From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, September 11, 2000, and in revised form, November 7, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our previous studies have focused on a family of
Saccharomyces cerevisiae nuclear pore complex (NPC)
proteins that contain domains composed of repetitive
tetrapeptide glycine-leucine-phenylalanine-glycine (GLFG) motifs. We
have previously shown that the GLFG regions of Nup116p and Nup100p
directly bind the karyopherin transport factor Kap95p during nuclear
protein import. In this report, we have further investigated potential
roles for the GLFG region in mRNA export. The subcellular
localizations of green fluorescent protein (GFP)-tagged mRNA
transport factors were individually examined in yeast cells
overexpressing the Nup116-GLFG region. The essential mRNA export
factors Mex67-GFP, Mtr2-GFP, and Dbp5-GFP accumulated in the nucleus.
In contrast, the localizations of Gle1-GFP and Gle2-GFP remained
predominantly associated with the NPC, as in wild type cells. The
localization of Kap95p was also not perturbed with GLFG overexpression.
Coimmunoprecipitation experiments from yeast cell lysates resulted in
the isolation of a Mex67p-Nup116p complex. Soluble binding assays with
bacterially expressed recombinant proteins confirmed a direct
interaction between Mex67p and the Nup116-GLFG or Nup100-GLFG regions.
Mtr2p was not required for in vitro binding of Mex67p to
the GLFG region. To map the Nup116-GLFG subregion(s) required for
Kap95p and/or Mex67p association, yeast two-hybrid analysis was used.
Of the 33 Nup116-GLFG repeats that compose the domain, a central
subregion of nine GLFG repeats was sufficient for binding either Kap95p or Mex67p. Interestingly, the first 12 repeats from the full-length region only had a positive interaction with Mex67p, whereas the last 12 were only positive with Kap95p. Thus, the GLFG domain may have the
capacity to bind both karyopherins and an mRNA export factor
simultaneously. Taken together, our in vivo and in
vitro results define an essential role for a direct Mex67p-GLFG
interaction during mRNA export.
To move between the nuclear and cytoplasmic compartments of a
eukaryotic cell, all molecules must pass through nuclear pore complexes
(NPCs)1 embedded in the
nuclear envelope. Ions, metabolites, and small proteins may diffuse
through an ~9-nm aqueous channel in the NPC. In contrast, the
movement of large macromolecules, including proteins and RNA, is
energy-dependent and facilitated (reviewed in Refs. 1-3).
The central channel of the proteinaceous NPC is formed by a
symmetrical, 8-fold assembly of spoke-like structures sandwiched between nuclear and cytoplasmic rings. Distinct filamentous structures extend from these rings on both the nuclear and cytoplasmic faces, with
the filaments on the nuclear side culminating in a basket-like structure. Overall, a vertebrate NPC measures ~200 nm from the tips
of the cytoplasmic filaments to the base of the nucleoplasmic basket
(4, 5). Unraveling the mechanism for active transport between the
cytoplasmic and nucleoplasmic NPC faces will require an in-depth
understanding of both the NPC itself and soluble transport factors.
The Saccharomyces cerevisiae NPC is built by the
oligomerization of over 30 different polypeptides collectively referred
to as nucleoporins (4, 6, 7). This includes three integral membrane
proteins, a subset of proteins with predicted coiled-coil domains,
several proteins with novel primary structure, and a family of 13 nucleoporins with phenylalanine-glycine (FG) repeat domains. All FG
repeat domains share the common feature of multiple FG dipeptide
repeats with variable length spacers (8). However, there are at least
two distinct subclasses: 1) glycine-leucine-phenylalanine-glycine (GLFG) repeat domains, which are separated by spacers that lack acidic
residues and are enriched in serine, threonine, glutamine, and
asparagine residues, and 2) phenylalanine-any amino
acid-phenylalanine-glycine (FXFG) repeat domains,
which have charged spacers. In vertebrates and yeast, different FG
nucleoporins reside in each of the NPC substructures, and there is
significant evidence for a direct involvement of FG nucleoporins in
both nuclear import and export (4, 6, 9).
Recently, several reports have documented in vitro
biochemical interactions between shuttling transport factors and
individual components of the NPC (reviewed in Ref. 9). In particular, there has been considerable focus on the interactions between FG repeat
nucleoporins and karyopherins (also known as importins/exportins). Karyopherins are a family of shuttling transport factors that recognize
specific nuclear import or export signals in their respective cargo
(reviewed in Refs. 2, 3, 10, and 11). All yeast FG nucleoporins have
been shown to bind at least one karyopherin in vitro
(reviewed in Ref. 9). Current models for the mechanism of docking and
translocation through the NPC are based on direct karyopherin-nucleoporin binding. Karyopherins also interact with the
small GTPase Ran, which acts as a molecular switch regulating the
association of karyopherins with their cargo and the NPC (2, 12). The
crystal structures of different karyopherins (or karyopherin domains)
complexed with either RanGTP or a FXFG nucleoporin were recently reported (13-15). A comparison of the structure of
importin- Several factors with specific roles in mRNA export have also been
shown to physically or genetically interact with nucleoporins. For
example, in S. cerevisiae, the RNA helicase Dbp5p directly associates with Nup159p (16). The mRNA export factor Gle2p
physically associates with Nup116p (17, 18), and gle2
mutants are synthetically lethal with a nup100 null mutant
(19). The essential factor Gle1p interacts with Dbp5p (20), and
gle1 mutants are synthetically lethal with either
rip1/nup42 or nup100 null mutants (19, 21). The
essential mRNA export factor Mex67p forms a heterodimeric complex
with Mtr2p, and Mtr2p mediates association with Nup85p (22). The
vertebrate Mex67p homologue, TAP, directly associates with vertebrate
Nup214p, Nup98p, and hCG1 (23, 24). Interestingly, all of these
nucleoporins, except Nup85p, are FG family members. Taken together, it
is intriguing that both karyopherins and mRNA export factors
interact with FG nucleoporins.
Although much progress has been made in defining interactions between
soluble transport factors and nucleoporins, the exact molecular
mechanisms for translocation through the NPC are still unknown. To
reveal critical events that mediate NPC translocation, we have analyzed
the interface between dynamic transport factors and two homologous
components of the S. cerevisiae NPC, the GLFG nucleoporins
Nup116p and Nup100p. Nup116p and Nup100p are localized on both sides of
the NPC (6, 25), and their C-terminal regions interact with Nup82p
(25). Nup82p plays a key role in the localization of Nup116p at the
cytoplasmic face of the NPC. Our previous studies have documented an
in vivo requirement for Nup116p and Nup100p in nuclear
transport (19, 26-29). The unique N-terminal region of Nup116p binds
the mRNA export factor Gle2p (17, 18), whereas the GLFG regions of
Nup116p and Nup100p directly bind Kap95p (27, 28) and other
karyopherins (20, 30-35). Interestingly, overexpression of the
Nup116-GLFG region inhibits mRNA export and cell growth (27). This
suggests an essential role for the GLFG region in mRNA export.
However, a direct role for karyopherins in mRNA export has not been
documented. We have found that the Nup116p and Nup100p GLFG regions
interact in vivo and in vitro with both the
karyopherin Kap95p (27, 28) and the mRNA export factor Mex67p (this
work). Furthermore, Kap95p and Mex67p bind to both common and distinct aspects of the Nup116-GLFG region.
Yeast Strains and Plasmids--
All yeast strains used in this
study are listed in Table I. The sequence
encoding the green fluorescent protein (GFP) was fused in frame before
the stop codons for the chromosomal MEX67, DBP5, GLE1, and
MTR2 genes. This was achieved using the gene integration method of Baudin et al. (36). Respective polymerase chain
reaction products were generated with oligonucleotides and a template
containing genes for GFP and the Schizosaccharomyces pombe
HIS5 (pGFP-HIS5; gift of J. Aitchison). The resulting DNA
fragments were introduced into SWY518 by standard transformation
methods and colonies selected on media lacking histidine. Correct
integration was confirmed by immunoblot and direct fluorescence
microscopy. The resulting strains were back-crossed to a wild type
yeast strain, and the GFP-tagged progeny was used in this study. For
GFP tagging the genomic copy of GLE2, a similar strategy was
used except the S. cerevisiae HIS3 gene served as the
selectable marker, and the polymerase chain reaction fragment was
transformed into the diploid SWY595. The resulting diploid was
sporulated and dissected to obtain SWY1920.
The plasmids used in this study are listed in Table
II and were maintained in either BL21
(pSW156, pSW304, and pSW329) or DH5 Microscopic Localization of Yeast Strains Overexpressing
Nup116-GLFG--
pSW163 (Nup116-GLFG under a galactose-inducible
promoter), pSW384 (Nup100-GLFG under a galactose-inducible promoter),
or pNLS-E1 (vector) were transformed into the respective GFP-tagged
strains by standard methods. Stationary phase cultures grown in
synthetic complete (SC) media lacking uracil ( Whole Cell Lysates and Immunoprecipitations--
Whole cell
lysates of SWY518 (MEX67) and SWY2131 (MEX67-GFP)
were prepared in 20 mM Tris-HCl, pH 6.5, 150 mM
NaCl, 5 mM MgCl2, 2% Triton X-100 by glass
bead lysis. Immunoprecipitations were conducted as described previously
(25). For immunoprecipitations, 1 µl of affinity-purified rabbit
polyclonal anti-GFP antibody (gift from M. Linder) was used per 100 µl of cell extract. Blots were probed with an anti-GFP antibody at a
1:1000 dilution (16 h, 4 °C), affinity-purified rabbit polyclonal
anti-Nup116-GLFG antibody WU956 (39) at a 1:2000 dilution (16 h,
4 °C), or affinity-purified rabbit polyclonal antibody WU600 (27)
raised against the Nup116 C-terminal region at a 1:1000 dilution (16 h,
4 °C). Bound antibodies were detected with alkaline
phosphatase-conjugated anti-rabbit IgG (Promega) diluted 1:7500 (1 h,
23 °C). After incubation with nitro blue tetrazolium and
5-bromo-4-chloro-3-indolyl-1-phosphate (Promega), the blots were
developed for color visualization.
Purification of Proteins from Bacteria and Soluble Binding
Assay--
For purification of the glutathione
S-transferase (GST) fusion proteins GST-Nup116-GLFG or
GST-Nup100-GLFG, Escherichia coli strains containing pSW304
or pSW433, respectively, were grown in 1 liter of LB-rich media
containing 100 µg/ml carbenicillin at 37 °C until the
A600 was between 0.9 and 1.0. Cultures were induced for 3.5 h with 0.3 mM
isopropyl-1-thio-
The maltose-binding protein (MBP) fusions with Mex67p (MBP-Mex67) and
full-length Nup116p (MBP-Nup116) were purified as above except the
thawed pellets were resuspended in 100 mM Tris, pH 7.5, 50 mM NaCl, 10 mM EDTA, 10 mM
The expression of polyhistidine (His6)-tagged Kap95p was
induced in bacteria as described above, and the protein was
subsequently purified on nickel-nitrilotriacetic acid-agarose (Qiagen)
as described by the manufacturer.
Proteins were dialyzed into either 20 mM Hepes-KOH, pH 7.0, 100 mM potassium acetate, 2 mM magnesium
acetate, 0.1% Tween 20, 5 mM Yeast Two-hybrid Analysis--
pSW332 and the various pSW291
constructs (Table II) were cotransformed into the yeast strain L40
(Table I), and pSW1254 and the various pSW291 constructs (Table II)
were cotransformed into the yeast strain PJ69-4A (Table I).
Transformants were selected on SC-leu-trp and assayed for two-hybrid
interaction by growth on SC-leu-trp-his (for L40) or
SC-leu-trp-his-ade (for PJ69-4A). All plasmids were checked for
specificity and ability to self-activate with pCH428 (pLexA-Orc2) (41),
pGBD-C1 (42), or pSE1111 (pGAD-Snf4) (43).
Mex67-GFP Accumulates in the Nucleus of Cells Overexpressing
Nup116-GLFG or Nup100-GLFG--
Previous studies have shown that
overexpression of the Nup116-GLFG region in yeast cells results in
nucleolar fragmentation, accumulation of polyadenylated RNA within the
nucleus, and cell lethality (27). The GLFG overexpression phenotype
does not result in any detectable NPC or nuclear envelope structural
perturbations, and the overexpressed Nup116-GLFG region localizes in
both the cytoplasm and nucleus (27). We previously suggested this
phenotype may be due to titration of an essential GLFG-interacting
factor away from the NPC. Recently, several S. cerevisiae
factors have been identified that are specifically required for
mRNA export and dispensable for the transport of proteins and other
classes of RNA (44). These factors include Gle1p, Gle2p, Dbp5p, Mex67p, and Mtr2p. All of these factors except Gle2p are essential, and in wild
type cells all localize predominantly at the NPC with some also showing
low levels of cytoplasmic or nucleoplasmic staining (19, 22, 29,
45-47). Therefore, each was a candidate for perturbation by the
overexpression of the Nup116-GLFG region.
To test directly whether the subcellular localizations of any of these
mRNA export factors were perturbed upon overexpression of
Nup116-GLFG, a panel of yeast strains harboring a respective GFP-tagged
transport factor was examined. Yeast strains were generated with the
sequence encoding GFP chromosomally integrated in-frame at the sequence
for the C terminus of the particular transport factor (see "Materials
and Methods"). A multicopy plasmid containing the Nup116-GLFG region
under control of the GAL10 promoter was introduced into each
strain. In all of the strains, overexpression of the Nup116-GLFG region
was confirmed by immunoblot, and the overexpressing strains failed to
grow on media containing galactose (data not shown). Nucleolar
fragmentation was documented for all strains by indirect
immunofluorescence microscopy with antibodies to Nop1p (data not
shown). After induction of GLFG overexpression, the localization of the
GFP-tagged factor was determined by direct fluorescence microscopy. The
results are summarized in Table III. In
wild type control cells, Mex67-GFP, Mtr2-GFP, and Dbp5-GFP were
predominantly localized in a punctate pattern at the nuclear periphery
(Fig. 1A and Table III),
identical to the published localizations (22, 45-47). Strikingly, in
Nup116-GLFG-overexpressing cells, Mex67-GFP, Mtr2-GFP, and Dbp5-GFP
accumulated within the nucleus, and the punctate pattern at the nuclear
periphery was absent (Fig. 1G and Table III). In contrast,
the localizations of Gle1-GFP and Gle2-GFP were not perturbed by
overexpression of the GLFG region (Table III). Gle1-GFP and Gle2-GFP
were localized predominantly at the nuclear rim in both control and
Nup116-GLFG-overexpressing cells. Because Nup116-GLFG has been
previously shown to interact directly with Kap95p (27, 28), we also
examined the localization of Kap95p by indirect immunofluorescence
microscopy. The distribution of Kap95p was not perturbed, and it was
localized in the nucleus, the cytoplasm, and at the NPC of both control
and GLFG-overexpressing cells (Table III). Thus, overexpression of
Nup116-GLFG specifically altered the distribution of Mex67-GFP,
Mtr2-GFP, and Dbp5-GFP.
Since Nup116p and Nup100p have a high degree of structural and
functional similarity, we investigated the localization of Mex67-GFP in
cells overexpressing the Nup100-GLFG region. The level of Mex67-GFP in
the nuclei of cells overexpressing Nup100-GLFG was greater than that in
the control cells (Fig. 1, compare I and K).
However, some peripheral punctate localization was detectable in a
fraction of the Nup100-GLFG-overexpressing cells (Fig. 1K). This contrasts with the Nup116-GLFG-overexpressing cells where Mex67-GFP was no longer localized in a punctate pattern at the nuclear
rim (Fig. 1G). These results suggest that Nup116-GLFG was
more effective than Nup100-GLFG at perturbing the localization of
Mex67-GFP. These results also correlate with our previous observation (27) that only Nup116-GLFG overexpression severely inhibits cell growth.
Mex67p and Nup116p Form a Complex in Vivo--
The mislocalization
of Mex67-GFP, Mtr2-GFP, and Dbp5-GFP in cells overexpressing the
Nup116-GLFG region suggested that these factors may directly or
indirectly associate in vivo with Nup116p. Izaurralde
and co-workers (23) recently reported that TAP, the vertebrate
homologue of Mex67p, associates with the vertebrate GLFG nucleoporin
Nup98p. Therefore, Mex67p was a good candidate for Nup116-GLFG and
Nup116p interaction. To investigate whether Mex67p and Nup116p
physically interact, we performed coimmunoprecipitation experiments
from whole cell yeast lysates. Cell lysates from wild type and
Mex67-GFP-expressing cells were incubated with an affinity-purified rabbit polyclonal antibody raised against GFP. Antibody-bound Mex67-GFP
and coprecipitating proteins were isolated with protein A-Sepharose,
and the unbound and bound fractions were separated by SDS-PAGE and
analyzed by immunoblotting. Immunoblotting with anti-GFP antibodies
showed that the antibodies recognized an ~105-kDa protein
representing Mex67-GFP. This band was present in the lysate from
Mex67-GFP cells but not in the lysate from the untagged cells (Fig.
2, lanes 1 and 2).
The anti-GFP antibody also recognized several endogenous yeast proteins
on immunoblots; however, the immunoprecipitation was specific for
Mex67-GFP as the 105-kDa band was the only one observed in the bound
fractions (Fig. 2, lanes 5 and 6). The
immunoisolation depleted a significant fraction of the Mex67-GFP, with
very little remaining in the unbound fraction (Fig. 2, lane
4).
To determine whether a GLFG protein(s) coprecipitated with Mex67-GFP,
the samples were probed with polyclonal antibodies raised against the
Nup116-GLFG region. The anti-GLFG antibody specifically recognized an
~120-kDa band in the bound fraction of the Mex67-GFP stain (Fig. 2,
lanes 9 and 10). Because the anti-Nup116-GLFG
antibody has the potential to react with other GLFG proteins in yeast, we confirmed that the ~120-kDa band was Nup116p by probing the samples with an antibody monospecific for the Nup116p C-terminal region. The anti-Nup116C antibody recognized the ~120-kDa band (Fig.
2, lane 14). Monospecific antibodies recognizing Nup100p are
not available, and we could not directly test for the presence of
Nup100p in the Mex67-GFP immunoprecipitate. Under the cell lysis
conditions used, Nup116p is solubilized into distinct subcomplexes, and
the immunoprecipitation does not represent mostly intact NPCs (25).
Thus, Mex67-GFP and Nup116p specifically associate in a complex in
yeast cells. However, not all of the Nup116p was coprecipitated with
Mex67-GFP (Fig. 2, compare lanes 10 and 14 with
8 and 12, respectively). This is consistent with
Nup116p being a binding site for multiple dynamic transport factors.
Bacterially Expressed Mex67p Directly Interacts with Nup116-GLFG
and Nup100-GLFG--
Although the coimmunoprecipitation results
suggested Mex67p and Nup116p may associate in vivo in a
complex, the interaction could be either direct or indirect with
another factor(s) bridging the two proteins. To test for direct
interaction, soluble binding assays were conducted with recombinant,
purified proteins. Based on the GLFG overexpression results (Fig. 1),
we predicted that the Nup116-GLFG domain would mediate a Mex67p-Nup116p
association. Mex67p was purified as an MBP fusion from bacteria (Fig.
3A). The purified MBP-Mex67
fraction contained a 117-kDa band corresponding to full-length
MBP-Mex67 and four smaller bands representing proteolytic products of
MBP-Mex67 (confirmed by immunoblot; data not shown and Fig.
3A). Nup116-GLFG was purified as a GST fusion and
immobilized on Glutathione-Sepharose. Purified MBP-Mex67, MBP (as a
negative control), or His6-Kap95 (as a positive
control) was incubated with either immobilized GST or GST-Nup116-GLFG.
Unbound and bound fractions were separated and analyzed by SDS-PAGE and
Coomassie staining. As in our previous studies (28), GST-Nup116-GLFG
bound His6-Kap95 (Fig. 3C, middle panel).
Neither GST nor GST-Nup116-GLFG bound MBP alone (Fig. 3B,
left and middle panels). Moreover, MBP-Mex67 did not
bind at all to GST alone (Fig. 3D, left panel). In contrast, binding of full-length MBP-Mex67 and its longest proteolytic product was detected with GST-Nup116-GLFG (Fig. 3D, middle panel).
MBP-Mex67 binding to GST-Nup116-GLFG was specific as it was competed by the addition of MBP-Nup116 (full-length) but not MBP alone
(Fig. 3E, right panel). These results demonstrate a direct
association between Mex67p and the Nup116-GLFG region.
Because Mex67-GFP was also perturbed in cells overexpressing
Nup100-GLFG, we investigated whether Mex67p and Nup100-GLFG directly interact in vitro. Nup100-GLFG was purified as a GST fusion
from bacteria and immobilized on Glutathione-Sepharose (Fig.
3A). GST-Nup100-GLFG did not bind MBP (Fig. 3B, right
panel). Binding of His6-Kap95 (Fig. 3C, right
panel) and full-length MBP-Mex67 (Fig. 3D, right panel)
was detected with GST-Nup100-GLFG. Therefore, Mex67p directly binds the
GLFG regions of Nup116p and Nup100p in vitro.
Others have shown (22) Mex67p and Mtr2p form a complex in yeast cells,
and Mtr2p mediates the association of Mex67p with the nucleoporin
Nup85p. We have not detected direct binding between bacterially
expressed, recombinant Mtr2p and either GST-Nup116-GLFG or
GST-Nup100-GLFG (data not shown). Therefore, Mex67p may bind some
nucleoporins directly and other nucleoporins through heterodimerization with Mtr2p (see "Discussion").
Mex67p and Kap95p Bind Distinct Regions of the Nup116-GLFG
Domain--
The Nup116-GLFG domain consists of 33 GLFG tetrapeptide
repeats separated by polar spacer regions (37). It is unknown whether all of the repeats and spacers are functionally or structurally equivalent. Because the GLFG region binds both Mex67p and Kap95p in vitro, we wanted to test whether each factor had the
capacity to interact with overlapping or distinct subregions of the
Nup116-GLFG domain. Yeast two-hybrid analysis was used to map the
Mex67p- and Kap95p-binding sites on the Nup116-GLFG domain. A series of sequential deletions of four or eight GLFG repeats from each end of the
Nup116-GLFG domain were generated and fused in-frame behind the
transcriptional activation domain of Gal4p (Gal4AD). These plasmids were cotransformed into reporter strains with plasmids expressing either Kap95p fused to the DNA binding domain of LexA (LexABD) or Mex67p fused to the DNA binding domain of Gal4p
(Gal4BD). The two-hybrid interaction was assayed by growth
on SC media lacking histidine (for Kap95p) or media lacking histidine
and adenine (for Mex67p). Both Kap95p and Mex67p resulted in positive
two-hybrid interactions with full-length Gal4AD-Nup116-GLFG
(Fig. 4, GLFG repeats 1-33).
Kap95p (27) and Mex67p (this work) also yielded a positive interaction
with Gal4AD-Nup100-GLFG in the two-hybrid assay (data not
shown). Deletion of eight Nup116-GLFG repeats from each end of the
full-length Nup116-GLFG region did not abolish the positive signal
(Fig. 4, GLFG repeats 1-25, 9-33, and 9-25). Subsequent deletions of four more repeats from each end also did not
abolish the positive interaction (Fig. 4, GLFG repeats 9-21, 13-25, and 13-21). Therefore, the central region
composed of Nup116-GLFG repeats 13-21 was sufficient for a two-hybrid
interaction with either Kap95p or Mex67p.
To test if the nine GLFG repeats from 13-21 were necessary for binding
Kap95p and Mex67p, Gal4AD fusions were generated with the
N- and C-terminal GLFG segments that flank GLFG repeats 13-21 (repeats 1-12 and 22-33, respectively) (Fig. 4). Strikingly,
GLFG repeats 1-12 were sufficient for a two-hybrid interaction with Mex67p, and GLFG repeats 22-33 were sufficient for interaction with
Kap95p but not vice versa. Therefore, although both transport factors
bind the central portion of the GLFG region, Mex67p alone associates
with the N-terminal portion of the GLFG region, and Kap95p alone
associates with the C-terminal portion.
To fully understand how the GLFG nucleoporins Nup116p and Nup100p
function in nuclear transport, it is critical to characterize their
protein interacting partners. Direct in vitro interactions between these GLFG regions and members of the karyopherin transport factor family have been definitively established (28, 30, 32, 33).
Genetic and in vivo evidence further supports a role for a
Kap95p-GLFG interaction during transport (28). Here we report in
vivo and in vitro documentation that the mRNA
export factor Mex67p directly interacts with the GLFG regions of
Nup116p and Nup100p. This conclusion is based on multiple pieces of
evidence. Mex67-GFP was mislocalized in yeast cells overexpressing the
Nup116p and Nup100p GLFG regions; Nup116p was coisolated from yeast
cell lysates in a complex with Mex67-GFP, purified recombinant
MBP-Mex67 and GST-Nup116-GLFG or GST-Nup100-GLFG directly bound
in vitro, and Mex67p yielded a positive two-hybrid
interaction with both GLFG regions. Interestingly, the localization of
Kap95p was not altered by overexpression of Nup116-GLFG. Moreover,
Mex67p and Kap95p exhibited differential two-hybrid interactions with
subregions of the Nup116-GLFG domain. Direct in vitro
interactions between the vertebrate Mex67p homologue, TAP, and the
vertebrate FG nucleoporins Nup214p and Nup98p have been recently
reported (23, 24). In addition, while our study on the homologous yeast
proteins was in progress, Hurt and coworkers (48) reported in
vitro interactions between the heterodimeric Mex67p-Mtr2p
complex and the FG domains from several different yeast nucleoporins.
This included in vitro binding of a recombinant Mex67p-Mtr2p
complex to the Nup116-GLFG region. Our studies independently
corroborate these findings and extend our knowledge in several
important ways. There are key implications for our results in terms of
both the mechanism of Mex67p action in mRNA export and the global
role of the Nup116p and Nup100p GLFG regions in nuclear transport.
In all other reported S. cerevisiae mutant phenotypes, wild
type Mex67p has only been observed to accumulate in the cytoplasm (22).
We have found that Mex67-GFP is mislocalized and accumulates in the
nucleus coincident with the overexpression of Nup116-GLFG, the
inhibition of mRNA export, and cell lethality (Fig. 1 and Ref. 27).
It is intriguing that Mex67-GFP accumulates in the nucleus (this study)
even though the overexpressed GLFG protein is in both the cytoplasm and
the nucleus (27). This suggests the GLFG region may preferentially
inhibit a Mex67p nuclear exit step, possibly involving Nup116p or
Nup100p on the nuclear face of the NPC. The overexpressed GLFG region
may also competitively inhibit Mex67p binding to another FG
nucleoporin. Nup1p, Nup2p, and Nup60p are FG nucleoporins localized
exclusively on the nuclear NPC side (6, 49); however, interactions with
Mex67p have not been documented. The other FG nucleoporins that to date
have been reported as interacting with the Mex67p-Mtr2p complex
in vitro are either localized exclusively on the cytoplasmic
side of the NPC (Rip1/Nup42p and Nup159p) or localized symmetrically on
both sides (Nsp1p) (4, 6, 48). Overall, our results provide novel
evidence for an essential in vivo Mex67p-GLFG interaction during mRNA export.
The molecular basis for the in vivo GLFG overexpression
phenotype (toxicity and mRNA export defects) may be partially due to sequestering Mex67p in the nucleus. However, we also observed accumulation of Mtr2-GFP and Dbp5-GFP in the nucleus of cells overexpressing the Nup116-GLFG region (Table III). Therefore, the phenotype could be a combined effect from coincident sequestering of
three essential mRNA export factors in the nucleus. Since Mex67p and Mtr2p physically interact (22), it is not unexpected that Mtr2-GFP
would accumulate in the nucleus through its association with Mex67-GFP.
We have not detected a direct binding between recombinant, bacterially
expressed Mtr2p and
Nup116-GLFG.2 Genetic or
biochemical associations between Dbp5p and GLFG nucleoporins or between
Mex67p/Mtr2p and Dbp5p have not been reported. Further studies will be
needed to examine Dbp5p interactions and reveal the mechanism of the
Dbp5p mislocalization in GLFG-overexpressing cells.
The recent study by Hurt and coworkers (48) concludes that in
vitro Mex67p binding to a FG nucleoporin requires Mtr2p and formation of the heterodimeric Mex67p-Mtr2p complex. They reported Mex67p or Mtr2p alone does not bind to Nup116-GLFG. In contrast, we
have found that Mex67p does independently interact in vitro with Nup116-GLFG or Nup100-GLFG. Therefore, Mtr2p is not strictly required for Mex67p binding to the GLFG regions. However, it is possible that Mtr2p increases the affinity of a Mex67p-GLFG
interaction. Our results showing a direct GLFG-Mex67p interaction more
closely parallel the results of vertebrate TAP directly binding
vertebrate FG nucleoporins (23). We believe that yeast Mex67p and
vertebrate TAP share a similar mechanism for interacting with GLFG nucleoporins.
Finally, our analysis of Mex67p versus Kap95p binding to the
Nup116-GLFG region suggests that the GLFG region has the capacity to
bind Mex67p and karyopherins simultaneously. These results indicate
that all of the GLFG repeats and spacer regions are not necessarily
functionally equivalent. This is surprising and should change the way
we think about the function of FG repeat domains. Most prior
investigations have treated subregions of FG repeat domains as
functionally representative of the full-length domains. The results in
this study now suggest that distinct subregions of the GLFG domain may
serve as unique binding sites for different transport factors.
How do such binding interactions between nucleoporins and transport
factors result in movement through the NPC portal? FG nucleoporins are
located on both the cytoplasmic and nuclear NPC faces as well as along
the central axis of the NPC (summarized in Refs. 4 and 6). Recent
models have suggested that karyopherins and mRNA export factors
could move from one FG repeat domain to another along the filaments and
central channel. Besides the potential distinct binding of transport
factors to subregions of an individual FG repeat domain, there is a
growing body of evidence that the FG repeat domains among the family
members are also not equivalent. In yeast cells, the GLFG region of
Nup116p cannot be functionally replaced with the FXFG region
of Nsp1p (27). Mutants of transport factors show contrasting
perturbations on their respective binding capacity for different FG
nucleoporins (13, 23, 28). For example, a yeast Kap95p mutant with
diminished GLFG interaction is not altered for FXFG
interaction (28). Deletions of vertebrate TAP that abolish binding to
Nup214p are not defective in vitro for binding Nup98p (23).
Thus, the interactions of a given transport factor with GLFG
versus FXFG repeats may be mechanistically
distinct in terms of facilitating, directing, or even propelling entry and exit through the NPC portal.
Another important emerging theme is that multiple different mRNA
export factors have binding sites on a single nucleoporin. Mex67p and
Gle2p have distinct binding sites on Nup116p in the GLFG and N-terminal
regions, respectively (this work and Refs. 17, 18, and 48).
Mex67p-Mtr2p and Dbp5p can both interact with Nup159p (16, 20, 48). The
fact that a single nucleoporin can bind more than one mRNA export
factor strongly suggests the particular factors may be required for a
common step in mRNA export. There is evidence that vertebrate TAP
and hGle2 may interact (23); however, we have not observed any
qualitative effects of yeast Gle2p on Mex67p binding to full-length
Nup116p.2 Finally, the characterization of a higher order,
cytoplasmically localized Nup116p-Nup82p-Nup159p complex suggests there
is a juxtaposition of binding sites for different mRNA export
factors at a discrete NPC substructure (25, 50). Taken together, these
interactions may reflect sequential steps in the mRNA export
mechanism. Revealing how Nup116p/Nup100p and GLFG binding influences
the movement of distinct transport factors through the NPC is a future goal.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
when complexed with either RanGTP or an FXFG
repeat region has suggested binding of RanGTP to importin-
may
induce a conformational change in importin-
that occludes the
FXFG repeat binding site. This would release importin-
from the nucleoporin. However, it is unclear if such a mechanism
contributes to vectoral movement along the central axis of the NPC or
to a terminal release step from the NPC.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Yeast strains
(all others).
Plasmids
ura) with 2% glucose
were pelleted, washed once in an equal volume of SC
ura, 2% raffinose and used to inoculate SC
ura, 2% raffinose at an initial
A600 of 0.015. When the
A600 reached 0.1 to 0.2, galactose (J. T. Baker Inc.) was added to one-half of the culture at a final
concentration of 2%. The raffinose and galactose cultures were
incubated 5 h at 30 °C. A small portion of the live cells was
removed for viewing direct fluorescence, and the remaining cells were
prepared for indirect immunofluorescence microscopy as described
previously (27, 37). To test for nucleolar fragmentation in the
GLFG-overexpressing strains, fixed cells were incubated overnight with
tissue culture supernatant monoclonal antibody D77 (38) (1:10) for
detection of Nop1p. Bound antibody was detected with a rhodamine donkey anti-mouse antibody (Chemicon; 1:400). For detection of Kap95p, a
rabbit polyclonal antibody (28) raised against Kap95p was used at a
1:20 dilution (16 h, 4 °C). Bound antibody was detected with a Texas
Red-conjugated donkey anti-rabbit antibody (Jackson Immunoresearch;
1:200). Images were collected with a × 100 objective on an
Olympus BX50 microscope using a Dage-MTI CCD-300-RC camera (Dage MTI,
Michigan City, IN).
-D-galactopyranoside and cell pellets
frozen at
70 °C. Thawed pellets were resuspended in ice-cold 20 mM sodium phosphate, pH 7.3, 150 mM NaCl, 10 mM EDTA, 0.1 mM DTT, 0.1 mM
phenylmethylsulfonyl fluoride, 0.1 µM pepstatin A, 0.1 µM leupeptin, 0.1 mM benzamidine, and 0.01%
sodium azide. 50 mg of lysozyme was added, and the suspensions were
incubated on ice for 30 min before sonication (15 s pulses, 15 s
rest for a total of 3 min). The suspension was clarified in a Sorvall
SS-34 rotor at 9000 × g for 30 min, and Triton X-100
was added to a final concentration of 1%. The clarified lysate was
loaded onto 2.5 ml of packed Glutathione-Sepharose 4B (Amersham
Pharmacia) equilibrated in 20 mM sodium phosphate, pH 7.3, 150 mM NaCl, 10 mM EDTA, 0.1 mM
DTT, 1% Triton X-100. After washing the column with 15 ml of 20 mM sodium phosphate, pH 7.3, 150 mM NaCl, 10 mM EDTA, 0.1 mM DTT, 1% Triton X-100, the same
buffer lacking Triton X-100 was used until the
A280 of the flow-through was 0. Finally, the
protein was eluted in 50 mM Tris, pH 9.0, 20 mM glutathione.
-mercaptoethanol, 5 µg/ml leupeptin, 1% aprotinin, 0.1 mg/ml
Pefabloc. After sonication, NaCl was added to a final concentration of
0.5 M, and the clarified lysate was loaded onto 4 ml of
packed amylose resin (New England Biolabs) previously equilibrated in
column buffer (10 mM sodium phosphate, 0.5 M
NaCl, pH 7.0, 0.25% Tween 20). The column was washed with 15 ml of
column buffer, followed by column buffer without detergent until the
A280 of the flow-through was 0. Finally, the
protein was eluted in the latter buffer containing 10 mM maltose.
-mercaptoethanol, 10%
glycerol (GST-Nup116-GLFG) or 20 mM Hepes-KOH, pH 6.8, 150 mM potassium acetate, 2 mM magnesium acetate, 0.1% Tween 20, 2 mM DTT, 0.1% casamino acids
(GST-Nup100-GLFG). Binding assays were conducted as described
previously (40) except 2 µg of protein was added per 10 µl of
packed Glutathione-Sepharose. Bound and unbound fractions were
separated by electrophoresis on 7.5 or 9% SDS-polyacrylamide gels and
detected by Coomassie staining.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Transport factor localization in galactose-inducible Nup116-GLFG
overexpression strains
View larger version (86K):
[in a new window]
Fig. 1.
Mex67-GFP accumulates in the nucleus of yeast
cells overexpressing the GLFG region. Yeast cells expressing
chromosomal Mex67-GFP were transformed with an empty vector (pNLS-E1)
(A-D), a vector with Nup116-GLFG under control of a
galactose-inducible promoter (pSW163) (E-H), or a vector
with Nup100-GLFG under control of a galactose-inducible promoter
(pSW384) (I-L). The cells were grown in raffinose (A,
B, E, F, I, and J) or shifted to growth in galactose
(C, D, G, H, K, and L) for 5 h before
visualization of direct fluorescence (A, C, E, G,
I, and K). The corresponding Nomarski panels are shown
to the right of the respective fluorescence panel (B,
D, F, H, J, and L).
View larger version (24K):
[in a new window]
Fig. 2.
Mex67-GFP coimmunoprecipitates Nup116p from
yeast cell lysates. Whole cell lysates from wild-type or
Mex67-GFP-expressing cells were immunoprecipitated with anti-GFP
antibodies. Input, unbound, and bound fractions
were separated by SDS-PAGE and immunoblotted with either anti-GFP
(A), anti-GLFG (B), or anti-Nup116C
(C) antibodies. The anti-GFP antibody recognizes several
endogenous yeast proteins on immunoblots. Mex67-GFP is indicated by
two asterisks. The large band at ~55 kDa in the bound fractions
represents IgG.
View larger version (63K):
[in a new window]
Fig. 3.
Mex67p and the GLFG region directly interact
in vitro. A, input for
B-D. 2 µg of purified, bacterially expressed MBP
(B), His6-Kap95 (C), or MBP-Mex67
(D) was incubated with either purified GST, GST-Nup116GLFG,
or GST-Nup100GLFG immobilized on Glutathione-Sepharose. The binding
reactions were incubated 45 min at room temperature, and the unbound
and bound fractions were separated by SDS-PAGE followed by Coomassie
staining. Competition binding experiments were conducted to test
specificity (E). 2 µg of purified, bacterially expressed
GST-Nup116-GFLG was pre-bound to Glutathione-Sepharose, and then
purified MBP-Mex67 alone, MBP-Mex67 and MBP, or MBP-Mex67 and
MBP-Nup116 (full-length) was added. Binding was analyzed as in
B-D. Asterisks indicate the respective
immobilized protein, and arrows denote the input protein.
The double arrow indicates full-length MBP-Mex67.
View larger version (24K):
[in a new window]
Fig. 4.
Mex67p and Kap95p interact with distinct
regions of the Nup116-GLFG domain. Plasmids expressing either
LexABD-Kap95 or Gal4BD-Mex67 were cotransformed
with the indicated Gal4AD-Nup116-GLFG fusion plasmid into
two-hybrid reporter strains. The two-hybrid interaction was assayed by
growth on synthetic complete media lacking leucine, tryptophan, and
histidine (LTH) for Kap95p or synthetic complete media
lacking leucine, tryptophan, histidine, and adenine (LTHA)
for Mex67p. Positive signal was scored after incubation at 30 °C for
5 days.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the following generous colleagues who have kindly shared their reagents: J. Aitchison for pGFP-HIS5; M. Bucci for the Gle2-GFP strain; K. Iovine for pSW433; S. Hollenberg and P. James for two-hybrid strains; C. Hardy, M. Carlson, and P. James for two-hybrid plasmids; H. Fried for pNLS-E1; J. Aris for anti-Nop1p monoclonals; and M. Linder for anti-GFP antibodies. We thank members of the Wente laboratory for discussion and comments on manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM51219 (to S. R. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Cell Biology
and Physiology, Washington University School of Medicine, 660 S. Euclid
Ave., St. Louis, MO, 63110. Tel.: 314-362-2713; Fax: 314-747-1259;
E-mail: swente@cellbio.wustl.edu.
Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M008311200
2 L. A. Strawn and S. R. Wente, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NPC, nuclear pore complex; FG, phenylalanine-glycine; Gal4AD, GAL4 transcriptional activation domain; Gal4BD, GAL4 DNA binding domain; GFP, green fluorescent protein; GLFG, glycine-leucine-phenylalanine-glycine; GST, glutathione S-transferase; LexABD, LexA DNA binding domain, MBP, maltose-binding protein; SC, synthetic complete; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Talcott, B., and Moore, M. S. (1999) Trends Cell Biol. 9, 312-318[CrossRef][Medline] [Order article via Infotrieve] |
2. | Nakielny, S., and Dreyfuss, G. (1999) Cell 99, 677-690[Medline] [Order article via Infotrieve] |
3. | Mattaj, I. W., and Englmeier, L. (1998) Annu. Rev. Biochem. 67, 265-306[CrossRef][Medline] [Order article via Infotrieve] |
4. | Stoffler, D., Fahrenkrog, B., and Aebi, U. (1999) Curr. Opin. Cell Biol. 11, 391-401[CrossRef][Medline] [Order article via Infotrieve] |
5. | Yang, Q., Rout, M. P., and Akey, C. W. (1998) Mol. Cell 1, 223-234[Medline] [Order article via Infotrieve] |
6. |
Rout, M. P.,
Aitchison, J. D.,
Suprapto, A.,
Hjertaas, K.,
Zhao, Y.,
and Chait, B. T.
(2000)
J. Cell Biol.
148,
635-651 |
7. | Doye, V., and Hurt, E. (1997) Curr. Opin. Cell Biol. 9, 401-411[CrossRef][Medline] [Order article via Infotrieve] |
8. | Rout, M. P., and Wente, S. R. (1994) Trends Cell Biol. 4, 357-365[CrossRef] |
9. | Ryan, K. J., and Wente, S. R. (2000) Curr. Opin. Cell Biol. 12, 361-371[CrossRef][Medline] [Order article via Infotrieve] |
10. | Pemberton, L. F., Blobel, G., and Rosenblum, J. S. (1998) Curr. Opin. Cell Biol. 10, 392-399[CrossRef][Medline] [Order article via Infotrieve] |
11. | Adam, S. A. (1999) Curr. Opin. Cell Biol. 11, 402-406[CrossRef][Medline] [Order article via Infotrieve] |
12. | Gorlich, D., and Kutay, U. (1999) Annu. Rev. Cell Dev. Biol. 15, 607-660[CrossRef][Medline] [Order article via Infotrieve] |
13. | Bayliss, R., Littlewood, T., and Stewart, M. (2000) Cell 102, 99-108[Medline] [Order article via Infotrieve] |
14. | Chook, Y. M., and Blobel, G. (1999) Nature 399, 230-237[CrossRef][Medline] [Order article via Infotrieve] |
15. | Vetter, I. R., Arndt, A., Kutay, U., Gorlich, D., and Wittinghofer, A. (1999) Cell 97, 635-646[Medline] [Order article via Infotrieve] |
16. |
Schmitt, C.,
Von Kobbe, C.,
Bachi, A.,
Pante, N.,
Rodrigues, J. P.,
Boscheron, C.,
Rigaut, G.,
Wilm, M.,
Seraphin, B.,
Carmo-Fonseca, M.,
and Izaurralde, E.
(1999)
EMBO J.
18,
4332-4347 |
17. |
Ho, A. K.,
Raczniak, G. A.,
Ives, E. B.,
and Wente, S. R.
(1998)
Mol. Biol. Cell
9,
355-373 |
18. |
Bailer, S. M.,
Siniossoglou, S.,
Podtelejnikov, A.,
Hellwig, A.,
Mann, M.,
and Hurt, E.
(1998)
EMBO J.
17,
1107-1119 |
19. | Murphy, R., Watkins, J. L., and Wente, S. R. (1996) Mol. Biol. Cell 7, 1921-1937[Abstract] |
20. |
Hodge, C. A.,
Colot, H. V.,
Stafford, P.,
and Cole, C. N.
(1999)
EMBO J.
18,
5778-5788 |
21. |
Stutz, F.,
Kantor, J.,
Zhang, D.,
McCarthy, T.,
Neville, M.,
and Rosbash, M.
(1997)
Genes Dev.
11,
2857-2868 |
22. |
Santos-Rosa, H.,
Moreno, H.,
Simos, G.,
Segref, A.,
Fahrenkrog, B.,
Pante, N.,
and Hurt, E.
(1998)
Mol. Cell. Biol.
18,
6826-6838 |
23. |
Bachi, A.,
Braun, I. C.,
Rodriques, J. P.,
Pante, N.,
Ribbeck, K.,
Von Kobbe, C.,
Kutay, U.,
Wilm, M.,
Görlich, D.,
Carmo-Fonseca, M.,
and Izaurralde, E.
(2000)
RNA (NY)
6,
136-158 |
24. |
Katahira, J.,
Sträßer, K.,
Podtelejnikov, A.,
Mann, M.,
Jung, J. U.,
and Hurt, E.
(1999)
EMBO J.
18,
2593-2609 |
25. |
Ho, A. K.,
Shen, T.,
Ryan, K. J.,
Kiseleva, E.,
Aach Levy, M.,
Allen, T. D.,
and Wente, S. R.
(2000)
Mol. Cell. Biol.
20,
5736-5748 |
26. | Wente, S. R., and Blobel, G. (1993) J. Cell Biol. 123, 275-284[Abstract] |
27. | Iovine, M. K., Watkins, J. L., and Wente, S. R. (1995) J. Cell Biol. 131, 1699-1713[Abstract] |
28. |
Iovine, M. K.,
and Wente, S. R.
(1997)
J. Cell Biol.
137,
797-811 |
29. | Murphy, R., and Wente, S. (1996) Nature 383, 357-360[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Aitchison, J. D.,
Blobel, G.,
and Rout, M. P.
(1996)
Science
274,
624-627 |
31. |
Hellmuth, K.,
Lau, D. M.,
Bischoff, F. R.,
Künzler, M.,
Hurt, E.,
and Simos, G.
(1998)
Mol. Cell. Biol.
18,
6374-6386 |
32. | Rout, M. P., Blobel, G., and Aitchison, J. D. (1997) Cell 89, 715-725[Medline] [Order article via Infotrieve] |
33. |
Marelli, M.,
Aitchison, J. D.,
and Wozniak, R. W.
(1998)
J. Cell Biol.
143,
1813-1830 |
34. | Damelin, M., and Silver, P. A. (2000) Mol. Cell 5, 133-140[Medline] [Order article via Infotrieve] |
35. |
Seedorf, M.,
Damelin, M.,
Kahana, J.,
Taura, T.,
and Silver, P. A.
(1999)
Mol. Cell. Biol.
19,
1547-1557 |
36. | Baudin, A., Ozier, K. O., Denouel, A., Lacroute, F., and Cullin, C. (1993) Nucleic Acids Res. 21, 3329-3330[Medline] [Order article via Infotrieve] |
37. | Wente, S. R., Rout, M. P., and Blobel, G. (1992) J. Cell Biol. 119, 705-723[Abstract] |
38. |
Henriquez, R.,
Blobel, G.,
and Aris, J. P.
(1990)
J. Biol. Chem.
265,
2209-2215 |
39. |
Bucci, M.,
and Wente, S. R.
(1998)
Mol. Biol. Cell
9,
2439-2461 |
40. | Rexach, M., and Blobel, G. (1995) Cell 83, 683-692[Medline] [Order article via Infotrieve] |
41. | Hardy, C. F. J. (1996) Mol. Cell. Biol. 16, 1832-1841[Abstract] |
42. |
James, P.,
Halladay, J.,
and Craig, E. A.
(1996)
Genetics
144,
1425-1436 |
43. | Yang, X., Hubbard, E. J., and Carlson, M. (1992) Science 257, 680-682[Medline] [Order article via Infotrieve] |
44. | Cole, C. N., and Hammell, C. M. (1998) Curr. Biol. 8, 368-372 |
45. |
Snay-Hodge, C. A.,
Colot, H. V.,
Goldstein, A. L.,
and Cole, C. N.
(1998)
EMBO J.
17,
2663-2676 |
46. | Tseng, S. S.-I., Weaver, P. L., Hitomi, M., Tartakoff, A. M., and Chang, T.-H. (1998) EMBO J. 17, 2652-2662 |
47. |
Segref, A.,
Sharma, K.,
Doye, V.,
Hellwig, A.,
Huber, J.,
Luhrmann, R.,
and Hurt, E.
(1997)
EMBO J.
16,
3256-3271 |
48. |
Strä9gerds]er, K.,
Babler, J.,
and Hurt, E.
(2000)
J. Cell Biol.
150,
695-706 |
49. |
Hood, J. K.,
Casolari, J. M.,
and Silver, P. A.
(2000)
J. Cell Sci.
113,
1471-1480 |
50. |
Bailer, S. M.,
Balduf, C.,
Katahira, J.,
Podtelejnikov, A.,
Rollenhagen, C.,
Mann, M.,
Pante, N.,
and Hurt, E.
(2000)
J. Biol. Chem.
275,
23540-23548 |
51. |
Bucci, M.,
and Wente, S. R.
(1997)
J. Cell Biol.
136,
1185-1199 |
52. | Guarente, L., Yocum, R. R., and Gifford, P. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7410-7414[Abstract] |