Deﬁning the essential functional regions of the ...jcs.biologists.org/content/joces/110/7/911.full.pdf · The presence of NPC/nuclear envelope perturbations suggests that Nup145p

911Journal of Cell Science 110, 911-925 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS8112

Defining the essential functional regions of the nucleoporin Nup145p

Jennifer L. T. Emtage, Mirella Bucci, Janis L. Watkins and Susan R. Wente*

Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, MO63110, USA

*Author for correspondence (e-mail: [email protected])

Studies of the essential nucleoporin Nup145p have shownthat its depletion is coincident with a block in RNA exportand that deletion of its amino-terminal domain results inclustering of nuclear pore complexes. To further define thefunctional domains of Nup145p, we have characterized apanel of nup145 mutants. Deletions from both the aminoterminus and the carboxy terminus resulted in temperaturesensitive mutants that accumulated polyadenylated RNA inthe nucleus at the non-permissive temperature. In addition,these mutants also displayed constitutive clustering ofnuclear pore complexes in localized patches of the nuclearenvelope. These results suggested that an internal region ofNup145p consisting of amino acids 593-893 is essential forfunction. Accordingly, when this region was deleted,growth was not supported at any temperature, whereas theregion alone was able to complement a null mutation when

expressed on a high copy plasmid. Previous studies havesuggested that Nup145p is cleaved into two polypeptides ofapproximately 65 and 80 kDa. Interestingly, our experi-ments suggest that cleavage occurs in vivo. However, asmall internal deletion of 17 amino acid residues thatabolished cleavage had no effect on cell growth. Therefore,cleavage is not necessary for Nup145p function. When asequence harboring the Nup145p cleavage site required forNup145p cleavage was inserted in a chimeric protein, it wasnot sufficient for mediating cleavage. Cleavage likelyrequires a second region from amino acid residues 247-524in addition to the cleavage site.

Key words: Nuclear pore complex, Nucleocytoplasmic transport,Trafficking, Clustering, Cleavage

SUMMARY

INTRODUCTION

The nucleus of a eukaryotic cell is separated from thecytoplasm by two membranes, and pores formed through thebilayers are necessary for all nucleocytoplasmic communica-tion. The nuclear pore complex (NPC) allows the passivediffusion of ions and small molecules and mediates the activetransport of large macromolecules with a diameter of up to 26nm (Feldherr et al., 1984). High resolution electron microscopyimages of vertebrate NPCs reveal an intricate structure with amolecular mass of 125 MDa (Reichelt et al., 1990). It has adistinct eightfold symmetry, and is comprised of multiple rings,a spoke network, a basket-like structure on the nuclear face,cytoplasmic filaments, and connections to the nuclear laminaand lattice (for reviews see Akey, 1995; Panté and Aebi, 1996).Functionally, yeast and vertebrate NPCs are highly homolo-gous: they recognize the same import and export signals(Osborne and Silver, 1993; Gerace, 1995), proteins in bothshare antibody epitopes and characteristic sequence motifs(Rout and Wente, 1994), and in one example to date, a geneencoding a vertebrate NPC protein complements a yeast NPCmutant background (Aitchison et al., 1995b).

Based on biochemical analysis of purified NPCs (Rout andBlobel, 1993) and on calculations from their total size, it isestimated that the NPCs may contain as many as a hundreddifferent proteins (called nucleoporins or NUPs). In the yeastSaccharomyces cerevisiae, genetic and biochemical strategies

to date have identified the genes encoding 21 nucleoporins (forreview see Rout and Wente, 1994; Doye et al., 1994; Grandi etal., 1995a; Hurwitz and Blobel, 1995; Grandi et al., 1995b;Pemberton et al., 1995; Li et al., 1995; Aitchison et al.,1995a,b; Gorsch et al., 1995; Kraemer et al., 1995; Heath etal., 1995; Siniossoglou et al., 1996). Only eight of these genesare essential. One of them, NUP145, encodes an essentialpolypeptide with a predicted molecular mass of 145 kDa(Fabre et al., 1994). NUP145 was independently isolated by itscross-reactivity with an antibody that recognizes other yeastnucleoporins (Wente and Blobel, 1994) and by its syntheticlethality with another nucleoporin, Nsp1p (Fabre et al., 1994).Nup145p can be divided into three structural regions. First, itsamino-terminal 220 amino acids contain 12 repeats of thetetrapeptide glycine-leucine-phenylalanine-glycine (GLFG), arepeating sequence element common to four other yeast nucle-oporins: Nup49p, Nup57p, Nup100p, and Nup116p. Together,these five proteins comprise the GLFG family (Wente et al.,1992; Wimmer et al., 1992; Grandi et al., 1995b). Second, themiddle 370 amino acids of Nup145p share homology with thecarboxy-terminal regions of Nup116p and Nup100p. The firsttwo regions are referred to together as the amino-terminal (N-terminal) region of Nup145p. Finally, the carboxy-terminal (C-terminal) region of Nup145p, which spans 727 amino acids, isunique.

Two possibly distinct NPC functions have been attributed toNup145p: maintaining proper NPC distribution and mediating

912 J. L. T. Emtage and others

RNA export. When Nup145p was depleted by placing NUP145under control of an inducible promoter, 30% of the cells hadaccumulated polyadenylated RNA in their nuclei after 3 hours;by 12 hours, almost all of the cells showed nuclear accumula-tion (Fabre et al., 1994). Inhibition of protein import into thenucleus lagged behind this RNA export defect. These resultssuggest that Nup145p has a primary role in RNA export.However, because the experimental strategy requiringNup145p depletion was dependent on the turnover of the wild-type protein, it is not possible to directly link the inability ofthe cells to export RNA to the loss of functional Nup145p.

Cells harboring a nup145∆N mutation, wherein the sequenceencoding the N-terminal region of Nup145p was deleted andthe LEU2 or URA3 gene inserted, were viable at all growthtemperatures but contained clusters of NPC-like structures in‘aggregated’ patches of the nuclear envelope (Wente andBlobel, 1994). This is in sharp contrast to the NPCs of wild-type cells which are distributed over the entire nuclear surfaceat a density of ~15 NPC/µm2, except for exclusion from areaswhere the nuclear and vacuolar membranes abut (Severs et al.,1976). The presence of NPC/nuclear envelope perturbationssuggests that Nup145p may play a role in the assembly ofNPCs. Because of the manner in which the nup145∆Nmutation was generated, it was not clear whether the constitu-tive clustering of nup145∆N NPCs was due to the absence ofthe N-terminal region of Nup145p or to a lower expressionlevel of the remaining C-terminal portion, which has noobvious promoter.

Interestingly, Nup145p in yeast cell extracts is proteolyzedinto two polypeptide fragments with apparent molecularmasses of ~80 kDa and ~65 kDa, corresponding to the C-terminal and N-terminal regions, respectively (Wente andBlobel, 1994). However, it is not known whether this reflectsspecific Nup145p cleavage in vivo or if it is a consequence ofcell breakage. If cleavage occurs in intact cells, the physiolog-ical consequences of producing exclusively full length proteincould prove insightful. The goal of the experiments in thispaper was to pinpoint the structural region(s) of Nup145pwhose absence or mutation results in NPC clustering, RNAexport, and/or non-cleavage phenotypes.

MATERIALS AND METHODS

Standard techniques were used for growth and transformation ofyeast, sporulation of diploids, dissection of tetrads, and extraction ofDNA from yeast (Kaiser et al., 1994). Strains were grown in YEPD(1% yeast extract, 2% bacto-peptone, 2% dextrose) unless plasmidselection required an appropriate selective minimal medium. Standardmethods were used for restriction digests, alkaline phosphatasetreatment of vectors, ligations, and all handling of bacterial strains,including growth, transformation with and extraction of DNA(Sambrook et al., 1989).

Yeast strainsThe yeast strains used in this study are listed in Table 1. Thenup145::LEU2 null allele was produced by transformation of PstI-digested pSW168 into the diploid strain W303. pSW168 was con-structed by ligating the 300 bp SacI/BamHI fragment of pSW165, the2 kbp PstI/SacI LEU2-bearing fragment of pJJ282 (Jones and Prakash,1990), and the 5.2 kbp BamHI/partial NsiI vector fragment of pSW102(Wente and Blobel, 1994). This construct removes amino acids 67

through 1,290 of Nup145p and replaces them with the LEU2 geneoriented in the opposite direction to NUP145. Leu+ transformantswere screened by Southern analysis to identify a heterozygous nullstrain (SWY203). Carboxy-terminal in-frame fusions of the five IgGbinding domains of Protein A to N-terminal regions of Nup145p wereconstructed using plasmid pProtA/HU and the strategy described byAitchison et al. (1995a,b).

Plasmid constructionThe 3′ end of NUP145 was amplified by PCR from Z1 λ DNA (Wenteand Blobel, 1994) using oligonucleotides 145-X (5′-TCGGGATCC-CCTTTGGCGGGACTTGGACTTTC-3′) and 145-Y2 (5′-GCTG-GATCCATTCAAGGCTACCACAGGTGGAGG-3′), and theresulting fragment was cut with AatII and BamHI and inserted intothe corresponding sites of pSW69 (Wente and Blobel, 1994) to formthe full length NUP145 gene in pBSKS (pSW181). The 6.2 kbpBamHI/SalI fragment bearing NUP145 was excised from pSW181and inserted into the corresponding sites of pRS316 and pRS313(Sikorski and Hieter, 1989) to form pSW190 (URA3) and pSW191(HIS3), respectively.

Deletions of NUP145 were made as follows (unless otherwisenoted pSW181 was used as the template for PCR). ForpSW303(nup145-58/HIS3), pSW191 was digested with XbaI toremove a 2.7 kbp fragment and the resulting vector religated. The first100 kDa (nup145-100) was placed in HIS3-CEN and URA3-2µvectors by inserting the 4.8 kbp EcoRI fragment from pSW69 intopRS313 and pRS426 (Sikorski and Hieter, 1989) to form pSW360 andpSW249, respectively. pSW363, (nup145∆GLFG/HIS3), was made ina three-step process. First, a 2.2 kbp BamHI/SalI fragment amplifiedby PCR using oligonucleotides T3 (5′-ATTAACCCTCACTAAAG-3′)and 145-P (5′-CCAGGATCCTATTAAACATAAGGTGGCTAC-3′),was inserted into the corresponding sites of pRS306 (Sikorski andHieter, 1989) to form pSW261. Next, the 3.7 kbp BamHI-digestedPCR product of oligonucleotides T7 (5′-AATACGACTCACTATAG-3′) and #161 (5′-TCGGGATCCCATTCCCAAGATCCGGT-3′) wasinserted into the BamHI site of pSW261 to form pSW280. Finally, the2.8 kbp SnaBI/MluI fragment from pSW280 replaced the corre-sponding fragment of pSW191 to form pSW363. pSW388(nup145∆NS/HIS3) was constructed by replacing the BamHI fragmentof pSW363 with the 2 kbp BamHI-digested PCR product made witholigonucleotides T7 and 145-X. pSW459 (nup145∆NL/HIS3) wasconstructed as pSW388, except that a 2.7 kbp BamHI-digested PCRproduct made with oligonucleotides T7 and 145-CS (5′-GGGGGATCCCGATGAAAGATACGACG-3′) was used. pSW540,(nup145∆524/592/HIS3), was constructed by ligating theBamHI/SalI-digested PCR product of oligonucleotides 145-D4 (5′-CTAGGATCCTAGAGGAACGAAATATTACAATTTTACC-3′) andT3 and the BamHI/XbaI-digested PCR product of oligonucleotides145-Y3 (5′-TGCTCTAGATTCAAGGCTACCACAGGTGGAGGTG-3′) and 145-X into XbaI/SalI-digested pRS313. pSW542,(nup145∆592/893/HIS3), was constructed in two steps as follows.First, pSW538 was constructed by inserting the BamHI/XbaI-digestedPCR product of oligonucleotides 145-D2 (5′-GCAGGATCCCTTC-TAACGAAATAGAACAAATATTTC-3′) and 145-Y3 into pRS313.Next, the BamHI/SalI-digested PCR product of oligonucleotides 145-D1 (5′-AGGGGATCCTATAGGATATATAGTTCATTTCCC-3′) andT3 was inserted into the corresponding sites of pSW538 to formpSW542. pSW543, (nup145∆592/608/HIS3), was constructed in twoanalogous steps. First, pSW539 was constructed by inserting theBamHI/XbaI-digested PCR product of oligonucleotides 145-D3 (5′-GCAGGATCCGGGGGTTAGTCAATGAAGAAGATGCGG-3′) and145-Y3 in pRS313. Next, the BamHI/SalI-digested PCR product ofoligonucleotides 145-D1 and T3 was inserted into the correspondingsites of pSW539 to form pSW543. pSW610, encoding amino acids593 through 893 on a high copy vector, was constructed by ligatingthe 3.0 kbp EcoRI fragment from pSW388 into pRS423 (Sikorski andHieter, 1989).

913Mapping the functional regions of Nup145p

Table 1. Strains and relevant genotypesStrain Relevant genotype Description

W303 MATa/MATα leu2-3,112/leu2-3,112 ura3-1/ura3-1 his3-11,15/his3-11,15 ade2-1/ade2-1trp1-1/trp1-1 can1-100/can1-100

SWY203 MATa/MATα nup145::LEU2/NUP145 leu2-3,112/leu2-3,112 ura3-1/ura3-1 his3-11,15/ W303 with one copy of NUP145 deleted and his3-11,15 replaced by LEU2

SWY211 MATa/MATα nup145::LEU2/NUP145 leu2-3,112/leu2-3,112 ura3-1/ura3-1 his3-11,15/ SWY203 + pSW191his3-11,15 pSW191 (NUP145/HIS3/CEN)

SWY476 MATa/MATα nup145::LEU2/NUP145 leu2-3,112/leu2-3,112 ura3-1/ura3-1 his3-11,15/ SWY203 + pSW249 his3-11,15 pSW249 (nup145-100/URA3/2µ)

SWY556 MATa/MATα nup145::LEU2/NUP145 leu2-3,112/leu2-3,112 ura3-1/ura3-1 his3-11,15/ SWY203 + pSW363 his3-11,15 pSW363 (nup145∆GLFG/HIS3/CEN)

SWY647 MATa/MATα nup145::LEU2/NUP145 leu2-3,112/leu2-3,112 ura3-1/ura3-1 his3-11,15/ SWY203 + pSW388 his3-11,15 pSW388 (nup145∆NS/HIS3/CEN)

SWY122 MATa nup145∆N::LEU2 leu2-3,112 his3-11,15 W303 derivative with the N-terminus of NUP145 replaced by LEU2 (Wente and Blobel, 1994)

SWY294 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW191 (NUP145/HIS3/CEN) Segregant of SWY211SWY389 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW190 (NUP145/URA3/CEN) SWY294 with pSW191 replaced by pSW190SWY513 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW249 (nup145-100/URA3/2µ) Segregant of SWY476SWY656 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW363 (nup145∆GLFG/HIS3/CEN) Segregant of SWY556SWY690 MATa nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW388 (nup145∆NS/HIS3/CEN) Segregant of SWY647SWY849 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW459 (nup145∆NL/HIS3/CEN) SWY389 with pSW190 replaced by pSW459SWY390 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW295 (nup145-A5/HIS3/CEN) SWY389 with pSW190 replaced by pSW295SWY535 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW369 (nup145-A5/URA3/CEN) SWY390 with pSW295 replaced by pSW369SWY391 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW296 (nup145-E6/HIS3/CEN) SWY389 with pSW190 replaced by pSW296SWY536 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW370 (nup145-E6/URA3/CEN) SWY391 with pSW296 replaced by pSW370SWY392 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW297 (nup145-L2/HIS3/CEN) SWY389 with pSW190 replaced by pSW297SWY537 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW371 (nup145-L2/URA3/CEN) SWY392 with pSW297 replaced by pSW371SWY393 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW298 (nup145-O1/HIS3/CEN) SWY389 with pSW190 replaced by pSW298SWY538 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW372 (nup145-O1/URA3/CEN) SWY393 with pSW298 replaced by pSW372SWY394 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW300 (nup145-R4/HIS3/CEN) SWY389 with pSW190 replaced by pSW300SWY540 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW374 (nup145-R4/URA3/CEN) SWY394 with pSW300 replaced by pSW374SWY395 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW302 (nup145-V8/HIS3/CEN) SWY389 with pSW190 replaced by pSW302SWY541 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW375 (nup145-V8/URA3/CEN) SWY395 with pSW302 replaced by pSW375SWY396 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW299 (nup145-R2/HIS3/CEN) SWY389 with pSW190 replaced by pSW299SWY539 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW373 (nup145-R2/URA3/CEN) SWY396 with pSW299 replaced by pSW373

SWY1333 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW190 (NUP145/URA3/CEN) SWY389 + pRS313pRS313 (HIS3/CEN)

SWY1334 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW190 (NUP145/URA3/CEN) SWY389 + pSW191pSW191 (NUP145/HIS3/CEN)

SWY1335 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW190 (NUP145/URA3/CEN) SWY389 + pSW540pSW540 (nup145∆524/592/HIS3/CEN)



SWY1364 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW190 (NUP145/URA3/CEN) SWY389 + pSW610pSW610 (nup145-33/HIS3/2µ)

SWY1349 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW191 (NUP145/HIS3/CEN) SWY1334 without pSW190SWY1350 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW543 (nup145∆592/608/HIS3/CEN) SWY1337 without pSW190SWY1351 MATα nup145::LEU2 leu2-3,112 ura3-1 his3-11,15 pSW540 (nup145∆524/592/HIS3/CEN) SWY1335 without pSW190

SWY1360 MATa leu2-3,112 ura3-1 his3-11,15 trp1-1 can1-100 pSW545 (GAL4BD-GFPS65T/TRP1) ADE2 W303a (M.B. and S.R.W., in press)+ pSW545

SWY1361 MATa leu2-3,112 ura3-1 his3-11,15 trp1-1 can1-100 pSW604 (GAL4BD-nup145- ADE2 W303a (M.B. and S.R.W., in press)GFPS65T/TRP1) + pSW604

SWY1396 MATa/MATα NUP145/nup145 (1-594) – Protein A:HIS3:URA3 ura3-1/ura3-1 W303 with one copy of NUP145 replaced by the his3-11,15/his3-11,15 first 594 amino acids of Nup145p fused to

Protein ASWY1397 MATa/MATα NUP145/nup145 (1-626) – Protein A:HIS3:URA3 ura3-1/ura3-1 W303 with one copy of NUP145 replacedby the

his3-11,15/his3-11,15 first 626 amino acids of Nup145p fused toProtein A

HF7c MATa ura3-52 his3-200 lys2-801 ade2-101 trp1-901 leu2-3,112 gal4-542 gal80-538 Gift from H. Feilotter, Cold Spring Harbor, NYLYS2::GAL1-HIS3 URA3::(GAL4 17mers)3-CYC1-lacZ

pSW545, which encodes a fusion of the green fluorescent protein(GFP) carboxy-terminal to the DNA binding domain of Gal4p wasconstructed by inserting a BamHI/EcoRI GFP-encoding fragmentfrom pRSETB-S65T (Heim et al., 1995) into pGBT8 (Bartel andFields, 1995). pSW604 was constructed by inserting the

EcoRI-digested PCR product of oligonucleotides CA (5′-GCCGAATTCGAGCTCTTCGGTAAAATTGTAATATTTCG-3′)and CB (5′-GCCGAATTCTTGTTTACTCAAATCGTCTTCATC-3′)into the EcoRI site of pSW545.

For pSW259, the N-terminal region of Nup145p (encoding residues


2-664) was fused to the DNA binding region of Gal4p by subcloninga BamHI/SacI-digested PCR product (generated with oligonucleotides#124 (5′-TCGGGATCCTTAATAAAAGTGTAAATAGTGGT-3′) and#112 (5′-CGAGAGCTCTTACCTGATAGACTTCC-3′)) into pGBT8(Bartel and Fields, 1995). For pSW253, the C-terminal region ofNup145p (encoding residues 593-1317) was fused to the activationdomain of Gal4p by subcloning the BamHI-digested PCR productgenerated from 145-X and 145-Y2 into pACTII (gift of S. Elledge).

Generation of temperature-sensitive nup145 allelespSW191 (NUP145 HIS3) was mutagenized in vitro with hydroxy-lamine for 16 hours at 37°C (Kaiser et al., 1994) and transformed intoSWY389. His+ transformants were selected at ambient temperature,replica-plated to medium containing 5-fluoroorotic acid (5-FOA), andthen tested for growth at 37°C on YEPD (Sikorski and Boeke, 1991).Temperature-sensitive colonies were tested for the ability to grow at37°C in the presence of pSW190 to identify mutations in other genesand on 5-FOA medium at 23°C to identify NUP145 null mutations.Plasmids were extracted from appropriate strains and retransformedinto SWY389 for further testing. These strains were allowed to losepSW190 and tested for growth at 37°C. From screening a total of12,000 transformants, seven strains (referred to as SWY390-SWY396) were unable to grow at 37°C and were designated temper-ature-sensitive nup145 alleles.

Gap repair and sequencing of the new temperature-sensitive nup145 allelespSW182, a fusion of the C-terminal region of Nup145p to glutathioneS-transferase (GST) behind the GAL1 promoter, was constructed byligating the BamHI-digested PCR product of oligonucleotides 145-Xand 145-Y2 from Z1 λ DNA into the BamHI site of pBJ382 (aGAL1/GST fusion in pRS424 provided by C. Hug). The temperature-sensitivity of strains SWY390-SWY396 was complemented by trans-formation with pSW182, indicating that the mutations were within theC-terminal region. pSW190 was cut with restriction enzymes delin-eating different regions of the C-terminal region of Nup145p andtransformed into strains SWY390-SWY396. Ura+ transformants wereselected at 23°C and then tested for growth at 37°C. SWY390,SWY394, and SWY396 were unable to grow at 37°C when pSW190was digested with MluI and AatII. The mutated area was furthernarrowed to the region between the MluI and AflII sites. SWY391-SWY393 and SWY395 were unable to grow when pSW190 wasdigested with MluI and AvrII, or with HindIII alone. The Ura+ tem-perature-sensitive strains were allowed to lose the HIS3-plasmid-borne nup145 alleles to form strains SWY535-SWY541. The relevantregions of the nup145/HIS3 plasmids were sequenced. nup145-L2,nup145-O1, nup145-V8, and nup145-A5 changed C to T to make glu-tamines #1193, 1147, 1091, and 1061, respectively, into stop codons.nup145-E6 changed C to A to make serine #1140 a stop codon.nup145-R4 deleted a T between amino acids #1012-1013; theframeshift results in a stop codon at #1013. nup145-R2 deleted the Cin amino acid #1017, which alters the amino acid sequence from#1017 and terminates 34 codons later.

MicroscopyCells were processed for immunofluorescence as described by Wenteet al. (1992) and for in situ hybridization with a digoxigenin-labeledoligonucleotide poly-(dT) probe as described (Wente and Blobel,1993). mAb414 (Davis and Blobel, 1986) was used for pore complexstaining, mAb D77 for Nop1p staining (Aris and Blobel, 1988), and5× diluted mAb B512 for tubulin staining (gift of J. Kilmartin).Fixation times were 15 minutes for staining with mAb414 and 1 hourfor all other antibodies. Samples were examined with an Olympusmicroscope through a ×100 objective. Photographs were taken usingan attached camera with Kodak T-MAX 400 film.

After fixation overnight in 2% glutaraldehyde and 2% formalde-hyde, cells were prepared for electron microscopy as described by

Wente and Blobel (1993) using the method preserving both proteinand membrane structures. Samples were viewed with a Zeiss-902electron microscope and photographs taken on Kodak electronmicroscopy film.

Immunoblotting and production of antiserum to the C-terminal regionThe C-terminal region of Nup145p was fused in frame behind themaltose binding protein (MBP) to make pSW184 by inserting theBamHI-digested PCR product of the 145-X2 (5′-TCGGGATC-CAATCCCTTTGGCGGGACTTGGACT-3′) and 145-Y oligonu-cleotides from Z1 λ DNA into the BamHI site of pMAL-cRI (Mainaet al., 1988). pSW184 was transformed into DH5α, and fusion proteinwas expressed and purified using amylose resin (New EnglandBiolabs) according to the manufacturer’s directions and sent toCocalico Biologicals, Inc. (Reamstown, PA) for production of rabbitantiserum WU599. For affinity purification of the antiserum, 10 mgof MBP and 7 mg of the fusion between MBP and the C-terminalregion of Nup145p were purified as described above and each wascoupled to 2 g CNBr-activated Sepharose 4B (Pharmacia Biotech,Uppsala, Sweden) according to the manufacturer’s directions. WU599was dialyzed into PBS (4°C) and centrifuged for 10 minutes at 9,000g. The supernatant was incubated with MBP-Sepharose 4B for 2hours, cleared of beads, and incubated with beads coupled to thefusion protein overnight. The beads were washed with 0.5 mM NaCl,20 mM sodium phosphate, pH 7.5, packed into a column, and elutedwith 0.1 M glycine-HCl, pH 2.8. The fractions were immediatelyadjusted to pH 7.5-8.0 with 1 M Tris base, dialyzed into PBS, andtitered.

Total yeast cell extracts and immunoblotting were conducted asdescribed (Iovine et al., 1995). Protein A blots were incubated with arabbit anti-mouse antibody (1:500 dilution; Cappel), and GFP blotswere incubated with a rabbit polyclonal antibody (1:500; Clonetech).Bands were visualized by the ECL system (Amersham Corp.)according to the manufacturer’s directions or by incubating the blotswith alkaline phosphatase-conjugated anti-rabbit antibody (Promega)and developing with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-1-phosphate.

RESULTS

Analysis of N-terminal Nup145p deletions In the previously reported nup145∆N::LEU2 mutation, theLEU2 gene replaced the sequence encoding amino acids 65through 549 of Nup145p (Wente and Blobel, 1994). Expressionof the C-terminal region was inferred from the fact that thenup145∆N::LEU2 cells are viable whereas a complete nup145null mutant is inviable (Fabre et al., 1994). Because the C-terminal region in the nup145∆N::LEU2 mutant has noobvious promoter, the NPC clustering phenotype could be dueto the absence of the N-terminal region, or due to a loweredexpression level (or stability) of the C-terminal region. Todirectly test whether the N-terminal region of Nup145p isrequired for maintaining proper NPC distribution, threedifferent deletions from the N terminus were constructed andplaced under the control of the endogenous NUP145 promoter(Fig. 1A). The nup145∆GLFG allele deleted the first 214amino acids containing all the GLFG repeats. The nup145∆NLallele deleted the first 562 amino acids, removing all of theGLFG repeat region and most of the middle region. Finally,the nup145∆NS allele deleted the first 592 amino acids,removing the entire N-terminal region. Strains carrying the N-terminal deletion alleles as their sole source of Nup145p were


A

B

893 nup145-100 (2µ)

nup145-L2

nup145-O1

nup145-E6

nup145-V8

nup145-A5

nup145-R2

nup145-R4

nup145-58 (2µ)

1192

1146

1139

1090

1060

1016

1012

551

+

+

+

+

+

+

+

+

NC

+

+

+

+

+

-

-

-

NA

+

+

ND

+

+

ND

ND

ND

NA

TS RNAEXPORT

-

-

+

ND

ND

nup145∆GLFG

nup145∆NS

nup145∆NL

ALLELE

NUP145

nup145∆N::LEU2

CLUSTERS

-

-

+

-

+

TS

-

-

+

-

-

1317591220

215

593

563

AAAAAAAAAAAAAAAAAAAA

LEU2

55064

rela

tive

A60

0

time at 37°C (hours)

0

1

2

3

4

5

6

7

8

C

nup145-100

nup145-V8

nup145-R4

nup145-A5

NUP145

nup145 NS∆

-2 0 2 4 6 8 10 12 14

Fig. 1. Mutant alleles of NUP145. (A and B) Diagrams of thepolypeptides encoded by the nup145 deletion mutations. Thedeletion positions are denoted by amino acid number. The GLFGrepeats are represented by boxes, the middle region is gray, and theC-terminal region is white. Clusters +, NPCs in clusters; clusters −,NPCs were evenly distributed. Temperature-sensitive (ts) −, growthat 37°C; ts +, no growth at 37°C. RNA export +, has a poly(A+)RNA export defect at 37°C; −, no export defect. ND, not done; NC,non-complementing; NA, not applicable. (C) Growth curves of thenup145 mutants at 37°C. NUP145 (SWY389), nup145-V8(SWY541), nup145-A5 (SWY535), nup145-R4 (SWY540),nup145-100 (SWY513), and nup145∆NS (SWY690) strains weregrown in YEPD at 30°C and shifted to 37°C at time 0. Strains weremaintained in logarithmic phase throughout the course of theexperiment by diluting as needed to keep the A600 under 0.8. Thus,the relative A600 is the actual A600 times the dilution factor(s).

assayed for growth at 37°C. The nup145∆NS cells showedimpaired growth at the high temperature (see Fig. 1C). Thestrains expressing the two milder N-terminal deletions,nup145∆GLFG and nup145∆NL (data not shown), were indis-tinguishable from wild type.

Indirect immunofluorescence was conducted to assesswhether the NPCs were clustering (Fig. 2A). Wild-type cellsshowed punctate nuclear rim staining, whereas the signal innup145∆N::LEU2 cells was concentrated in discrete foci (NPCclusters). Removal of the GLFG region had no detectable effecton the surface distribution of the pore complexes (not shown).This is in agreement with the published characterization of aProtein A-Nup145p fusion which removed the GLFG region(Fabre et al., 1994). The nup145∆NL cells also appearedsimilar to wild type. The NPC staining in the nup145∆NS cellswas overall very weak, and therefore a definitive evaluation ofthe presence or absence of NPC clusters was determined bythin-section electron microscopy. Pore complexes in wild-type

and mutant cells are represented by electron dense patches thatspan the double nuclear membranes. In wild-type cells (seeFig. 5A), the NPCs were generally found singly distributedaround the entire circumference of the nuclear envelopesection. When thin sections of nup145∆NS cells wereexamined (Fig. 2B), clusters of NPC-like structures wereobserved (large arrowheads). These NPCs were grouped inlocalized patches of the nuclear envelope and closely resemblestructures reported for nup145∆N::LEU2 cells. The clusterswere characterized by multiple NPC-like structures assembledin grape-like aggregates (both micrographs, at ~11:00).Besides the NPC clusters, another distinct morphological per-turbation was observed (small arrowheads, left micrograph). Inapparently NPC-free areas, the nuclear envelope sometimesappeared discontinuous and as a lace-like meshwork. This wasin contrast to the clearly continuous bilayer observed in wild-type cells.

NPC clustering was not observed in the nup145∆NL cells,


Fig. 2. Characterization of the N-terminal Nup145p deletions.(A) Examination of NPC distribution. NUP145 (SWY294),nup145∆NL (SWY849), nup145∆NS (SWY690), andnup145∆N::LEU2 (SWY122) strains were grown at 30°C,fixed, permeabilized, and stained with antibodies against theNPC and fluorescein-linked secondary antibodies. DNA wasstained with DAPI. Bar, 2 µm. (B) Examination by electronmicroscopy of NPC clusters in nup145∆NS cells. The

nup145∆NS strain (SWY690) wasgrown at 30°C and processed forthin section electron microscopy.Large arrowheads point toclusters of NPCs. Smallarrowheads point to unusualmembrane structures in A. n,nucleus; c, cytoplasm. Bar, 0.25µm. (C) Poly(A+) RNA exportphenotype of the nup145∆NSstrain. NUP145 (SWY294) andnup145∆NS (SWY690) cells weregrown to early logarithmic phaseat 30°C and shifted to 37°C for 5hours. Cells were fixed,permeabilized, and stained with adigoxigenin-linkedoligonucleotide poly-(dT) probeplus fluorescein-labeled anti-digoxigenin antibodies. DNA wasstained with DAPI. Bar, 5 µm.

B

and therefore the absence of the N-terminal region alone wasprobably not responsible for the NPC clustering phenotypes.To test whether the protein levels of the C-terminal region werealtered in the nup145∆N::LEU2 and nup145∆NS cells, proteinswere extracted from equivalent cell numbers and immunoblot-

ted with a polyclonal Nup145p C-terminal specific antibody.The C-terminal region from wild-type cells and from both ofthe mutants migrated as an ~90 kDa fragment (Fig. 3A). Anunrelated yeast protein of greater molecular mass was also rec-ognized by the antibody. Both of the mutants contained sig-


203 -

118 -

86 -

51.6 -

non-specific

C-term

C-term*

A B

203 -

118 -

86 -

51.6 -

C-term

non-specific

NU

P14

5

nu

p14

5∆N

::L

EU

2

nu

p14

5∆N

S 2

3°C

nu

p14

5∆N

S 3

7°C

NU

P14

5 2

3°C

nu

p14

5-R

4 2

3°C

NU

P14

5 3

7°C

nu

p14

5-R

4 3

7°C

Fig. 3. Expression levels of Nup145p C-terminal region in deletion mutants. nup145 strains were grown at 23°C and shifted to 37°C for 1 hourbefore harvesting where indicated; otherwise, they were grown at 23°C. Proteins were extracted from equal numbers of cells, separated bySDS-PAGE, and transferred to nitrocellulose. The blot was probed with an affinity-purified polyclonal antibody raised against the C-terminalregion of Nup145p. Bands were visualized using the ECL system. (A) NUP145 (SWY294), nup145∆NS (SWY690), and nup145∆N::LEU2(SWY122) strains were used. (B) NUP145 (SWY294) and nup145-R4 (SWY394) strains were used.

nificantly less C-terminal polypeptide than that in the wild-typesample.

Because the nup145∆NS strain was temperature-sensitive, itwas also assayed for nuclear export capacity. The localizationof poly(A)+ RNA was monitored by in situ hybridization witha digoxigenin-labeled poly(dT)30 oligonucleotide probe aftershifting to growth at 37°C. The staining in wild-type cells wasdiffuse and cytoplasmic (Fig. 2C). However, in nup145∆NScells, the fluorescent signal became predominantly nuclear,reflecting a block in poly(A)+ RNA export. By immunoblot-ting analysis, the protein level of the C-terminal region was notaltered with growth of the nup145∆NS cells at 37°C (Fig. 3A).Thus, the nup145∆NS cells exhibited an NPC clusteringphenotype, a lowered level of the C-terminal region, and a tem-perature dependent RNA export defect.

Isolation of temperature-sensitive C-terminalNup145p truncations To further dissect the regions of Nup145p necessary for NPCfunction, a panel of temperature-sensitive nup145 mutationswas generated. Gap repair analysis revealed that all the tem-perature sensitive mutations resided in the sequence encodingthe C-terminal region. The new alleles were sequenced and allwere mutations which led to premature truncations, removing200 to 300 amino acids from the C terminus (Fig. 1B). Thephenotypes of a 424 amino acid C-terminal truncation(nup145-100) and a 766 amino acid C-terminal truncation(nup145-58) were also examined. The nup145-100 allelesustained the viability of a nup145 null strain, but only on ahigh copy 2µ plasmid. In contrast, the nup145-58 allele on a2µ plasmid did not rescue a nup145 null strain. The nup145-100 strain was strongly temperature-sensitive lethal and growthwas impaired even at 23°C.

The doubling time and viability of cells harboring the nup145temperature-sensitive alleles were examined at 37°C (Fig. 1C).The mutations fell into 2 classes: those whose growth actually

ceased, and those whose growth merely slowed dramaticallyand were still dividing after 25 hours. The degree of growthimpairment at 37°C correlated with the extent of the truncation.nup145-100, nup145-R2 and nup145-R4 strains contained thelargest truncations, and correspondingly, their growth ceased.As determined by plating cells at the permissive temperature,<5% of nup145-R2 and nup145-R4 cells were viable after 16hours at 37°C, versus ~25% for the milder mutants.

Characterization of the nup145 C-terminal truncationmutants The distribution of NPCs in cells harboring the C-terminaltruncations was examined by indirect immunofluorescencemicroscopy. Wild-type and nup145-O1 cells exhibited even,punctate, nuclear rim staining (see Fig. 4), as did nup145-L2and nup145-E6 (not shown). However, the NPCs in nup145-V8 cells were localized into discrete foci at the permissivegrowth temperature. Alleles with more extensive truncationsalso showed a constitutive NPC clustering phenotype (forexample, nup145-A5 and nup145-R4 in Fig. 4). Therefore, theabsence of 227 or more of the C-terminal residues resulted indistinct NPC clustering.

To analyze the ultrastructure of NPC clusters in cells withextensive C-terminal truncations, thin section electronmicroscopy was conducted on nup145-R4 and nup145-100cells. In wild-type cells (Fig. 5A), the NPCs were distributedaround the entire circumference of the nuclear envelope cross-section. In contrast, both mutants contained clusters of NPCs.The NPCs were often grouped in a single layer of the nuclearenvelope (Fig. 5B,C). These may represent NPCs concentratedin a patch of nuclear envelope (appearing as linear in thesection). In other sections, the clusters appeared in grape-likeaggregates in regions where the nuclear envelope was highlyconvoluted (Fig. 5E,F,H,G). Both types of clustered structurescould result in the concentrated anti-NPC signal detected byimmunofluorescence microscopy.


Fig. 4. The NPCs cluster constitutively in strains carrying C-terminally truncated Nup145p. NUP145 (SWY294), nup145-O1(SWY393), nup145-V8 (SWY395), nup145-A5 (SWY390), andnup145-R4 (SWY394) strains were grown at 30°C and processed forimmunofluorescence with antibodies against the NPCs as in Fig. 2A.

The effect of representative C-terminal truncations onpoly(A)+ RNA export was also examined. Cells were shiftedto 37°C for 30 minutes and probed with poly(dT). In contrastto wild type, nuclear accumulation of poly(A)+ RNA wasdetected in a significant fraction of all four mutants tested(nup145-V8, nup145-A5, nup145-R4, and nup145-100 in Fig.6). However, at no time point was the export block at 37°Cobserved in 100% of the cells. This lack of penetrance suggestseither that the export block is an indirect effect, or that theaccumulated poly(A)+ RNA is not stable over time.

To test whether the C-terminally truncated polypeptideswere expressed, immunoblot analysis was conducted with the

most severe nup145-R4 allele (Fig. 3B). A cross-reactivepolypeptide with a molecular mass of ~55 kDa correspondeddirectly to the product predicted for the nup145-R4 allele, anda band at the wild-type position (90 kDa) was absent. A directcomparison to the levels of the C-terminal region in wild-typecells could not be made because of the polyclonal nature of theantibody. However, the signal was not reduced dramatically.After growth at 37°C for 1 hour, the truncated polypeptide inthe nup145-R4 cells was present but the level was lowerrelative to cells grown at 23°C.

The severe N- and C-terminal truncations result inpleiotropic defectsIn addition to their strong temperature sensitivity, cells carryingalleles with the most severe truncations (nup145-100, nup145-R4, and nup145∆NS) displayed numerous morphologicaldefects. Cells of all three mutants were noticeably larger thanwild-type cells at all growth temperatures, and DAPI stainingand thin section electron micrographs revealed that the mutantnuclei were large and irregularly-shaped (data not shown). Inaddition, nup145-100 and nup145∆NS strains had an increasedtendency to be anucleate or binucleate, and in the case ofnup145∆NS, even trinucleate. The mutant cells were alsoanalyzed by indirect immunofluorescence microscopy withantibodies against the nucleolar Nop1p and tubulin. At 30°C,the nucleolus in the mutant strains had a wild-type appearance(cresent-shaped body occupying one edge of the nucleus).However, after growth at 37°C, the nucleolar staining innup145-100 or nup145∆NS cells fragmented into multiple foci(data not shown). The tubulin staining revealed a markedlylower percentage of mutant cells with very long spindles andmany of the cells had short spindles spanning their nuclei, withpossible orientation defects in regard to bud position (data notshown). The growth defect of the nup145-R4 strain was sup-pressed at 37°C on media of high osmolarity (containing 1.2M sorbitol or 0.9 M NaCl) (data not shown). Similarpleiotropic defects have been reported in several nucleoporinmutant strains (Bogerd et al., 1994; Aitchison et al., 1995a;Heath et al., 1995; Iovine et al., 1995). These are probablysecondary defects arising from the inability to properlytransport substrates through the NPCs, and not directly relatedto any single nucleoporin. Interestingly, neither nup145-100nor nup145∆NS cells showed a protein import defect at 37°C(data not shown).

Defining the essential region of Nup145p The results from the N-terminal and C-terminal truncationmutations suggest that an internal region from amino acid 593to 893 may be essential for Nup145p function. The differingphenotypes between the nup145∆NL and nup145∆NS mutantssuggest that the region from amino acid 563 to 593 may alsobe important. Peptide sequencing analysis of the two Nup145pfragments places the cleavage site between amino acids 524and 606 (Wente and Blobel, 1994). Therefore, both of thesepotentially important spans are in the region of Nup145p wherea cleavage event may be occurring. To map the essential regionof Nup145p and to assess whether cleavage is important forfunction, three different in frame internal deletion mutationswere constructed, as diagrammed in Fig. 7A: nup145∆524/592removed residues 524 to 592 (inclusive), nup145∆592/608removed residues 592 to 608, and nup145∆592/893 removed


Fig. 5. Examination by electronmicroscopy of NPC clusters innup145-R4 and nup145-100 cells.NUP145 (SWY294), nup145-R4(SWY540), and nup145-100(SWY513) cells were grown at30°C and processed for thinsection electron microscopy.Small arrowheads indicate singleNPCs; large arrowheads point toclusters of NPCs. (A) NUP145;(B,D-F) nup145-R4; (C,G-I)nup145-100. n, nucleus; c,cytoplasm. Bar, 0.25 µm.

residues 592 to 893. These alleles were expressed behind theNUP145 promoter on a CEN plasmid and transformed into anup145 null strain containing NUP145 on a URA3 vector. Theresulting strains were tested for their ability to lose theNUP145/URA3 vector by growing them on medium contain-ing 5-FOA, which selects against Ura+ cells. Cells with eithernup145∆524/592 or nup145∆592/608 were viable at all testedtemperatures (Fig. 7B). In contrast, cells with thenup145∆592/893 allele were not viable at any growth temper-ature. To confirm this result, we expressed amino acids 593

through 893 behind the NUP145 promoter on a high copyvector and assayed its ability to complement the nup145 nullallele. As shown in Fig. 7C, cells were viable when thisconstruct was the sole source of Nup145p. Therefore, a 300amino acid span at the beginning of the C-terminal region isessential for Nup145p function.

Nup145p cleavage occurs in vivo but is not requiredfor functionThe nup145∆524/592 and nup145∆592/608 alleles were


Fig. 6. mRNA export is compromised in mutants carrying C-terminaltruncations of Nup145p at the non-permissive temperature. NUP145(SWY294), nup145-V8 (SWY541), nup145-A5 (SWY535), nup145-R4 (SWY540), and nup145-100 (SWY513) cells were grown to earlylogarithmic phase at 30°C and shifted to 37°C for 30 minutes beforeprocessing as for Fig. 2C..

further analyzed for perturbations of NPC distribution and forsynthesis of Nup145p. By indirect immunofluorescence local-ization, the nup145∆524/592 cells had distinct NPC clusterswhile the nup145∆592/608 cells appeared like wild type (datanot shown). Immunoblot analysis revealed thatnup145∆524/592 cells expressed a cleaved ~90 kDa C-terminal polypeptide at a lower protein level relative to wildtype (Fig. 8A, lanes 1 and 2). Interestingly, a ~90 kDa C-terminal polypeptide was absent in the nup145∆592/608 celllysates, and instead a polypeptide of ~140 kDa was observed(Fig. 8A, lane 3). This suggested that the cleavage site resides

between residues 592 and 608, but that cleavage is notnecessary for Nup145p function.

To further investigate the physiological significance ofNup145p cleavage, an assay was designed to test whethercleavage actually occurs in vivo or whether it is a consequenceof cell breakage. If cleavage occurs in vivo, a fusion proteinwith the C-terminal region of Nup145p replaced by the IgGbinding region of Protein A should show differential subcellu-lar localization depending on whether the cleavage site ispresent. If the cleavage site is absent, the Protein A domainshould be targeted to the NPC via attachment to the N-terminalregion. Alternatively, if cleavage occurs in vivo and thecleavage site is present, the Protein A domain should beseparated from the N-terminal region and thus found through-out the cell. Nup145p fusion proteins with Protein A insertedeither after residue 594 (before the cleavage site) or afterresidue 626 (after the cleavage site) were expressed in wild-type cells and analyzed by immunoblotting for the Protein Adomain (Fig. 8A). Nup145p(1-594)-ProtA appeared uncleavedand migrated with the predicted molecular mass of ~80 kDa(lane 7), whereas Nup145p(1-626)-ProtA showed a band forcleaved Protein A at ~30 kDa (lane 8). Indirect immunofluo-rescence analysis for the Protein A domain showed that theProtein A localization for the uncleaved construct was pre-dominantly nuclear/nuclear rim (Nup145p(1-594)-ProtA; Fig.8B, lower left). Interestingly, the signal was localized through-out the cell for Protein A expressed from the construct that wascleaved (Fig. 8B, lower right). Therefore, cleavage of theNup145p(1-626)-ProtA fusion was occurring in vivo.

To test whether the cleavage site of Nup145p was sufficientfor conferring cleavage of heterologous protein, chimericreporter proteins were constructed and assayed for differentialsubcellular localization. Sequence encoding amino acids 512to 627 of Nup145p was fused in frame between the DNAbinding region of Gal4p (Gal4BD) (encoding the first 147residues of Gal4p) and the green fluorescent protein (GFPS65T).If the Nup145p region can mediate cleavage of this chimera invivo, the GFP fluorescent signal should localize throughout thecell because nuclear localization of GFP is presumablydependent on fusion to the Gal4BD region and the nuclear local-ization signal therein (Silver et al., 1984). As expected, theGFP signal from the chimera protein without the Nup145pinsert (Gal4BD-GFPS65T) was localized to the nucleus (Fig. 8B,upper left). However, the chimera with the cleavage site(Gal4BD-Nup145-GFPS65T) also showed GFP signal predomi-nantly localized in the nucleus (Fig. 8B, upper right).Immunoblotting with antibodies recognizing the GFP domainconfirmed that the Gal4BD-Nup145-GFPS65T chimeric fusionwas not cleaved (Fig. 8A, lane 5). Thus, the region of Nup145pfrom residues 592-604 is not sufficient for conferring cleavage.

Because the N-terminal and C-terminal regions of Nup145pare apparently cleaved in vivo into separate molecules, we usedthe two-hybrid assay to assess whether the two halves physi-cally interact. The N-terminal region was fused to Gal4BD andthe C-terminal region was fused to the activation domain ofGal4p (Gal4AD). If the two regions interact, transcriptionalactivation of a lacZ gene with upstream Gal4p DNA-bindingsites will occur via coincident juxtaposition of the Gal4BD andGal4AD domains (Bartel and Fields, 1995). The Gal4BD-Snf1pand Gal4AD-Snf4p fusions were used as a positive interactioncontrol (Yang et al., 1992), and also as specificity controls


A

Fig. 7. An internal span of 300 amino acids is essential. (A) Diagramof the polypeptides encoded by the nup145 internal deletionmutations. Notations as in Fig. 1A, including: alive +, complementsthe nup145 null mutation at 23°C; alive −, does not complement.Cleaved +, the ~90 kDa fragment is formed; cleaved −, uncleavedNup145p observed. (B) Growth phenotypes of the internal deletionmutants. nup145 null strains carrying a NUP145/URA3 plasmid plusan empty HIS3 vector or the NUP145, nup145∆524/592,nup145∆592/608, or nup145∆592/893 alleles in a HIS3 vector(SWY1333-1337) were streaked on SD-ura-his and 5-FOA plates at23°C. Those which grew on 5-FOA (now SWY1349-1351 becausethe URA3 plasmid is gone) were streaked on a SD-his plate at 37°C,along with a nup145-R4 strain (SWY394) as a temperature sensitivecontrol. (C) An internal span of 300 amino acids is sufficient tosupport growth. NUP145 (SWY1334), nup145-33 (SWY1364), andcontrol (SWY389) strains were streaked on a 5-FOA plate at 23°C.

when combined with the Nup145p fusions. The plasmids wereco-transformed into a reporter yeast strain, and the level of β-galactosidase expression was measured using a color filterassay (Fig. 9). The Gal4BD-N-term and Gal4AD-C-term fusionsdid not show detectable activation capacity in combinationwith the respective Snf fusions. Interestingly, blue signal wasdetected when the N-terminal and C-terminal fusions werecombined, suggesting that the two halves of Nup145p interactin this assay. However, a direct protein-protein interactionremains to be demonstrated.

DISCUSSION

To define the structural regions of the nucleoporin Nup145presponsible for the mRNA export and clustering defectsobserved in previous studies (Fabre et al., 1994; Wente andBlobel, 1994), we have characterized a series of deletionmutations. We report that a distinct 300 amino acid region fromresidues 593 through 893 at the beginning of the C-terminaldomain is essential for cell viability. At least two other regionsare also important for function. Interestingly, it appears that thecleavage of Nup145p into N- and C-terminal fragments occursbecause in vivo cleavage of a Nup145p N-terminal fusion toProtein A was observed. However, there was no phenotypewhen the breakpoint between the two halves of Nup145p waseliminated and full length product was synthesized. Therefore,the conversion of Nup145p into two polypeptides is notrequired for NPC function. This is not surprising in light of our


A

1 2 3

203

118

86

51.6

34.1

M r

WT

∆5

92

-60

8

∆5

24

-59

2

M r

**

*

Nup145p C-term

81

48.3

33.629

6 7 8

WT

Nu

p1

45

p (

1-5

94

) -

Pro

t A

Nu

p1

45

p (

1-6

26

) -

Pro

t A

M r

*

**

Protein A

86

51.6

34.129

4 5

M r

Ga

l4B

D -

GF

PS

65

T

*

*

Ga

l4B

D -

Nu

p1

45

- G

FP

S6

5T

GFP

594Prot A

626Prot A

GFP-S65T

512 627Nup145p

GAL4BDGFP-S65TGAL4BD

B

GFP

Protein A

DAPI

Fig. 8. Characterization of Nup145pcleavage. (A) Immunoblot analysis ofcleavage was conducted as for Fig. 3except that the left and middle blotswere visualized using alkalinephosphatase-linked secondaryantibodies. The blot on the left showssamples from strains WT (SWY1349),∆524-592 (SWY1350), and ∆592-608(SWY1351) grown at 23°C, andprobed with an affinity-purifiedpolyclonal antibody against the C-terminal region of Nup145p. Thesingle star marks the ~140 kDauncleaved nup145∆592/608 protein(lane 3), and the double star marks thecleaved ~90 kDa wild-type (lane 1)and nup145∆524/592 protein (lane 2).The middle blot shows samples fromstrains Gal4BD-GFPS65T (SWY1360)and Gal4BD-Nup145-GFPS65T(SWY1361) grown at 30°C, andprobed with a polyclonal anti-GFPantibody. The single stars mark therespective positions of full length,uncleaved chimeric fusion proteins. Aband of ~30 kDa for cleaved GFP isnot present in lane 5. The blot on theright shows samples from strains WT(W303), Nup145p(1-594)-ProtA(SWY1396), and Nup145p(1-626)-ProtA (SWY1397) probed with apolyclonal rabbit anti-mouse antibodyrecognizing Protein A. Non-specificbands are present in all samples, thesingle star marks the position of theuncleaved Nup145p(1-594)-ProtAfusion (lane 7), and the double starmarks the cleaved product from theNup145p(1-626)-ProtA fusion (lane8). (B) Fluorescence analysis of invivo cleavage. For the GFP images(upper set), SWY1360 (left) andSWY1361 (right) were grown at 30°Cin SD-trp medium to early logarithmicphase, fixed for 10 minutes in 75 mMNaCl/ 3.7% formaldehyde/ 0.25 µg/mlDAPI, and washed with 100 mMpotassium phosphate, pH 6.5. For theProtein A immunofluorescence fields(lower set), SWY1396 (left) andSWY1397 (right) were grown at 30°Cand processed for immunofluorescencewith antibodies against the Protein Adomain as in Fig. 2A. Bar, 5 µm.

finding that the two halves of Nup145p can interact with eachother.

Previous studies of Nup145p have suggested two differenthypotheses for the observed fragmentation into N- and C-terminal polypeptides: that Nup145p biogenesis requirescleavage, or that the cleavage is an indirect consequence of cellbreakage. The experiments in this report have demonstratedthat cleavage does in fact occur in vivo, and it is not simply a

protein extraction artifact. However, the fact that the productof nup145∆592/608 allele is not cleaved and supports wild-type growth and NPC distribution reveals that processing is notrequired for Nup145p function in vivo. In terms of the site ofcleavage, although the region spanning residues 593-607 wasnecessary, it was not sufficient for processing. This isevidenced by the lack of cleavage in the fusion between Gal4BDand GFP, and by the published characterization of two different


Gal4BD

Snf1

N-term

Snf1

N-term

filter assayGal4AD

Snf4

Snf4

C-term

C-term

Fig. 9. Nup145p N-terminal and C-terminal regions interact in thetwo hybrid assay. Yeast strain HF7c was cotransformed with theplasmids expressing the indicated Gal4BD and Gal4AD constructs. β-galactosidase activity was detected with a color filter assay (Breedenand Nasmyth, 1985).

ProteinA-Nup145p fusions (Fabre et al., 1994). The previousstudy reported that when Protein A replaced the GLFG regionof Nup145p (residues 1-247), the resulting fusion protein wascleaved. However, when Protein A replaced most of the N-terminal region (residues 1-551) the fusion was not cleaved.Considering our internal deletion of residues 524-592 wascleaved, additional information for cleavage may reside in theN-terminal region between residues 247 and 524. Alterna-tively, the fusion of the heterologous GFP and Protein Adomains immediately N-terminal to the cleavage site may non-specifically inhibit proteolysis.

Preliminary conclusions regarding the analysis of thenup145∆N::LEU2 phenotype attributed the clusteringphenotype to the lack of the N-terminal region of Nup145p(Wente and Blobel, 1994). The phenotypes of the N-terminal,C-terminal, and internal deletion mutations in this reportfurther suggest that at least two different regions (between563-593 and 893-1090) are important for maintaining NPCdistribution. The contrasting phenotypes of the nup145∆NLand nup145∆NS alleles implicate the region between residues563 and 593; this is corroborated by clustering in the strainlacking residues 524 to 592. However, it appears that theabsence of the N-terminal region per se is not responsible forthe clustering phenotype. First, the strain carrying thenup145∆NL allele lacks the initial 562 amino acids, or mostof the N-terminal region, but displayed a wild-type distribu-tion of NPCs. Second, all three of the clustering mutationsfrom N-terminal and internal deletion analysis coincidentallylowered the protein level of the C-terminal region in yeastcell lysates. Finally, expression of the N-terminal region(nup145-58) in nup145∆N::LEU2 cells did not rescue theclustering phenotype, while expression of the C-terminalregion (in the form of nup145∆NS) does (unpublished obser-vations). We have not characterized an N-terminal deletionmutation that clusters and has wild-type levels of the C-terminal region. These results suggest that lowered levels ofthe C-terminal region contribute to the NPC clusteringphenotype. Truncations from the C terminus of Nup145p alsoresulted in a constitutive NPC clustering phenotype, indicat-

ing that the entire C-terminal region must be present forproperly spaced NPCs.

Because the nup145∆NS and nup145∆524/592 alleles wereexpressed under the control of the endogenous NUP145promoter, the lower levels of C-terminal region probably reflectan instability of the translated product. The same instabilityprobably applies to the nup145∆N::LEU2 product, and theeven lower protein level in the nup145∆N::LEU2 strain maybe attributed to the lack of a true promoter. Comparison of themutations suggests that the initiation of translation in thenup145∆N::LEU2 mutant occurs before amino acid 592;otherwise, the strain would have the pleiotropic defects asso-ciated with the nup145∆NS allele. Because the nup145∆NLallele which initiates at amino acid 562 also behaves essentiallyas wild type, the methionine residues at positions 564, 583, and586 are all possible initiation sites for the nup145∆N::LEU2C-terminal region. It is not likely that translation initiateswithin the LEU2 fragment, because a similar deletion whichremoves the same residues but instead has inserted the URA3gene confers the same phenotype (Wente and Blobel, 1994).Interestingly, high-copy expression of nup145-58 (encodingthe N-terminal 551 residues) does not complement thenup145∆NS growth defect (unpublished observations). Thus,the amino acid sequences between residues 551 and 593(removed in the nup145∆NS and nup145∆524/592 alleles) maybe required for stabilizing the C-terminal region.

Mutations in genes encoding six different nucleoporin genesresult in NPC clustering: NUP145 (Wente and Blobel, 1994),NUP133 (Doye et al., 1994; Pemberton et al., 1995; Li et al.,1995), NUP159 (Gorsch et al., 1995), NUP120 (Aitchison etal., 1995a; Heath et al., 1995), NUP84, and NUP85 (Sinios-soglou et al., 1996). Two distinct types of NPC clustering havebeen reported in these mutant strains; clusters with convolutedmembranes, and clusters in linear arrays. In all the previouslyreported clustering mutants except nup159, both types ofclusters have also been shown to coexist. It is possible thatmore than one mechanism may lead to clustering. However, itis more likely that the two different types of clusters merelyrepresent different severities of the same perturbation.

For the above listed nucleoporin genes, there are alsomutated alleles which block RNA export. The RNA exportdefects do not reflect a general collapse of NPC function, sinceprotein import is unaffected (see above references). Thus, thesesix nucleoporins may act together in mediating RNA exportand maintaining proper NPC distribution. Mutational studiesof these other NUPs have also not clearly separated the struc-tural regions required for the NPC clustering and RNA exportphenotypes. In most cases, clustering is constitutive whileRNA export is temperature sensitive, which may meanclustered NPCs can only export RNA at the permissive tem-perature. However, two exceptions exist. nup159 mutantsdisplay both NPC clustering and a mild RNA export defect atthe permissive temperature (Gorsch et al., 1995). By 15minutes after the shift to 37°C, RNA export is blocked. Afteran hour at 37°C, the clustered NPCs largely return to a normalconfiguration. However, at this time, Nup159p is no longerdetectable (Gorsch et al., 1995). Thus, the two nup159 pheno-types do not become separable until Nup159p is absent. Cellsexpressing an N-terminal deletion allele of nup133 haveclusters but are only mildly temperature sensitive with a mildRNA export defect (Doye et al., 1994). The data from this


Nup145p study imply that the full C-terminal region is requiredto maintain viability and mRNA export capacity at all temper-atures. Interestingly, lower C-terminal protein levels alone donot affect growth and export capacity, because thenup145∆N::LEU2 strain is not compromised.

It is possible that NPC clustering and RNA export defectsare both manifestations of failure of some underlying processwhich this group of nucleoporins mediates. We have previouslyspeculated that the formation of clusters may be due to changesin the attachment of NPCs (and the nuclear envelope) to anunderlying nuclear scaffold which maintains their fairly evenspacing (Wente and Blobel, 1994). RNA may utilize the sameintranuclear structures to be transported from their sites ofsynthesis to the NPCs (reviewed by Xing et al., 1993). Suchcoincident perturbation in mutants of nucleoporins thatmaintain NPC-nuclear interactions would therefore beexpected.

In conclusion, the results from mutational analysis suggestthat all essential Nup145p functions are mediated by its C-terminal region, with the N-terminal region possibly mediatinga non-essential cleavage event. The essential region spanningresidues 593 and 893 of Nup145p can now be targeted forfuture analysis. It will also be critical to determine bothNup145p’s nearest neighbor protein-protein interactions and itssubstructural localization within the NPC.

We thank G. Raczniak and A. Wilson for helping with the produc-tion of the antibody specific for the Nup145p C-terminal region; J.Aitchison, M. Rout, and G. Blobel for pProtA/HU; R. Murphy forpSW545; M. Levi and L. LaRose for technical assistance with EMexperiments; H. Piwnica-Worms for use of her Olympus fluorescencemicroscope. We are grateful to C. Hardy and members of the Wentelaboratory for critical discussion of the results, and to C. Cole for com-municating unpublished results. J. L. T. Emtage performed this workas a Fellow of the Missouri Affiliate of the American Heart Associa-tion. This work was supported by an RO1 grant from the NationalInstitutes of Health, No. GM51219-02, to S. R. Wente.

REFERENCES

Aitchison, J. D., Blobel, G. and Rout, M. P. (1995a). Nup120p – a yeastnucleoporin required for NPC distribution and mRNA transport. J. Cell Biol.131, 1659-1675.

Aitchison, J. D., Rout, M. P., Marelli, M., Blobel, G. and Wozniak, R. W.(1995b). Two novel related yeast nucleoporins Nup170p and Nup157p –complementation with the vertebrate homologue Nup155p and functionalinteractions with the yeast nuclear pore-membrane protein Pom152p. J. CellBiol. 131, 1133-1148.

Akey, C. W. (1995). Structural plasticity of the nuclear pore complex. J. Mol.Biol. 248, 273-293.

Aris, J. P. and Blobel, G. (1988). Identification and characterization of a yeastnucleolar protein that is similar to a rat liver nucleolar protein. J. Cell Biol.107, 17-31.

Bartel, P. and Fields, S. (1995). Analyzing protein-protein interactions usingtwo-hybrid system. Meth. Enzymol. 254, 241-263.

Bogerd, A. M., Hoffman, J. A., Amberg, D. C., Fink, G. R. and Davis, L. I.(1994). nup1 mutants exhibit pleiotropic defects in nuclear pore complexfunction. J. Cell Biol. 127, 319-332.

Breeden, L. and Nasmyth, K. (1985). Regulation of the yeast HO gene. ColdSpring Harbor Symp. Quant. Biol. 50, 643-650.

Davis, L. I. and Blobel, G. (1986). Identification and characterization of anuclear pore complex protein. Cell 45, 699-709.

Doye, V., Wepf, R. and Hurt, E. C. (1994). A novel nuclear pore proteinNup133p with distinct roles in poly(A)+ RNA transport and nuclear poredistribution. EMBO J. 13, 6062-6075.

Fabre, E., Boelens, W. C., Wimmer, C., Mattaj, I. W. and Hurt, E. C. (1994).

Nup145p is required for nuclear export of mRNA and binds homopolymericRNA in vitro via a novel conserved motif. Cell 78, 275-289.

Feldherr, C. M., Kallenbach, E. and Schultz, N. (1984). Movement of akaryophilic protein through the nuclear pores of oocytes. J. Cell Biol. 99,2216-2222.

Gerace, L. (1995). Nuclear export signals and the fast track to the cytoplasm.Cell 82, 341-344.

Gorsch, L. C., Dockendorff, T. C. and Cole, C. N. (1995). A conditional alleleof the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes bothrapid cessation of mRNA export and reversible clustering of nuclear porecomplexes. J. Cell Biol. 129, 939-955.

Grandi, P., Emig, S., Weise, C., Hucho, F., Pohl, T. and Hurt, E. C. (1995a).A novel nuclear pore protein Nup82p which specifically binds to a fraction ofNsp1p. J. Cell Biol. 130, 1263-1273.

Grandi, P., Schlaich, N., Tekotte, H. and Hurt, E. C. (1995b). Functionalinteraction of Nic96p with a core nucleoporin complex consisting of Nsp1p,Nup49p and a novel protein Nup57p. EMBO J. 14, 76-87.

Heath, C. V., Copeland, C. S., Amberg, D. C., Delpriore, V., Snyder, M. andCole, C. N. (1995). Nuclear pore complex clustering and nuclearaccumulation of poly(A)+ RNA associated with mutation of theSaccharomyces cerevisiae rat2/nup120 gene. J. Cell Biol. 131, 1677-1697.

Heim, R., Cubitt, A. B. and Tsien, R. Y. (1995). Improved green fluorescence.Nature 373, 663-664.

Hurwitz, M. E. and Blobel, G. (1995). NUP82 is an essential yeastnucleoporin required for poly(A)+ RNA export. J. Cell Biol. 130, 1275-1281.

Iovine, M. K., Watkins, J. L. and Wente, S. R. (1995). The GLFG repetitiveregion of the nucleoporin Nup116p interacts with Kap95p, an essential yeastnuclear import factor. J. Cell Biol. 131, 1699-1713.

Jones, J. S. and Prakash, L. (1990). Yeast Saccharomyces cerevisiaeselectable markers in pUC18 polylinkers. Yeast 6, 363-366.

Kaiser, C., Michaelis, S. and Mitchell, A. (1994). Methods in Yeast Genetics.Plainview, NY: Cold Spring Harbor Laboratory Press, NY.

Kraemer, D. M., Strambio de Castillia, C., Blobel, G. and Rout, M. P.(1995). The essential yeast nucleoporin NUP159 is located on thecytoplasmic side of the nuclear pore complex and serves in karyopherin-mediated binding of transport substrate. J. Biol. Chem. 270, 19017-19021.

Li, O., Heath, C. V., Amberg, D. C., Dockendorff, T. C., Copeland, C. S.,Snyder, M. and Cole, C. N. (1995). Mutation or deletion of theSaccharomyces cerevisiae RAT3/NUP133 gene causes temperature-dependent nuclear accumulation of poly(A)+ RNA and constitutiveclustering of nuclear pore complexes. Mol. Biol. Cell 6, 401-417.

Maina, C. V., Riggs, P. D., Grandea, A. G., Slatko, B. E., Moran, L. S.,Tagliamonte, J. A., McReynolds, L. A. and Guan, C. (1988). A vector toexpress and purify foreign proteins in Escherichia coli by fusion to, andseparation from, maltose binding protein. Gene 74, 365-373.

Osborne, M. A. and Silver, P. A. (1993). Nucleocytoplasmic transport in theyeast Saccharomyces cerevisiae. Annu. Rev. Biochem. 62, 219-254.

Panté, N. and Aebi, U. (1996). Molecular dissection of the nuclear porecomplex. Crit. Rev. Biochem. Mol. Biol. 31, 153-199.

Pemberton, L. F., Rout, M. P. and Blobel, G. (1995). Disruption of thenucleoporin gene NUP133 results in clustering of nuclear pore complexes.Proc. Nat. Acad. Sci. USA 92, 1187-1191.

Reichelt, R., Holzenburg, A., Buhle, E. J., Jarnik, M., Engel, A. and Aebi,U. (1990). Correlation between structure and mass distribution of the nuclearpore complex and of distinct pore complex components. J. Cell Biol. 110,883-894.

Rout, M. P. and Blobel, G. (1993). Isolation of the yeast nuclear pore complex.J. Cell Biol. 123, 771-783.

Rout, M. P. and Wente, S. R. (1994). Pores for thought: nuclear pore complexproteins. Trends Cell Biol. 4, 357-365.

Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: aLaboratory Manual. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY.

Severs, N. J., Jordan, E. G. and Williamson, D. H. (1976). Nuclear poreabsence from areas of close association between nucleus and vacuole insynchronous yeast cultures. J. Ultrastruct. Res. 54, 374-387.

Silver, P. A., Keegan, L. P. and Ptashne, M. (1984). Amino terminus of yeastGAL4 gene product is sufficient for nuclear localization. Proc. Nat. Acad.Sci. USA 81, 5951-5955.

Sikorski, R. S. and Boeke, J. D. (1991). In vitro mutagenesis and plasmidshuffling: from cloned gene to mutant yeast. Meth. Enzymol. 194, 302-318.

Sikorski, R. S. and Hieter, P. (1989). A system of shuttle vectors and yeast hoststrains designed for efficient manipulation of DNA in Saccharomycescerevisiae. Genetics 122, 19-27.


Siniossoglou, S., Wimmer, C., Rieger, M., Doye, V., Tekotte, H., Weise, C.,Emig, S., Segref, A. and Hurt, E. C. (1996). A novel complex ofnucleoporins, which includes Sec13p and a Sec13p homolog, is essential fornormal nuclear pores. Cell 84, 265-275.

Wente, S. R., Rout, M. P. and Blobel, G. (1992). A new family of yeast nuclearpore complex proteins. J. Cell Biol. 119, 705-723.

Wente, S. R. and Blobel, G. (1993). A temperature-sensitive NUP116 nullmutant forms a nuclear envelope seal over the yeast nuclear pore complexthereby blocking nucleocytoplasmic traffic. J. Cell Biol. 123, 275-284.

Wente, S. R. and Blobel, G. (1994). NUP145 encodes a novel yeast glycine-leucine-phenylalanine-glycine (GLFG) nucleoporin required for nuclearenvelope structure. J. Cell Biol. 125, 955-969.

Wimmer, C., Doye, V., Grandi, P., Nehrbass, U. and Hurt, E. C. (1992). Anew subclass of nucleoporins that functionally interact with nuclear poreprotein NSP1. EMBO J. 11, 5051-5061.

Xing, Y., Johnson, C. V., Dobner, P. R. and Lawrence, J. B. (1993). Higherlevel organization of individual gene transcription and RNA splicing. Science259, 1326-1330.

Yang, X., Hubbard, E. and Carlson, M. (1992). A protein kinase substrateidentified by the two-hybrid system. Science 257, 68-72.

(Received 30 September 1996 – Accepted 17 January 1997)

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