(Received for publication, May 9, 1995)
From the
We have identified a new yeast nucleoporin of 159 kDa that we
term NUP159. Immunofluorescence microscopy with a monospecific
monoclonal antibody against NUP159 gave the punctate nuclear rim
staining characteristic of nucleoporins. Immunogold electron microscopy
with isolated yeast NEs yielded decoration of only the cytoplasmic side
of the nuclear pore complex. The gene encoding NUP159 is essential,
and, like some other nucleoporins, NUP159 contains a coiled-coil domain
as well as a domain of repeated motifs. Five segments of NUP159,
covering its entire length, were expressed in Escherichia
coli. The repeat motif-containing segment was found to bind a
nuclear transport substrate in the presence of vertebrate cytosolic
extract containing nuclear transport factors. This segment also bound S-labeled mammalian karyopherin
, one such transport
factor that mediates the docking of substrates to the nuclear pore
complex. These data establish a direct biochemical link between the
repeat motif domain of a yeast nucleoporin, transport factors,
(specifically karyopherin
), and nuclear transport substrates. Its
cytoplasmic aspect implies a role for NUP159 in nuclear import.
Bidirectional transport of macromolecules across the nuclear
envelope proceeds via the nuclear pore complex (NPC). ()Several soluble transport factors that are required for
the uptake of transport substrate into the nuclei of
digitonin-permeabilized mammalian cells have now been isolated (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) ,
and progress has been made in characterizing the myriad of NPC proteins
(collectively termed nucleoporins) ((11, 12, 13, 14, 15, 16) ;
for review see (17) and (18) ). Most interestingly, a
biochemical link between the isolated transport factors and a subgroup
of nucleoporins has been established. Thus, a heterodimeric complex,
termed karyopherin(6, 7) or
importin(5, 8) , has been shown to bind to nuclear
transport substrates via its
subunit (7, 10) and
to bind repeat motif-containing nucleoporins via its
subunit(6, 10, 15) . Moreover, the transport
factor Ran (1, 2) has been shown to bind to the
mammalian nucleoporin Nup358, which is a component of the
>50-nm-long fibers that extend from the NPC into the
cytoplasm(16) . It has been proposed that the repeat
motif-containing nucleoporins form a stationary phase of multiple
docking sites that extend from the cytoplasmic to the nucleoplasmic
side of the NPC (6, 15) and that a Ran GTP/GDP cycle
controls docking and release to and from these binding sites from the
mobile phase of transport factors and
substrates(1, 2) .
Although there are also numerous repeat motif nucleoporins in Saccharomyces cerevisiae (for review see (17) and (18) ), a biochemical connection to soluble transport factors has so far not been established. Furthermore, although some vertebrate nucleoporins (including a few that contain repeat motifs) have been localized to substructures of the NPC (for review see (17) and (18) ), such sublocalization has not yet been achieved for any of the nucleoporins of yeast.
Here we report the identification of a new yeast nucleoporin of 159 kDa, termed NUP159. By immunoelectron microscopy, we sublocalized NUP159 to the cytoplasmic side of the NPC. NUP159 contains numerous short peptide repeats, similar to those found in other nucleoporins. We have expressed five segments of NUP159, covering its entire length, in Escherichia coli and shown that only the repeat motif-containing segment of NUP159 serves in mammalian transport factor-mediated binding of import substrate.
Microsequencing of a 50-kDa polypeptide present in the
highly enriched NPC fraction of yeast (19) yielded a peptide
sequence that was identical to residues 1106-1118 (Fig.1)
of an uncharacterized ORF YIL115C (EMBL/GenBank
/DDBJ
accession number Z38125) located on chromosome IX of S.
cerevisiae. The ORF is predicted to code for a protein of 1460
amino acids and a M
of 158,923. As this protein is
a nucleoporin (see below) we have termed it NUP159, consistent with
conventional nomenclature(28, 30) .
Figure 1: Sequence analysis. A, amino acid sequence obtained from a polypeptide in a fraction of highly enriched NPCs shows identity to an unknown ORF coding for a protein of 158,923 kDa that we have termed NUP159. Numbers to the right and to the left indicate the position of the peptide sequence in the ORF. B, NUP159 contains 28 repeat motifs. Besides the nucleoporin characteristic motif XFXFG, there are three other motifs; SAFG, PASG, and PSFG. The numbers indicate the first amino acid position of the repeated motif.
To investigate
if this gene is essential for cell growth, we transformed a linearized
fragment containing a LEU2-marked deletion allele into a
diploid strain to replace one copy by homologous recombination.
Leu transformants were verified by Southern analysis
and sporulated, and the tetrads were dissected. All tetrads yielded
only two viable segregants, both of which were Leu
,
suggesting that the NUP159
::LEU2 segregants were not viable (Fig.2).
Figure 2: Structural features of NUP159. A, NUP159 was expressed in E. coli in five different segments, p159-1 to p159-5. Segment p159-3 is the repeat motif domain, p159-4 contains the epitope recognized by the anti-NUP159 mAb 165C10, and p159-5 contains the putative coiled-coil domain. B, disruption of NUP159. The diagram shows the construct for the disruption of the gene by a LEU2 marker. Arrows indicate the direction of transcription of NUP159 and the marker. C, tetrad dissection. All tetrads (1-7) yielded only two viable segregants among four spores (a-d).
As with many nucleoporins, NUP159 contains a region with repeated motifs (Fig.1) including the XFXFG motif found in several other nucleoporins (for review see (17) and (18) ) as well as three additional related repeat motifs, namely PSFG, PASG, and SAFG (Fig.1). A computer homology search revealed that the motifs PSFG and SAFG were also found in another uncharacterized yeast ORF of 430 amino acids (GenPept accession number Z48784, ORF 9346.04c). NUP159 is 65% similar to this potential yeast nucleoporin across 20% of its length and 56% similar to Nup214 across 19% of its length(25, 33) . NUP159 is rich in serines (14%) and in potential phosphorylation sites. NUP159 also contains a potential coiled-coil domain (34) (Fig.2), which may be involved in homo- or heterodimerization, a feature found in some other nucleoporins (for review see (17) and (18) ).
For further analysis,
we expressed five segments, p159-1 to p159-5, covering the entire
length of NUP159 in E. coli ( Fig.2and 3A, a). Segment 3 contains the repeat motifs, and the C-terminal
segment 5 contains the potential coiled-coil domain of NUP159. We
screened a panel of monoclonal antibodies that had been obtained by
immunization of mice with NEs. One of these monoclonal antibodies (mAb
165C10) reacted with NUP159, specifically with the expressed segment
p159-4 (residues 877-1222) (Fig.3A, c). This
segment contains none of the repeat motifs. In contrast, the
polyspecific mAb 414 recognized the repeat motif domain (segment 3) (Fig.3A, b). mAb 165C10 reacted with a single
polypeptide of 190 kDa in a whole yeast spheroplast lysate or in yeast
nuclei (Fig.3B), indicating that the mAb is
monospecific for Nup159. During the preparation of NPCs and NEs, NUP159
appeared to be extraordinarily protease-sensitive. One breakdown
product appeared to be the 50-kDa polypeptide from which the
peptide sequence data were derived. For this reason we used the haploid
protease-deficient strain cl3-ABYS-86. Fractions from spheroplasts to
NEs were subjected to SDS-PAGE and transferred to nitrocellulose.
NUP159, detected with the monoclonal antibody, cofractionated
absolutely with the NEs (data not shown).
Figure 3:
Characterization of anti-NUP159 mAb
165C10. A, NUP159 was expressed in E. coli in five
different segments (p159-1 to p159-5), and the fractions containing the
expressed proteins (the inclusion body containing pellet for segments
1, 2, 4, and 5 (lanes 1, 2, 4, and 5, respectively) and the cytosol-containing supernatant for
segment 3 (lane 3) were subjected to a 12.5% SDS-PAGE. Arrows indicate the
isopropyl-1-thio--D-galactopyranoside-induced proteins (a). The same fractions were transferred to nitrocellulose (b and c) and incubated with the polyspecific mAb 414 (b), which recognizes the repeat motif domain (segment 3) in
NUP159. The NUP159-specific protein amounts of yeast spheroplasts (a) and nuclei (b) were separated by 7.5% SDS-PAGE,
transferred to nitrocellulose, and stained with mAb
165C10.
We used mAb 165C10 to localize NUP159. Formaldehyde-fixed yeast spheroplasts were incubated with the mAb for immunofluorescence microscopy, and a nucleoporin-characteristic punctate nuclear rim staining was observed (Fig.4).
Figure 4: Immunolocalization of NUP159. Formaldehyde-fixed yeast cells stained with the anti-NUP159 mAb 165C10 showed the punctate nuclear rim staining that is typical for nucleoporins (top). Coincident DAPI staining is shown (bottom). Bar, 5 µm.
For immunoelectron microscopy, isolated NEs were incubated with mAb 165C10, and the labeling was visualized with gold-labeled secondary antibodies. By using this pre-embedding immunolabeling technique in yeast, it was possible to sublocalize NUP159 with greater accuracy than post-embedding labeling methods (Fig.5). All of the 196 gold particles in the micrographs that could be unequivocally assigned to NPCs were found exclusively on their cytoplasmic side. In the cases of NPCs clearly sectioned perpendicular to their midplane (and hence to midplane of the NE), the shortest distance from the center of each gold particle to the midplane was measured, and the average of these measurements was 41 ± 15 nm (n = 41) (Fig.5). Another nucleoporin was localized with this technique to both sides of the NPC (data not shown); hence the lack of signal on the nuclear face of the NPC is not a result of general inaccessibility of this face. NPCs were often multiply labeled, with as many as seven gold particles each.
Figure 5: Immunoelectron microscopy. A, NEs were incubated with mAb 165C10, and the labeling was visualized with gold-labeled secondary antibodies. NUP159 was localized only on the cytoplasmic side of the NPC. C, cytoplasm; N, nucleoplasm; bar, 200 nm. B, the distance (d) from the center of each gold particle to the midplane of its associated NPC was measured for NPCs clearly sectioned perpendicular to that plane (and hence to the plane of the NE) (left); these were found to average 41 ± 15 nm (n = 41) (right).
Mammalian repeat motif-containing nucleoporins have been shown to
bind transport substrate in a reaction mediated by cytosolic
subfraction A (6, 32) . For one of these nucleoporins,
Nup98, the binding site has been mapped to its repeat motif-containing
domain(15) . The active component of fraction A in nuclear
import has been shown to be the heterodimeric
karyopherin(6, 7, 10, 15) . Further
analysis has revealed that binding to the repeat motif-containing
nucleoporins is via the subunit of karyopherin(10) . The
cytoplasmic exposure of NUP159, a repeat motif nucleoporin, makes it a
candidate for such import-related binding. To test whether the repeat
motif segment 3 of NUP159 also functions as a docking site, the five E. coli-expressed segments of NUP159 were incubated with a
transport substrate and Xenopus ovary cytosolic subfraction
A(32) . Only the peptide repeat-containing segment 3 of NUP159
bound transport substrate in a fraction A-dependent fashion (Fig.6A; for controls see Fig. 6B).
Likewise,
S-labeled recombinant rat karyopherin
bound exclusively to the repeat motif segment 3 of NUP159 (Fig.6C).
Figure 6:
Ligand blotting assay. A,
fractions containing all five expressed segments of NUP159 (Fig.3A, a) were separated by SDS-PAGE and
transferred to nitrocellulose as in Fig.3A, a (lanes 1-5). The membrane was incubated with Xenopus fraction A and nuclear location sequence-conjugated
human serum albumin. Binding was detected by incubation with a
secondary antibody against human serum albumin and I-labeled protein A. Only the expressed protein in lane 3, which is the domain with the repeat motifs, binds the
conjugate in the presence of fraction A. B, a blot of the
fraction containing segment 3 was incubated with the conjugate alone (lane 6), with fraction A and unconjugated human serum albumin (lane 7), or with the nuclear location sequence-conjugate and
fraction A (lane 8). C, the five expressed segments
of NUP159 were subjected to SDS-PAGE, transferred to nitrocellulose as
in A and incubated with
S-labeled rat karyopherin
.
NUP159 is the first of the yeast nucleoporins that has so far been sublocalized. Given the uncertainties associated with secondary immunolabeling, the epitope may be between 30 and 50 nm from the central plane of the NPC. We therefore expect NUP159 to project some distance into the cytoplasm. This suggests that NUP159 may be a component of the short cytoplasmic fibers that emanate from the NPC (35) . Diffuse structures of 20-30 nm attached to the NPC have been shown in section electron micrographs of pelleted yeast nuclei that could be collapsed fibers(19) . In vertebrate cells, three nucleoporins have so far been sublocalized to these fibers: Nup358(16) , Nup214 (25) , and Tpr(11) . Of these, Nup358 and Nup214 are repeat motif-containing nucleoporins. Because of the sequence similarities of NUP159 and Nup214 and because of their similar locations, the repeat module of yeast NUP159 could be functionally homologous to that of mammalian Nup214. Besides the XFXFG repeats that are also shared by some other nucleoporins (for review see Refs. 17 and 18), NUP159 contains the repeat motifs PSFG, PASG, and SAFG. The motifs PSFG and SAFG were found also in another potential yeast nucleoporin. Motif SAFG is especially similar to the motif SVFG in Nup214(25) . All of these repeats are located in a central domain (segment 3) of NUP159.
The ability of the NUP159 repeat motif
domain to bind transport substrate in a karyopherin-dependent fashion
in an overlay assay is similar to that recently demonstrated for the
repeat domain in rat Nup98(15) . Our data indicate that there
is a functional conservation of NPC transport components amongst the
eukaryotes, because Xenopus karyopherin or rat karyopherin
were both able to interact with the repeat motif domain of a
yeast nucleoporin. It is likely that the repeat motif domains of other
yeast nucleoporins will also form an array of multiple docking sites
across the NPC(6, 15) .