The Yeast Nucleoporin Nup53p Specifically Interacts Protein Import

The Yeast Nucleoporin Nup53p Specifically Interacts Protein Import
Molecular Biology of the Cell
Vol. 11, 3885–3896, November 2000
The Yeast Nucleoporin Nup53p Specifically Interacts
with Nic96p and Is Directly Involved in Nuclear
Protein Import
Birthe Fahrenkrog,*† Wolfgang Hübner,‡ Anna Mandinova,* Nelly Panté,§㥋
Walter Keller,‡ and Ueli Aebi*¶
*M. E. Müller Institute for Structural Biology, and ‡Department of Cell Biology, Biozentrum,
University of Basel, CH-4056 Basel, Switzerland; and §Institute of Biochemistry, Federal Institute of
Technology Zürich (ETHZ), CH-8092 Zürich, Switzerland
Submitted March 10, 2000; Revised June 27, 2000; Accepted September 7, 2000
Monitoring Editor: Pamela A. Silver
The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore
complex (NPC) which, in yeast, is composed of ⬃30 different proteins (nucleoporins). Preembedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near
or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear
protein import. To more specifically explore the potential role of Nic96p in nuclear protein import,
we performed a two-hybrid screen with NIC96 as the bait against a yeast genomic library to
identify transport factors and/or nucleoporins involved in nuclear protein import interacting with
Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple
locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p
is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear
import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.
INTRODUCTION
The nuclear pore complex (NPC) is a large supramolecular
assembly that spans the double membrane of the nuclear
envelope (NE) and mediates bidirectional nucleocytoplasmic transport (Izaurralde and Adam, 1998; Mattaj and
Englmeier, 1998; Ohno et al., 1998). Using amphibian oocytes
extensive electron microscopic analyses has unveiled the
principal structural organization of the ⬃125 MDa vertebrate NPC (Panté and Aebi, 1996; Stoffler et al., 1999). To a
large extent the yeast NPC appears to be designed according
to the same architectural principles except that its linear
dimensions appear to be ⬃15% smaller (Fahrenkrog et al.,
1998) and its mass only amounts to ⬃60 MDa (Rout and
Blobel, 1993; Yang et al., 1998). Compared with vertebrate
NPC three-dimensional (3-D) reconstructions (cf. Akey and
Radermacher, 1993), 3-D reconstruction of the yeast NPC
exhibits more tenuous cytoplasmic and nuclear ring moieties (Yang et al., 1998).
Present addresses: †European Molecular Biology Laboratory,
Meyerhofstr.1, D-69117 Heidelberg, Germany; 㛳Department of
Zoology, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
¶
Corresponding author. E-mail address: [email protected]
© 2000 by The American Society for Cell Biology
The vertebrate NPC is composed of in excess of 50 different proteins, termed nucleoporins (Nups), whereas the yeast
NPC is thought to consist of ⬃30 –50 different nucleoporins
(Doye and Hurt, 1997; Stoffler et al., 1999; Rout et al., 2000).
To date, ⬃20 vertebrate and ⬃30 yeast nucleoporins, i.e.,
presumably all yeast nucleoporins (Rout et al., 2000), have
been identified and characterized. Localization by immunogold-electron microscopy (immunogold-EM) in both vertebrate and yeast has revealed that these nucleoporins mainly
reside at the cytoplasmic and the nuclear periphery of the
NPC, many of them having dual locations in a near-symmetrical manner relative to the central plane of the NPC
(Panté and Aebi, 1996; Stoffler et al., 1999; Rout et al., 2000).
Ions and small molecules can traverse the NPC by passive
diffusion, whereas proteins, RNAs, and ribonucleo protein
(RNP) particles are transported through the NPC by a signal-mediated mechanism. More specifically, the nuclear import of cargoes harboring a classical nuclear localization
signal (NLS) is mediated by a soluble dimeric receptor consisting of an adaptor subunit, called importin ␣ in vertebrates and Srp1p in yeast, and the actual receptor subunit,
called importin ␤ in vertebrates and Kap95p in yeast. The
adaptor subunit recognizes the cargo’s NLS, whereas the
receptor subunit recognizes distinct nucleoporins and interacts with various transport factors (e.g., NTF2; Nehrbass and
3885
B. Fahrenkrog et al.
Blobel, 1996) as it escorts the cargo-adaptor complex from
the cytoplasm through the NPC into the nucleus (Corbett
and Silver, 1997; Fabre and Hurt, 1997; Izaurralde and
Adam, 1998; Mattaj and Englmeier, 1998; Ohno et al., 1998).
Other adaptors, e.g., the importin ␣-like snurportin involved
in the import of U snRNPs, and receptors, e.g., the importin
␤-like transportin involved in import of hnRNP A1, have
been identified and characterized, and the related transport
pathways have been elucidated (Izaurralde and Adam, 1998;
Mattaj and Englmeier, 1998; Ohno et al., 1998). Similarly,
nuclear export is mediated by the formation of a heterotrimeric complex consisting of the cargo harboring a nuclear
export signal (NES), the export receptor, and Ran-GTP (Izaurralde and Adam, 1998; Mattaj and Englmeier, 1998; Ohno et
al., 1998). The first NES has been identified in the HIV-1 Rev
protein together with its export receptor CRM1 (exportin):
Both Rev and CRM1 are involved in the export of unspliced
viral RNA (Izaurralde and Adam, 1998; Mattaj and Englmeier, 1998; Ohno et al., 1998). Other export receptors have
been identified, for example, exportin-t or TAP, whereas the
export signals for many RNA export pathways, especially
those for mRNA export, have remained elusive (Izaurralde
and Adam, 1998; Mattaj and Englmeier, 1998; Ohno et al.,
1998).
Nic96p is an essential nucleoporin in yeast: it has been
identified by its interaction with Nsp1p which, in turn, is the
first yeast nucleoporin that has been identified and molecularly characterized (Hurt, 1988; Nehrbass et al., 1990; Grandi
et al., 1993). Affinity purification of ProtA-Nsp1p by IgGSepharose chromatography identified Nic96p as a copurifying constituent (Grandi et al., 1993). Additionally, mutations
in NSP1 and NIC96 were found to be synthetically lethal
(Grandi et al., 1995). Biochemically, both Nic96p and Nsp1p
belong to one subcomplex of the yeast NPC, i.e. the Nsp1p
complex, which also contains the nucleoporins Nup49p and
Nup57p (Grandi et al., 1993, 1995; Schlaich et al., 1997).
Nic96p resides about the cytoplasmic and the nuclear periphery of the central channel in a near-symmetrical manner,
as well as near or at the distal ring of the nuclear basket
(Fahrenkrog et al., 1998; Fahrenkrog et al., 2000). Hence,
Nic96p and Nsp1p closely colocalize in all three sites (Fahrenkrog et al., 1998, 2000). Evidently, both nucleoporins,
Nic96p and Nsp1p, are involved in protein import into the
nucleus (Nehrbass et al., 1993; Grandi et al., 1995). However,
although Nsp1p interacts with distinct soluble factors involved in nuclear import, i.e. importin ␤ and Ran (Stochaj et
al., 1998; Seedorf et al., 1999), the direct interaction of Nic96p
with nuclear import factors has remained elusive.
To gain more insight into the specific role of Nic96p in
nuclear protein import, e.g., to identify transport factors
interacting with Nic96p, we performed a two-hybrid screen
with NIC96 as the bait against a yeast genomic library.
According to this assay, we found Nic96p not to interact
with transport factors, but with the nucleoporin Nup53p, a
nucleoporin that has recently been identified independently
in a synthetic lethal screen with POM152p (Marelli et al.,
1998). Nup53p is located at the cytoplasmic and the nuclear
periphery of the central NPC framework, and at the nuclear
basket. Although mutations of NUP53 cause no obvious
structural alterations of the yeast NPC, they lead to defects
in nuclear protein import. Molecularly, the role of Nup53p
in NLS-mediated protein import involves its direct interac3886
tion with the NLS-receptor Kap95p. In contrast, NES-mediated nuclear export appears not to be impaired by the disruption of NUP53.
MATERIALS AND METHODS
Yeast Strains and Media
The yeast strains used in this study are listed in Table 1. All strains
were grown at 30°C, unless otherwise stated. Media and genetic
methods, including mating, sporulation, and tetrad dissection were
as described elsewhere (Guthrie and Fink, 1991). Yeast cells were
transformed by using the lithium acetate method (Gietz et al., 1992).
Plasmids
The following yeast plasmids were used: pUN100-NOP1::ProtA-TEV
(pNOPPATA1L; Hellmuth et al., 1998); YEp13-NIC96 (Grandi et al.,
1993; kindly provided by Ed Hurt, Biochemie-Zentrum, Heidelberg,
Germany); pAS2(⌬⌬) and pACT2 (Harper et al., 1993; FromontRacine et al., 1997); pPS815(pADH-NLS-GFP-lacZ), pPS1372(pADHNLS-NES-GFP-GFP), pPS1494(pGAL-REV-GFP), kindly provided
by Jennifer Hood and Pamela Silver (Dana Farber Institute, Boston,
MA); pLDB419(pYAP1-GFP LEU2 2 ␮), kindly provided by Anita
Corbett (Emory University School of Medicine, Atlanta, GA) and
Laura Davis (Brandeis University, Waltham, MA). pAS2-NIC96, a
polymerase chain reaction (PCR) amplification of NIC96 open reading frame (ORF) extending from nucleotide ⫹1 to ⫹2590 inserted
into BamHI-NcoI cut pAS2. The PCR product was exchanged against
a BsmI fragment of genomic NIC96 from YEp13-NIC96. pACT2, 2
␮/LEU2 based yeast genomic library. pNOPPATA1L-NUP53, PCR
product of NUP53 ORF extending from nucleotide ⫹1 to ⫹1428
inserted into NcoI-BamHI cut pNOPPATA1L.
Yeast Two-Hybrid Screen
The yeast two-hybrid screen, with NIC96 as the bait against a yeast
genomic library (Table 1), was performed exactly as described
(Fromont-Racine et al., 1997)
Gene Disruption and Recombinant Protein A
Tagging of NUP53
NUP53 deletion constructs were prepared by replacing nucleotides
⫺10 to ⫹500 with the TRP1 selectable marker gene generated by
PCR. nup53::TRP1 was transformed into the diploid BMA41 strain
(Baudin-Baillieu et al., 1997) and selected on SD-W plates (Rothstein,
1991). Trp1⫹ transformants were characterized for correct integration of nup53::TRP1 at the NUP53 locus by PCR analysis. BMA41
diploid heterozygous for NUP53 were sporulated and subsequently
dissected by tetrad analysis. For recombinant protein A tagging, the
NUP53 gene was amplified by PCR, thereby generating an NcoI and
a BamHI site at the 5⬘ and the 3⬘ end, respectively. The resulting PCR
product was sequenced and inserted into NcoI-BamHI cut
pNOPPATA1L. The resulting plasmid was transformed into the
⌬nup53 strain and selected on SD-LW plates (Gietz et al., 1992).
Immunogold-EM
Preparation and in situ immunolocalization of ProtA-Nup53p was
performed by pre-embedding labeling yeast cells with an antiprotein A antibody directly conjugated to 8-nm colloidal gold as
described previously (Fahrenkrog et al., 1998).
EM
To evaluate the morphology of the ⌬nup53 strain, yeast cells were
transformed into spheroplasts, washed twice in 0.1 M potassium
phosphate buffer, pH 6.5, and fixed in 2% glutaraldehyde for 1 h, all
Molecular Biology of the Cell
Nup53p Is Involved in Nuclear Import
Table 1. Yeast strains
Strain
CG-1945
Y187
BMA41
BMA41/1a
⌬nup53
ProtA-Nup53p
BMA41 (NLSGFP)
⌬nup53 (NLSGFP)
⌬nup53 (NESNLS-GFP)
BMA41/1a
(NES-NLSGFP)
⌬nup53 (RevGFP)
BMA41/1a
(Rev-GFP)
BMA41/1a
(Yap1p-GFP)
⌬nup53 (Yap1pGFP)
xpo1-1
xpo1-1 (Yap1pGFP)
xpo1-1 (NESNLS-GFP)
xpo1-1 (RevGFP)
nup49-313
nup49-313
(NLS-GFP)
Genotype
Source/references
Mata, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3, 112, gal4-542, gal80538, LYS2⬋GAL1-HIS3, URA3⬋(GAL4 17mers)3-CYC1-lacZ, cyhr2
Mat␣, ura3-52, his3, ade2-101, trp1-901, leu2-3, 112, met⫺, gal4⌬, gal80⌬,
URA3⬋GAL1-lacZ
Mata/␣, ade2-1/ade2-1, leu2-3, 112/leu2-3, 112, ura3-1/ura3-1, trp1⌬/trp1⌬, his3-11,
15/his3-11, 15, can1-100/can1-100
Mata, ade2-1, leu2-3, 112, ura3-1, trp1⌬, his3-11, 15, can1-100
Mata, ade2-1, leu2-3, ura 3-1, trp1⌬, his3-11, 15, can1-100, nup53⬋TRP1
Mata, ade2-1, leu2-3, ura 3-1, trp1⌬, his3-11, 15, can1-100, nup53⬋TRP1 (pUN100NOP1⬋ProtA-TEV-NUP53)
Mata/␣, ade2-1/ade2-1, leu2-3, 112/leu2-3, 112, ura3-1/ura3-1, trp1⌬/trp1⌬, his3-11,
15/his3-11, 15, can1-100/can1-100 (pADH-NLS-GFP-lacz)
Mata, ade2-1, leu2-3, ura 3-1, trp1⌬, his3-11, 15, can1-100, nup53⬋TRP1 (pADHNLS-GFP-lacZ)
Mata, ade2-1, leu2-3, ura 3-1, trp1⌬, his3-11, 15, can1-100, nup53⬋TRP1 (pADHNLS-NES-GFP-GFP)
Mata, ade2-1, leu2-3, 112, ura3-1, trp1⌬, his3-11, 15, can1-100 (pADH-NLS-NESGFP-GFP)
Stan et al. (1994)
Song et al. (1994)
Baudin-Baullieu et al. (1997)
Baudin-Baullieu et al. (1997)
This study
This study
This study
This study
This study
This study
Mata, ade2-1, leu2-3, ura 3-1, trp1⌬, his3-11, 15, can1-100, nup53⬋TRP1 (pGalRev-GFP)
Mata, ade2-1, leu2-3, 112, ura3-1, trp1⌬, his3-11, 15, can1-100 (pGal-Rev-GFP)
This study
Mata, ade2-1, leu2-3, 112, ura3-1, trp1⌬, his3-11, 15, can1-100 (pLDB419)
This study
Mata, ade2-1, leu2-3, ura 3-1, trp1⌬, his3-11, 15, can1-100, nup53⬋TRP1 (pLDB419)
This study
ade2-1, ura3-1, his3-11,15, trp1-1, leu2-3, 112, can1-100, xpo1⬋LEU2 (pKW456)
ade2-1, ura3-1, his3-11,15, trp1-1, leu2-3, 112, can1-100, xpo1⬋LEU2 (pKW456,
pLDB419)
ade2-1, ura3-1, his3-11,15, trp1-1, leu2-3, 112, can1-100, xpo1⬋LEU2 (pADH-NESNLS-GFP-GFP)
ade2-1, ura3-1, his3-11,15, trp1-1, leu2-3, 112, can1-100, xpo1⬋LEU2 (pGal-RevGFP)
Mat␣, ade2, ade3, his3, leu2, ura3, nup49⬋TRP1 (pUN90-nup49-313)
Mat␣, ade2, ade3, his3, leu2, ura3, nup49⬋TRP1 (pUN90-nup49-313) (pADH-NLSlacZ-GFP)
Stade et al. (1997)
This study
steps as described (Fahrenkrog et al., 1998). After 1-h postfixation in
1% osmium tetroxide, the yeast cells were processed for electron
microscopy (Fahrenkrog et al., 1998). Thin-sections were cut on a
Reichert Ultracut ultramicrotome (Reichert-Jung Optische Werke,
Vienna, Austria) by using a diamond knife (Diatome, Biel, Switzerland). The sections were collected on collodion-coated copper grids
and stained with 6% uranyl acetate for 1 h followed by 2% lead
citrate for 2 min. Specimens were inspected and electron micrographs recorded with a Hitachi H-7000 transmission electron microscope (Hitachi Ltd., Tokyo, Japan) operated at an acceleration
voltage of 100 kV.
Affinity Purification of ProtA-TEV-Nup53p
ProtA-TEV-Nup53p was affinity purified from a ⌬nup53 strain
transformed with recombinant Nup53p that was amino-terminally
tagged with two IgG binding domains of Staphylococcus aureus
protein A followed by a cleavage site for the TEV protease, by
IgG-Sepharose chromatography (Hellmuth et al., 1998; Senger et al.,
1998). The lysis buffer used contained either 100 mM potassium
acetate for the elution of Nic96p or 300 mM potassium acetate for
the elution of Kap95p. The NPC-containing fraction (2 ␮l) and 100
␮l of the eluate, respectively, were precipitated in acetone, resuspended in 20 ␮l of gel-loading buffer, and analyzed by SDS-PAGE,
Vol. 11, November 2000
This study
This study
This study
Grandi et al. (1995)
This study
followed by Coomassie blue staining and Western blotting by using
an anti-Nic96p antibody (Grandi et al., 1995) and an anti-Kap95p
antibody (Koepp et al., 1996), respectively. Finally, the blot was
stained by a secondary antibody conjugated with alkaline-phosphatase.
In Vivo Protein Import Assay
An in vivo protein import assay was performed as described
(Shulga et al., 1996). In this assay, logarithmically growing yeast
cells constitutively expressing NLS-green fluorescent protein (GFP)
(i.e., ⌬nup53 [NLS-GFP]; Table 1) as a reporter cargo were treated
with sodium azide and deoxyglucose to block NLS-mediated protein import. At steady state, this block yields an approximately even
distribution of the NLS-GFP cargo in the cytoplasm and the nucleus
by passive diffusion of NLS-GFP across the NPC. After removal of
the metabolic inhibitors the cells recover immediately in fresh medium, and the NLS-GFP cargo is reimported into the nucleus in an
active manner. The relative import rates in the mutant and the
wild-type strain were compared by counting the cells that exhibited
NLS-GFP nuclear accumulation as a function of time. For immunogold-EM, the assay was performed in the same way: after 0, 5, and
15 min of active reimport, the cells were fixed by addition of 2%
paraformaldehyde, pH 6.5, and prepared for EM as described else-
3887
B. Fahrenkrog et al.
where (Fahrenkrog et al., 1998). Cells were labeled with a polyclonal
anti-GFP antibody (a kind gift from Jennifer Hood and Pamela
Silver) directly conjugated to 8-nm colloidal gold.
In Vivo Protein Export Assays
In a first protein export assay a GFP reporter was fused simultaneously to the NLS of the simian virus 40 large T antigen and the
NES of the protein kinase inhibitor PKI (i.e. NES-NLS-GFP; Table 1;
Stade et al., 1997). For this purpose, the ⌬nup53 and BMA41 and
xpo1-1 control strain were transformed with a plasmid expressing
the NES-NLS-GFP reporter. The cells were grown in selective media
and the subcellular location of the reporter was determined by
confocal laser scanning microscopy.
In a second protein export assay the ⌬nup53 and BMA41 and
xpol1-1 control strain were transformed with a construct where a
GFP reporter was fused to the HIV-1 Rev protein (i.e., Rev-GFP;
Table 1; Taura et al., 1998), grown in selective medium containing
glucose. After shifting the cells to galactose-containing medium for
4 h, the subcellular location of the Rev-GFP reporter was analyzed
by confocal laser scanning microscopy.
In a third protein export assay the ⌬nup53, BMA41, and xpo1–1
cells were transformed with the plasmid pLDB419 expressing the
Yap1p-GFP reporter (Table 1; Yan et al., 1998). The cells were grown
in selective medium and the subcellular location of Yap1p-GFP was
determined under steady-state conditions or after treatment of cells
with 1.5 mM diamide (i.e. to induce oxidative stress) for 15 h by
confocal laser scanning microscopy.
RESULTS
Nic96p Interacts with the Yeast Nucleoporin Nup53p
by a Two-Hybrid Screen
Based on the known involvement of Nic96p in nuclear protein
import (Grandi et al., 1995) and its multiple locations about the
cytoplasmic and the nuclear periphery of the central channel
and near or at the distal ring of the nuclear basket (Fahrenkrog
et al., 1998; Fahrenkrog et al., 2000), we set out to gain more
insight into the molecular interactions Nic96p may experience
while a cargo is traversing the NPC on its way from the
cytoplasm into the nucleus. Hence, we carried out a yeast
two-hybrid screen with full-length NIC96 (i.e., fused in frame
to the GAL4 DNA binding domain) as the bait. To achieve this,
the strain CG1945 containing the bait plasmid was mated to the
strain Y187 containing FRY1 libraries (Table 1). One hundred
and forty clones exhibited activation of the two reporter genes
HIS3 and lacZ; 96 of these positive clones were sequenced and
8 of these were identified as the ORF YMR153w by a database
search of the yeast genome. Recently, YMR153w has been
identified to encode the yeast nucleoporin Nup53p by a synthetic lethality screen with POM152 (Marelli et al., 1998). The
interactions between NIC96 and the prey NUP53 resided in
two overlapping fragments in the N-terminal domain of
NUP53, classifying this fusion as an A1 fusion (Fromont-Racine
et al., 1997). Because NIC96 alone did not activate the transcription of the reporter genes, we conclude that the interaction
between Nic96p and Nup53p is specific. A database search
with NUP53 revealed putative homologous ORFs and ESTs in
various species, e.g., human, mouse, Xenopus laevis, Caenorhabditis elegans, Drosophila melanogaster, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Arabidopsis thaliana, and Gossypium
hirsutum, with the highest homologies among these ORFs residing in their central domains, thus indicating high conservation of these ORFs from yeast to higher eukaryotes and even
plants (our unpublished results; Marelli et al., 1998).
3888
Ultrastructural Localization of Nup53p by
Immunogold-EM
Nup53p has been previously localized to the nuclear rim by
immunofluorescence microscopy and to both the cytoplasmic and the nuclear face of the NPC by immunogold-EM
(Marelli et al., 1998; Rout et al., 2000). To more precisely
localize Nup53p at the ultrastructural level within the yeast
NPC to distinct NPC substructures, we performed immunogold-EM with the ProtA-Nup53p strain (Table 1). For this
purpose, we constructed a yeast strain that is disrupted for
NUP53 (i.e., ⌬nup53; see MATERIALS AND METHODS;
Table 1). In this strain we then replaced its disrupted NUP53
gene by a plasmid-borne version of NUP53 fused to two IgG
binding domains of the S. aureus protein A to the 5⬘ end of
its ORF. Pre-embedding labeling of spheroplasted yeast cells
with an anti-protein A antibody directly conjugated to 8-nm
colloidal gold revealed that Nup53p resides at the cytoplasmic and the nuclear periphery of the central NPC framework (Figure 1A, top and middle), as well as at the fibrils
forming the nuclear basket (Figure 1A, bottom). Quantification of the gold particle distribution (Figure 1B) with respect
to the central plane of the NPC revealed that 55% of the gold
particles were detected at distances of 25–50 nm from the
central plane (30.2 ⫾ 6.4 nm). Together with the corresponding radial distances of 30 –50 nm (31.5 ⫾ 7.9 nm) this locates
Nup53p to the cytoplasmic face of the central framework
rather than to the cytoplasmic fibrils. In the latter case the
gold particles would have been detected at radial distances
ranging from 0 to ⬃60 nm due to the high flexibility of the
cytoplasmic fibrils. In addition, 45% of all gold particles
were depicted on the nuclear face of the NPC, with ⬃40% at
vertical distances of ⫺20 to ⫺40 nm (⫺24.3 ⫾ 4.5 nm) and
⬃60% at vertical distances of ⫺50 to ⫺100 nm (⫺68.3 ⫾ 15.4
nm), corresponding to the nuclear periphery of the central
framework and the nuclear basket fibrils, respectively. The
gold particles found on the nuclear side of the NPCs were
detected 1) at radial distances ranging from 30 to 50 nm
(34.7 ⫾ 12.1 nm), thus labeling the nuclear face of the central
framework; and 2) at distances of 20 –30 nm (26.5 ⫾ 10.4
nm), thus labeling an epitope residing at the nuclear basket.
Nup53p and Nic96p Closely Colocalize
As displayed schematically in Figure 1C, based on the previously published immunogold-EM localization of Nic96p
(Fahrenkrog et al., 1998, 2000), both Nup53p and Nic96p
exhibit three distinct locations within the yeast NPC. The
corresponding “location clouds” are centered about the average distances (x, y) of each epitope of Nup53p and Nic96p
normal to the central plane (y-axis) and perpendicular to the
central eightfold symmetry axis (x-axis) of the NPC (Fahrenkrog et al., 2000). The radii of the elliptical location clouds
are defined by the respective SDs from the mean of the
distances from the central plane and the central eightfold
symmetry axis. Although the location clouds of Nic96p and
Nup53p do not exactly coincide, they closely colocalize.
Overall, the Nup53p epitopes reside at higher radii compared with Nic96p. As illustrated schematically in Figure
1C, the Nup53p and Nic96p epitopes come close enough so
that the two nucleoporins may indeed physically interact
(see below) at all three distinct sites.
Molecular Biology of the Cell
Nup53p Is Involved in Nuclear Import
Figure 1. Immunogold-EM localization of Nup53p in ProtA-Nup53p cells (i.e., ⌬nup53[ProtA-Nup53p] strain; Table 1). (A) Triton
X-100-extracted spheroplasts were preimmunolabeled with a polyclonal anti-protein A antibody directly conjugated to 8-nm colloidal gold.
Shown are selected examples of gold-labeled NPCs in cross-sections along the NE. The antibody labeled the cytoplasmic (top) and the nuclear
(middle) periphery of the central framework, and the nuclear basket (bottom). c, cytoplasm; n, nucleus. (B) Quantitative analysis of the gold
particle distribution associated with the NPCs in the ⌬nup53(ProtA-Nup53p) strain. For quantitative analysis 94 gold particles were scored.
(C) Schematic representation of the close colocalization of Nic96p and Nup53p within the yeast NPC by their respective “location clouds”
(Fahrenkrog et al., 2000). Accordingly, Nic96p is located about the cytoplasmic and the nuclear periphery of the central channel, and near or
at the distal ring of the nuclear basket. Similarly, Nup53p resides on the cytoplasmic and the nuclear face of the central framework (i.e., close
to or at the base of the cytoplasmic and the nuclear fibrils), and near the distal end of the nuclear basket fibrils. Scale bar, 100 nm (A).
Nup53p and Nic96p Physically Interact
To confirm that Nup53p and Nic96p do indeed physically
interact as suggested by the yeast two-hybrid screen,
Nup53p was affinity-purified from yeast cells, and analyzed
for copurifying components. To do so, recombinant Nup53p
was amino-terminally tagged with two IgG binding domains of S. aureus protein A followed by a cleavage site for
the TEV protease, and transformed into the ⌬nup53 strain
(Table 1). ProtA-TEV-Nup53p was purified from this strain
by IgG-Sepharose chromatography under nondenaturating
conditions. Nup53p together with bound proteins was released from the IgG-Sepharose column upon incubation
with TEV protease. The yeast cells after enzymatic removal
of the cell wall followed by homogenization in lysis buffer
containing 0.5% Tween 20 were further fractionated by centrifugation. The resulting supernatant containing the NPC
fraction, which was applied to the IgG-Sepharose column,
and the eluate of the column after digestion with TEV protease were analyzed by SDS-PAGE followed by Coomassie
blue staining and by Western blotting with an anti-Nic96p
antibody (Figure 2). Although it was not possible to identify
any specific copurifying components from the Commassie
Vol. 11, November 2000
blue stained gel (our unpublished results), Western-Blot
analysis with a polyclonal anti-Nic96p antibody clearly
demonstrated the specific interaction between Nup53p and
Figure 2. Affinity purification of ProtA-TEV-Nup53p from
⌬nup53(ProtA-TEV-Nup53p) cells (Table 1) reveals interaction with
Nic96p. ProtA-TEV-Nup53p was affinity-purified by IgG-Sepharose
chromatography and Nup53p was released by TEV-mediated proteolytic cleavage. Western blot analysis with a polyclonal antiNic96p antibody reveals specific interaction of Nup53p with
Nic96p. Lane 1, NPC-containing fraction of a wild-type control
strain (BMA41); lane 2, eluate derived from the wild-type control
strain; lane 3, NPC-containing fraction of the strain expressing
ProtA-TEV-Nup53p; and lane 4, eluate derived from the ProtATEV-Nup53p.
3889
B. Fahrenkrog et al.
Figure 3. Thin-section electron micrographs of ⌬nup53 cells (B) and of a wild-type BMA41 control strain (A) (for the cell strains see Table
1). Cells were grown at 30°C, fixed, embedded, and processed for thin-section electron microscopy. The morphology of the nucleus, the NE,
and the NPCs in the ⌬nup53 cells appears indistinguishable from that of the wild-type cells. Arrows mark the nuclear basket of the NPCs.
Scale bars, 100 nm (A and B).
Nic96p (Figure 2). Nic96p was present in the NPC-containing fraction of the yeast strain expressing ProtA-TEVNUP53p (Figure 2, lane 3) as well as in the eluate from the
IgG Sepharose beats after TEV protease digestion (Figure 2,
lane 4). This interaction of Nic96p with Nup53p is specific
because in the wild-type BMA41 control strain (Table 1)
Nic96p could be detected in the NPC-containing fraction of
this strain (Figure 2, lane 1), but not in the eluate after
IgG-Sepharose chromatography (Figure 2, lane 2). Hence,
Nic96p does not unspecifically bind to the IgG beads but
rather via ProtA-TEV-NUP53p.
nup53 Deletion Strains Are Viable and Exhibit No
Obvious Structural Abnormalities of Their NPCs
To examine the phenotype of the nup53 null strain (i.e.
⌬nup53; see MATERIALS AND METHODS; Table 1), a
Trp1⫹ transformant containing the disrupted NUP53 gene
was selected, sporulated, and tetrads were dissected. All
four tetrads were viable, indicating that NUP53 is not essential. Trp⫹ haploids lacking NUP53 grow at similar rates than
those observed in the presence of wild-type NUP53 at temperatures ranging from 15°C to 37°C (our unpublished results). To further characterize the phenotype of the ⌬nup53
null strain, we examined its morphology by thin-section
electron microscopy. For this purpose, ⌬nup53 cells were
grown in YPAD medium at 30°C and processed for EM. As
documented in Figure 3, the morphology of the NE and the
NPCs of the ⌬nup53 cells (Figure 3B) appear indistinguishable from those of the wild-type BMA41 control cells (Figure
3A). Additionally, in ⌬nup53 cells grown at 37°C the morphology of the NE and the NPCs also appear indistinguishable from those of the wild-type BMA41 cells grown under
the same conditions (our unpublished results). These observations indicate that Nup53p is not required for specifying
NPC assembly and structure, at least not to the extent that
could be detected at the resolution level provided by embedding/thin sectioning EM.
3890
Disruption of NUP53 Attenuates Nuclear Protein
Import
Next we set out to gain more insight into the possible
functional role of Nup53p in nucleocytoplasmic transport.
To test whether the ⌬nup53 strain is impaired in nuclear
protein import, we performed an in vivo import assay
(Shulga et al., 1996). In this assay, logarithmically growing
yeast cells constitutively expressing an NLS-GFP reporter
cargo (i.e. ⌬nup53 [NLS-GFP]; Table 1) were treated with
sodium azide and deoxyglucose to block NLS-mediated nuclear protein import. At steady state, this block yields an
even distribution of the GFP signal in the cytoplasm and the
nucleus by passive diffusion of NLS-GFP across the NPC.
After removal of the metabolic inhibitors by placing the cells
in fresh glucose-containing medium, they recover from the
block and the NLS-GFP is actively imported into the nucleus. The relative import rates in the ⌬nup53 and the wildtype BMA41 strain were compared by counting the cells that
exhibited NLS-GFP nuclear accumulation as a function of
time (Figure 4, A and B). As illustrated in Figure 4A, the
⌬nup53 cells displayed a strongly attenuated import rate of
the NLS-GFP reporter (left) compared with that of the wildtype BMA41 strain (middle). A nup49 –313 control strain that
is known to be defective in NLS-dependent nuclear protein
import accumulated the NLS-GFP reporter predominantly
in the cytoplasm (Figure 4A, right). Quantification of the
relative import rates for the BMA41 control strain and the
⌬nup53 strain revealed that for the BMA41 control strain a
relative accumulation rate of GFP inside the nucleus of
13.5%/min, whereas the ⌬nup53 strain yielded an accumulation rate of only 3%/min (Figure 4B). However, protein
import is not completely blocked in the ⌬nup53 strain because under steady-state conditions as well as after ⬃5 h of
removing the sodium azide/deoxyglucose block the NLSGFP reporter does accumulate in the nucleus (our unpublished results). Immunogold-EM with a polyclonal anti-GFP
antibody directly conjugated to 8-nm colloidal gold further
revealed that the NLS-GFP reporter cargo preferentially accumulates in the nuclear basket (Figure 4C), indicating that
although the NLS-GFP cargo can traverse the central pore it
Molecular Biology of the Cell
Nup53p Is Involved in Nuclear Import
Figure 4. Nup53p mediates nuclear import of a NLS-GFP reporter. (A) Intracellular localization of the NLS-GFP reporter in ⌬nup53,
wild-type BMA41 control cells, and nup49-313 control cells (Table 1) after azide and deoxyglucose treatment (i.e., 0 min) and after recovery
from the drug treatment in a glucose-containing medium (i.e., 15 and 120 min). Shown are confocal fluorescence micrographs (left) and
coincident fluorescence/differential interference contrast images (right). (B) Quantification of the relative import rates in the ⌬nup53 and the
wild-type BMA41 control cells by counting cells harboring nuclear fluorescence as a function of time. (C) Immunogold-EM reveals that the
NLS-GFP reporter accumulates within the nuclear basket of the yeast NPCs in the ⌬nup53 cells (for quantification see Table 2). (D) Nup53p
specifically interacts with the yeast nuclear import receptor Kap95p. For this purpose, ProtA-TEV-Nup53p was affinity purified by
IgG-Sepharose chromatography and analyzed for copurifying components. Shown is a Western blot analysis with a polyclonal anti-Ka95p
antibody revealing the specific interaction of Nup53p with Kap95p. Lane 1, NPC-containing fraction of a wild-type control strain (BMA41);
lane 2, eluate derived from the wild-type control strain; lane 3, NPC-containing fraction of the strain expressing ProtA-TEV-Nup53p; and lane
4, eluate derived from the ProtA-TEV-Nup53p. Scale bar, 100 nm (C).
gets caught in the nuclear basket so that its release from the
NPC into the nucleoplasm is strongly attenuated albeit not
completely inhibited. As displayed in Table 2, quantitation
of the gold particle distribution reveals a slower accumulation of the NLS-GFP reporter in the nucleus of the ⌬nup53
cells compared with that of the BMA41 control cells after
drug removal and initiation of signal-mediated import of the
NLS-GFP reporter. Taken together, the results obtained by
immunogold-EM (Figure 4C and Table 2) are in good agreement with those obtained by immunofluorescence microscopy (Figure 4, A and B).
Nup53p Interacts with Kap95p
To address the question of whether Nup53p’s involvement
in NLS-dependent nuclear protein import is via a direct
Vol. 11, November 2000
interaction with a nuclear import factor, we affinity-purified
Nup53p from yeast cells by IgG-Sepharose chromatography
under nondenaturing conditions, exactly as described
above, and analyzed the copurifying constituents for transport factors. Western blot analysis was performed with antibodies directed against the yeast nuclear protein import
receptor Kap95p (importin ␤) and the small GTPase Gsp1p
(i.e. the yeast homologue of vertebrate Ran), respectively. As
documented in Figure 4D, we found Nup53p to specifically
interact with Kap95p. Kap95p could be detected in the NPCcontaining fraction of the ⌬nup53 strain (Figure 4D, lane 3)
and in the eluate after IgG-Sepharose chromatography (Figure 4D, lane 4). The NPC-containing fraction of the BMA41
control strain also included Kap95p (Figure 4D, lane1), but
not so its eluate after IgG-Sepharose chromatography (Fig3891
B. Fahrenkrog et al.
Table 2. Protein import assay
Yeast strain
BMA41 (NLS-GFP)
⌬nup53 (NLS-GFP)
Cytoplasmic⬊nuclear ratio
before drug treatment
Cytoplasmic⬊nuclear ratio
immediately after drug removal
Cytoplasmic⬊nuclear
ratio 5 min after
drug removal
1⬊4
1⬊4
1⬊1
1⬊2
1⬊5
1⬊3
Quantitation of the distribution of 8-nm gold particles coupled to anti-GFP antibody within wild-type (i.e., BMA41) and mutant (i.e., ⌬nup53)
cells (Table 1) after pre-embedding immuno-gold EM (see MATERIALS AND METHODS; for selected examples in a ⌬nup53 background,
see Figure 4C). Twenty-two cells each were analyzed for the BMA41 (NLS-GFP) and the ⌬nup53 (NLS-GFP) strain. Quantitation was
performed by a stereological counting method (Lucocq, 1992).
ure 4D, lane 2). Under the same isolation conditions no
interaction with Gsp1p was depicted (our unpublished results).
Nup53p Does Not Participate in NES-mediated
Nuclear Protein Export
The involvement of Nup53p in the import of NLS proteins
(this study) and ribosomal proteins (Marelli et al., 1998)
prompted us to also evaluate a potential role of Nup53p in
nuclear protein export. For this purpose, three protein export assays were evaluated, all involving GFP as a fluorescent reporter. All three assays required conditions enabling
the protein cargo to be imported into the nucleus, so that in
a second step its export could be evaluated.
First, we performed a competition assay (Stade et al., 1997)
in the ⌬nup53 and the BMA41 control strain by introducing
a GFP reporter consisting of two GFP moieties, which was
simultaneously fused to the NES of the protein kinase inhibitor and the NLS of the SV40 large T antigen (NES-NLSGFP; Table 1). Because the observed NLS-mediated protein
import defect in ⌬nup53 cells is relatively weak, we assume
that such an NES-NLS-GFP reporter can be imported into
the nucleus in a ⌬nup53 background, and that this assay will
therefore provide insight into the potential role of Nup53p
in NES-dependent protein export. As illustrated by confocal
laser scanning microscopy in Figure 5 (left), in both the
⌬nup53 strain and the wild-type BMA41 the NES-NLS-GFP
reporter accumulated in the cytoplasm in cells grown at
30°C because evidently under steady-state conditions the
action of the NES dominates over that of the NLS, whereas
in a xpo1-1 control strain, which is defect in nuclear protein
export, the NES-NLS-GFP reporter accumulated inside the
nucleus.
In a second export assay we transformed the ⌬nup53 and
the BMA41 control strain with a construct where a GFP
reporter was fused to the HIV-1 Rev protein (Table 1), which
harbors both an NES and an NLS (Fischer et al., 1995; Wen et
al., 1995). Expression of the Rev-GFP reporter in these cells
was induced by shifting to galactose-containing medium
and analyzed by confocal laser scanning microscopy. As
documented in Figure 5 (right), in both the ⌬nup53 strain
and the wild-type BMA41 the Rev-GFP reporter accumulated in the cytoplasm at 30°C, whereas the same reporter
accumulated in the nucleus of the xpo1-1 control strain.
Additionally, when the growth temperature of either the
⌬nup53 or the wild-type BMA41 cells was shifted to 37°C
3892
(i.e., for 2 h), the GFP signal of both the NES-NLS-GFP and
the Rev-GFP reporter also accumulated in the cytoplasm
(our unpublished results).
A third export assay involved localization of a Yap1p-GFP
reporter. The Yap1p protein is a yeast activator protein-1like transcription factor that activates genes that are required
for the response to oxidative stress (Kuge and Jones, 1994;
Kuge et al., 1997; Yan et al., 1998). Yap1p is normally cytoplasmic and translocates to the nucleus after addition of
oxidants to the growth medium (Kuge et al., 1997). Yap1p
harbors an NES-like sequence and its cytoplasmic location is
dependent on Xpo1p/Crm1p (Yan et al., 1998). To test
whether the location of Yap1p is altered in a ⌬nup53 background, we transformed the ⌬nup53 strain and a wild-type
control (BMA41) with full-length Yap1p fused to GFP and
determined the location of this reporter in the presence and
absence of the oxidant diamide. As shown in Figure 6, the
Yap1p-GFP is located in the cytoplasm of ⌬nup53 and
BMA41 cells in the absence of diamide (left), but translocates
to the nucleus in both ⌬nup53 and BMA41after incubation
with diamide (right). In a xpo1-1 control, the Yap1p-GFP
accumulates in the nucleus without treating the cells with
diamide. These experiments demonstrate that the Yap1pGFP reporter can enter the nucleus in a ⌬nup53 background
under oxidative stress and hence can be actively exported by
Xpo1p under steady-state conditions.
Hence, based on three protein export assays, i.e., involving NES-NLS-GFP, Rev-GFP, and Yap1p-GFP as synthetic
export cargoes, we conclude that NES-mediated nuclear
protein export is not impaired by the disruption of NUP53.
The NES-NLS-GFP reporter is imported into the nucleus in
a NLS-dependent manner via the importin ␣/␤ pathway
(Stade et al., 1997; Taura et a., 1998), whereas the Rev-GFP
reporter is imported into the nucleus by direct interaction of
the Rev NLS with importin ␤ (Truant and Cullen, 1999).
Therefore, the export assays underscore the results from the
nuclear protein import assay (see above; Figure 4), suggesting that the NLS-mediated import in the ⌬nup53 strain is not
completely blocked but rather attenuated compared with
the wild-type control strain BMA41.
DISCUSSION
To eventually arrive at a more structure-based understanding of the functional involvement of the NPC in nucleocytoplasmic transport it is necessary to identify and locate in
Molecular Biology of the Cell
Nup53p Is Involved in Nuclear Import
Figure 5. NES-mediated protein export monitored in ⌬nup53 cells and in a wild-type BMA41 control strain (Table 1). An NLS-NES-GFP or
a Rev-GFP fusion construct was expressed in ⌬nup53 or BMA41 cells. The localization of the GFP reporter was analyzed after growing the
cells at 30°C revealing accumulation of the GFP signal in the cytoplasm, thus indicating that the presence of Nup53p is not necessary for
NES-mediated protein export. Shown are confocal fluorescence and coincident fluorescence/differential interference contrast images to
localize the GFP reporters relative to the cell periphery.
3-D the nucleoporins that do participate in distinct transport
steps, and to determine how these nucleoporins interact
with their neighbors and with transport factors. Toward this
goal, we have identified and characterized the yeast nucleoporin Nup53p as a physical neighbor of Nic96p within the
yeast NPC. Primary sequence analysis and secondary structure prediction have revealed that both Nup53p and Nic96p
harbor heptad repeat segments and thus are potential
coiled-coil–forming proteins (cf. Lupas et al., 1991). This
finding, in turn, suggests that the interaction between these
two nucleoporins is mediated by their coiled-coil domains.
In fact, by the two-hybrid screen we found that it was the
N-terminal domain of Nup53p consisting of two long coiledcoil stretches starting at amino acid 47 and 124, respectively,
that is interacting with Nic96p. Moreover, we have documented that Nup53p is directly involved in NLS-dependent
nuclear protein import by its specific interaction with
Kap95p, yet its absence from the NPC does not appear to
significantly interfere with NES-mediated nuclear protein
export nor with NPC assembly and/or structural integrity.
Based on the multiple locations of Nic96p and Nup53p
within the 3-D architecture of the NPC (Figure 1C) the
spatial separation of nucleoporins that are evidently constituents of the central framework or of the cytoplasmic or
nuclear fibrillar periphery of the NPC is not as stringent as
it might be expected. Nic96p, for example, is located about
Vol. 11, November 2000
the cytoplasmic and the nuclear periphery of the central
channel in a near-symmetrical arrangement with respect to
the central plane of the NPC, and near or at the distal ring of
the nuclear basket (Figure 1C; Fahrenkrog et al., 1998, 2000;
Stoffler et al., 1999; Rout et al., 2000). Because these multiple
locations of Nic96p line the transport route of cargoes on
their way into or out of the nucleus, they suggest that
Nic96p directly participates in nucleocytoplasmic transport.
In fact, temperature-sensitive nic96 mutants, although causing cytoplasmic accumulation of an NLS-containing reporter
protein, do not exhibit an obvious export defect (Grandi et
al., 1995). Nevertheless, as yet there has been no biochemical
evidence for a direct interaction of Nic96p with factors involved in protein import, despite the interaction of Nic96p
with Pse1p, the import receptor for the transcription factor
Pho4p as recently demonstrated by fluorescence resonance
energy transfer analysis (Damelin and Silver, 2000). In agreement with these earlier findings, we also failed to establish a
direct interaction of Nic96p with protein import factors by
the yeast two-hybrid system. Therefore, it is conceivable that
the import defect observed in NIC96 mutant strains is a
secondary effect, in the sense that Nic96p does not actually
physically interact with the cargo complex. Instead, absence
of Nic96p might cause loss or destabilization of the Nsp1p
complex from the central framework of the NPC in temperature-sensitive nic96 mutants because Nic96p anchors the
3893
B. Fahrenkrog et al.
Figure 6. Yap1p-GFP export is not impaired in a ⌬nup53 background. Yeast strains BMA41, ⌬nup53, or xpo1-1 (Table 1) were transformed
with pLDB419(Yap1-GFP), grown in selective medium in the presence or absence of the oxidant diamide. GFP fusion proteins are visualized
by confocal fluorescence and coincident fluorescence/differential interference contrast optics.
Nsp1p complex within the yeast NPC (Grandi et al., 1995;
Schlaich et al., 1997; Bucci and Wente, 1998). Hence, these
results demonstrate that location of a particular nucleoporin
along the route followed by cargo in or out of the nucleus
does not necessarily imply a direct role of this nucleoporin
in nucleocytoplasmic transport.
Our immunogold-EM analysis has revealed three distinct
locations of Nup53p within the 3-D architecture of the NPC
(Figure 1). It is a constituent of the central framework of the
NPC (i.e., in a near-symmetrical way at its cytoplasmic and
nuclear periphery), and it resides at the nuclear basket, an
NPC substructure that is directly involved in nucleocytoplasmic transport (cf. Bastos et al., 1996; Shah et al., 1998;
Nakielny et al., 1999; Ullman et al., 1999). Therefore, our
immunolocalization of Nup53p is consistent with previous
publications, which localized Nup53p to both faces of the
NPC, but not precisely to any NPC substructures (Marelli et
al., 1998; Rout et al., 2000). Surprisingly, although Nup53p is
part of the central framework of the yeast NPC, a nup53 null
strain exhibited no obvious morphological alterations of its
NPCs, at least not at the level of embedding/thin-sectioning
EM. Structural alterations of the NPC or its spatial distribution within the NE, such as NPC clustering, are known from
mutations in various nucleoporins, e.g., Nup85p and
Nup145p (Siniossoglou et al., 1996). Both of these nucleoporins are constituents of the Nup84p complex (Siniossoglou et
al., 1996) which, in turn, forms part of the cytoplasmic fibrils
of the yeast NPC (Fahrenkrog et al., 1997; Stoffler et al., 1999;
Siniossoglou et al., 2000). Therefore, it remains elusive why,
3894
on the one hand, mutations in nucleoporins that are part of
the central framework do not necessarily cause structural
defects of the NPC in the resulting mutant strain, whereas,
on the other hand, mutations in nucleoporins that are constituents of the cytoplasmic fibrils or the nuclear basket
cause such drastic structural alterations as NPC clustering.
The absence of any obvious structural alterations of the
NPC in the ⌬nup53 strain and the physical interaction of
Nup53p with the yeast nuclear protein import receptor
Kap95p suggest that, different from Nic96p (see above),
Nup53p does play a direct role in nuclear protein import. In
this context, the interaction of a number of nucleoporins
with importin ␤-like proteins appears to be mediated by
phenylalanine– glycine (FG) repeats within the amino acid
sequence of these nucleoporins (Seedorf et al., 1999; Stoffler
et al., 1999). Although Nup53p harbors four separated FG
sequence motifs within its amino acid sequence, it does
clearly not represent an FG-repeat containing nucleoporin.
Hence, whereas the interaction between Nup53p and
Kap95p may indeed involve one or several of these FGs,
there must be additional amino acids specifying this interaction.
Location of Nup53p at the nuclear basket (Figure 1A) and
accumulation of an NLS-GFP reporter, most likely in the
nuclear basket in a nup53 null strain (Figure 4C), support a
model in which Nup53p is involved in a late step of nuclear
protein import. However, because NUP53 is not essential
and protein import is not completely inhibited in the ⌬nup53
strain (Figure 4B), other nucleoporins residing at the nuclear
Molecular Biology of the Cell
Nup53p Is Involved in Nuclear Import
basket, e.g., Nup1p, the presumed yeast homologue of vertebrate Nup153 (Moroianu et al., 1997), must be able to at
least partially substitute for Nup53p in its absence. This may
be even more so in the case of cargo export, because NESmediated protein export is not noticeably impaired by the
disruption of NUP53 (Figures 5 and 6).
Taken together, we have structurally and functionally
identified and characterized the yeast nucleoporin Nup53p
that physically (i.e., both by a yeast two-hybrid screen and
biochemically) interacts with the essential yeast nucleoporin
Nic96p. Nup53p resides near-symmetrically (i.e. relative to
the central plane of the NPC) at the cytoplasmic and the
nuclear periphery of the central framework, and at the nuclear basket fibrils of the yeast NPC. Although deletion of
NUP53 causes no obvious morphological defects, nuclear
protein import is attenuated significantly in a nup53 null
strain. Due to its specific interaction with the yeast import
receptor Kap95p, Nup53p must play a direct functional role
in nuclear protein import. In contrast, Nup53p does not
appear to play a significant role in cargo export because its
absence does not significantly interfere with NES-mediated
protein export.
Fahrenkrog, B., Aebi, U., and Panté, N. (1997). Yeast nuclear pore
complexes (NPCs): dissecting their molecular architecture. Mol.
Biol. Cell 8, 236a.
ACKNOWLEDGMENTS
Grandi, P., Schlaich, N., Tekotte, H., and Hurt, E.C. (1995). Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p. EMBO J. 14,
76 – 87.
We thank Drs. Jennifer Hood and Pamela Silver for providing
several plasmids and the antibodies against GFP and Kap95p, Dr.
Ed Hurt for providing the antibody against Nic96p, Drs. Anita
Corbett and Laura Davis for the Yap1p-GFP plasmid, and Drs.
Françoise Stutz and Karsten Weis for the xpo1-1 strain. We also
thank Dr. Markus Dürrenberger for help with confocal laser scanning microscopy, Ursula Sauder for help with preparing samples for
EM, Robert Wyss for help with Figure 1C, and Hedi Frefel and
Marlies Zoller for expert photographic work. This work was supported by a research grant from the Human Frontier Science Program (HFSP), and by the Kanton Basel-Stadt and the M. E. Müller
Foundation of Switzerland.
REFERENCES
Akey, C.W., and Radermacher, M. (1993). Architecture of the Xenopus nuclear pore complex revealed by 3-dimensional cryo-electron
microscopy. J. Cell Biol. 122, 1–19.
Fahrenkrog, B., Aris, J.P., Hurt, E.C., Panté, N., and Aebi, U. (2000).
Comparative localization of protein A tagged and endogenous yeast
nuclear pore complex proteins by immunoelectron microscopy. J.
Struct. Biol., 129, 295–305.
Fahrenkrog, B., Hurt, E.C., Aebi, U., and Panté, N. (1998). Molecular
architecture of the yeast nuclear pore complex: localization of
Nsp1p subcomplexes. J. Cell Biol. 143, 577–588.
Fischer, U., Huber, J., Boelens, W.C., Mattaj, I.W., and Lührmann, R.
(1995). The HIV-1 Rev activation domain is a nuclear export signal
that accesses an export pathway used by specific cellular RNAs. Cell
82, 475– 483.
Fromont-Racine, M., Rain, J.C., and Legrain, P. (1997). Toward a
functional analysis of the yeast genome through exhaustive twohybrid screens. Nat. Genet. 16, 277–282.
Gietz, D., St. Jean, A., Woods, R.A., and Schiestl, R.H. (1992). Improved method for high efficiency transformation of intact yeast
cells. Nucleic Acids Res. 20, 1425.
Grandi, P., Doye, V., and Hurt, E.C. (1993). Purification of NSP1
reveals complex formation with “GLFG”nucleoporins and a novel
nuclear pore protein NIC96. EMBO J. 12, 3061–3071.
Guthrie, C., and Fink, G.R. (1991). Guide to Yeast Genetics and
Molecular Biology. San Diego: Academic Press.
Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K., and Elledge, S.J.
(1993). The p21 CdK-interacting protein Cip1 is a potent inhibitor of
G1 cyclin-dependent kinases. Cell 75, 805– 816.
Hellmuth, K., Lau, D.M., Bischoff, F.R., Künzler, M., Hurt, E., and
Simos, G. (1998). Yeast Los1p has properties of an exportin-like
nucleocytoplasmic transport factor for tRNA. Mol. Cell. Biol. 18,
6374 – 6386.
Hurt, E.C. (1988). A novel nucleoskeletal-like protein located at the
nuclear periphery is required for the life cycle of Saccharomyces
cerevisiae. EMBO J. 7, 4324 – 4334.
Izaurralde, E., and Adam, S.A. (1998). Transport of macromolecules
between the nucleus and the cytoplasm. RNA 4, 351–364.
Bastos, R., Lin, A., Enarson, M., and Burke, B. (1996). Targeting and
function in mRNA export of the nuclear pore complex protein
Nup153. J. Cell Biol. 134, 1141–1156.
Koepp, D.M., Wong, D.H., Corbett, A.H., and Silver, P.A. (1996).
Dynamic localization of the nuclear import receptor and its interaction with transport factors. J. Cell Biol. 133, 1163–1176.
Baudin-Baillieu, A., Guillemet, E., Cullin, C., and Lacroute, F. (1997).
Construction of a yeast strain deleted for the TRP1 promotor and
coding region that enhances the efficiency of the polymerase chain
reaction-disruption method. Yeast 13, 353–356.
Kuge, S., and Jones, N. (1994). YAP1 dependent activation of TRX2
is essential for the response of Saccharomyces cerevisiae to oxidative
stress by hyperperoxides. EMBO J. 13, 655– 664.
Bucci, M., and Wente, S.R. (1998). A novel fluorescence-based genetic strategy identifies mutants of Saccharomyces cerevisiae defective
for nuclear pore complex assembly. Mol. Biol. Cell 9, 2439 –2461.
Corbett, A.H., and Silver, P.A. (1997). Nucleocytoplasmic transport
of macromolecules. Microbiol. Mol. Biol. Rev. 61, 193–211.
Damelin, M., and Silver, P.A. (2000). Mapping interactions between
nuclear transport factors in living cells reveals pathways through
the nuclear pore complex. Mol. Cell 5, 133–140.
Doye, V., and Hurt, E. (1997). From nucleoporins to nuclear pore
complexes. Curr. Opin. Cell Biol. 9, 401– 411.
Fabre, E., and Hurt, E.C. (1997). Yeast genetics to dissect the nuclear
pore complex and nucleocytoplasmic trafficking. Annu. Rev. Genet.
31, 277–313.
Vol. 11, November 2000
Kuge, S., Jones, N., and Nomoto, A. (1997). Regulation of yAP-1
nuclear localization in response to oxidative stress. EMBO J. 16,
1710 –1720.
Lucocq, J. (1992). Quantitation of gold labeling and estimation of
labeling efficiency with a stereological counting method. J. Histochem. Cytochem. 40, 1929 –1936.
Lupas, A., Van Dyke, M., and Stock, J. (1991). Predicting coiled coils
from protein sequences. Science 252, 1162–1164.
Marelli, M., Aitchinson, J.D., and Wozniak, R.W. (1998). Specific
binding of the karyopherin Kap121p to a subunit of the nuclear pore
complex containing Nup53p, Nup59p, and Nup170p. J. Cell Biol.
143, 1813–1830.
Mattaj, I.W., and Englmeier, L. (1998). Nucleocytoplasmic transport:
the soluble phase. Annu. Rev. Biochem. 67, 265–306.
3895
B. Fahrenkrog et al.
Moroianu, J., Blobel, G., and Radu, A. (1997). RanGTP-mediated
nuclear export of karyopherin ␣ involves its interaction with the
nucleoporin Nup153. Proc. Natl. Acad. Sci. USA, 94, 4451– 4456.
Nakielny, S., Shaikh, S., Burke, B., and Dreyfuss, G. (1999). Nup153
is an M9-containing mobile nucleoporin with a novel Ran-binding
domain. EMBO J. 18, 1982–1995.
Nehrbass, U., and Blobel, G. (1996). Role of the nuclear transport
factor p10 in nuclear import. Science 272, 120 –122.
Nehrbass, U., Fabre, E., Dihlmann, S., Herth, W., and Hurt, E.C.
(1993). Analysis of nucleo-cytoplasmic transport in a thermosensitive mutant of nuclear pore protein NSP1. Eur. J. Cell Biol. 62, 1–12.
Nehrbass, U., Kern, H., Mutvei, A., Horstmann, H., Marshallsay, B.,
and Hurt, E.C. (1990). NSP1: a yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxyterminal domain. Cell 61, 979 –989.
Ohno, M., Fornerod, M., and Mattaj, I.W. (1998). Nucleocytoplasmic
transport: the last 200 nanometers. Cell 92, 327–336.
Panté, N., and Aebi, U. (1996). Molecular dissection of the nuclear
pore complex. Crit. Rev. Biochem. Mol. Biol. 31, 153–199.
Rothstein, R. (1991). Targeting, disruption, replacement, and allele
rescue: integrative DNA transformation in yeast. Methods Enzymol.
194, 281–301.
Rout, M.P., Aitchinson, J.D., Suprapto, A., Hjertaas, K., Zhao, Y.,
and Chait, B.T. (2000). The yeast nuclear pore complex: composition,
architecture and transport mechanism. J. Cell Biol. 148, 635– 651.
Rout, M.P., and Blobel, G. (1993). Isolation of the yeast nuclear pore
complex. J. Cell Biol. 109, 2641–2652.
Schlaich, N.L., Häner, M., Lustig, A., Aebi, U., and Hurt, E.C. (1997).
In vitro reconstitution of a heterotrimeric nucleoporin complex consisting of recombinant Nsp1p, Nup49p, and Nup57p. Mol. Biol. Cell
8, 33– 46.
Seedorf, M., Damelin, M., Kahana, J., Taura, T., and Silver, P.A.
(1999). Interactions between a nuclear transporter and a subset of
nuclear pore complex proteins depend on Ran GTPase. Mol. Cell.
Biol. 19, 1547–1557.
Saccharomyces cerevisiae: a role for heat shock protein 70 during
targeting and translocation. J. Cell Biol. 135, 329 –339.
Siniossoglou, S., Lutzmann, M., Santos-Rosa, Leonard, K., Müller,
S., Aebi, U., and Hurt, E. (2000). Structure and assembly of the
Nup84p complex. J. Cell Biol. 149, 41–54.
Siniossoglou, S., Wimmer, C., Rieger, M., Doye, V., Tekotte, H.,
Weise, C., and Hurt, E.C. (1996). A novel complex of nucleoporins,
which includes Sec13p and a Sec13p homolog, is essential for normal nuclear pores. Cell 84, 265–275.
Song, H.Y., Dunbar, J.D., and Donner, D.B. (1994). Aggregation of a
intracellular domain of the type 1 tumor necrosis factor receptor
defined in the two-hybrid system. J. Biol. Chem. 269, 22492–22495.
Stade, K., Ford, C.S., Guthrie, C., and Weis, K. (1997). Exportin 1
(Crm1p) is an essential nuclear export factor. Cell 90, 1041–1050.
Stan, R., McLaughlin, M.M., Cafferkey, R., Johnson, R.K., Rosenberg,
M., and Livi, G.P. (1994). Interaction between FKBP12-rapamycin
and TOR involves a conserved serine residue. J. Biol. Chem. 269,
32027–32030.
Stochaj, U., Héjazi, M., and Belhumeur, P. (1998). The small GTPase
Gsp1p binds to the repeat domain of the nucleoporin Nsp1p. Biochem. J. 330, 412– 427.
Stoffler, D., Fahrenkrog, B., and Aebi, U. (1999). The nuclear pore
complex: from molecular architecture to functional dynamics. Curr.
Opin. Cell. Biol. 11, 391– 401.
Taura, T., Krebber, H., and Silver, P.A. (1998). A member of the
Ran-binding protein family, Yrb2p, is involved in nuclear protein
export. Proc. Natl. Acad. Sci. USA 95, 7427–7432.
Truant, R., and Cullen, B.R. (1999). The arginine-rich domains
present in human immunodeficiency virus type 1 Tat and Rev
function as direct importin ␤-dependent nuclear localization signals. Mol. Cell. Biol. 19, 1210 –1217.
Ullman, K.S., Shah, S., Powers, M.A., and Forbes, D.J. (1999). The
nucleoporin Nup153 plays a critical role in multiple types of nuclear
export. Mol. Biol. Cell 10, 649 – 64.
Senger, B., Simos, G., Bischoff, F.R., Podtelejnikov, A., Mann, M.,
and Hurt, E. (1998). Mtr10p functions as a nuclear import receptor
for the mRNA-binding protein Npl3p. EMBO J. 17, 2196 –2207.
Wen, W., Meinkoth, J.L., Tsien, R.Y., and Taylor, S.S. (1995). Identification of a signal for rapid export of proteins from the nucleus.
J. Cell Biol. 133, 4 –14.
Shah, S., Tugendreich, S., and Forbes, D. (1998). Major binding sites
for the nuclear import receptor are the internal nucleoporin Nup153
and the adjacent nuclear filament protein Tpr. J. Cell Biol. 141,
31– 49.
Yan, C., Lee, L.H., and Davis, L.I. (1998). Crm1p mediates regulated
nuclear export of a yeast AP-1-like transcription factor. EMBO J. 17,
7416 –7429.
Shulga, N., Roberts, P., Gu, Z.Y., Spitz, L., Tabb, M.M., Nomura, M.,
and Goldfarb, D.S. (1996). In vivo nuclear transport kinetics in
3896
Yang, Q., Rout, M.P., and Akey, C. (1998). Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and
evolutionary implications. Mol. Cell 1, 223–234.
Molecular Biology of the Cell
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement