Proc. Natl. Acad. Sci. USAVol. 91, pp. 1519-1523, February 1994Cell Biology

The human CAN protein, a putative oncogene product associatedwith myeloid leukemogenesis, is a nuclear pore complex proteinthat faces the cytoplasm

(wheat germ agglutinin-binding protein/monospecific antibodies/immunofluorescence/immunoelectron microscopy)

DoRis KRAEMER, RICHARD W. WOZNIAK, GUNTER BLOBEL, AND AURELIAN RADULaboratory of Cell Biology, The Rockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10021

Contributed by Gunter Blobel, November 12, 1993

ABSTRACT We have carried out partial amino acid se-quence analysis of a putative nuclear pore complex protein(nucleoporin) of rat that reacts with wheat germ agglutinin andwith the polyspecific monoclonal antibody 414. Surprisingly,these partial amino acid sequence data revealed a high degreeof similarity with the human CAN protein, the completecDNA-derived primary structure ofwhich was reported by VonLindern et al. [Von Lindern, M., Fornerod, M., van Baal, S.,Jaegle, M., de Wit, T., Bulis, A. & Grosveld, G. (1992) Mol.CeU. Biol. 12, 1687-1697]. The CAN protein has been proposedto be a putative oncogene product associated with myeloidleukemogenesis. Its subcellular localization was not estab-lished. To confirm that the putative rat nucleoporin is indeeda homolog of the human CAN protein and to determine itssubcellular localization, we expressed a 39-kDa internal seg-ment of the 213,790-Da human CAN protein in Escherichia coliand raised monospecific antibodies, which reacted with theputative rat nucleoporin. Immunofluorescence microscopy ofHeLa cells gave a punctate nuclear surface staining patterncharacteristic of nucleoporins, and immunoelectron micros-copy yielded specific decoration of the cytoplasmic side of thenuclear pore complex. This suggests that the protein is part ofthe short fibers that emanate from the cytoplasmic aspect of thenuclear pore complex. In agreement with previously proposednomenclature for nucleoporins, we propose the alternativeterm nup214 (mcleoporin of 214 kDa) for the CAN protein.

Only 3 of the estimated 100 or more nuclear pore complex(NPC) proteins [collectively referred to as nucleoporins (1)]have so far been molecularly characterized in vertebratecells. These are p62 (2, 3), nuplS3 (4), and nuplS5 (5). Byusing monospecific antibodies, nuplS5 (5) and p62 (2) werefound by immunoelectron microscopy to be located symmet-rically on the nucleoplasmic and cytoplasmic aspects of theNPC. In contrast, nuplS3 was demonstrated to be locatedasymmetrically on only the nucleoplasmic side of the NPC(4), specifically on the terminal ring of the nuclear basket (6).NuplS3 has been shown to contain four Cys-Cys-type zincfinger motifs and to bind to DNA in a zinc-dependent fashion(4). It has been proposed to function in the three-dimensionalorganization of the chromatin (4). The functions of p62 andnupl55 are unknown.Two groups of vertebrate nucleoporins have so far been

distinguished. One ofthem comprises about a dozen proteins,including p62 and nuplS3, that contain single N-acetylglu-cosamine (GlcNAc) residues attached to serine or threonineresidues (refs. 7-14; for review, see ref. 15). As a conse-quence, these proteins react with wheat germ agglutinin(WGA) (1, 4, 7, 9-13). The other group, comprising the bulkof the vertebrate nucleoporins and so far represented by

nupl55, does not react with WGA (5). The WGA-reactivenups share repetitive sequence motifs with each other thatare conserved across various species, including yeast (2-4,16-21). These sequence motifs appear to be part of theepitope that is recognized by monoclonal antibodies (mAbs)(4, 7, 14, 16-22), which may explain the observed polyspec-ificity of these mAbs. To molecularly characterize additionalnucleoporins, we focused on one of the previously describedWGA- and mAb 414-reactive proteins of the rat liver nuclearenvelope that has an apparent molecular mass (based onmobility in SDS/PAGE) of =-225 kDa [previously estimatedto have a molecular mass of -210 kDa (4)]. In subcellularfractionation, p225 cofractionated with nuclear envelopes (4)and, like p62, nupl53, and nup155, was extractable byurea/EDTA (5). Based on these criteria, p225 thereforeappeared to be a strong candidate for an NPC protein. Inpreparation for cDNA cloning, we obtained partial aminoacid sequence of internal peptides. Surprisingly, these aminoacid sequences matched the cDNA-deduced amino acidsequence of the human CAN protein, a putative oncogeneproduct of 213,790 Da that is associated with leukemogenesis(23). Although not previously noted, the CAN protein se-quence showed degenerate XFXFG pentapeptide motifs.This motif can be considered a nucleoporin signature since itis also found in p62 (2, 3, 16), nuplS3 (4), and several yeastnucleoporins (17-20), clearly supporting the candidacy ofCAN as a nucleoporin. However, although no data have beenpublished on the cellular localization of the CAN protein, itwas suggested to be a cytoplasmic protein (23). To defini-tively sublocalize the CAN protein, we obtained antibodiesthat react monospecifically with the WGA- and mAb 414-reactive rat p225. In immunofluorescence ofHeLa cells theseantibodies gave the punctate nuclear surface staining patterncharacteristic for nucleoporins (1). Immunogold electronmicroscopy showed exclusive decoration of NPCs, estab-lishing that the CAN protein is a nucleoporin. In keeping withprevious suggestions for nomenclature, we propose the al-ternative term nup214 (for nucleoporin of 214 kDa). Inter-estingly, we found that nup214 is as3ymmetrically localized ononly the cytoplasmic side ofthe NPC, suggesting that it mightbe a component of the NPC-attached short fibers projectingan estimated 35-50 nm into the cytoplasm (24, 25).

MATERIAL AND METHODSProtein Sequencing and Analysis. WGA-binding proteins

from rat liver nuclear envelopes were prepared as described(5). The proteins were concentrated in a Centricon micro-concentrator (Amicon), loaded on a5% SDS/polyacrylamidegel (26), electrophoretically transferred (27) for 20 hr at 60 Vto poly(vinylidene difluoride) membrane (Millipore), and

Abbreviations: WGA, wheat germ agglutinin; NPC, nuclear porecomplex; mAb, monoclonal antibody.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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detected with amido black. A strip of the membrane with the225-kDa protein was cut out and digested with endoprotein-ase Lys C (Sigma), and the resulting fragments were sepa-rated by reversed-phase HPLC and subjected to automatedEdman degradation (28).

Five peaks ofthe HPLC profile were analyzed; one yieldeda single sequence, one produced a double sequence, andthree produced triple sequences. After protein homologysearches with the single sequence in the GenBank data basesusing the BLAST program (29), a high similarity to the humanprotein CAN (23) was found. After this the mixed double andtriple sequences could also be separated and all ofthem couldbe aligned to the sequence of CAN. Some of the sequencesappeared in different HPLC peaks. Similarity was measuredby using the similarity matrix of Henikoff and Henikoff (30).Characterization of the cDNA-deduced protein sequenceswas performed on a Macintosh personal computer usingsoftware obtained from DNAstar (Madison, WI) and MacPattern (31).

Expression and Purification of a 39-kDa Fragment of CAN.For the purpose of expressing a 38,906-Da fragment of thehuman CAN protein we have synthesized a 1093-bp PCRproduct encoding amino acids 818-1175. Nondegeneratesense (5'-CATCAGTATGTGAAATTTGCT-3') (nt 2546-2566) and antisense (5'-GGAGATGGCTTAAGGGAATT-TAT-3') (nt 3599-3619) oligonucleotide primers containingadditional restriction sites for EcoRI (5') and HindIII (3')were synthesized according to the published sequence of can(23). Together with these primers, a human Agtll cDNAlibrary (32) was used as template to synthesize the corre-sponding region ofthe can gene using the PCR. Amplificationwas conducted as described (5) except that the annealingtemperature was increased to 480C. The reaction product ofthe expected size was gel purified, cut with EcoRI andHindIII (New England Biolabs), and subcloned into thepET21b plasmid (Novagen) in frame with six C-terminalhistidine residues. The resulting plasmid was designatedpET21b/p39. Sequencing of the insert was performed ac-cording to the dideoxy chain-termination method (33) usingSequenase 2.0 (United States Biochemical).The p39/6xHis fusion protein was expressed in the Esch-

erichia coli cell line BL21(DE3) (Novagen). Cells were cul-tured in 50 ml of LB medium containing 50 ,ug of ampicillin(Sigma) per ml at 37TC until the OD60 reached 0.6-1.0. Forinduction, 1 mM isopropyl P-D-thiogalactoside (BoehringerMannheim) and, 15 min later, 250 p.g of rifampicin (Boeh-ringer Mannheim) per ml were added and the incubation wascontinued at 370C for 2.5 hr. The cells were harvested bycentrifugation at 5000 x g for 5 min at 40C and the pellet wassuspended in 12 ml of buffer A (5 mM imidazole/0.5 MNaCl/20 mM Tris-HCl, pH 7.9). Lysozyme (100 pg/ml)(United States Biochemical) and 0.05% Triton X-100 (Boeh-ringer Mannheim) were also added. After incubation at 300Cfor 15 min the cells were disrupted by sonication. All of thefollowing steps were carried out at 40C. After centrifugationfor 20 min at 12,000 x g the pellet was suspended in 10 ml of6 M urea in buffer A and the suspension was incubated for 1hr. Insoluble cell debris was removed by centrifugation at40,000 x g for 20 min. The supernatant was loaded on 2.5 mlof Ni2+-nitrilotriacetic acid-agarose (Qiagen, Chatsworth,CA), which was previously equilibrated with 7.5 ml of 6 Murea in buffer A. The column was washed with 25 ml of 6 Murea in bufferA and 15 ml of20mM imidazole/0.5 M NaCl/20mM Tris-HCl, pH 7.9/6 M urea, and the protein was elutedwith 5 ml of 0.5 M imidazole/0.5 M NaCl/20 mM Tris HCl,pH 7.9/6M urea (Novagen pET system manual). The proteinconcentration was determined according to Bradford (34).

Anti-p39 Antibodies. Two hundred micrograms (first injec-tion) and 300 pg (in three more injections) of purified p39/6xHis were injected into rabbits to raise polyclonal antibodies

200-

,4 nup 153

116 -

97-

66 ___

FIG. 1. Isolation of p225, a putative nucleoporin. Proteins of a 2M urea/i mM EDTA extract of purified rat liver nuclear envelopeswere fractionated on WGA Sepharose. The WGA-reactive proteinswere separated by SDS/PAGE on a 5% acrylamide gel and stainedwith Coomassie blue. Lane 1, size marker proteins; lane 2, WGA-reactive proteins. The masses of marker proteins in lane 1 areindicated to the left in kDa. The arrow indicates p225 and arrowheadspoint to the nucleoporins p62 and nuplS3.

(Rockland, Gilbertsville, PA). The serum was affinity puri-fied using p39/6xHis bound to nitrocellulose (35). For im-munolocalization the affinity-purified antibodies were con-centrated 5-fold in a Centricon microconcentrator (Amicon).Immunoblot Analysis. After electrophoretical transfer of

SDS/PAGE-separated proteins to nitrocellulose membrane(27), blots were incubated with primary p39 antibodies [di-luted 1:10 in 5% nonfat dry milk/0.1% Tween 20 in phos-phate-buffered saline (PBS)] and visualized with alkalinephosphatase-conjugated anti-rabbit IgG (Promega) under thesame conditions as described previously (4), except thatsolutions were buffered with PBS.Imnunofluorescence Microscopy. HeLa cells were grown

on coverslips in modified Eagle's medium (16) until they weresubconfluent. After washing for 2 min with ice-cold PBS theywere fixed for 10 min with 2% paraformaldehyde in PBS,permeabilized for 2 min with 0.5% Triton X-100 in PBS, and

EAYNQKII I I

131 EAKOOK

KXDDSLPVGVTIDYTNFV11111:11 :1111

356 KSDDSLPMGVVVDYTNOV

KXXLEXTSPVXXXS11 11

554 KPTLEST PVPSVS

KXXEVRSTAP111111

955 KTPPVRSTAP

KXFFSFGNQQT1111:111

1436 KTSFSFGSQQT

KXPFSSASXGFGSTPTLN111111 111111900 KNPFSSASGGFGSTATSN

KXFGFGAAPVFAS11111111

1966 KTGGFGAAPVFGS

KXFGEGTAXGXXGAF111111

2010 KVFGEGTAAASAGGF

rat

136 human

rat

373 human

rat

562 human

rat

9641 human

rat

1447 human

rat

1917 human

rat

1978 human

rat

2024 human

FIG. 2. Comparison of partial amino acid sequences of rat p225with similar sequences of the human CAN protein. Numbers to theleft and right of human sequences indicate their positions in thecomplete amino acid sequence of the human CAN protein; 1,identical amino acids;:, similar amino acids. Five peaks oftheHPLCprofile of p225 digestion with endoproteinase Lys C were analyzed.

Proc. Nad. Acad. Sci. USA 91 (1994)

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Proc. Natl. Acad. Sci. USA 91 (1994) 1521

XFXFG:481 VFSFG519 TFSFV545 SFSFG

$2LKUATVTGEPPSYUGSDI1KAAPGPGPSPPSKASLAPTPAASPVAPSAA

1213 PFSFS PSGT1222 GFNFG ITPTP5sNFTAAQGATPSTKEMOPDAFjGGGSKPS

YEA I PE.UPPSG I TSASNTTPGEPAASSMRPVAPSGTALSTT.SKLETPPSKLGELLFPS3LAGETLGSFSGLRVGOADDSTKPTNKA,5TSLTSTOPTKTSGVPSGFNFTAPPVLGKHTEPPVTS&ATTTSVAPPAATSTSSTSLPVTSAG3IGV SFGGTSLSAGKT

1438 SFSFG SOOTNSTVPPSAPPPTTAATPLPTSFPTLSFGSLLj$ATTPSLPMSAGRSTEEATI5ALPEKPGDSEVSASAASLLEEQOSAOLPOAPPOTSDSVKKEPVLAOPAVSNSGTAASTSLTVALSAEATPATTGVPDARTEAVPPAS.FOTAVTAAA I£AGPVAVET.STP I AgTTS I VAPGPSAEAAAFGTVTSGSOPPAA=SAFNOLTNNTATAPSATOVAASTAPSLFG0OTGSTASTAAATPOV.UGFWPAFGTTAPTTFGQAOSASAAFSOPGFSSVPAFG0PASTPTSTSGAASTSM

1783 SFSFG OQ$PNTGGGLFGOSNAPAFGQSPGFGOGGGTSAATTTAATS

1833 GFSFC OA,NTGOAASTGGOOs5 GSGNTGRGGGFFSGLGGKPSODAANKNPF,5ASGGFGSTATSNTSNLFGNSGAKTFGGFASFGEQKPTGTF£GGGSVASO

1959 GFGFS SPNKTGGFGAASPPTFGGSPGFGGVPAFGSAPAFTSPLGSTGGEGTAAASAG

2023 GFGFG $&UNTTSFGTLASONAPTFGSLSOOTTOUGFSGTG

2072 GFSFG SNNSFGGWRS

SVFG bovine serum albumin/0.05% Tween 20 in PBS. Antibodybinding was detected with Texas red-labeled goat anti-rabbitIgG (Organon). Coverslips were washed and mounted in asolution of 1 mg of p-phenylenediamine per ml in 90%oglycerol (pH 8.0) and examined as described (5).Immunoelectron Microscopy. HeLa cells were processed to

prepare frozen ultrathin sections (4, 36). The sections wereincubated first with the concentrated affinity-purified anti-

1409 AVFG p39 antibodies and then with 10-nm gold-conjugated second-ary antibodies as described (4).

1589

16431672

1719173017411770

1817

1840184918601872

SVPG

SVFAPVFG

GVFGSVFGSVFSSVFG

SVFG

SGFGSVFGI VFGSVFG

1974 PVFG2010 KVFG

2054 SGFG2064 SGFG

2081 SVOG

RESULTSAs previously described (5), purified rat liver nuclear enve-lopes contain dozens ofperipheral membrane proteins. Manyof them are potential nucleoporins. These proteins can beextracted by urea/EDTA and further subfractionated onWGA Sepharose. About a dozen of these potential nucle-oporins bind to WGA (5). The proteins also react polyspe-cifically with various mAbs (4). These mAbs decorate theNPC in immunoelectron microscopy. Fig. 1 shows the WGA-reactive proteins separated by SDS/PAGE and stained withCoomassie blue (lane 2). Among them are the already mo-lecularly characterized nucleoporins p62 (2, 3) and nupl53 (4)(lane 2, arrowhead). In preparation for the molecular cloningof a protein of -225 kDa (p225) (indicated by arrow in lane2), p225 was subjected to proteolytic cleavage with endopro-

FIG. 3. Repetitive peptide motifs in the human CAN protein. Thepentapeptide XFXFG is a nucleoporin "signature" motif. The tet-rapeptide motif SVFG (on the right) and the tripeptide motif FGG(indicated in bold letters in the middle column) have so far not beendescribed for other nucleoporins. FGQ is part of the hexapeptidemotif GGLFGQ in NUP100 and NUP116 (20, 21). Polyserines areunderlined. Numbers indicate the first amino acid position ofXFXFG and SVFG motifs, respectively, in the human CAN proteinsequence.

blocked for 1 hr with 2% bovine serum albumin/0.05%Tween 20 in PBS. The cells were probed further with theconcentrated affinity-purified p39 antibodies for 1 hr at roomtemperature and washed five times for 5 min with 0.1%

A B1 2 3 1

66 -

45 - _

- 200

- 116

FIG. 4. Expression of a 39-kDa internal segment of the humanCAN protein in E. coli and characterization of antibodies raisedagainst the expressed protein. (A) Lane 1, Coomassie blue-stainedsize markers; lane 2, Coomassie blue-stained 39-kDa internal seg-ment of the CAN protein expressed in E. coli and subsequentlypurified; lane 3, nitrocellulose blot of protein in lane 2 probed withaffinity-purified rabbit anti-p39 antibodies and visualized with alka-line phosphatase-coupled anti-rabbit IgG. (B) Lane 1, nitrocelluloseblot of SDS/PAGE-separated polypeptides of rat liver nuclear en-velopes probed with affmiity-purified rabbit anti-p39 antibodies. Thearrowhead indicates p225. The positions of markers are labeled (inkDa).

FIG. 5. Immunofluorescence microscopy of HeLa cells. Cellsgrown on coverslips were fixed in paraformaldehyde, permeabilizedwith Triton X-100, and probed with affinity-purified anti-p39 anti-bodies. Focusing on the nuclear periphery (Upper) or the nuclearsurface (Lower) yielded nuclear rim staining and a punctate nuclearsurface staining pattern, respectively, characteristic for nucleopor-ins. (Bar = 5 Am.)

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teinase Lys C. The resulting peptides were separated byHPLC and some of them were subjected to amino acidsequencing. Surprisingly, a comparison of the obtainedamino acid sequences with those in the data banks showed ahigh degree of similarity with the cDNA-deduced amino acidsequence of the human CAN protein throughout the entirelength of the molecule (Fig. 2). This suggested that the ratp225 is the homolog of the human CAN protein (23). Al-though the subcellular localization ofthe humanCAN proteinhas not been established, it has been suggested to be acytoplasmic protein (23), clearly at odds with it being anucleoporin.An analysis of the human CAN protein sequence (Fig. 3)

revealed the presence of 11 redundant XFXFG motifs thatare present in vertebrate p62 (2, 3, 16) and nuplS3 (4) as wellas in various yeast nucleoporins (17-19). This motif is likelyto be part of the epitope reactive with mAb 414 (20). Inaddition, there is one other tetrapeptide motifSVFG (see Fig.3) and the repetitive motif FGG that have so far not beenreported for other molecularly characterized nucleoporins.The tripeptide motif FGQ is part of the GGLFGQ motif inNUP100 and NUP116 (20, 21). Especially the C-terminal

a

portion (amino acids 1213-2090) is unusually rich in serines(21%), threonines (12%), and prolines (9,%) (23). Serines andthreonines, which are located less than three residues from aproline, have been proposed to be potential sites for GlcNAcaddition (8). The amino acid sequence of CAN contains 32potential phosphorylation sites (7 for protein kinase C, 24 forcasein kinase II, and 1 for cAMP-dependent protein kinase).Although the presence in the human CAN protein of the

XFXFG motifwas consistent with it being a nucleoporin, thelow abundance of this motif (relative to that in other nucleo-porins) as well as its considerable degeneracy made it farfrom certain that the human CAN protein is a nucleoporin. Todefinitively localize the human CAN protein it was thereforenecessary to prepare monospecific antibodies. To obtainthese we expressed a 39-kDa segment ofthe protein (residues818-1175) in E. coli (Fig. 4A, lane 2). This segment lacks theXFXFG motif that is part of the mAb 414-reactive epitopeand the other repetitive motifs. Antibodies to this segmentshould therefore be monospecific for the human CAN pro-tein. Indeed, the obtained antibodies reacted with the E.coli-expressed CAN protein segment (Fig. 4A, lane 3) andrecognized a single 225-kDa protein in rat liver nuclear

sI~~~~~i..FiG. 6. Immunoelectron microscopy of HeLa cells. Frozen ultrathin sections of HeLa cells were probed with affinity-purified anti-p39 and

binding was detected with 10-nm gold-coupled secondary antibodies. Nucleoplasm (N) and cytoplasm (C) are indicated. (a) Section through apart ofa HeLa cell. Arrows point to gold particles decorating the cytoplasmic side of the NPCs. (b-d) Transverse sections with their cytoplasmicand nucleoplasmic sides aligned up and down, respectively. Gold exclusively decorated the cytoplasmic side of the NPCs. (Bars = 200 nm ina and 100 nm in b-d.)

1522 Cell Biology: Kraemer et al.

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Proc. Natl. Acad. Sci. USA 91 (1994) 1523

envelopes (Fig. 4B, lane 1). In addition, this antibody spe-cifically recognized the mAb 414-reactive p225 polypeptideisolated from rat liver nuclear envelopes (data not shown).These monosoedific antibodies were next used for indirect

immunofluorescence microscopy of paraformaldehyde-fixedand Triton X-100-permeabilized HeLa cells grown on cov-erslips (Fig. 5). Focusing on the nuclear rim (Fig. 5 Upper) orthe nuclear surface (Fig. 5 Lower) revealed a punctatenuclear rim and surface staining pattern that is characteristicfor nucleoporins (1). Cytoplasmic staining was not detected.To definitively localize p225 to the NPC we carried out

immunoelectron microscopy. Frozen ultrathin sections ofHeLa cells were incubated with the monospecific antibodiesand then with 10-nm gold-conjugated secondary antibodies.The gold particles decorated NPCs (Fig. 6). Most strikingly,they decorated only the cytoplasmic side of the NPC (Fig. 6).Thus we conclude that the human CAN protein is a nucleo-porin that is asymmetrically localized on the cytoplasmic sideof the NPC.

DISCUSSIONThe human CAN protein, a putative oncogene associatedwith myeloid leukemogenesis (23, 37), has been shown hereby immunoelectron microscopy to be a nucleoporin. Consis-tent with previous nomenclature, we therefore suggest thealternative term nup214 for this protein.nup214 is only the fourth vertebrate nucleoporin that has

been localized to the NPC (see Introduction). Its exclusivelocalization to the cytoplasmic side of the NPC suggests that.it may be part ofthe short "fibers" that extend 35-50 nm fromthe cytoplasmic aspect of the NPC (assuming that the proteincomposition of the cytoplasmic and the nucleoplasmic rings isidentical). These fibers are the only macromolecular struc-tures so far known to be asymmetrically associated with theNPC on its cytoplasmic side. nup214 and nuplS3 cofractionatequantitatively with nuclear envelopes. Thus, although nupl53is located at the "extreme" nucleoplasmic end of the NPC andnup214 at its cytoplasmic perimeter, neither of them appearsto be lost from the NPC during isolation of nuclear envelopes.This knowledge will be useful for further structural andfunctional analysis of components of the NPC in the contextof isolated nuclear envelopes.The function of nup214 remains to be determined. One

possibility is that this protein serves as a docking site in thereceptor-mediated import of substrates across the NPC (38,39). In fact, binding of gold-decorated nuclear import sub-strate to what could be such fibers has been reported previ-ously (40).An understanding of the function of nup214 may also help

in elucidating its presumed assc~iation with myeloid leuke-

a NH3

b NH3

C NH,

nup 214

812 A 813

nup 214DEK1l 349

SET!1 270

nup 214

COOH2090

i COOH2090

^,COOH

FIG. 7. Schematic representation of nup214 (CAN) and the twochimeric proteins that result from chromosome translocation orfusion associated with myeloid leukemogenesis. As a result ofchromosome translocation or fusion, the C-terminal part of nup214(CAN) (a) on chromosome 9 is linked to the N-terminal part of theDEK protein (chromosome 6) (b) or fused to the SET protein(chromosome 9) (c). Numbers indicate the positions ofresidues at theN and C termini and at the breakpoints.

mogenesis. In Fig. 7 are summarized previously publisheddata (23, 37). This work showed that two distinct chromo-some aberrations, a translocation and a fusion (both charac-teristic for myeloid leukemogenesis), yielded two chimericproteins. Because each of these chimeric proteins shares acommon C-terminal part derived from the C terminus ofnup214 (Fig. 7), nup214 (rather than the N-terminal DEK andSET proteins) was suggested to be the putative oncogeneproduct (23, 37). If nup214 were to play an important role inthe control ofnucleocytoplasmic traffic, it is conceivable thatan imbalance in traffic control could yield a proliferativeadvantage to the cell (39).

We thank Helen Shio for preparation of the immunoelectronmicroscopy specimens, Mike Rout for critical reading of the manu-script, and members of the Rockefeller University Protein Sequenc-ing Facility, especially Joseph Fernandez, for protein sequencing.D.K. was supported by a postdoctoral fellowship (Kr 1412/1-1) fromthe Deutsche Forschungsgtmeinschaft.

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