and incubated on
ice for 10 min. Proteins were precipitated with trichloroacetic acid,
washed with acetone, resuspended in SDS sample buffer, sonicated, and
separated by SDS-polyacrylamide gel electrophoresis.
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RESULTS |
Expression of POM152 Deletions and Mapping of the mAb118C3 Binding
Site--
A series of POM152 deletion mutations were
constructed to examine the functions of different domains of Pom152p.
Schematic representations of these deletions are shown in Fig.
1. The majority of these constructs
contain two tandem repeats of an epitope derived from the HA antigen
inserted after amino acid residue 295 (12) that are recognized by the
monoclonal antibody mAb 12CA5. Plasmids containing POM152-HA
and the different deletion variants were introduced into a
pom152-null strain (PMY17). Synthesis of the encoded
proteins was evaluated by Western blotting of total cell extracts using
mAb 12CA5 (Fig. 2A). With the
exception of the p1-1219 construction, which lacks the HA tag, each of
the deletion variants was detected with mAb 12CA5 and was visible
migrating at an apparent molecular mass near that predicted by their
sequence.

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Fig. 1.
Schematic representations of the Pom152p
deletion variants. Names of constructions are shown to the
left. Numbers indicate the positions of the amino
acid residues in Pom152p. and indicate the approximate position
of the two potential transmembrane segments (residues 111-131 and
176-195, respectively; see Fig. 3). The asterisk indicates
the location of the HA epitope.
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Fig. 2.
A, expression of Pom152p-HA deletion
variants. Total cell lysates derived from a pom152-null
strain (PMY17) expressing the indicated deletion constructs were run on
an 8% SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose membranes, and probed with the anti-HA monoclonal
antibody mAb 12CA5. Molecular mass markers are indicated in
kilodaltons. B, localization of the Pom152p epitope
recognized by the monoclonal antibody mAb118C3. Total cell lysates
derived from a pom152-null strain (PMY17) expressing the
indicated deletion constructs were processed as described in
A and probed with the mAb118C3. The asterisk
denotes the position of the p 295-1036 protein. Molecular mass
markers are indicated in kilodaltons.
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Using these deletion variants, we have mapped by Western blotting the
region of Pom152p that is recognized by mAb118C3 (Fig. 2B),
a previously identified monoclonal antibody that binds specifically to
Pom152p (21). We observed that C-terminal truncations as short as 118 amino acid residues (including p1-1219 and p1-1025-HA) abolished
mAb118C3 binding (Fig. 2B). Conversely, all deletion constructions that contained the last 301 amino acid residues of
Pom152p (including p170-1337-HA, p
176-263-HA, and
p
295-1036) bound to mAb118C3, suggesting that this antibody binds to
an epitope within this region. Moreover, taking into account that
variant p1-1219 is functionally expressed in yeast (see the
complementation data below), it is likely that the epitope for mAb118C3
is located within the last 118 amino acid residues of Pom152p.
Topology of Pom152p in the Pore Membrane--
On the basis of its
primary structure, we previously proposed that Pom152p is a type II
integral membrane protein containing a single transmembrane segment
(TMS) with a pore-side N-terminal domain and a lumenal C-terminal
segment (12). However, this and other plausible models have not been
experimentally tested.
As a first step in defining its topology, we have attempted to identify
the region(s) of Pom152p that acts as a transmembrane segment(s).
Hydropathy analysis of Pom152p (Fig.
3A) revealed two hydrophobic
regions of sufficient length to serve as TMSs extending from amino acid
residues 111-131 and 176-195. To test the ability of each of these
regions to act as a membrane anchor, alkaline extraction experiments
were performed on crude nuclear fractions isolated from yeast
expressing deletion constructions of POM152 encoding one or
both of the potential TMSs. Consistent with previous results (12),
full-length Pom152p was resistant to extraction and was quantitatively
present in the membrane pellet, whereas peripheral nucleoporins,
including Nup116p, were extracted (Fig. 3B). Similarly, a
truncated form of Pom152p containing the N terminus and both of the
potential TMSs, p1-301-HA, was resistant to alkaline extraction and
remained associated with the membrane. However, the ability of Pom152p
to integrate into the membrane was dependent on the 176-195 segment
alone. A deletion construct lacking the 111-131 segment and containing
the 176-195 segment (p170-1337-HA) was resistant to alkaline
extraction. Conversely, a deletion mutant lacking the 176-195 segment
and containing the 111-131 segment was quantitatively extracted from
the membrane (p
176-263-HA; Fig. 3B). These results
suggest that Pom152p contains a single TMS. We therefore concluded that
the molecule is divided into three segments: (a) an
N-terminal segment (consisting of amino acid residues 1-175),
(b) a TMS (amino acid residues 176-195), and (c)
a long, C-terminal segment (amino acid residues 196-1337).

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Fig. 3.
A, hydropathy plot of Pom152p.
Hydropathic values of individual amino acid residues were averaged
within a 23-amino acid residue window as described by Kyte and
Doolittle (26) using the Strider program. Arrows indicate
the position of two hydrophobic peaks centered within two potential
transmembrane segments extending from amino acid residues 111-131
(1) and 176-195 (2). B, membrane
integration of Pom152p deletion constructs. Crude nuclei were isolated
from PMY17 cells expressing various POM152 deletion
constructs. Nuclei were then extracted with 20 mM NaOH.
After centrifugation, polypeptides in aliquots of the supernatant
(S) and the membrane pellet (P) fractions were
analyzed by Western blotting using the anti-HA antibody mAb 12CA5
( -HA). The efficiency of the extraction was evaluated by
probing the p1-301-HA-containing samples with an anti-GLFG polyclonal
antibody ( -GLFG) (the position of Nup116p is indicated).
Molecular mass markers are indicated in kilodaltons.
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The presence of a single membrane anchoring domain suggested four
possible orientations for Pom152p in the membrane (Fig. 4). Models I and II represent a situation
in which the hydrophobic segment crosses the membrane, whereas in
models III and IV the anchoring domain loops into the membrane. To
assess the topology of Pom152p, we performed limited protease digestion
experiments of NEs containing Pom152p-HA. We predicted that the
cisternal domain of Pom152p should be largely protected by the nuclear
membrane, whereas a cytoplasmic, pore-exposed segment would be
accessible to the protease and would be at least partially
degraded.

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Fig. 4.
Hypothetical models for the topology of
Pom152p in the pore membrane. Four potential topological models
for Pom152p within the pore membrane are shown. N and
C indicate the position of the N terminus and the C terminus
of the protein. and indicate the approximate positions in
Pom152p-HA of epitopes recognized by the monoclonal antibodies mAb118C3
and mAb 12CA5, respectively. A thick black line denotes the
transmembrane segment.
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As shown in Fig. 5, the digestion of
intact NEs with trypsin produced a fragment of Pom152p-HA with an
apparent molecular mass ~7 kDa less than that of the wild-type
protein (termed Pom152p-7). The Pom152p-7 fragment was largely
resistant to proteolysis at various concentrations of trypsin, and it
was recognized by both mAb 12CA5 and mAb118C3. Because these antibodies
bind epitopes that lie near opposite ends of the C-terminal segment, it
is likely that Pom152p-7 contains the majority, if not all, of this
segment. Under similar conditions, the nucleoporins Nup53p and Nup188p were rapidly degraded (data not shown). The Pom152p-7 fragment, however, was quickly degraded in the presence of Triton X-100. In this
case, several distinct fragments were detected with mAb 12CA5 and
mAb118C3, suggesting that the C terminus was now accessible to the
protease. To a far lesser degree, these smaller fragments were also
present in intact NEs treated with high concentrations of trypsin (50 and 500 µg/ml). This likely reflects the accessibility of the
protease to the perinuclear cisterna in unsealed NEs.

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Fig. 5.
Accessibility of Pom152p to exogenous
protease. NEs were isolated from PMY17 cells containing Pom152p-HA
and treated with different concentrations of trypsin in the absence
( ) or presence (+) of 1% Triton X-100. After incubation for 15 min
at 0 °C, all digests were terminated by the addition of
trichloroacetic acid. The trichloroacetic acid precipitates were
analyzed by Western blotting using the monoclonal antibody mAb118C3 or
mAb 12CA5. Molecular mass markers are indicated in kilodaltons.
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To further evaluate whether the Pom152p-7 fragment is the result of a
cleavage at the N terminus or the C terminus, we performed similar
trypsin digestion experiments on nuclear fractions isolated from cells
expressing Pom152p-HA or a deletion variant lacking the N terminus
(p170-1337-HA). As shown in Fig.
6A, the molecular mass of
p170-1337-HA was unaffected by trypsin treatment, whereas full-length
Pom152p was cleaved to the Pom152p-7 form. These results demonstrate
that the cleavage that generates Pom152p-7 occurs at the N terminus and
that the TMS and the C terminus are protected. Interestingly, the
protease-resistant Pom152p-7 truncation migrated more slowly than the
p170-1337-HA deletion, suggesting that the Pom152-7 fragment contains
a portion of the N terminus.

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Fig. 6.
A, resistance of p170-1337-HA to trypsin
digestion. Crude nuclei were isolated from PMY17 cells containing
Pom152p-HA and p170-1337-HA (lacking the N terminus). Samples were
treated with (+) or without ( ) trypsin (50 µg/ml). Immediately
after addition (0) or after a 15-min incubation at 0 °C
(+), all digests were terminated by the addition of trichloroacetic
acid. The trichloroacetic acid precipitates were analyzed by Western
blotting using the monoclonal antibody mAb 12CA5 ( -HA).
B, membrane association of the Pom152p-7 trypsin fragment.
NEs containing Pom152p-HA were treated with (+) or without ( ) trypsin
(100 µg/ml). Trypsin-digested membranes were then subjected to
alkaline extraction. After centrifugation, aliquots of the supernatant
(S) and membrane pellet (P) fractions were
analyzed by Western blotting using mAb 12CA5 ( -HA).
Molecular mass markers are indicated in kilodaltons.
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On the basis of these results, we would predict that Pom152p-7 is
anchored to the membrane. To test this, alkaline extractions were
performed on NEs after treatment with a high concentration of trypsin
(100 µg/ml). As shown in Fig. 6B, Pom152p-7 was present within the pellet fraction, suggesting that it was anchored to the
membrane. Similarly, smaller fragments recognized by mAb 12CA5 were
also bound to the membrane, which is consistent with the location of
the HA epitope near the TMS.
The results of the experiments described above are consistent with a
model for the topology of Pom152p in which the N terminus is located on
the pore side of the membrane and the C terminus is positioned in the
lumen of the NE (Fig. 4, II). However, we could not exclude
the possibility that the C-terminal segment is positioned at the pore
side of the membrane and is highly protected from proteolysis by other
NPC proteins (Fig. 4, IV). Two observations argue against
this model: (a) Pom152p binds concanavalin A, suggesting that it is glycosylated (12); and (b) concanavalin A
binding to Pom152p can be abolished by treatment with endoglycosidase H
(data not shown). The specificity of this enzyme for oligosaccharide modifications made in the lumen of the endoplasmic reticulum/NE suggests that in fact a domain of Pom152p lies within the NE lumen. To
clearly establish that the C-terminal segment is glycosylated, Endo H
treatment was performed on Pom152p-HA- and p170-1337-HA-containing nuclear fractions. The presence of N-linked oligosaccharides
was assessed by a change in the molecular mass of the proteins after Endo H digestion. As shown in Fig. 7,
Endo H treatment reduced the molecular masses of both Pom152p-HA and
p170-1337-HA by ~7 kDa. This reduction in mass corresponds to the
elimination of three to four polysaccharide chains from the
N-glycosylation sites. This is in agreement with the number
of potential sites (four) in the C-terminal region. These results
confirm that the C-terminal segment is N-glycosylated and is
localized in the lumen. Finally, in similar experiments performed with
a Pom152p mutant lacking the TMS (p
176-263-HA; Fig. 7), Endo H
treatment had no effect on the protein's mass, suggesting that the TMS
is necessary for translocation of the C-terminal segment into the lumen
of the endoplasmic reticulum/NE (Fig. 7). Together, these data
demonstrate that Pom152p is a type II integral membrane protein.

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Fig. 7.
Endoglycosidase H treatment of Pom152-HA and
deletion variants. Crude nuclei from PMY17 cells containing
Pom152p-HA, p170-1337-HA or p 176-263-HA (lacking the TMS) were
either mock-digested or treated with Endo H for 16 h at 37 °C.
After precipitation with trichloroacetic acid, the samples were
analyzed by Western blotting using the monoclonal antibody mAb 12CA5
( -HA). Molecular mass markers are indicated in
kilodaltons.
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Domain-specific Complementation of Pom152p Synthetic Lethal
Mutants--
We have previously identified a battery of mutants
allelic to the nucleoporin genes NUP59, NUP170,
NUP188, and NIC96, which are dependent on the
presence of Pom152p for their viability (13, 14, 16). This may reflect
the ability of Pom152p to perform a function similar to these
nucleoporins and/or stabilize structures effected by various
nucleoporin mutations. To determine what regions of Pom152p are
required to complement these various mutants, different deletion
constructions were tested for their ability to replace wild-type
Pom152p. Because each of the mutant strains is dependent on a
URA3-containing plasmid, pCH1122-POM152 (URA3),
they fail to grow on 5-FOA-containing plates (13, 14). Functional
complementation of these mutants by various truncated forms of Pom152p
was scored by their ability to rescue growth on 5-FOA-containing
plates. The results of these experiments are summarized in Table
II. As previously shown (13, 14, 16), all
of these mutants could be complemented by wild-type Pom152p (pPom152p).
In addition, strains containing the mutant alleles nic96-7
(psl7), nup170-21 (psl21), and nup59-40 (psl40)
were each rescued by all deletion variants containing the N terminus
and the TMS (amino acids residues 1-301), suggesting that this region
is sufficient for complementation. The TMS region, however, was not
strictly required to complement psl7 (nic96-7) and psl40
(nup59-40) because the construct p
176-263, which lacks
the TMS but contains the N terminus, also rescued growth on
5-FOA-containing media. However, in the absence of the TMS, the
majority of the C-terminal domain was required, because deletions in
this segment (p1025 and p424) abolished complementation of psl7 and
psl40. For the complementation of the psl21 (nup170-21) mutant, the N terminus and the TMS were both necessary and sufficient. Surprisingly, strains containing mutations allelic to NUP188
(psl4 and psl44) were rescued only by full-length POM152.
Although capable of complementing other psl mutants, all deletions of
the C terminus that we tested failed to complement psl4 and psl44.
These results suggest an important role for the lumenally disposed
C-terminal domain.
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Table II
Complementation of psl mutants with pom152 deletions mutants
The plasmid pRS315 alone or containing the indicated constructs was
introduced into various POM152 synthetic lethal mutants. The
resulting strains were then tested for growth on 5-FOA-containing
plates at 30 °C. A plus sign indicates growth, and a minus sign no
growth.
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DISCUSSION |
The data presented in this study provide experimental evidence for
the topology of Pom152p. In addition, we have used this information to
identify functional domains of Pom152p that are necessary for its
genetic interactions with other NPC proteins. Using Pom152p and various
deletion constructs in combination with alkaline extraction, protease
protection, and endoglycosidase H assays, we have demonstrated that
Pom152p is a type II integral membrane protein with its N-terminal 175 amino acid residues positioned within the nuclear pore and its
C-terminal 1141 amino acid residues located within the lumen of the NE.
Pom152p is anchored to the membrane by a single TMS extending from
amino acid residues 176-195. This segment is both necessary and
sufficient for its attachment to the membrane. Adjacent to the TMS, two
positively charged amino acid residues are positioned on its N-terminal
side, and two negatively charged amino acid residues flank the
C-terminal side. This charge distribution flanking the TMS is similar
to that observed in other type II membrane proteins and has been used
as a tool for predicting membrane protein topology (22-24).
As is the case for other type II membrane proteins, Pom152p does not
contain a cleavable N-terminal signal sequence for integration into the
endoplasmic reticulum membrane (12). Thus, the TMS of Pom152p is likely
to function both as a signal sequence and as a stop-transfer sequence
to integrate the protein into the membrane. Our data showing that all
of the constructs containing the TMS are integrated into the membrane
support this idea. Moreover, if a small region containing the TMS is
deleted, the remainder of the protein (p
176-263-HA) is no longer
resistant to alkaline extraction, and the C-terminal segment is not
glycosylated (Figs. 3 and 7).
The N-terminal 175 amino acid residues of Pom152p are located on the
pore side of the nuclear envelope membrane. This region is thus
positioned to directly interact with nucleoporins that compose the core
domain of the NPC. These are likely to include a subset of the abundant
nucleoporins that have been localized to the NPC core including
Nup188p, Nup170p, Nup157p, and Nic96p (13, 14). Consistent with this
observation, in mutants that are allelic to the NUP59,
NUP170, NUP188, and NIC96 genes and require
Pom152p for viability, the N terminus of Pom152p is uniformly required
for complementation. Interestingly, we observed that the N-terminal
truncation of Pom152p that is resistant to trypsin (Pom152p-7) migrates
on SDS-polyacrylamide gels with an apparent mass larger than a deletion
mutant lacking the N terminus (p170-1337-HA). This result can not be
explained by the positioning of trypsin cleavage sites (data not
shown). Instead, it is likely due to the partial protection of the N
terminus, perhaps by its association with other NPC proteins or the
periphery of the pore membrane.
Whereas the N terminus was necessary for the complementation of all of
the POM152 synthetic lethal mutants that we examined, the
requirements for the TMS and the lumenally disposed C-terminal segment
were dependent on the particular nucleoporin mutants examined. For
example, the complementation of the nic96-7 and
nup59-40 mutant alleles required the N terminus in
combination with either the TMS or the complete C-terminal segment. The
necessity for either of these two segments suggests that they can act
indirectly to sequester two or more N-terminal segments in a
conformation that is capable of binding nucleoporins. For wild-type
Pom152p, the spatial orientation of N termini may be established by
lateral interactions between adjacent TMSs (either homotypic or
heterotypic) and the C-terminal segments of neighboring Pom152p
molecules. By comparison, the complementation of the
nup170-21 mutant strictly required both the N terminus and
the TMS, suggesting that integration into the membrane is essential for
rescuing this mutant. Such varying requirements for the context in
which the N terminus is presented may reflect the involvement of
Pom152p in different stages of NPC biogenesis.
It is likely that the large lumenal domain of Pom152p (~85% of the
molecule) contributes a significant amount of mass to NPC structures
located in the lumen of the NE. In this regard, it is similar to the
mammalian pore membrane protein gp210 (5, 8), which has 95% of its
mass positioned on the lumenal side of the membrane. This region of
gp210 likely contributes to lumenal structures such as the lumenal
spokes or radial arms, and it has been proposed that this region plays
a role in pore formation and the maintenance of NPC structure (5, 8,
10). Moreover, antibodies to the lumenal domain of gp210 partially
inhibit nuclear import of classical nuclear localization
signal-containing substrates (25). Surprisingly, the complementation of
mutants allelic to NUP188 requires full-length Pom152p
including the lumenally disposed C-terminal segment. Even a deletion as
short as 118 amino acid residues from the C terminus abolishes
complementation. These results further underscore the importance of the
lumenal region of Pom152p in the function of its N terminus. Moreover,
they represent a functional link between structures located within the
lumen of the NE and those positioned on the pore side of the membrane.