©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Essential Yeast Nucleoporin NUP159 Is Located on the Cytoplasmic Side of the Nuclear Pore Complex and Serves in Karyopherin-mediated Binding of Transport Substrate (*)

(Received for publication, May 9, 1995)

Doris M. Kraemer (§) Caterina Strambio-de-Castillia Günter Blobel (¶) Michael P. Rout

From theLaboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have identified a new yeast nucleoporin of 159 kDa that we term NUP159. Immunofluorescence microscopy with a monospecific monoclonal antibody against NUP159 gave the punctate nuclear rim staining characteristic of nucleoporins. Immunogold electron microscopy with isolated yeast NEs yielded decoration of only the cytoplasmic side of the nuclear pore complex. The gene encoding NUP159 is essential, and, like some other nucleoporins, NUP159 contains a coiled-coil domain as well as a domain of repeated motifs. Five segments of NUP159, covering its entire length, were expressed in Escherichia coli. The repeat motif-containing segment was found to bind a nuclear transport substrate in the presence of vertebrate cytosolic extract containing nuclear transport factors. This segment also bound S-labeled mammalian karyopherin beta, one such transport factor that mediates the docking of substrates to the nuclear pore complex. These data establish a direct biochemical link between the repeat motif domain of a yeast nucleoporin, transport factors, (specifically karyopherin beta), and nuclear transport substrates. Its cytoplasmic aspect implies a role for NUP159 in nuclear import.


INTRODUCTION

Bidirectional transport of macromolecules across the nuclear envelope proceeds via the nuclear pore complex (NPC). (^1)Several soluble transport factors that are required for the uptake of transport substrate into the nuclei of digitonin-permeabilized mammalian cells have now been isolated (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) , and progress has been made in characterizing the myriad of NPC proteins (collectively termed nucleoporins) ((11, 12, 13, 14, 15, 16) ; for review see (17) and (18) ). Most interestingly, a biochemical link between the isolated transport factors and a subgroup of nucleoporins has been established. Thus, a heterodimeric complex, termed karyopherin(6, 7) or importin(5, 8) , has been shown to bind to nuclear transport substrates via its alpha subunit (7, 10) and to bind repeat motif-containing nucleoporins via its beta subunit(6, 10, 15) . Moreover, the transport factor Ran (1, 2) has been shown to bind to the mammalian nucleoporin Nup358, which is a component of the >50-nm-long fibers that extend from the NPC into the cytoplasm(16) . It has been proposed that the repeat motif-containing nucleoporins form a stationary phase of multiple docking sites that extend from the cytoplasmic to the nucleoplasmic side of the NPC (6, 15) and that a Ran GTP/GDP cycle controls docking and release to and from these binding sites from the mobile phase of transport factors and substrates(1, 2) .

Although there are also numerous repeat motif nucleoporins in Saccharomyces cerevisiae (for review see (17) and (18) ), a biochemical connection to soluble transport factors has so far not been established. Furthermore, although some vertebrate nucleoporins (including a few that contain repeat motifs) have been localized to substructures of the NPC (for review see (17) and (18) ), such sublocalization has not yet been achieved for any of the nucleoporins of yeast.

Here we report the identification of a new yeast nucleoporin of 159 kDa, termed NUP159. By immunoelectron microscopy, we sublocalized NUP159 to the cytoplasmic side of the NPC. NUP159 contains numerous short peptide repeats, similar to those found in other nucleoporins. We have expressed five segments of NUP159, covering its entire length, in Escherichia coli and shown that only the repeat motif-containing segment of NUP159 serves in mammalian transport factor-mediated binding of import substrate.


MATERIALS AND METHODS

Peptide Sequencing

Highly enriched NPCs were fractionated and prepared for peptide sequencing as described(13, 19, 20) .

S. cerevisiae Strains

Diploid strain DF5 (MATa/MATalpha trp1-1/trp1-1 ura3-52/ura3-52 his3-Delta200/his3-Delta200 leu2-3, 112/leu2-3, 112 lys2-801/lys2-801)(21) , diploid strain W303 (MATa/MATalpha ade2-1/ade2-1 ura3-1/ura3-1 his3-11, 15/his3-11, 15 trp1-1/trp1-1 leu2-3, 112/leu2-3, 112 can1-100/can1-100), and haploid strain cl3-ABYS-86 (MATalpha pra1-1 prb1-1 prc1-1 cps1-3 ura3Delta5 leu2-3, 112 his) with point mutations in proteinase A and B and carboxypeptidase Y and S (22) were used.

Disruption of NUP159

Two PCR products of NUP159 (A and B) were obtained by PCR with genomic DNA from S. cerevisiae (Promega, Madison, WI) as a template. PCR product A contained the upstream flanking region with 339 base pairs of non-coding and 60 base pairs of coding sequence of NUP159. The primers contained additional engineered SacII and XbaI restriction sites. The second PCR product (B) contained a 423-base pair flanking region downstream and additional BamHI and SmaI linkers. For the LEU2 marker we digested plasmid pJJ252 (23) with XbaI and BamHI. PCR product A, the LEU2 marker, and PCR product B were sequentially cloned into pBluescript II. The whole construct (PCR product A, LEU2 marker, and PCR product B) was linearized by digestion with SacII and SmaI, introduced into the diploid strain DF5, and Leu transformants were selected. Targeted integration was verified by Southern blot. Sporulation and dissection of the resulting strain were as described(24) .

Immunoblot Analysis

Proteins were subjected to SDS-PAGE, transferred to nitrocellulose, and incubated with the primary antibodies as described(25) . After incubation with horseradish peroxidase-linked anti-mouse IgG (1:3000), labeling was detected with the ECL system (Amersham Corp.).

Expression of Five Different Segments of NUP159

NUP159 was divided into five fragments (p159-1, amino acid residues 1-175; p159-2, residues 176-440; p159-3, residues 441-876; p159-4, residues 877-1222; p159-5, residues 1223-1460), and each corresponding DNA segment was cloned by PCR and inserted between the EcoRI and NotI sites of the pET21-b vector (Novagen, Madison, WI) containing a tag of six histidines. The constructs were transformed into E. coli BL21(DE3). All proteins were expressed in inclusion bodies except for p159-3, which was found as a soluble product in the cytosol (Novagen, pET manual).

Preparation of Yeast Nuclei and Nuclear Envelopes (NEs)

The preparation of spheroplasts and nuclei (26) was modified by adding chymostatin, antipain, leupeptin (2.5 µg/ml each), and a protinin (15-30 microunits/ml) (Sigma) as additional protease inhibitors. Yeast NEs were prepared from the protease-deficient strain cl3-ABYS-86 as described (modification of a previous method (27) ). (^2)

Monoclonal Antibodies

The anti-NUP159 monoclonal antibody (mAb) 165C10 was identified by screening a panel of monoclonal antibodies. They were raised by immunizing mice with a yeast nuclear envelope fraction.^2 The polyspecific mAb 414 has been described(28) .

Immunofluorescence

Diploid cells of the strain W303 were prepared for immunofluorescence microscopy as described (29, 30) and incubated with the mAb 165C10 for 12 h at 4 °C. The primary antibody was detected with Cy3-labeled polyclonal donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).

Immunoelectron Microscopy

NEs were centrifuged onto the bottom of a well of a microtiter plate(26, 31) , washed in bt-Me(2)SO (bt-Me(2)SO = 10 mM BisTris-Cl, pH 6.50, 0.1 mM MgCl(2), and 20% (v/v) Me(2)SO) and fixed for 5 min at 25 °C with 4% formaldehyde in bt-Me(2)SO. After another wash with bt-Me(2)SO, the wells were incubated for 5 min at 25 °C in 1 mM glycine in bt-Me(2)SO and preblocked for 5 min in IEM buffer (IEM buffer = 5 mg/ml bovine serum albumin, 0.5 phosphate-buffered saline, 1 mM MgCl(2), 0.1 mM CaCl(2), 0.1 mM ZnCl(2), 0.02% NaN(3), 90 µg/ml phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin A, 2.5 µg/ml chymostatin, 2.5 µg/ml antipain, 2.5 µg/ml leupeptin, and 30 microunits/ml aprotinin). All subsequent washes and incubations were performed in IEM buffer. Tissue culture supernatant from the monoclonal cell line mAb 165C10 was diluted 1:3 with IEM buffer, added to the wells, and incubated for 1 h at 25 °C. The wells were washed four times, and a 1:10 dilution of 10-nm gold-labeled affinity-purified goat anti-mouse IgG (Sigma) was added and incubated for 1 h at 25 °C. The wells were washed again as above, then washed twice in 100 mM potassium phosphate, pH 6.5, and 1 mM MgCl(2), and fixed for 12 h at 4 °C with 2.5% glutaraldehyde. The wells were further processed for electron microscopy as described(26, 31) .

Ligand Blotting Assay

For the overlays, fragments p159-1 to p159-5 were transferred to nitrocellulose. The further overlay with Xenopus fraction A (32) and the nuclear localization signal of the SV-40 T antigen coupled to human serum albumin and S-labeled karyopherin beta were as described(6, 10, 15) .


RESULTS

Microsequencing of a 50-kDa polypeptide present in the highly enriched NPC fraction of yeast (19) yielded a peptide sequence that was identical to residues 1106-1118 (Fig.1) of an uncharacterized ORF YIL115C (EMBL/GenBank/DDBJ accession number Z38125) located on chromosome IX of S. cerevisiae. The ORF is predicted to code for a protein of 1460 amino acids and a M(r) of 158,923. As this protein is a nucleoporin (see below) we have termed it NUP159, consistent with conventional nomenclature(28, 30) .


Figure 1: Sequence analysis. A, amino acid sequence obtained from a polypeptide in a fraction of highly enriched NPCs shows identity to an unknown ORF coding for a protein of 158,923 kDa that we have termed NUP159. Numbers to the right and to the left indicate the position of the peptide sequence in the ORF. B, NUP159 contains 28 repeat motifs. Besides the nucleoporin characteristic motif XFXFG, there are three other motifs; SAFG, PASG, and PSFG. The numbers indicate the first amino acid position of the repeated motif.



To investigate if this gene is essential for cell growth, we transformed a linearized fragment containing a LEU2-marked deletion allele into a diploid strain to replace one copy by homologous recombination. Leu transformants were verified by Southern analysis and sporulated, and the tetrads were dissected. All tetrads yielded only two viable segregants, both of which were Leu, suggesting that the NUP159Delta::LEU2 segregants were not viable (Fig.2).


Figure 2: Structural features of NUP159. A, NUP159 was expressed in E. coli in five different segments, p159-1 to p159-5. Segment p159-3 is the repeat motif domain, p159-4 contains the epitope recognized by the anti-NUP159 mAb 165C10, and p159-5 contains the putative coiled-coil domain. B, disruption of NUP159. The diagram shows the construct for the disruption of the gene by a LEU2 marker. Arrows indicate the direction of transcription of NUP159 and the marker. C, tetrad dissection. All tetrads (1-7) yielded only two viable segregants among four spores (a-d).



As with many nucleoporins, NUP159 contains a region with repeated motifs (Fig.1) including the XFXFG motif found in several other nucleoporins (for review see (17) and (18) ) as well as three additional related repeat motifs, namely PSFG, PASG, and SAFG (Fig.1). A computer homology search revealed that the motifs PSFG and SAFG were also found in another uncharacterized yeast ORF of 430 amino acids (GenPept accession number Z48784, ORF 9346.04c). NUP159 is 65% similar to this potential yeast nucleoporin across 20% of its length and 56% similar to Nup214 across 19% of its length(25, 33) . NUP159 is rich in serines (14%) and in potential phosphorylation sites. NUP159 also contains a potential coiled-coil domain (34) (Fig.2), which may be involved in homo- or heterodimerization, a feature found in some other nucleoporins (for review see (17) and (18) ).

For further analysis, we expressed five segments, p159-1 to p159-5, covering the entire length of NUP159 in E. coli ( Fig.2and 3A, a). Segment 3 contains the repeat motifs, and the C-terminal segment 5 contains the potential coiled-coil domain of NUP159. We screened a panel of monoclonal antibodies that had been obtained by immunization of mice with NEs. One of these monoclonal antibodies (mAb 165C10) reacted with NUP159, specifically with the expressed segment p159-4 (residues 877-1222) (Fig.3A, c). This segment contains none of the repeat motifs. In contrast, the polyspecific mAb 414 recognized the repeat motif domain (segment 3) (Fig.3A, b). mAb 165C10 reacted with a single polypeptide of 190 kDa in a whole yeast spheroplast lysate or in yeast nuclei (Fig.3B), indicating that the mAb is monospecific for Nup159. During the preparation of NPCs and NEs, NUP159 appeared to be extraordinarily protease-sensitive. One breakdown product appeared to be the 50-kDa polypeptide from which the peptide sequence data were derived. For this reason we used the haploid protease-deficient strain cl3-ABYS-86. Fractions from spheroplasts to NEs were subjected to SDS-PAGE and transferred to nitrocellulose. NUP159, detected with the monoclonal antibody, cofractionated absolutely with the NEs (data not shown).


Figure 3: Characterization of anti-NUP159 mAb 165C10. A, NUP159 was expressed in E. coli in five different segments (p159-1 to p159-5), and the fractions containing the expressed proteins (the inclusion body containing pellet for segments 1, 2, 4, and 5 (lanes 1, 2, 4, and 5, respectively) and the cytosol-containing supernatant for segment 3 (lane 3) were subjected to a 12.5% SDS-PAGE. Arrows indicate the isopropyl-1-thio-beta-D-galactopyranoside-induced proteins (a). The same fractions were transferred to nitrocellulose (b and c) and incubated with the polyspecific mAb 414 (b), which recognizes the repeat motif domain (segment 3) in NUP159. The NUP159-specific protein amounts of yeast spheroplasts (a) and nuclei (b) were separated by 7.5% SDS-PAGE, transferred to nitrocellulose, and stained with mAb 165C10.



We used mAb 165C10 to localize NUP159. Formaldehyde-fixed yeast spheroplasts were incubated with the mAb for immunofluorescence microscopy, and a nucleoporin-characteristic punctate nuclear rim staining was observed (Fig.4).


Figure 4: Immunolocalization of NUP159. Formaldehyde-fixed yeast cells stained with the anti-NUP159 mAb 165C10 showed the punctate nuclear rim staining that is typical for nucleoporins (top). Coincident DAPI staining is shown (bottom). Bar, 5 µm.



For immunoelectron microscopy, isolated NEs were incubated with mAb 165C10, and the labeling was visualized with gold-labeled secondary antibodies. By using this pre-embedding immunolabeling technique in yeast, it was possible to sublocalize NUP159 with greater accuracy than post-embedding labeling methods (Fig.5). All of the 196 gold particles in the micrographs that could be unequivocally assigned to NPCs were found exclusively on their cytoplasmic side. In the cases of NPCs clearly sectioned perpendicular to their midplane (and hence to midplane of the NE), the shortest distance from the center of each gold particle to the midplane was measured, and the average of these measurements was 41 ± 15 nm (n = 41) (Fig.5). Another nucleoporin was localized with this technique to both sides of the NPC (data not shown); hence the lack of signal on the nuclear face of the NPC is not a result of general inaccessibility of this face. NPCs were often multiply labeled, with as many as seven gold particles each.


Figure 5: Immunoelectron microscopy. A, NEs were incubated with mAb 165C10, and the labeling was visualized with gold-labeled secondary antibodies. NUP159 was localized only on the cytoplasmic side of the NPC. C, cytoplasm; N, nucleoplasm; bar, 200 nm. B, the distance (d) from the center of each gold particle to the midplane of its associated NPC was measured for NPCs clearly sectioned perpendicular to that plane (and hence to the plane of the NE) (left); these were found to average 41 ± 15 nm (n = 41) (right).



Mammalian repeat motif-containing nucleoporins have been shown to bind transport substrate in a reaction mediated by cytosolic subfraction A (6, 32) . For one of these nucleoporins, Nup98, the binding site has been mapped to its repeat motif-containing domain(15) . The active component of fraction A in nuclear import has been shown to be the heterodimeric karyopherin(6, 7, 10, 15) . Further analysis has revealed that binding to the repeat motif-containing nucleoporins is via the beta subunit of karyopherin(10) . The cytoplasmic exposure of NUP159, a repeat motif nucleoporin, makes it a candidate for such import-related binding. To test whether the repeat motif segment 3 of NUP159 also functions as a docking site, the five E. coli-expressed segments of NUP159 were incubated with a transport substrate and Xenopus ovary cytosolic subfraction A(32) . Only the peptide repeat-containing segment 3 of NUP159 bound transport substrate in a fraction A-dependent fashion (Fig.6A; for controls see Fig. 6B). Likewise, S-labeled recombinant rat karyopherin beta bound exclusively to the repeat motif segment 3 of NUP159 (Fig.6C).


Figure 6: Ligand blotting assay. A, fractions containing all five expressed segments of NUP159 (Fig.3A, a) were separated by SDS-PAGE and transferred to nitrocellulose as in Fig.3A, a (lanes 1-5). The membrane was incubated with Xenopus fraction A and nuclear location sequence-conjugated human serum albumin. Binding was detected by incubation with a secondary antibody against human serum albumin and I-labeled protein A. Only the expressed protein in lane 3, which is the domain with the repeat motifs, binds the conjugate in the presence of fraction A. B, a blot of the fraction containing segment 3 was incubated with the conjugate alone (lane 6), with fraction A and unconjugated human serum albumin (lane 7), or with the nuclear location sequence-conjugate and fraction A (lane 8). C, the five expressed segments of NUP159 were subjected to SDS-PAGE, transferred to nitrocellulose as in A and incubated with S-labeled rat karyopherin beta.




DISCUSSION

NUP159 is the first of the yeast nucleoporins that has so far been sublocalized. Given the uncertainties associated with secondary immunolabeling, the epitope may be between 30 and 50 nm from the central plane of the NPC. We therefore expect NUP159 to project some distance into the cytoplasm. This suggests that NUP159 may be a component of the short cytoplasmic fibers that emanate from the NPC (35) . Diffuse structures of 20-30 nm attached to the NPC have been shown in section electron micrographs of pelleted yeast nuclei that could be collapsed fibers(19) . In vertebrate cells, three nucleoporins have so far been sublocalized to these fibers: Nup358(16) , Nup214 (25) , and Tpr(11) . Of these, Nup358 and Nup214 are repeat motif-containing nucleoporins. Because of the sequence similarities of NUP159 and Nup214 and because of their similar locations, the repeat module of yeast NUP159 could be functionally homologous to that of mammalian Nup214. Besides the XFXFG repeats that are also shared by some other nucleoporins (for review see Refs. 17 and 18), NUP159 contains the repeat motifs PSFG, PASG, and SAFG. The motifs PSFG and SAFG were found also in another potential yeast nucleoporin. Motif SAFG is especially similar to the motif SVFG in Nup214(25) . All of these repeats are located in a central domain (segment 3) of NUP159.

The ability of the NUP159 repeat motif domain to bind transport substrate in a karyopherin-dependent fashion in an overlay assay is similar to that recently demonstrated for the repeat domain in rat Nup98(15) . Our data indicate that there is a functional conservation of NPC transport components amongst the eukaryotes, because Xenopus karyopherin or rat karyopherin beta were both able to interact with the repeat motif domain of a yeast nucleoporin. It is likely that the repeat motif domains of other yeast nucleoporins will also form an array of multiple docking sites across the NPC(6, 15) .


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from the Deutsche Forschungsgemeinschaft (Kr 1412/1-1) and by funds from Fritz Thyssen Stiftung (Az.2.12).

To whom correspondence should be addressed: Laboratory of Cell Biology, Howard Hughes Medical Inst., The Rockefeller University, New York, NY 10021. Tel.: 212-327-8096; Fax: 212-327-7880.

^1
The abbreviations used are: NPC, nuclear pore complex; NE, nuclear envelope; PAGE, polyacrylamide gel electrophoresis; ORF, open reading frame; PCR, polymerase chain reaction; mAb, monoclonal antibody.

^2
Strambio-de-Castillia, C., Blobel, G., and Rout, M. P. (1995) J. Cell Biol., in press.


ACKNOWLEDGEMENTS

We thank members of the Rockefeller University Biopolymer Facility, especially Joseph Fernandez, for protein sequencing. We also thank Eleana Sphicas for assistance in performing the electron microscopy studies, Dr. Aurelian Radu for the S-labeled karyopherin beta, Dr. Erica Johnson for critical reading of the manuscript, and Dr. D. H. Wolf for the strain cl3-ABYS-86.


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