The NUP1 gene encodes an essential component of the yeast nuclear pore complex

Cell, Vol. 61, 965-976,

June

15, 1990, Copyright

0 1990 by Cell Press

The NW7 Gene Encodes an Essential of the Yeast Nuclear Pore Complex Laura I. Davis’ and Gerald R. Fink’t ’ Whitehead institute for Biomedical Research t Massachusetts Institute of Technology Cambridge, Massachusetts 02132

Summary Monoclonal antibodies generated against a family of related nuclear pore complex proteins (nucleoporins) from rat liver nuclei cross-react with several proteins in the yeast S. cerevisiae and show punctate nuclear envelope staining similar to the pattern seen in mammalian cells. We have cloned a gene encoding one of these proteins (NUPf) and have confirmed the localization of the NUPI protein to the pore complex by immunofluorescence, using an epitope-tagged construct to differentiate it from other members of this family. The NUPl protein is essential for cell viability, and over-expression from the yeast GAL10 promoter prevents further cell growth. The central domain of NUPl consists of a series of degenerate repeats similar to those found in the nucleoskeletal protein NSPl, a protein that cross-reacts with monoclonal antibodies against NUPl. We propose that the repetitive domain is a feature common to the nucleoporins. Introduction The nuclear pore complex is a conserved component of the eukary!tic nuclear envelope. With an overall diameter of ~1200 A (Unwin and Milligan, 1982) and a molecular weight estimated to be on the order of 10s daltons (Krohne et al., 1978) the pore complex can be viewed as a small organelle. Electron microscopic studies suggest that the pore provides the sole avenue for transport of protein and RNA between nucleus and cytoplasm (Stevens and Swift, 1966; Feldherr et al., 1984; Dworetzky and Feldherr, 1988). An aqueous channel with an ~4.5 nm radius through the center of the pore allows passive diffusion of ions and macromolecules below 45-60 kd (Paine et al., 1975; Lang et al., 1986), but the pore complex is thought to specifically regulate transport of larger macromolecules. Nucleocytoplasmic protein transport has been the subject of numerous studies (for review see Dingwall and Laskey, 1986). Proteins destined for the nucleus contain one or more short, usually basic, nuclear localization sequences within the mature polypeptide that are necessary for accumulation within the nucleus and are sufficient to achieve nuclear localization of cytosolic proteins to which they are fused (see, for example, Kalderon et al., 1984a, 1984b; Lanford and Butel, 1984; Hall et al., 1984). The transport process has been dissected into two separable events: binding at the nuclear pore complex, which is dependent upon a functional nuclear localization signal, fol-

Component

lowed by ATP-dependent translocation across the pore (Richardson et al., 1988; Newmeyer and Forbes, 1988). Several monoclonal antibodies (MAbs) have been described that recognize a group of nuclear proteins (Davis and Blobel, 1986, 1987; Snow et al., 1987; Park et al., 1987), all of which are modified by the addition of monosaccharidic GlcNAc residues O-linked to serine and threonine (Holt and Hart, 1988; Holt et al., 1987; Hanover et al., 1987; Davis and Blobel, 1987). Many of the antibodies show a striking punctate staining pattern of the nuclear envelope by immunofluorescence. Their specificity for the nuclear pore complex has been demonstrated by immunoelectron microscopy of either rat liver nuclei (Davis and Blobel, 1986) or nuclear envelope fractions (Snow et al., 1987). The antigens recognized by these antibodies have been termed nucleoporins (Davis and Blobel, 1986). The MAbs cross-react with various subsets of the proteins; some recognize all of them, whereas others are specific for one or a few polypeptides. The sugar moiety apparently forms all or part of the epitope recognized by some of the antibodies (Holt et al., 1987; Park et al., 1987). However, others are able to recognize unglycosylated forms of the proteins (L. I. D. and G. Blobel, unpublished data and this report). The existence of a common epitope independent of the sugar moiety implies that the primary structure of some or all of these proteins may be related. Evidence is rapidly accumulating that the nucleoporins play an essential role in nucleocytoplasmic transport. Featherstone et al. (1988) showed that anti-nucleoporin MAbs blocked efflux of 5s RNA as well as import of nucleoplasmin (a protein whose import characteristics are well studied) in vivo. Furthermore, the lectin wheat germ agglutinin (WGA), which binds the nucleoporins via their sugar residues, has also been shown to inhibit protein transport (Finlay et al., 1987). The observation that WGA blocked the translocation event, but did not interfere with binding of proteins at the pore complex (Newmeyer and Forbes, 1988), suggested that the nucleoporins are required during the translocation step but not for binding. However, recent studies have shown that Xenopus nuclear pores formed from in vitro extracts that had been depleted of the nucleoporins were defective not only for transport but also for binding of proteins at the pore (Finlay and Forbes, 1990). Thus, the nucleoporins may be involved, directly or indirectly, in both steps of the transport pathway. Recently, Akey and Goldfarb (1989) have used WGA and the pore-specific MAb 414 to further localize the nucleoporins to the central region of the pore, within a discrete structure that they termed the transporter. They also found that colloidal particles adsorbed with nucleoplasmin were bound at the transporter. In some cases the gold particles were found in the peripheral half of this structure, whereas in others they were located directly above an apparent central pore. They proposed that nucleoplasmin first binds at the periphery and is then translocated

Cdl 966

Figure 1. Specificity MAbs in Rat Liver

of the Anti-Nucleoporin

(A) Rat liver nuclear envelopes (5 ODss,, equivalents per lane; see Davis and Blobel, 1966) were extracted with 1% Briton X-100, 0.5 M NaCl (see Experimental Procedures). The extract was subjected to SDS-PAGE and the separated proteins blotted to nitrocellulose. Blots were incubated as described in Experimental Procedures with either MAb 350 (lane l), 414 (lane 2) or 306 (lane 3) and then with a rabbit anti-mouse IgG antibody followed by ‘251-labeled protein A. Bands were visualized by autoradiography. (B) Indirect immunofluorescence was performed on unfixed, Triton X-lOO-extracted But falo rat liver tissue culture cells as previously described (Davis and Blobel, 1966) using MAb 306. The top panel shows a focal plane through the center of the nucleus. In the bottom panel, the focal plane was tangent to the surface of the nuclear envelope.

through the center of the transporter. The localization of the nucleoporins to this structure is consistent with a role in transport. Although little is known about the yeast nuclear pore complex, the highly organized structure is similar to that of vertebrate cells (Allen and Douglas, 1989) and the signals that mediate protein import into the nucleus appear to be shared. For example, nuclear localization signals of yeast proteins are similar to those of complex eukaryotes (Hall et al., 1984; Dingwall et al., 1988; Moreland et al., 1987) and proteins fused to the SV40 large T antigen nuclear localization sequence are efficiently imported into the yeast nucleus in vivo (Nelson and Silver, 1989). Recently, an in vitro assay has been described in which isolated yeast nuclei were able to import both SV40 large T antigen and nucleoplasmin in a process that was ATP dependent and required a functional nuclear localization sequence (Kalinich and Douglas, 1989). Furthermore, one of the anti-nucleoporin MAbs has been found to cross-react with a yeast nuclear envelope protein (Aris and Blobel, 1989). These observations suggest that the pore complexes of yeast are structurally and functionally similar to those of complex eukaryotes. In this paper, we describe the identification of a group of yeast nuclear envelope proteins that is recognized by MAbs raised against mammalian nucleoporins. These MAbs stain the yeast nuclear envelope with a punctate pattern similar to that seen in mammalian cells, indicating that they are also specific for pore complex proteins in yeast. We isolated an essential gene encoding one of these proteins, which we have designated NUPI. We further show that a previously characterized yeast nuclear protein, encoded by the NSP7 gene (Hurt, 1988) is also a member of this family, as it is recognized by many of the MAbs. The two proteins have similar repeated sequences within a central domain, a motif that probably constitutes the cross-reactive epitope recognized by the MAbs and may represent a general feature of the nucleoporins.

Results Antibodies against Rat Liver Nuclear Pore Proteins Cross-React with Yeast Nuclear Antigens Three MAbs (MAbs 350,414, and 308) were chosen from a panel of antibodies generated against rat liver nucleoporins (Davis and Blobel, 1988, 1987, and unpublished data). When used to probe blots of rat liver nuclear envelope extracts, these MAbs showed varying degrees of crossreactivity among members of this protein family. MAb 350 showed the broadest cross-reactivity (Figure lA, lane l), with a pattern very similar to that obtained by probing with WGA (Davis and Blobel, 1987). The previously characterized anti-pore complex MAb 414 (Davis and Blobel, 1988) and MAb 306 showed more restricted reactivity than MAb 350. The patterns obtained with these MAbs were similar: both recognized predominantly p62 and a polypeptide just below it, but differed somewhat in their relative affinities for several minor bands (Figure lA, lanes 2 and 3). The pattern of immunofluorescence staining seen with MAb 306 in Buffalo rat liver cells was identical to that previously shown for MAb 414 (Davis and Blobel, 1988). Typical of antibodies directed against pore complex antigens, MAb 306 stained the rim of the nucleus in a finely punctate pattern (Figure 1B). No internal nuclear staining was evident. MAb 350 showed a pattern similar to that of 414 and 308 (data not shown). These three MAbs were used to probe blots of whole cell extracts and isolated nuclei from yeast strain F901 (Figure 2). MAb 350 recognized several proteins in whole cell extracts (Figure 2, lane 1). Those of molecular sizes 44, 51, 65, and a doublet at ~100 kd were highly enriched in the nuclear fraction (Figure 2, lane 2). An abundant 53 kd polypeptide found in whole cell extracts was not present in the nuclear fraction. This protein could be a cytosolic component, a degradation product of one of the nuclear proteins, or a loosely associated nuclear protein that is extracted during the isolation procedure. MAb 414 rec-

NUP7 Encodes 967

a Yeast

Nuclear

350 r-l-~

.

Pore Complex

Protein

mAb

414

306

DAPI

.*

.

Figure 2. The Anti-Nucleoporin S. cerevisiae

MAbs

Recognize

Several

Proteins

in

Whole yeast cell extracts obtained by glass bead lysis (lanes 1, 3, and 5) or isolated yeast nuclei (lanes 2, 4, and 6) were subjected to SDS-PAGE and the separated proteins blotted to nitrocellulose filters. Slots were probed with the anti-nucleoporin MAbs 350 (lanes 1 and 2), 414 (lanes 3 and 4) or 306 (lanes 5 and 6) as described in Experimental Procedures. The positions of the 130 and 100 kd nuclear proteins and the nonnuclear 53 kd protein are shown. Arrowheads on the left indicate the positions of molecular size markers as follows (in daltons): myosin, (200,000), E. coli 6-galactosidase (116,250), rabbit muscle phosphorylase b (97,400) bovine serum albumin (66,200) hen egg white albumin (42,699). Figure 3. Punctate Staining of the Yeast Nuclear nofluorescence with MAb 306

ognized a subset of the antigens identified by MAb 350, consisting of the 100 kd nuclear proteins (Figure 2, lane 4) and the 53 kd nonnuclear polypeptide (Figure 2, lane 3). These results are in agreement with those published by Aris and Blobel (1989). In contrast, MAb 306 specifically recognized three proteins highly enriched in the nuclear fraction (Figure 2, lane 6): the 100 kd doublet as well as a 130 kd protein that was undetectable in whole cell extracts (Figure 2, lane 5). The latter was also weakly recognized by MAb 350. MAb 306 was used in immunofluorescence because this MAb was unique in its specificity for proteins enriched in nuclei. lmmunofluorescence was performed on a tetraploid yeast strain (LDYl), because the relatively large size of these cells facilitated visualization of nuclear structure (Figure 3). MAb 306 stained the rim of the yeast nucleus with a punctate pattern that looked very similar to that seen in Buffalo rat liver cells (see Figure 1B) and is typical of antibodies specific for the nuclear pore complex. No staining of internal nuclear components was observed. We infer from these results that, as is the case in mammalian cells, MAb 306 is specific for proteins of the nuclear pore complex in yeast. A comparison of the staining pattern of MAb 306 with that of DNA as visualized by DAPI (4,6-diamidino-2-phenylindole) shows that the nuclear envelope encloses a

Envelope

by Immu-

Indirect immunofluorescence was performed on tetraploid yeast strain LDYl, as described in Experimental Procedures, using MAb 306 (diluted 1:lO from an ammonium sulfate concentrate of culture supernatant) and a DEAF-labeled goat anti-mouse IgG. The left panel shows DTAF staining. DAPI staining is shown on the right.

slightly larger area than that stained with DAPI. In particular, adomain within the nucleus, most likely the nucleolus, stains very poorly with DAPI. Using MAb 306 as a marker, we have been able to examine more closely the exact positioning of the nuclear envelope throughout the cell cycle (Figure 4). Unbudded and small budded cells (Figure 4a) have a round nuclear envelope. The first evidence of nuclear migration is a protrusion of the envelope toward the bud neck (Figures 4b and 4~). The nuclear envelope then invades the bud as a very narrow elongated structure well before any DAPI staining can be visualized crossing the neck (Figure 4d). The appearance of the envelope as it begins to enter the bud often suggests attachment to an underlying filament structure, which could consist of cytoplasmic microtubules. As migration into the bud continues, the envelope staining closely follows that of DAPI (Figures 4e and 4f). During chromosome separation, the nuclear envelope can be seen to stretch across the cell along the entirety of the spindle (Figures 4g and 4h). No large budded cells were found in which the nuclear mem-

Cell 966

branes did not appear continuous. The absence of this class of cells suggests that scission of the nuclear membranes is closely tied to cytokinesis. Single cells were often found that retained an extension of the envelope (Figure 4i) that could be the remnants of the elongated envelope found in Figures 4g and 4h. Examination of hundreds of cells by light microscopy reemphasizes the conclusion, reached by electron microscopy, that the yeast nuclear envelope remains intact throughout mitosis. Isolation of a Gene Encoding the 130 kd Protein lmmunoscreening was performed using a yeast genomic hgtll library (Young and Davis, 1983; see Experimental Procedures) with the less specific but higher affinity MAb 350, because preliminary attempts using either MAb 308 or 414 failed to identify any positive clones. This screen yielded 22 positive clones, which fell into 13 distinct

groups by restriction analysis. Protein blots of extracts from isopropyl B-D-thiogalactopyranoside-induced lysogens were then probed with MAbs 414, 308, and 350. Six fusion proteins were recognized only by MAb 350, three by MAbs 350 and 414, and three by all the MAbs. One clone produced a f3-galactosidase fusion protein of ~210 kd that was recognized by 350 and 308, but not by 414 (Figure 5B), and thus could encode the 130 kd antigen. This clone (Figure 5A) was selected for further characterization. The 5’end of the gene was obtained by integration into the yeast chromosome followed by excision with BamHl (Figure 5A, see Experimental Procedures). From the resulting 10 kb BamHl fragment, a 5.5 kb EcoRl fragment was sequenced. A single long open reading frame was found, capable of encoding a protein with a predicted molecular size of 113 kd. The gene was designated NUP7 (for nuclear pore).

NUPl 969

Encodes

a Yeast

Nuclear

Pore Complex

Protein

130,

mla

100,

‘.

12

.I

3

4

Figure 6. Overexpression of NUP7 Results in an Increase in the Amount of the 130 kd Nuclear Protein Recognized by MAb 306 SW...

1

Figure

5. Isolation

‘H

2

3

4

of the NUP7 Gene

(A) 5gtll clone 9-1, encoding part of the NUPl gene, was obtained by immunoscreening. The position of the open reading frame is shown below. The EcoRI-Hindlll fragment used to make a frpE::NUPl fusion is also shown. Disruption of the NUPl gene was accomplished by replacing the 1.4 kb Bsu361 fragment in the middle of the coding region with the MA3 gene. (B) The full-length NUP7 gene was obtained by integration of a plasmid (pLD9-lb) containing the 9-l insert and a URA3 marker at the chromosomal NUP7 locus. The full-length gene was recovered with the marked plasmid by excision with BamHl as shown. Also shown is the map position of NUPI on chromosome 15, relative to ADE2 (chromosomal distances are not to scale). (C) A whole cell lysate was made from an induced E. coli strain lysogenie for the Igtll 9-1 clone. The lysate was subjected to SDS-PAGE and the separated proteins blotted to nitrocellulose. The blots were probed with either MAb 350 (lane I), 414 (lane 2) 306 (lane 3) or with no primary antibody (lane 4) as described in Experimental Procedures. The position of the 210 kd 6-galactosidase fusion protein is noted at the left.

To determine which of the proteins recognized by MAb 306 was encoded by the NUPl gene, we overproduced NUP7 by placing it under control of the inducible yeast GAL10 promotor on plasmid pLDl-5. Yeast cells harboring pLDl-5 were induced to overexpress NlJPl by growth on galactose. Protein blots of whole cell extracts from induced versus uninduced cells, probed with MAb 306, are shown in Figure 6. In uninduced cells (Figure 6, lane 1) or cells transformed with the control plasmid (pDAD1; Figure

Yeast strain L2612 was transformed with either pLDl-5 (pDAD1 containing the NUPl coding region fused to the yeast GAL10 promotor, lanes 1 and 2) or pDAD1 alone (lanes 3 and 4) and grown overnight on synthetic medium lacking uracil and with 0.1% glucose added. They were then diluted and grown to mid-log phase in the same medium. Cells were washed and resuspended in the same medium, except that glucose was replaced with 2% galactose. Samples were removed at 0 (lanes 1 and 3) and 5 (lanes 2 and 4) hr after galactose induction. Whole cell extracts were made by glass bead lysis and subjected to SDS-PAGE. Separated proteins were transferred to nitrocellulose, and the blots were probed with MAb 306 as described in Experimental Procedures. The positions of the 130 kd and 100 kd nuclear proteins are shown at the left.

6, lanes 3 and 4) only the 100 kd antigens could be detected. However, after 5 hr of growth on galactose, the 130 kd protein was visible as the major reactive band in cells harboring pLDl-5 (Figure 6, lane 2). No change in the levels of the 100 kd proteins was seen in galactose-induced cells, indicating that these are not degradation products of the 130 kd antigen. M/P7 Is an Essential Gene A deletion of NUP7 was created by removal of a 1.5 kb 6~~361 fragment from the middle of the open reading frame (see Figure SA) and replacement of this segment with the selectable marker URA3. This construct, nupl7::URA3, was then transformed into diploid strain LDY2 and produced stable, viable Ura+ transformants that exhibited no obvious growth defect. Southern analysis of several of these showed that the disrupted copy was integrated at the NUP7 locus and had replaced the resident gene (data not shown). The transformed diploid (LDY3) was sporulated, and 80 tetrads were analyzed. In 78 cases, only two viable spores were recovered from each

1 121 241 361 481 601 721 841 961 1

GTTATAGGGACAGAGAGTGAGCGACMTTTTTAGTCATTCATGTCTTC-CACTTCTTCTGT~TGTCTTCTCCACGTGTCGAARAGAGATCGTTTTCTTCCACTTT-TCATTCTT “SSNTSSVMSSPRVEKRSFSSTLKSFF

1321 108 1441 148 1561 188 228 1801 268

AGACCTACG~GCAGACATCiACTCTAATA~GTTATCGAAkCTC~ DLRADINSNRLSNPQKNLLLKGPASTVAKTAPIQESFVPN

TCTACTTTT-GGACCAGTTCCACAGTTGCAAARRCT

1921 308 2041 348 2161 388 2281 428

CCAARAGGCTMTAAAACTAGGCTGTCGACMTACTGT~CCTTCCACMCTTTATTC~TTTTGGTGG~-TCAGAT~CCGTTACTT~TGCCAGTC~CCTTTT-~TTGG-~~ -ANKTKA”DNT”PSTTLFNFGGKSDTVTSASOPFKFGK~ 1 ATCCGAAAARAGTGAAAATCTACAGMT~A~CGCGCCT SEKSENHTESDAPPKSTAPiFSFGKOEENGDEGDDENEPK

2401 468

MGARRRAGGCGTTTACCTGTTAGCGAGGATACARACACC R K R R L P" S EDT N T K

2521 508

* ACCMGCTTTGTCTTTGGTGCARGTGATAAGCRAGCTGAACGG PSP"FGISDKOAEGTPLFTFGKKAC"TS~ICSSAOFTFG~

P

11 F

D

F

G

K

+

G

D

0

K

E

T

K

K

G

E

SE

K

D

A

S

Gy,

AAAAAAAGCTGATGTAA-GRAGW\ATATTGACTCCTCTGCATTACCTTTGGTAA

2641 548 2761 588 2881 628 3001 668 3121 708

TTTCACCTTTGGCGGTTCCACAACAAATAATAC~CMCCACTAGCAC-CCATCTTTTAGTTTTGGGGCTCCCGAGTCGAT-GTCGACAGC~GTACAGCGGCAGC-TACGGA FTFGGSTTNNTTTTSTKPSFSPGAPESMKSTASTAAANTE

3241 748

GAAGCTATCAAATGGCTTTTCCTTTACAAAGTTCAATCACMT-G-GTC-CTCTCC~CTTCTTTCTTCGATGGTTCTGCTTCCTC~CGCCGATTCCTGTCTTGGGT~GCC KLSNGFSFTKFNHNKEKSNSPTSFFDGSASSTP IPVLGKP

3361 788 3481 828

MCAGACGCTACTGGTMTACMCATCT-TCTGCATTTTCATTCGGTACTGCTMCACCMTGGTACCMTGCCTCAGC-CTCCACATCATTCTCGTTTMCGCCCCTGCTACTGG TDATGNTTSKSAFSFGTANTNGTNASANSTSFSFNAF'ATG mu361 TAACGGCACAACTACTACTTCCAATACCTCAGGAACCAATGGAGCGGGCTCGGCATTTGG NGTTTTSNTSGTNIAGTFNVGKPDQSIASGNTNGACSAFG

3601 868

CTTTTCGAGCTCAGGMCAGCAGCMCTGGTGCAGCTTCTMTC~TCTTCATTT~TTTTGG-CMTGGTGCAGGGGGTCTCMTCCTTTTACATCAGC~CTTCGTCAACTMTGC .?SSSGTAATGAASNOSSFNFGNNGAGGLNPFTSATSSTNA

3121 908

TAATGCTGGTTTATTCAATACCTCCTTCCACGAATGCACGCCTGGTGGCGGCTCTGTATTTAATATGAA NAGLFNKPPSTNAONVNVPSAFNFTGNNSTPGGGSYFNMN

3841 948

CGGCAACACTAATGCTMTACGGTGTTTGCCGGCTCTMTMCCMCCACATCMTCGC-CCCCATCTTTCMTAC-CAGCTCATTCACGCCATCMCAGTTCCT~TATT~TTT -TNANT”FAGSNNQPHQSQTPSFNTNSSFTPSTVPNINF

3961 988

TAGCGGATTGAATGGCGGAATTACCGC~CCGCGACCATT~GGCC~GTGATATATTTGGTGCG~TGCTGCCTCTGGTTCC~TTC-CGT~C~TCCATCATCCATTT~ FGANAASGSNSNVTNPSSIF SGLNGGITNTATNALRPSDI

4081 1028

TGGGGGGGCAGGTGGTGTGCCGACAACTTCTTCTTTTGGGCAGCCGCAGTCAGCCCCTMTCAGATGGG~TGGGMC-TMTGGCATGAGCATGGGCGGTGGTGTTRTGGCGAACAGARA GGAGGVPTTSFGQPQSAPWQMGMGTNNGMSMGGGVMANRK

4201 1068 4321 4441 4561 4681 4801 4921

Figure

7. Nucleotide

and Deduced

Amino

Acid Sequence

Numbering starts at the EcoRl site. The g-amino-acid influenza HA epitope was inserted. The star indicates disruotion are also shown.

of the Region

Containing

the NW’7

Gene

repeats are underlined. The vertical arrowhead denotes the site at which the g-amino-acid the C-terminus of the Vpf::NUP7 fusion. The Bsu361 sites that were used to create the nupl-7

M/P7

Encodes

a Yeast

Nuclear

Pore Complex

Protein

971

tetrad, all of which were Ura-. Examination of nupl-7 spores revealed that, in general, these cells failed to complete the first mitotic division and were arrested at the large budded stage. To rule out the possibility that an intact NUP7 gene is required solely for spore germination, we examined the viability of haploid vegetative cells that contain the nupl7::URASdisruption. pLDi-1, acentromere-containing plasmid bearing an intact copy of NUf 7 and a LEU2 selectable marker, was introduced into diploid LDY3. Sporulation and tetrad dissection of Leu+ transformants allowed recovery of spores containing NUP7 on the plasmid and nupl-7 on the chromosome. No viable haploid segregants that had lost the plasmid-borne NUP7 could be obtained, indicating that NUP7 is also essential for vegetative growth of cells. We also found that overexpression of NW7 is lethal to cells. Cells harboring pLDl-5, containing the GAL::NUP7 fusion, but not those with pDAD1 alone (see above), were incapable of growth on galactose-containing medium. Microscopic analysis of cells grown under such conditions revealed the accumulation of many cells that had two or more buds. Thus, alterations in the copy number of NW7 may interfere with proper completion of cell division. The NUPl Protein Has Similarity to the Nucleoskeletal Protein NSPl The nucleotide and deduced amino acid sequence of the NW7 gene (Figure 7) revealed a single long open reading frame that could encode a 1,076 amino acid protein with a predicted molecular size of 113,471 daltons. A search of GenBank nucleic acid data base using TFASTA (Pearson and Lipman, 1988) revealed no proteins with significant homology to NUPI. Notably, there was also no homology found between NUPl and the published C-terminal sequence of the 62 kd mammalian nucleoporin (D’Onofrio et al., 1988). The NUPl protein has a large number of charged residues and is basic in nature, with a predicted isoelectric point of 10.2. It can be divided into three domains. The N-terminal region (residues l-320) contains -25% charged residues with a net positive charge of 22. In contrast, the C-terminal domain (residues 990-1076) is relatively devoid of charge, except for the presence of seven basic amino acids at the C-terminus. The middle portion of the NUPl protein consists of 28 degenerate g-amino-acid repeats, separated by short stretches that contain high charge density (Figure 8A, left). The structural characteristics of the NUPl repetitive domain are very similar to those of NSPl (Figure 8A, right). NSP7 encodes a protein migrating at 100 kd on SDS gels and was identified using an antibody raised against yeast nucleoskeletal components (Hurt, 1988). The NSPl protein contains 22 repeats within its central domain that are similar to, but more highly conserved than, those of NUPl. The intervening regions of the NSPl central domain also contain a high density of charged residues. Secondary structure predictions (Chou and Fasman, 1978) indicated that in both cases the repeats are generally predicted to

form 8 sheets and the intervening regions to form a helices (Figure 8B). Figure 8C shows the Eisenberg hydrophobic moment calculations for each protein were it to assume a 6 sheet conformation. For both NUPl and NSPl, each of the repeats shows a significant hydrophobic moment, owing to alternating polar and apolar residues. It has been suggested that one factor in determining secondary structure may be amphiphilicity, since many protein segments assume the periodic structure (either a helix or 8 sheet) that maximizes their hydrophobic moment (Eisenberg et al., 1984). Thus, although the ChouFasman predictions for j3 sheet formation within the repeats are not extremely high (particularly in the case of NSPl), the amphiphilicity that would be generated in the 8 sheet conformation could stabilize such a structure. Despite the fact that the last nine repeats of NUPl have very little sequence conservation and the intervening stretches are almost devoid of charged residues, this region shows secondary structure predictions similar to the more conserved repeats. NSP7 Encodes the 100 kd Protein Recognized by the Anti-Nucleoporin MAbs The similarity between NUP7 and NSP7 suggested that NSP7 could encode one of the 100 kd antigens recognized by the anti-nucleoporin MAbs. Overexpression of NSP7 from a high copy plasmid (YEpl3/NSP7-4) led to a significant increase in the amount of one of the 100 kd polypeptides recognized by MAb 306, relative to the isogenic control (Figure 9A, lanes 1 and 2). The absence of the upper band of the 100 kd doublet in strain 811, which carries a deletion of almost all of the repetitive domain of NSPI (Figure 9A, lane 3) provides direct evidence that NSP7 encodes this protein. Although this mutant NSPl protein is expressed as an -50 kd protein that is recognized by polyclonal antibody against NSPl (Nehrbass et al., 1990) MAb 306 does not recognize the mutant protein, suggesting that the cross-reactive epitope between NUPI and NSPl lies within the central domain of each protein. Further evidence of the relationship between these two proteins was obtained by making a polyclonal antiserum against an N-terminal region of the NUPl protein extending into the repetitive domain. The antibody was obtained by injecting mice with an ~250 amino acid segment of the NUPl protein ending at residue 509 (starred residue, Figure 7) fused in frame to the Escherichia coli trpE gene. When used to probe protein blots of isolated yeast nuclei, the polyclonal anti-NUPl antibody exhibited a pattern very similar to that of MAb 306, recognizing not only the NUPl protein, but also NSPl and the other 100 kd protein (Figure 9B, lanes 4 and 5). This antibody was then used to probe blots of whole cell extracts from the same strains as those shown in Figure 9A (Figure 98, lanes 6-8). The results are similar to those obtained with MAb 306 (see Figure 9A). These observations further emphasize the antigenic relatedness of these two proteins and show that immunization with a polypeptide containing only a small region within the central domain is sufficient to generate a cross-reactive antibody.

Cell 972

NUPl A

NSPI

343 362 381 403 419 445 481 507 522 540 568 588 611 633 654 668 686 705 724 750

TVGFDFIKD KSSVEMGKT TLSFNFSQK TTLFNFGGK SQPFKFGKT APIFSFGKQ KPLFDFGKT KPSFVFGAS TPLFTFGKK SAOFTFGKA KPiFTFGQS KPTFSFSKS KPSFSFPGK KPTFSFTEP KPSFTFASS KPLFSFGKS NTSFSFTKP PPSFTFGGS KPSFSFGAP SNGFSFTKF

EPKKDKESIVLP NETPSKKTSPKATSSAGAVF DKSTKTAEAP ANKTKAVDNTVPS SDTVTSA SEKSENHTESDAPPKST EENGDEGDDENEPKRKRRLPVSEDTNT GDQKETKKGESEKDASG DKQAEG ADVTSNIDS ATAKETHTKPSETPATIVK TSENKISEGSA EEERKSSPISNEAA PVDVQAPTDDKTL AQKDSSWSEPK KTSQP DAAiEPPGS PANETDKRPT TTNNTTTTST ESMKSTASTAAANTEKL NHNKEKSNS

768 797 816 842 863 882 904 926 941

PTSFFDGSA KSAFSFGTA STSFSFNAP AGTFNVGKP GSAFGFSSS QSSFNFGNN STNANAGLF PSAFNFTGN GSVFNMNGN

SSTPIPVLGKPTDATGNTTS NTNGTNASAN ATGNGTTTTSNTSGTNI DQSIASGNTNGA GTAATGAASN GAGGLNPFTSATS NKPPSTNAQNVNV NSTPGG

Figure

8. NUPl

and NSPl

Have

Similar

Central

176 195 214 234 263 281 300 319 338 357 376 395 414 433 452 471 490 509 528 547 566 585

KPAFSFGAT KPAFSFNSS TTGFSFGSQ KPSLSFGSG KPALSFGTA TPSFSFGAY KPAFSFGAK KPAFSFGAK KPAFSFGAK KPAFSFGAK KPAFSFGAK KPAFSFGAK KPAFSFGAK KPAFSFGAK KPAFSFGAK KPAFSFGAK KPAFSFGTK KPAFSFGAK KPAFSFGAK KSAFSFGSK KAAISFGAK KPAFTFGAQ

NLFGATANAN TNDDKKTEPD VGNKTDAQAP LGGNKTVNEAA SAGANPAGASQPEPTTNEPA TSDNKTTNT SDENKAGATS PEEKKDDNSS SNEDKQDGTA PAEKNNNETS SDEKKDGDAS PDENKASATS PEEKKDDNSS SNEDKQDGTA PAEKNNNETS SDEKKDGDAS SDEKKDiiSS SNEKKDSGSS PDEKKNDEVS ANEKKESDES PTGKEEGDGA PEEQKSSDTS KDNEKKTEES

Domains

(A) The deduced amino acid sequences of the central domain of NUPl (left) and NSPI (right) are shown, with the g-amino-acid repeats placed in register. (6) Chou-Fasman secondary structure predictions (window = 4) for NUPl (left panel, residues 330-950) and NSPl (right panel, residues 160-640). The ordinate shows Pp (solid line) and P, (dotted line) values between 0.8 and 1.4. Predictions are considered significant above a value of 1.0. (C) Eisenberg hydrophobic moment calculations for the same segments of NUPI and NSPI shown in (6). The ordinate shows hydrophobic moment values from 0 to 0.75 calculated for interresidue angle = 140-180“ (B sheet, window = 4). The lines below correspond to the approximate positions of each repeat.

Epitope Tagging of NUPl Localization of NUPl with MAb 306 is ambiguous because this antibody also recognizes NSPl and the other 100 kd polypeptide. Antibody specific for NSPl was previously shown to stain the periphery of the yeast nucleus (Hurt, 1988) and thus clearly contributes to the punctate staining pattern obtained with MAb 306. Critical evidence for the localization of NUPl to the nuclear pore requires an antibody that recognizes only NUPl. Unambiguous localization of NUPl to the nuclear envelope was obtained by tagging it with a foreign epitope (Munro and Pelham, 1987; Field et al., 1988). A sequence encoding a g-amino-acid epitope derived from the influenza hemagglutinin (HA) antigen (FLU) was inserted within the middle domain of NUPl (see arrow, Figure 7). A plasmid carrying the NUP7::FLU gene (pLDl-3) was introduced into diploid strain LDY3, heterozygous for the nupl-7 disruption (see previous section). Transformants were sporulated and the resulting tetrads dissected. The NUPl::FLU protein was able to complement a nupl-7 disruption, as evidenced by the recovery of viable Ura+Leu+ spores (Leu+ indicating the presence of the NUP7::fLUcontaining plasmid and Ura+ indicating that the cells re-

tained the chromosomal nupl-7 disruption marked with URA3). The recovery of NUPl::FLU spores with normal growth rates shows that the addition of this epitope to the protein does not interfere with NUPl function. Yeast extracts made from one of the Ura+Leu+ spores (LDYSO) and from a control strain (L2612) harboring a wild-type NUP7 gene were assayed for the presence of the HA epitope by Western blot analysis using a MAb directed against the epitope (12CA5; Field et al., 1988) as a probe. MAb 12045 recognized a 130 kd protein in cells harboring pLDl-3, but not in cells harboring the control plasmid pLDl-1 (data not shown). Yeast strain LDYSO was then used for immunofluorescence microscopy with anti-FLU MAb 12CA5 (Figure lOa). This antibody demonstrated specific, punctate staining of the rim of the nucleus in LDYSO, but not in a control strain lacking the tagged protein (Figure 10~). The punctate staining observed with the FLU epitope, though less intense than that of MAb 306, showed the identical localization within the nuclear periphery. The resolution of this procedure was validated by comparing the staining with that of an internal nuclear protein, RNA polymerase. In strain 2277, which contains an epitope tagged copy of

NUPI 973

Encodes

a Yeast

1

2

Nuclear

3

Pore Complex

4

5

Protein

6

Figure 9. NSF’7 Encodes One of the 100 kd Proteins the Anti-Nucleoporin MAbs

76 Recognized

by

(A) Whole yeast extracts were prepared by glass bead lysis from the following isogenic strains: lane 1, wild-type control (strain JR26-146 x JU4-2; Hurt, 1966); lane 2, NSP7 overproducer (strain TF2 harboring YEpl3/NSPI-4; Hurt, 1968); lane 3, nspf deletion mutant (strain 611 harboring pSB32/nsplA, deleted for amino acid residues 231-615). Equal amounts of extract from each strain (~50 ug of protein per lane) were subjected to SDS-PAGE and the separated proteins blotted to nitrocellulose. Blots were probed with MAb 306 as described in Experimental Procedures. The positions of NUPI and the 100 kd doublet are shown at the left. Note that in these strains the two polypeptides at 100 kd are poorly resolved. Also the amount of NUPl appears to be increased in this strain background, because MAb 306 was able to detect it in whole cell extracts. (8) A fusion was made between the E. coli trpEgene and a region of NUP7 that encodes part of the repetitive domain, ending at amino acid 508 (see star, Figure 7) and beginning approximately 250 amino acids up from this site. The exact location of the 3’ EcoRl junction is unclear because this site originated during construction of the hgtll library and is not present in the full-length clone that was sequenced. A mouse polyclonal antibody was made against the fusion protein as described in Experimental Procedures. The antibody was used at a dilution of 1500 to probe blots of isolated yeast nuclei (25 frg per lane, lane 5). MAb 306 was used in parallel (lane 4) to show the positions of NUPI and the 100 kd doublet. The polyclonal antibody was then used to probe blots from the same extracts shown in (A) (lanes 6-6).

RPB3 (P Kolodziej and R. Young, unpublished data), MAb 12CA5 showed homogeneous staining throughout the nucleus, which was almost indistinguishable from DNA staining as visualized with DAPI (Figure lob). Thus, even though the yeast nucleus is small, it is possible to clearly distinguish intranuclear staining from staining of the nuclear envelope. These data show that the staining pattern observed with MAb 306 is indeed representative of the cellular localization of the NUPl protein. Discussion We have identified

several yeast proteins that are homo-

Figure MAb

10. Localization

of NUP1::HA

and RPB3::HA

Using an Anti-HA

Indirect immunofluorescence was performed using the anti-HA MAb 12CA5 (1:200 dilution of ascitis fluid), as described in Experimental Procedures. DTAF staining is shown at the left, DAPI staining at the right. (a) Strain L2612 harboring pLDl-4 (a CEN plasmid containing the NUP7::HA construct). (b) Strain 2277, which has the chromosomal copy of RPB3 replaced with an RPB3::HA construct. (c) Strain L2612 control.

logs of the mammalian nucleoporins. These proteins are recognized by one or more of the nucleoporin-specific MAbs and are highly enriched in isolated yeast nuclei. Furthermore, the MAbs that recognize them (exemplified by MAb 306) show punctate staining of the yeast nuclear envelope by immunofluorescence. The pattern is virtually identical to that seen in mammalian cells and is indicative of specificity for the pore complex. MAb 306 provides a useful immunoffuorescence marker to investigate the behavior of the entire nucleus during cell division in yeast. Previous studies have depended on DAPI staining, which we have found does not accurately represent nuclear positioning, as the nuclear envelope encloses a larger area than that stained with DAPI. We observed, for example, that a narrow extension of the envelope invades the bud well before DAPI staining can be detected in the bud neck. This extension very often looks as though it were attached to a filament, which may serve to guide the migration of the nucleus into the bud. The spindle itself is probably not involved in this event, because at this stage the spindle is usually entirely within the mother cell, oriented at a diagonal to the bud neck. However, the cytoplasmic microtubules that extend across the bud neck at this stage could guide early nuclear migration. Double immunolabeling experiments using MAb 306 and anti-

Cell 974

tubulin antibodies will be used to investigate whether the envelope extension colocalizes with cytoplasmic microtubules. Oneof the yeast nucleoporin homologs, NUPl, encodes a 130 kd protein recognized by two of our MAbs. Both of these antibodies, as well as a polyclonal antibody made against a trpE::NUP7 fusion gene product, also recognized two other nuclear proteins at 100 kd. Thus, we could not be certain that the punctate nuclear envelope staining that these antibodies demonstrated in immunofluorescence accurately represented the location of each of the proteins recognized on blots. To solve this problem, we used an ‘epitope tagging” procedure (Munro and Pelham, 1987; Field et al., 1988) in which a g-amino-acid influenza HA epitope was inserted within the NUPl protein. A MAb directed against the HA epitope localized the NUPl::HA construct by immunofluorescence. The pattern we obtained was the same as that found with the anti-nucleoporin MAbs, confirming the location of NUPl. The successful in situ localization of NUPl suggests that epitope tagging may be generally applicable as a means of uniquely localizing individual members of a family of antigenitally related proteins, or proteins for which antibodies that work well in immunofluorescence are not available. The deduced amino acid sequence of NlJP7 showed similarity to that of a previously characterized yeast gene, NSP7. NSPl was identified using a polyclonal antibody raised against an insoluble yeast nucleoskeletal fraction and was localized to the periphery of the nucleus by immunofluorescence (Hurt, 1988). We show here that NSPl is one of the 100 kd nuclear proteins recognized by our MAbs and propose that it is another of the yeast nucleoporin homologs. The observation that NSPl is localized to the yeast nuclear pore complex by immunoelectron microscopy (Nehrbass et al., 1990) supports this contention and confirms that the punctate immunofluorescence staining observed with MAb 306 indicates specificity for the pore complex. The similarity between NUPl and NSPl lies entirely within the central region of the two proteins. In each case, this domain consists of a series of repeats of a g-aminoacid sequence, flanked by short, highly charged segments. The repeats themselves are well conserved in NSPl, but are more degenerate in NUPl. The sequences separating the repeats are not conserved and share only the presence of numerous charged residues. Within the central domain the two proteins show similar secondary structure predictions. In both cases the repeats are predicted to form short, amphipathic 6 sheets with the flanking regions generally expected to form a helices. Interestingly, even in regions of NUPl where the primary sequence conservation is quite low (see Figure 8, residues 768949), there is preservation of alternating polar and apolar residues within the repeats and the structural predictions are similar. These observations suggest that this domain constitutes an element, common to the two proteins, whose function depends on secondary structural characteristics rather than primary sequence. If the secondary structure predictions are correct, it may consist of an amphipathic 8 barrel with highly charged helices or coils at either end.

A search of GenBank using the consensus KPAFSFGK (four mismatches allowed) did not yield any proteins that contain more than one iteration of a similar sequence. Like members of other protein families with shared structural elements, such as intermediate filaments, NUPl and NSPl have very different N- and C-terminal domains. These domains may specify different functions for the two proteins. The observation that both proteins are essential indicates that they do not have overlapping functions. Knowledge of the primary sequence of NUPl and NSPl provides some insight into the basis for cross-reactivity of the anti-nucleoporin MAbs, almost all of which recognize more than one of these proteins. For some of the MAbs it appears that the GlcNAc residues found on all of the mammalian nucleoporins constitute a major part of the epitope, because reactivity is lessened or abolished upon deglycosylation (Holt et al., 1987; Park et al., 1987). However, this is not the case for the three MAbs described in this report, since they react with hgtll fusion proteins synthesized in E. coli. This conclusion is consistent with previous observations that these MAbs still recognize the nucleoporins after enzymatic deglycosylation (L. I. D. and G. Blobel, unpublished data). It is therefore likely that the common epitope lies within the protein itself. Since the only similarity between NUPl and NSPl lies within the g-amino-acid repeats, and deletion of the central repetitive domain from NSPl abolishes reactivity with all of our antibodies, we conclude that the shared epitope lies within the repeats. This raises the possibility that there are multiple epitopes on each protein, which could explain the strong immunofluorescence signal that the anti-nucleoporin MAbs produce in both mammalian and yeast cells, even though the nucleoporins are not abundant proteins. The cross-reactivity between the antibodies suggests that they may recognize a general characteristic such as secondary structure, rather than a specific sequence, because there is little absolute sequence conservation between NUPl and NSPl repeats. This is particularly true in the region of NUPl that we used to make a polyclonal antibody, yet this antibody still cross-reacted with NSPl. If the repetitive domain is a feature common to the nucleoporins, then the varying degrees of cross-reactivity exhibited by the MAbs could be explained if each MAb has a slightly different stringency for recognition. For example, some antibodies might require the perfect repeats within NSPl for recognition, whereas others may also recognize the degenerate repeats of NUPl. In support of this, we note that all three antibodies described here (and several others that we have tested) recognize NSPl, but only a subset of these recognize NUPl. We do not know whether the yeast proteins recognized by our MAbs are modified by the addition of O-linked GlcNAc. Both NSPl and NUPl show aberrant migration on SDS gels, which could result from glycosylation or could simply be due to their unusually high positive charge or secondary structure. We have probed protein blots of isolated yeast nuclei with WGA and were unable to obtain a reproducible signal. Furthermore, Kalinich and Douglas (1989) observed no inhibition of protein import into isolated yeast nuclei when incubated in the presence

NUPI 975

Encodes

a Yeast

Nuclear

Pore Complex

Protein

of WGA. This could mean that these proteins are not glycosylated in yeast, or are modified with different sugars. Alternatively, the enzyme preparations used to remove the cell wall may contain an activity capable of removing the sugar residues. We note that if the central domain of NUPl forms a 9 barrel, then one face would be very rich in serine and threonine since these residues are often found at every other amino acid position within the repeats. Glycosylation at these residues might result in a cluster of GlcNAcs that would be expected to bind WGA well, because this lectin has a much higher affinity for multiple GlcNAc residues than it does for the monosaccharide. Appropriate levels of both NUPl and NSPl are essential for cell growth, and alterations in expression appear to affect the ability of cells to complete mitosis. Spores containing a nupl-7 disruption usually fail to complete the first mitotic division and arrest as large budded cells. The same phenotype was observed in an nspl deletion (Hurt, 1988). Overexpression of NUPl placed under control of the GAL10 promotor is also lethal. Cells containing a GAL::NUFV fusion stop growing, and many appear to arrest with two or more buds when plated on galactose-containing medium. The lethality due to overexpression could be explained if proper stoichiometry of the components that make up the pore complex is necessary for the large amount of nuclear envelope assembly that is presumably required during cell division. Our data suggest that, in addition to NUP7 and NSP7, there are probably several other yeast genes that belong to the nucleoporin family. NUP7 was one of a number of unique clones that we isolated by hgtll screening with the anti-nucleoporin MAbs. We are now characterizing these other clones to see which, if any, encode related proteins. The identification of genes encoding the nucleoporins opens the way to genetic analysis of nuclear pore complex function. Conditional mutations within the NUP7 gene will provide the tools to investigate the function of the nucleoporins themselves and to identify other proteins with which NUPl interacts. Experimental

Procedures

Plasmids Ligations were generally performed using DNA fragments in low melt agarose (Struhl, 1983). All other DNA manipulations, including preparation of plasmid DNA by alkaline lysis, were done according to Maniatis et al. (1982). Bacterial transformation was performed using the method of Hanahan (1985). E. coli strain DH5a was used for all transformations unless otherwise noted. pLD9-1: A 4 kb EcoRl insert from Igtll clone 9-1 subcloned into the EcoRl site of pGEM 7 (Promega). The orientation is such that the BamHl site in the polylinker is at the 5’ end of the insert. pLD9-la: A Bsu361 fragment in pLD9-1 replaced with a 1.1 kb Hindlll fragment containing a functional URAB gene via blunt-end ligation. pLD9-lb: A 1.1 kb Hindlll fragment containing the URA3gene cloned into the Xhol site of pLD9-1 via blunt-end ligation. pLD9-lc: A 750 bp EcoRI-Hindlll fragment of pLD9-la (the EcoRl site originally formed the junction between the laci’gene and the insert in hgtll) cloned into the EcoRI-Hindlll sites in PATH 11 (Koerner et al., 1990). This generates an in-frame gene fusion between the E. coli trpE gene and NUP7. pLD1: A 5.5 kb EcoRl fragment containing the entire NUP7 coding region, cut from the 11.5 kb fragment recovered after integration of

pLD9-lb and excision with BarnHI, cloned into the EcoRl site of pUCll6 (Vieira and Messing, 1987). pDAD1: pCGS109 (a kind gift of J. Schaum and J. Mao, Collaborative Research), into which a polylinker was cloned 3’to the GAL10 promotor (D. Miller, D. Pellman, and G. R. F., unpublished data). pLDl-5: A SnaBI-Xhol fragment of pLD1 containing the entire NUP7 gene in addition to 80 bp b’and ~1.5 kb 3’to the coding region, ligated into pDAD1 at Sal1 and Xhol sites within the polylinker. pLDl-1: A 4.3 kb Nhel-Pvull fragment of pLD1 containing the NW7 gene blunt-end ligated into the Spel-Sall sites of pRS315 (Sikorski and Hieter, 1989). pLDl-3: pLDl-1 with FLU epitope coding sequence inserted at nucleotide 2377 (Figure 7). YEpl3/NSPl-4: A 3.5 kb fragment containing the entire NSPl coding region inserted into the BamHl site of YEpl3 (Hurt, 1988). pSB32: An ARS-CEN plasmid containing nsplb, deleted for amino acid residues 233-615 (U. Nehrbass and E. C. Hurt, unpublished data). Yeast Strain Construction and Growth Conditions Standard media preparation, yeast cell culture, and tetrad analysis were carried out according to Sherman et al. (1986). Yeast transformation was done using the lithium acetate procedure of Ito et al. (1983) (see Table 1). Preparation of Cell and Subcellular Fractions and lmmunoblottlng Rat liver nuclei were purified according to published procedures (Blobel and Potter, 1966) with modifications (Davis and Blobel, 1986). Nuclear envelopes were prepared by digestion of nuclei with DNAase (Dwyer and Blobel, 1976) and the envelopes were extracted with 10% sucrose, 10 mM triethanolamine-HCI (pH 7.4) 5 mM MgClp, 1% Triton X-100,500 mM NaCI, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol on ice for 10 min. The supernatant was recovered after centrifugation in a TL-100.2 rotor at 50,000 rpm for 10 min. This procedure quantitatively extracts the WGA binding proteins of the nuclear envelope fraction (Snow et al., 1967). Yeast nuclei were purified and prepared for electrophoresis as described by Aris and Blobel(1988). Whole cell extracts from yeast were prepared by glass bead lysis directly into trichloroacetic acid (Ohashi et al., 1982). For immunoblotting, samples were electrophoresed through 8.5% SDS-polyacrylamide gels (Laemmli, 1970) and blotted electrophoretitally to nitrocellose membranes. The blots were probed with primary antibody for 4 hr at room temperature in incubation buffer (phosphatebuffered saline, 0.1% Triton X-100,0.01% SDS, 2% bovine serum albumin). MAbs 306 and 350 were diluted 1:lO and 150, respectively, from ammonium sulfate concentrates of culture supernatant. MAb 414 was diluted 1:3 from culture supernatant. The polyclonal antibody against the CfpE::NUPl fusion gene product was diluted 1500. After incubation with primary antibody, the blots were washed over a 30 min period with three changes of wash buffer (phosphate-buffered saline, 0.1% Triton X-100,0.01% SDS) and then incubated for 1 hr with affinity-purified rabbit anti-mouse antibody(Jackson Laboratories) diluted 1:500 in incubation buffer. Blots were washed as before and incubated for 1 hr with affinity-purified tz51-labeled protein A (100 uCi/ml, Amersham), diluted I:2000 in incubation buffer. Blots were washed as before, dried, and subjected to autoradiography. lmmunofluoreecence Microscopy Indirect immunofluorescence was performed on unfixed Buffalo rat liver cells, after in situ extraction as previously described (Davis and Blobel, 1986). For immunofluorescence of yeast cells, a modification of published procedures (Aris and Blobel, 1988; Pringle et al,, 1990) was used. Late log phase cultures were diluted in the evening and grown overnight to approximately 1 x 10’ cells/ml. They were then chilled on ice for 10 min. Forty percent formaldehyde, freshly prepared in water from paraformaldehyde, was added directly to the cells in growth medium to a final concentration of 4%. Cells were fixed on ice for 2 hr and then washed twice with solution B (100 mM potassium phosphate buffer [pH 7.51, 1.2 M sorbitol) and resuspended to 1 x 108 cells/ml in solution B containing 30 HIM 8-mercaptoethanol and protease inhibitors: 5

Cdl 976

Table

1. Yeast

Strain

Construction

and Growth

Conditions

Strain

Genotype

L2612 F608 9933-2oc 6296-2D F901 L464 L2499 LDYl

Mata can1 cyh2 leu2-3, 112 trpl-289 ura3-52 MATa adel-100 his4-519(fs) /eu2-3, 112 ura3-52 MATa h&3-200 leu2-3, 112 trpld 1 ura3-52 MATa ade2-1 can1 ura3-1, 2 MATa pep4-3, prbl-1122 prcl-407 trpl ura3-52 MATa/MATa lysl-l(fJAA)/lysl-l(lJAA) MATalMATa am1 l/am 1 cryl/cryl his4A5/his4A29 MATaIMATa ; I ; +- I -+ + I +

LDYP LDY3 LDY4 LDY41 LDY50 2277 JR26-148 TF2 Bll

x JU4-2

Iysl-l(UAA)/iysl-l(UAA)

MATa/MATa argl Vargll cryl/cryl his4AVhis4A29 + I + MATalMATa +/adel-100 canl/+ cyh2/+ +/his4-519(fs) leu2-3, 112/leu2-3, 112 trpl-289/+ ura3-52/ura3-52 MATalMATa +/adel-100 canl/+ cyh.V+ +/his4-519(fs) leu2-3, 112/leu2-3, 112 trpl-289/+ ura3-52/ura3-52 nupl-l::URA3/+ MATa can1 cyh2 leu2-3, 112 trpl-289 ura3-52 pLD9-lb(URA) MATa ade2-1 urad pLD9-lb(URA) MATa leu2-3, 112 lrpl-289 ura3-52 nupl-1::URAd (pLDl-3) MATa lys2-128d ura3-52 leu2-3, 112 HIS4-912d RPElSl::LEUP MATa/MATa ade2-l/ade2 +/adeS canl-lOO/canl-100 his3/+ +/his4 leu2-3, 112/ieu2-3, 112 lysl-l/lysl-1 ura3-52/ura3-52 adebl canl-100 his4 leu2-3, 112 lysl-1 trpl ura3-52 nspl::URA-3 (YEpl3/NSPl-4) ade2 canl-100 his4 1~2-3, 112 lysl-1 trpl ura3-52 nspl::URAS (pSB32lnspA)

All strains originated from an S266C background. LDYl was constructed by mating L484 with L2499. and LDYP from mating F808 and L2612. LDY3 was derived from LDYP by one-step integration of an EcoRl fragment of pLD9-la containing nupl-l::URA3. LDY50 was derived from LDY2 by sporulation after transformation with pLDl-3. LDY4 was derived from L2612 by integration of pLD9-1 b. LDY41 is an ascospore from a cross between LDY4 and 8296-2D and was constructed for mapping purposes.

pg/ml each of leupeptin, pepstatin, antipain, and aprotinin; 1 pg/ml chymostatin; and 0.1 mM phenylmethylsulfonyl fluoride. Oxalolyticase (Enzogenetics) was added to a final concentration of 0.1 mglml, and cells were digested at 30°C until approximately 70% were spheroplasted, usually about 10 min. Spheroplasts were diluted with ice-cold solution B plus protease inhibitors and centrifuged for 5 min. They were resuspended to 1 x 108 cells/ml and pipetted onto polylysine-coated round cover slips that had been placed in wells of a 24-well dish (Corning). After 20 min, the cover slips were washed gently and incubated sequentially with 100% methanol (5 min at -2OOC) and then solution B plus protease inhibitors plus 0.1% Triton X-100 (5 min at room temperature). Wells were washed twice with solution B after each of these steps. They were then incubated with solution B plus protease inhibitors, 2% BSA, and 2% normal goat serum (incubation buffer) for 2 hr at 30°C. First antibody incubation was done at 30°C for 2 hr in incubation buffer (see figure legends for dilutions), after which wells were washed four times for 5 min each in solution B. The secondary antibody was a DlAF-conjugated affinity-purified rabbit anti-mouse IgG (Jackson Labs; DTAF: 5-[(4,6-dichlorotriazin-2y)aminojfluorescein) diluted 150 in incubation buffer. After incubation with secondary antibody for 2 hr at room temperature, the cover slips were washed in the same manner, except that 300 mM NaCl was included in the wash buffer. Cover slips were then incubated with 0.5 pglml DAPI in solution B for 10 min and washed three times in solution 8. The cover slips were mounted on slides with 90% glycerol containing 1 mg/ml p-phenylene diamine buffered to pH 8.0. Slides were viewed with a Zeiss axioscope microscope. Kodak T-MA% 400 film was used for all photomicroscopy. lmmunoscreening of ?.gtll Library A yeast genomic library made from Saccharomyces cerevisiae S288C DNA (Young and Davis, 1983) was used for immunoscreening by established procedures (Huynh et al., 1985). E. coli strain Y1090 was infected with I.gtll recombinants and plated on 15 petri dishes of 150 mm (-2 x lo5 plaques per plate). An ammonium sulfate cut of MAb 350 supernatant was diluted 1:30 for incubation with nitrocellulose replica filters. Positive colonies were detected using a biotinylated secondary antibody and an avidin-biotinylated peroxidase complex (Vector Labs).

Probable positive clones were plaque purified and rescreened with antibody. Twenty-two clones remained positive after tertiary screening. These were subcloned into pGEM 7 (Promega) and analyzed by restriction mapping, which reduced the number of distinct clones to 13. The clones were further classified by overproducing the IacZ fusion protein from each clone using the lysogenic strain Y1089. Preparation of lysogens and induction were performed as described (Snyder et al., 1987). Cells were lysed directly into SDS-PAGE buffer, sonicated, boiled, and electrophoresed. The separated proteins were blotted onto nitrocellulose and the blots probed with either MAb 350, 306, or 414 as described above. Recovery of Full-Length NW1 Clone To obtain the 5’ end of the gene, integration/excision was carried out as described (Roeder and Fink, 1980). pLD9-lb was integrated into yeast strain L2612 by transformation to Ura+. DNA was isolated from four transformants and checked by Southern blot analysis using the NUP7 clone as a probe. All four showed restriction patterns indicative of integration within the NUPl gene. DNA from one transformant (LDY4) was cut with BamHI, ligated, and used to transform E. coli strain DH5a to ampR. Transformants were restriction mapped and found to contain an 11.5 kb insert representing the original 4 kb clone plus 7.5 kb upstream. DNA Sequencing A series of unidirectional nested deletions of pLD1 was made in both orientations with exonuclease Ill according to Henikoff (1984), except that Exo VII was used instead of Sl nuclease. The deletion series was transformed into strain JM109. Dideoxy sequencing and gradient gel electrophoresis were performed as described (Sanger et al., 1977; Biggin et al., 1983). Sequence editing and analysis were performed using the UWGCG programs (Devereux et al., 1984). Genetic Mapplng Southern blot analysis of genomic S288C DNA indicated that NUPl is a single copy gene (data not shown). It was assigned to chromosome 15 by Southern blot hybridization to yeast DNA resolved by OFAGE electrophoresis (Carle and Olson, 1987). For genetic mapping, strain LDY4 (ADE2 ura3-52, containing URA3 integrated adjacent to the NUPl

;J+P7

Encodes

a Yeast

Nuclear

Pore Complex

Protein

gene) was crossed to strain 829628 (ade2-7 ura3). A total of 126 tetrads were analyzed for uracyl and adenine auxotrophy, yielding 69 parental ditypes, 37 tetratypes, and 0 nonparental ditypes with a map distance calculated to be 14.7 CM between NUPl and ADE2. A three point cross performed between LDY41 (adell ure3-52, containing URA3 integrated adjacent to the NUP7 gene) and 9933-20C (hi@200 ura3-52) determined that ADE2 lies between NUP7 and HIS3, and thus that NUPI is on the centromeric side of ADE2. This places NUPI within 3 CM of 7sm 6740. The position of these two genes relative to one another was not ascertained.

G. R. F. is an American Cancer Society Professor of Genetics. L. I. D. was supported by a postdoctoral fellowship from the Helen Hay Whitney Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advsrtisemenf” in accordance with 16 USC. Section 1734 solely to indicate this fact. Received

February

12, 1989; revised

March

29, 1990.

Rekrences Preparation of Antibody to the NUPl Gene Product E. coli harboring pLDQ-lc produce a fusion protein containing the first 324 amino acids of the TrpE protein fused to approximately 260 amino acids of NUPl. To prepare fusion protein for injection, the frpEoperon was induced with indolacrylic acid (Spindler et al., 1984) and cells were fractionated into soluble and insoluble fractions (Keorner et al., 1990). All of the 65 kd fusion protein was recovered in the soluble fraction. Material resulting from 40 ml of original culture was precipitated with 10% trichloroacetic acid. The precipitate was dissolved in SDS sample buffer and electrophoresed through a preparative 7.0% SDSpolyacrylamide gel. The gel was stained for 15 min in 1% Coomassie blue in water and then destained with several changes of water. The band corresponding to the fusion protein was excised and finely minced with a razor blade. Two volumes of electrophoresis buffer containing 0.1% SDS was added, and the gel pieces were forced back and forth through an 18 gauge needle until resistance was negligible. The gel pieces were incubated in the buffer overnight at 68OC in a 15 ml conical tube. After centrifugation, the supernatant was carefully removed and concentrated using a centricon concentrator (Amicon). The final volume was brought to 200 nl, and an equal volume of synthetic adjuvant (RIBI Immunochemical) was added. This material was used to inject two mice intraperitoneally. Mice were boosted in the same manner 1 and 2 months later, and orbital blood was taken 7 days after the last boost. Overexpression of NUPl from the GAL10 Pmmotor Yeast strain L2612 was transformed to Ura+ with either pDAD1 or pLDi-5. To induce overexpression, cells were grown overnight in liquid synthetic medium containing 0.1% glucose and lacking uracyl. They were then diluted 1:lO in the same medium and grown to an OD2s0 of 0.3. Cells were washed twice with minimal medium and then resuspended to the same density in uracyl-lacking medium containing either 2% galactose (induced) or 2% glucose (repressed). Cells (50 nl) were removed from each culture at 0 and 5 hr. Whole extracts were made as described above. Epitope Tagging Site-directed mutagenesis was used to insert the sequence encoding a Q-amino-acid influenza HA epitope (Wilson et al., 1984) in frame within the NUP7 gene (see arrow, Figure 7). A 61 bp oligonucleotide containing the epitope plus 17 bp of NUPI sequence on either side was used to direct the insertion, using a kit produced by Amersham. The kit was used as directed with two exceptions: DH5u cells were used instead of TGl, and primer annealing was carried out in a 70°C block for 5 min, which was then cooled to room temperature over 30 min. Insertion was confirmed by restriction analysis with Aatll, which cuts within the inserted sequence. Acknowledgments We are grateful to all of the members of the laboratory for helpful discussions and technical advice, particularly Vivian Berlin, Per Ljungdahl, and David Miller, as well as Fred Dietrich who provided the OFAGE blot. We are grateful also to David Miller and David Pellman for providing the pDAD1 plasmid. We thank Peter Kolodziej and Richard Young (Whitehead Institute, Cambridge, MA) for strain 2277 and for providing MAb 12CA5. We are grateful to Ulf Nehrbass and Eduard Hurt (European Molecular Biology Laboratory, Heidelburg) for providing strains JR26-14B x JU4-2, TF2, and Bll, and for communicating results prior to publication. We also thank Gunter Blobel, in whose laboratory the antibodies used in this study were made. This work was supported by National Institutes of Health grant GM40266 (G. R. F.).

Akey, C. W., and Goldfarb, D. S. (1969). Protein import through the nuclear pore complex is a multistep process. J. Cell Biol. 109, Q7t-982. Allen, J. L., and Douglas, complex in Saccharomyces 702, 95-108.

M (1989).

cerevisiae.

Organization of the nuclear J. Ultrastruct. Mol. Struct.

pore Res.

Aris, J. P, and Blobel, G. (1986). Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J. Cell Biol. 107, 17-31. Aris, J. P., and Blobel, G. (1989). Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins. J. Cell Biol. 708, 2059-2067. Biggin, M. D., Gibson, T. J., and Hong, G. F. (1983). gels and 35S label as an aid to rapid DNA sequence Proc. Natl. Acad. Sci. USA 80, 3963-3965. Elobel, method

Buffer gradient determination.

G., and Potter, V. R. (1966). Nuclei from rat liver: isolation that combines purity with high yield. Science 754, 1662-1665.

Carle, G. F., and Olson, M. V. (1967). Orthogonal-field-alternation electrophoresis. Meth. Enzymol. 755, 468-482.

gel

Chou, P Y., and Fasman, G. D. (1978). Prediction of the secondary structure of proteins from their amino acid sequence. Adv. Enzymol. 47; 45-147. Davis, L. I., and Blobel, G. (1966). Identification and characterization of a nuclear pore complex protein. Cell 45, 699-709. Davis, L. I., and Blobel, G. (1967). The nuclear pore complex contains afamilyof glycoproteins that includes ~62: glycosylation through a previously unidentified pathway. Proc. Natl. Acad. Sci. USA 84, 75527556. Devereux. J., Haeberli, P., and Smithies, 0. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucl. Acids Res. 72, 387-395. Dingwall, C., and Laskey, R. A. (1986). Protein cleus. Annu. Rev. Cell Biol. 2, 367-390.

import

into the cell nu-

Dingwall, C., Robbins, J., Dilworth, S. M., Roberts, B., and Richardson, W. D. (1968). The nucleoplasmin nuclear localization sequence is larger and more complex than that of SV-40 large T antigen. J. Cell Biol. 107, 841-849. D’Onofrio. M., Starr, C. M., Park, M. K., Holt, G. D., Haltiwanger, R. S., Hart, G. W., and Hanover, J. A. (1986). Partial cDNA sequence encoding a nuclear pore protein modified by O-linked N-acetylglucosamine. Proc. Natl. Acad. Sci. USA 85, 9595-9599. Dworetzky, S., and Feldherr, C. (1968). Translocation gold particles through the nuclear pores of oocytes. 575-564.

of RNA-coated J. Cell Biol. 706,

Dwyer, N., and Blobel, G. (1976). A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei. J. Cell Biol. 70, 581-591. Eisenberg. D., Weiss, R. M.. and Terwilliger, T. C. (1984). The hydrophobic moment detects periodicity in protein hydrophobicity. Proc. Natl. Acad. Sci. USA 87, 140-144. Featherstone, C., Darby, M. K., and Gerace, L. (1988). A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo. J. Cell Biol. 707, 1289-1297. Fetdherr, C. M., Kallenbach, a karyophilic protein through 99, 2216-2222.

E., and Schultz, N. (1984). Movement of the nuclear pores of oocytes. J. Cell Biol.

Field, J., Nikawa, J., Broek. D., MacDonald, B., Rodgers, I. A., Lerner, R. A., and Wigler, M. (1968). Purification

L., Wilson, of a RAS-

Cell 978

responsive adenylyl cyclase complex from Seccharomyces cefevisiae by use of an epitope addition method. Mol. Cell. Biol. 6, 2159-2165.

Nelson, M., and Silver, P. (1989). Context affects nuclear protein localization in Saccharomyces cefevisieie. Mol. Cell. Biol. 9, 384-389.

Finlay, D. Ft., and Forbes, D. J. (1990). Reconstitution altered nuclear pores: transport can be eliminated 60, 17-29.

Newmeyer, D. D., and Forbes, D. J. (1988). Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell 52, 641-653.

of biochemically and restored. Cell

Finlay, D. Ft., Newmeyer, D. D., Price, T. M., and Forbes, D. J. (1987). Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores. J. Cell Biol. 704, 189-200. Hall, M. N., Hereford, L., and Herskowitz, f3-galactosidase to the nucleus in yeast.

I. (1984). Targeting Cell 36, 1057-1065.

of E. coli

Hanahan, D. (1985). Techniques for transformation of E. co/i. In DNA Cloning, Vol. I, D. M. Glover, ed. (Oxford, England: IRL Press), pp. 109-135. Hanover, J. A., Cohen, C. K., Willingham, M. C., and Park, M. K. (1987). O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins. J. Biol. Chem. 262, 9887-9695. Henikoff, S. (1964). Unidirectional digestion with exonuclease Ill creates targeted breakpoints for DNA sequencing. Gene 28, 351-359. Holt, G. D., and Hart, G. W. (1986). The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel proteinsaccharide linkage, O-linked GlcNAc. J. Biol. Chem. 267, 8049-8057. Holt, G. D., Snow, C. M., Senior, A., Haltiwanger, Ft., Gerace, L., and Hart, G. W. (1987). Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine. J. Cell Biol. 104, 1157-1164.

Ohashi, A., Gibson, J., Gregor, I., and Schatz, G. (1982). Import teins into mitochondria. J. Biol. Chem. 257, 13042-13047. Paine, i?, Moore, L., and Horowitz, ability. Nature 254, 109-114.

S. (1975). Nuclear

envelope

of properme-

Park, M. K., D’Onofrio, M., Willingham, M. C., and Hanover, J. A. (1987). A monoclonal antibody against a family of nuclear pore proteins (nucleoporins): O-linked N-acetylglucosamine is part of the immunodeterminant. Proc. Natl. Acad. Sci. USA 84, 64626466. Pearson, sequence

W. R., and Lipman, D. J. (1988). Improved tools for biological comparison. Proc. Natl. Acad. Sci. USA 85, 2444-2448.

Pringle, J. R., Adams, lmmunofluorescence

A. E. M., Drubin, D. G., and Haarer, B. K. (1990). methods for yeast. Meth. Enzymol., in press,

Richardson, W. D., Mills, A. D., Dilworth, S. M., Laskey, R. A., and Dingwall, C. (1988). Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell 52, 655-664. Roeder, G. S., and Fink, G. R. (1980). DNA rearrangements with a transposable element in yeast. Cell 27, 239-249. Sanger, F., Nicklen, with chain-terminating 5467.

associated

S., and Coulson, A. R. (1977). DNA sequencing inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-

Hurt, E. C. (1988). A novel nucleoskeletal-like protein located at the nuclear periphery is required for the life cycle of Seccharomyces cefevisiee. EMBO J. 7, 4323-4334.

Sherman, F., Hicks, J. B., and Fink, G. R. (1986). Methods in Yeast Genetics (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory).

Huynh, T. V., Young, R. A., and Ft. W. Davis (1965). Constructing and screening cDNA libraries. In DNA Cloning Techniques: A Practical Approach, Vol. 1, D. Glover, ed. (Oxford, England: IRL Press), pp. 49-78.

Sikorski, R. S., and Hieter, P. (1969). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in S. cerevisiae. Genetics 722, 19-27.

Ito, H., Fukada, Y., Murata, K., and Kimura, A. (1983). Transformation of intact yeast cells with alkali cation. J. Bacterial. 153, 163-168.

Snow, C. M., Senior, A.. and Gerace, L. (1967). Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J. Cell Biol. 704, 1143-I 158.

Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E. (1984a). A short amino acid sequence able to specify nuclear location. Cell 39, 499-509. Kalderon, D., Richardson, W. D., Markham, A. F., and Smith, A. E. (1964b). Sequence requirements for nuclear location of simian virus 40 IargeT antigen. Nature 377, 499-509. Kalinich, J. F., and Douglas, M. G. (1969). In vitro translocation through the yeast nuclear envelope: signal dependent transport requires ATP and calcium. J. Biol. Chem. 264, 17979-17989. Keorner, T. J., Hill, J. E., Myers, A. M., and Tzagoloff, A. (1990). Highexpression vectors with multiple cloning sites for construction of trpf fusion genes: PATH vectors. Meth. Enzymol., in press.

Snyder, M., Elledge, S.. Sweetser, D., Young, R. A., and Davis, R. W. (1987). Xgtll: gene isolation with antibody probes and other applications Meth. Enzymol. 154, 107-126. Spindler, K. R., Rosser, D. S. E., and Berk, A. J. (1964). Analysis of adenovirus transforming proteins from early regions 1A and 1B with antiserum to inducible fusion antigens produced in Escherichia co/i. J. Virol. 49, 132-141. Stevens, cytoplasm

B. J., and Swift, H. (1966). RNA transport from nucleus in chironomus salivary glands. J. Cell Biol. 37, 55-77.

Struhl, K. (1963). Direct Gene 26, 231-242.

selection

for gene replacement

to

events in yeast.

Krohne, G., Werner, W., and Scheer, U. (1978). The major polypeptides of the nuclear pore complex. Exp. Cell Res. 176, 85-102.

Unwin, P T., and Milligan, R. A. (1962). A large particle associated with the perimeter of the nuclear pore complex. J. Cell Biol. 93, 83-75.

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 880-665.

Vieira, J., and Messing, J. (1967). Production mid DNA. Meth. Enzymol. 753, 3-11.

Lanford, R. E., and Butel, J. S. (1984). Construction tion of an SV40 mutant defective in nuclear transport 37, 801-813.

Wilson, I. A., Niman, H. L., Houghten, R. A., Cherenson, A. R., Connolly, M. L., and Lerner, R. A. (1984). The structure of an antigenic determinant in a protein. Cell 37, 767-776.

Lang, I., Scholz, nucleocytoplasmic

and characterizaof T antigen. Cell

M., and Peters, R. (1966). Molecular mobility and flux in hepatoma cells. J. Cell Biol. 702, 1183-1190.

Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). Moreland, R. B., Langevin, G. L., Singer, R. H., Garcia, R. L., and Hereford, L. M. (1987). Amino acid sequences that determine the nuclear localization of yeast histone 28. Mol. Cell. Biol. 7, 4046-4057. Munro, S., and Pelham, H. R. B. (1987). A C-terminal secretion of luminal ER proteins. Cell 48, 699-907.

signal

prevents

Nehrbass, U., Kern, H., Mutvei, A., Horstmann, H., Marshallsay, B., and Hurt, E. (1990). NSPl: a yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxy-terminal domain. Cell 67, this issue.

of single-stranded

Young, R. A., and Davis, R. W. (1983). Yeast RNA polymerase isolation with antibody probes, Science 222, 776-762. GenBank

Accession

The accession M33632.

number

plas-

II genes:

Number for the sequence

reported

in this paper

is