The SONB^NUP98 Nucleoporin Interacts With the NIMA Kinase in Aspergillus nidulans

Corresponding author: Stephen A. Osmani, Ohio State University, 804 Riffe Bldg., 496 W. 12th Ave., Columbus, OH 43210., osmani.2{at}osu.edu (E-mail)

Communicating editor: M. ZOLAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Aspergillus nidulans NIMA kinase is essential for mitotic entry. At restrictive temperature, temperature-sensitive nimA alleles arrest in G₂, before accumulation of NIMA in the nucleus. We performed a screen for extragenic suppressors of the nimA1 allele and isolated two cold-sensitive son (suppressor of nimA1) mutants. The sonA1 mutant encoded a nucleoporin that is a homolog of yeast Gle2/Rae1. We have now cloned SONB, a second nucleoporin genetically interacting with NIMA. sonB is essential and encodes a homolog of the human NUP98/NUP96 precursor. Similar to NUP98/NUP96, SONB^NUP98/NUP96 is autoproteolytically cleaved to generate SONB^NUP98 and SONB^NUP96. SONB^NUP98 localizes to the nuclear pore complex and contains a GLEBS domain (Gle2 binding sequence) that binds SONA^GLE2. A point mutation within the GLEBS domain of SONB1^NUP98 suppresses the temperature sensitivity of the nimA1 allele and compromises the physical interaction between SONA^GLE2 and SONB1^NUP98. The sonB1 mutation also causes sensitivity to hydroxyurea. We isolated the histone H2A-H2B gene pair as a copy-number suppressor of sonB1 cold sensitivity and hydroxyurea sensitivity. The data suggest that the nucleoporins SONA^GLE2 and SONB^NUP98 and the NIMA kinase interact and regulate nuclear accumulation of mitotic regulators to help promote mitosis.

THE cell cycle is regulated at multiple levels to ensure the fidelity of both DNA replication and the faithful segregation of chromosomes at mitosis. The NIMA kinase is the prototypic member of the NIMA-related kinase (NRK or NEK) family and is essential for mitotic entry in Aspergillus nidulans (OSMANI et al. 1991 ; YE et al. 1995 ). Temperature-sensitive never in mitosis (nimA) alleles arrest the cell cycle in G₂ with uncondensed DNA and interphase microtubules (OSMANI et al. 1987 ). During G₂, NIMA is only weakly active as a kinase and requires multiple phosphorylations occurring downstream of NIMX^cdc2/NIME^cyclinB activation to achieve a fully active mitotic state (YE et al. 1995 ). During NIMA activation and mitotic entry, NIMA accumulates in the nucleus where it carries out its mitotic functions (DE SOUZA et al. 2000 ). We have previously demonstrated that NIMA genetically interacts with sonA1^GLE2 (suppressor of nimA1), a member of the Gle2/Rae1 family of WD-repeat proteins that localize predominantly to the nuclear pore complex (NPC; BROWN et al. 1995 ; MURPHY et al. 1996 ; WU et al. 1998 ). In the presence of the sonA1 mutation, nimA1 mutants that normally arrest in G₂ are able to enter mitosis, at least in part by allowing access of NIMX^cdc2/NIME^cyclinB to the nucleus (WU et al. 1998 ).

The NPC is a multi-protein structure embedded in the nuclear envelope that provides highly regulated access of proteins and nucleic acids to and from the nucleus (reviewed in WENTE 2000 ; ROUT and AITCHISON 2001 ). Recent research has greatly expanded our knowledge of the structure and makeup of the NPC (ALLEN et al. 2001 ; ROUT et al. 2000 ; CRONSHAW et al. 2002 ). Yeast and vertebrate NPCs have a similar structural organization although the vertebrate NPC is significantly larger. Interestingly, two-thirds of nucleoporins are conserved between yeast and vertebrates although overall similarity of individual proteins is not high (CRONSHAW et al. 2002 ). Protein transport through the NPC is typically facilitated by binding of a cargo molecule containing a specific import or export sequence to a specific import or export protein (importin, karyopherin, exportin, or transportin), which shuttles the cargo into or out of the nucleus (reviewed in CHOOK and BLOBEL 2001 ). A subset of NPC proteins (nucleoporins/NUPs), containing multiple FG, GLFG, or GXFG repeats, is thought to bind import/export proteins carrying their cargo and to facilitate transport through the NPC (RADU et al. 1995 ; REXACH and BLOBEL 1995 ; reviewed in RYAN and WENTE 2000 ; CHOOK and BLOBEL 2001 ). Energy for this process is provided by the Ran GTPase system. In the case of nuclear import, cytoplasmic Ran-GDP promotes cargo binding to karyopherins and nuclear Ran-GTP promotes release of cargo following transport into the nucleus (reviewed in ROUT and AITCHISON 2001 ; DASSO 2002 ).

During mitosis in vertebrate cells, the nuclear envelope and NPC disassemble, allowing the spindle to form and likely negating the need for regulated nuclear transport during mitosis. In contrast, A. nidulans and many other simple eukaryotes undergo a closed mitosis in which the nuclear envelope and NPCs remain intact. An interesting question in cell biology is how nuclear transport of mitotic regulators occurs during a closed mitosis.

Here we describe the cloning of A. nidulans sonB. An allele of sonB was first identified as an extragenic suppressor of the nimA1 allele. SONB^NUP98/NUP96 is an FG repeat nucleoporin and a homolog of the human NUP98/NUP96 precursor. Like human NUP98/NUP96, SONB^NUP98/NUP96 contains an autoproteolytic cleavage domain and is processed into distinct N-terminal (SONB^NUP98) and C-terminal (SONB^NUP96) proteins. SONB^NUP98 contains a GLEBS (GLE2-binding sequence) domain that binds SONA^GLE2, and the sonB1 allele contains a point mutation within this domain that compromises SONA^GLE2 binding. sonB1 mutants also display sensitivity to hydroxyurea (HU), suggesting a role in S-phase progression. We isolated the histone H2A-H2B gene pair as a copy-number suppressor of sonB1 phenotypes. Our data suggest that SONA^GLE2 and SONB^NUP98/NUP96 play an important role in the mitotic-specific nuclear entry of NIMA and other regulators of mitosis.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

General techniques:
Media and general techniques for culture of A. nidulans, protein extraction, Western analysis, transformation, immunofluorescence, and 4',6-diamidino-2-phenylindole (DAPI) staining for the chromosome mitotic index were as previously described (OSMANI et al. 1987 , OSMANI et al. 1991 , OSMANI et al. 1994 ; OAKLEY and OSMANI 1993 ; YE et al. 1995 ; WU et al. 1998 ; DE SOUZA et al. 2000 ). Immunoprecipitation of SONA^GLE2-hemagglutinin (HA) was performed using the 12CA5 antibody (Roche) from HK extracts (YE et al. 1995 ). SONB^NUP98/NUP96 antibodies were generated against the following peptides: B1, CQQQPQPMQQSQPAPGS; B2, CLRSIRKNGTPNGVNGV; B3, CEEGESHNQSTLPADRD and affinity purified (Bethyl Laboratories, Montgomery, TX). Cells were grown in 35-mm glass-bottom Microwell dishes (Matteck, Ashland, MA) for confocal microscopy, which was carried out using a Nikon TE300 inverted microscope configured with an Ultraview spinning-disk confocal system (Perkin-Elmer, Norwalk, CT) controlled by Ultraview software.

A. nidulans strains:
Strains used in this study were GR5 (pyrG89; wA3; pyroA4), R153 (wA3; pyroA4), CDS40 (pyrG89; wA2; pyroA4; sonB1^NUP98/NUP96), CDS36 (pyrG89; wA2, nimA1; sonB1^NUP98/NUP96), CDS62 (pyrG89; wA2; pyroA4; sonB1^NUP98/NUP96; pyr4⁺, sonA^GLE2-2xHA C-term tag, extra copy), CDS108 (pyrG89; wA3; pyroA4; pyr4⁺, sonA^GLE2-2xHA C-term tag extra copy), LPW42 (pyrG89; wA2; sonA1^GLE2, pyr4⁺, sonA^GLE2-2xHA C-term tag, multiple copies), CDS91 (pyrG89; wA3; pyroA4; pyr4⁺, alcA::sonB^NUP98/NUP96), CDS170 (pyrG89, gpdp::StuA C-term-DsRedT4 [nuclear localization sequence (NLS)-DsRed]; wA3; pyroA4; green fluorescent protein (GFP)-sonB^NUP98/NUP96), CDS70 (pyrG89; wA2, pyr4⁺, nimA1-4xHA; pyroA4; nicB2 or nicA2), CDS38 (pyrG89; pyr4⁺, extra copy histone H2A-H2B; pyroA4; sonB1^NUP98/NUP96). GR5 was transformed with pLW26 containing sonA^GLE2-HA (WU et al. 1998 ) to generate CDS108 and pCDS20 containing alcA::sonB^NUP98/NUP96 to generate CDS91. CDS40 was transformed with pLW26 to generate CDS62 and with pRG3-H2A-H2B to generate CDS38. LPW3 (pyrG89; wA2, nimA1; pyroA4; nicB2 or nicA2) was transformed with pLW41 containing an N-terminally truncated version of nimA1 C-terminally tagged with four copies of the HA epitope to generate CDS70. CDS165 (pyrG89; wA3; pyroA4; GFP-sonB^NUP98/NUP96) was generated by transforming GR5 with pCDS26 containing a GFP-sonB tagging construct followed by selection for plasmid loss with 5-fluoroorotic acid assessed by loss of the pyr4 marker to leave single-copy GFP-sonB^NUP98/NUP96 under control of its own promotor. CDS170 was generated by cotransformation of CDS165 with a plasmid containing pyrG and pJH19 containing STUA C-term-DsRedT4 (NLS-DsRed). Protein expression of HA-tagged and GFP-tagged constructs was confirmed by immunoblotting of extracts using the 3F10 (Roche) or 12CA5 anti-HA antibodies or the JL-8 anti-GFP antibody (CLONTECH, Palo Alto, CA).

Cloning sonB:
sonB1 mutants were isolated in the same screen described for sonA1 (WU et al. 1998 ). Complementation of sonB1 was carried out by transformation of CDS40 with a pRG3-AMA1 plasmid library, a kind gift from Greg May (OSHEROV and MAY 2000 ), selecting for transformants complemented for growth at 20°. Plasmids recovered from complemented strains by genomic DNA preparation and Escherichia coli transformation were retested for their ability to complement CDS40. Complementing plasmids were subjected to random transposon insertion using the GPS-genome priming system (New England Biolabs, Beverly, MA) and sequenced using transposon-based primers according to the manufacturer's instructions in combination with primer walking. sonB cDNA was obtained by rapid amplification of cDNA ends (RACE)-PCR using the Marathon cDNA amplification kit (CLONTECH), and the 5' and 3' RACE-PCR products were sequenced. To determine the sonB1 mutation, three overlapping segments of sonB1 were amplified from CDS40 genomic DNA by PCR and sequenced. Contig assembly and sequence comparisons were carried out using DNAStar software. Partially complementing plasmids were sequenced by primer walking and all contained the histone H2A-H2B gene pair.

Plasmid constructs:
Plasmid pCDS20 was generated by PCR amplification of two halves of sonB cDNA containing the unique BsiWI site in an overlapping region. Full-length sonB was subcloned into the pAL5 expression vector (DOONAN et al. 1991 ) using the BsiWI site to ligate the two halves of sonB. To generate pCDS26, a PCR product containing 869 bp of the sonB promotor region and genomic DNA encoding the first 844 amino acids of SONB^NUP98/NUP96 was cloned into pRG3 (WARING et al. 1989 ) and mutagenized to introduce NsiI and BglII sites at the start codon to insert plant-adapted GFP (FERNANDEZ-ABALOS et al. 1998 ) in frame. Primers 241 bp 3' of the histone H2B stop codon and 405 bp 3' of the histone H2A stop codon were used to PCR amplify a 2.4-kb fragment containing the histone H2A-H2B gene pair, which was cloned into pRG3 to generate pRG3-H2A-H2B. If PCR amplification was used to clone constructs, sequence fidelity was verified by sequencing.

Targeted disruption of sonB:
Targeted disruption of sonB was performed using standard techniques (DE SOUZA et al. 1999 ) to transform GR5 with pCDS17 containing an internal sonB^NUP98 2651-bp EcoRI/PstI genomic DNA fragment in pRG3, which contains the pyr4 selectable marker. Transformants were selected for growth in the absence of uridine and uracil. Homologous integration at the sonB locus results in duplication with one copy lacking 2954 bp of its 3' end, including its normal termination and processing sequences, and the other copy lacking a promoter and 969 bp of the 5' coding sequence. A 3' truncation occurs prior to the autoproteolytic cleavage site. Putative heterokaryons containing both sonB⁺; pyr4^- nuclei and sonB^-; pyr4⁺ nuclei were identified by streaking uninucleate conidiospores and looking for colony growth on nonselective, but not on selective, media. The presence of a wild-type allele of sonB was confirmed by PCR of heterokaryon genomic DNA using sonB-specific primers in regions disrupted by homologous integration. The presence of the disrupted sonB allele was confirmed using a pRG3-specific primer and a sonB-specific primer outside the region of duplication.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of sonB1 as an extragenic suppressor of nimA1:
To isolate nimA interacting genes, we undertook a screen to isolate extragenic suppressors of the nimA1 temperature-sensitive allele. At restrictive temperature, nimA1 mutants arrest the cell cycle in late G₂ and thus our screen should identify mutations that relieve this G₂ arrest and allow cells to enter mitosis. Following mutagenesis of a nimA1 strain (WU et al. 1998 ), we isolated two independent mutants in which the ability to suppress nimA1 temperature sensitivity also conferred a cold-sensitive phenotype to allow subsequent cloning by complementation of the cold sensitivity (Fig 1). We previously identified the first of these mutants, sonA1, as encoding a homolog of the Gle2/Rae1 protein family of NPC proteins (WU et al. 1998 ). The second mutant, sonB1, suppressed nimA1; however, the sonB1 + nimA1 double mutant is not completely wild type at 42° as sonB1 mutants themselves do not grow as well as wild type (Fig 1). We also tested the nimA5 and nimA7 alleles for genetic interaction with sonB1. In contrast to suppression of the nimA1 allele by sonB1, the nimA5 and nimA7 alleles both displayed synthetic lethal interactions with sonB1 (data not shown). Thus sonB1 is an allele-specific suppressor of the nimA1 allele.

View larger version (71K): In this window In a new window Download PPT slide   Figure 1. Suppression of nimA1 by sonB1. Colony growth phenotype of wild-type (R153), nimA1 (LPW3), nimA1 + sonB1 (CDS36), and sonB1 (CDS40) strains at 20°, 32°, and 42°, showing both suppression of nimA1 by sonB1 at 42° and cold sensitivity of sonB1.

We cloned sonB by complementation of sonB1 cold sensitivity after transformation of a plasmid-based genomic DNA library (OSHEROV and MAY 2000 ). We recovered four complementing plasmids and found that they encoded an overlapping sequence with similarity to domains found in NPC proteins. To determine the coding sequence, we performed 5' and 3' RACE-PCR. Comparison of cDNA and genomic DNA sequences determined that we had isolated a single gene containing four introns and encoding a 1962-amino-acid protein with a predicted molecular weight of 208 kD (accession no. AY223678). We confirmed that we had cloned sonB by sequencing the mutant sonB1 allele and identifying a single point mutation (see below).

Sequence analysis indicates that SONB is a member of the NUP98/NUP96 precursor family of nucleoporins displaying 35.5 and 19.4% amino acid identity to uncharacterized gene products in Neurospora crassa and Caenorhabditis elegans, respectively, 23.3% identity to Schizosaccharomyces pombe NUP189, and 18.3% identity to human NUP98/NUP96. As with other members of this family, sonB contains an N terminus enriched with FG repeats containing 53 within the first third (584 amino acids) of the gene product, 5 of which are GLFG repeats (Fig 2A).

View larger version (27K): In this window In a new window Download PPT slide   Figure 2. Autoproteolytic cleavage of SONBNUP98/NUP96. (A) A schematic diagram showing full-length unprocessed SONBNUP98/NUP96. Vertical lines indicate the relative position of FG repeats. The three amino acids at the site of cleavage are indicated. The GLEBS and autoproteolytic cleavage domains conserved in members of the NUP98/NUP96 precursor family are also indicated. (B) Alignment (ClustalW, Biology Workbench; http://workbench.sdsc.edu/) of the amino acid sequence surrounding the three amino acids (shown in boldface type) at the predicted autoproteolytic cleavage sites of A. nidulans (SONBNUP98/NUP96; AY223678), an uncharacterized gene in N. crassa (EAA31174), S. pombe (NUP189; SPAC1486.05), S. cerevisiae (NUP145; P49687), and human (NUP98/NUP96 precursor; AF071076.1). Human and rat NUP98/NUP96 (NP_112336.1) are identical over this region. Identical (*), conserved strong groups (:), and conserved weak groups (.) are indicated. The full autoproteolytic cleavage domain is not shown for simplicity. (C) The predicted SONBNUP98 and SONBNUP96 proteins generated by autoproteolytic cleavage. The relative positions of the epitopes used to generate the antibodies Ab-B1, Ab-B2, and Ab-B3 are indicated. (D) CDS91 containing alcA::sonB was grown to log phase under repressing conditions (Ac, acetate). SONBNUP98/NUP96 was then expressed by media exchange to inducing conditions (EtOH, ethanol) for 2 hr. Immunoblotting of lysates using antibodies Ab-B1, Ab-B2, and Ab-B3 identify the predicted SONBNUP98 and SONBNUP96 autoproteolytic products. However, full-length SONBNUP98/NUP96 was not detectable. The 100-kD band recognized by Ab-B1 and the 70-kD band recognized by Ab-B2 were not induced by alcA induction, indicating that they are nonspecific cross-reactive bands.

Two spatially conserved domains within the sonB gene product have been previously characterized and are of particular interest. One has been identified as a GLEBS in Saccharomyces cerevisiae NUP116 (BAILER et al. 1998 ) and in human NUP98 (PRITCHARD et al. 1999 ) and is capable of binding the SONA homologs GLE2 and hRae1, respectively. A second conserved domain is involved in autoproteolytic cleavage of mammalian NUP98/NUP96 precursors to generate the distinct NUP98 and NUP96 proteins (FONTOURA et al. 1999 ; ROSENBLUM and BLOBEL 1999 ) and a similar cleavage in S. cerevisiae NUP145 has also been demonstrated (TEIXEIRA et al. 1999 ).

SONB^NUP98/NUP96 is cleaved to generate two distinct proteins:
The predicted site of autoproteolytic cleavage in SONB^NUP98/NUP96 (His-Tyr-Thr; Fig 2A and Fig B) differs from mammalian NUP98/NUP96 (FONTOURA et al. 1999 ; ROSENBLUM and BLOBEL 1999 ), budding yeast NUP145 (TEIXEIRA et al. 1999 ), and S. pombe Nup189 (TANGE et al. 2002 ) in which this sequence is His-Phe-Ser (Fig 2B). However, these are highly conservative substitutions and would still likely allow catalysis of cleavage by the proposed mechanism (ROSENBLUM and BLOBEL 1999 ). To determine whether SONB^NUP98/NUP96 is processed into the predicted SONB^NUP98 and SONB^NUP96 autoproteolytic cleavage products (Fig 2C), we generated antibodies to three independent peptide sequences in SONB^NUP98/NUP96 (Fig 2C). We next placed sonB cDNA under the control of the regulatable alcA promoter and transformed this construct into A. nidulans. Cells were first allowed to grow to log phase before induction of SONB^NUP98/NUP96 expression. Upon induction, a 104-kD doublet was recognized by the two antibodies (Ab-B1 and Ab-B2) raised against independent regions in SONB^NUP98 (Fig 2D). The size of this band is consistent with SONB^NUP98/NUP96 being cleaved at the predicted site. The third antibody, Ab-B3, raised against a region in SONB^NUP96, recognized a specifically induced 120-kD band (Fig 2D). The 120-kD band is not recognized by either of the two N-terminal antibodies and was specifically induced in all transformants examined. This suggests that the SONB^NUP96 migrates more slowly by SDS-PAGE than the predicted 104 kD does, as was previously observed for mammalian NUP96 (FONTOURA et al. 1999 , FONTOURA et al. 2001 ). Both the 104-kD SONB^NUP98 and 120-kD SONB^NUP96 bands were completed by the appropriate immunogenic peptide (data not shown). No bands migrating at the predicted 208 kD for full-length SONB^NUP98/NUP96 were recognized by any of the three peptide-specific antibodies (Fig 2D). These data strongly suggest that, similar to mammalian NUP98/NUP96 and budding yeast NUP145, SONB^NUP98/NUP96 is also autoproteolytically processed. Thus, the conservative substitutions of Tyr for Phe and Thr for Ser at the predicted site of cleavage in SONB^NUP98/NUP96 (Fig 2D) still allow autoproteolytic cleavage, and this may further be supported by substitution of a Thr for a Ser at the predicted cleavage site in an uncharacterized N. crassa homolog (Fig 2B).

sonB has essential functions in A. nidulans:
To determine whether sonB has an essential function(s) in A. nidulans, we performed a targeted gene disruption using the pyr4 gene as a nutritional marker (DE SOUZA et al. 1999 ). If sonB is essential, disruptions can be maintained by formation of a heterokaryon in which pyr4⁺ nuclei containing a disrupted sonB allele, and pyr4^- nuclei containing the wild-type sonB allele, exist in the same cytoplasm (OSMANI et al. 1994 ). Such heterokaryons would produce uninucleate conidiospores containing either (a) the disrupted allele displaying the disruption phenotype or (b) the wild-type allele that will not germinate on selective media due to lack of the pyr4 nutritional marker. Conidiospores from putative heterokaryons unable to form colonies on selective media (Fig 3A) were germinated for 18.5 hr at 32° and examined by DAPI staining for DNA. This revealed ungerminated conidiospores with single nuclei (wild-type sonB⁺; pyr4^-; Fig 3B, arrowhead) and short germlings (sonB disruption; pyr4⁺; Fig 3B). The sonB disruptants all displayed highly disorganized DNA that was fragmented with varying levels of DNA condensation (Fig 3B). The presence of a wild-type allele and a disrupted allele was confirmed by PCR analysis of DNA prepared from the heterokaryon (see MATERIALS AND METHODS). These data indicate that sonB has an essential function(s) in A. nidulans and is required for normal nuclear morphology.

View larger version (53K): In this window In a new window Download PPT slide   Figure 3. Phenotypes associated with sonB disruption and overexpression. (A) Spores from a heterokaryon (containing both sonB+; pyr4- nuclei and nuclei with the sonB disruption that are pyr4+) grow in the presence (YAGUU), but not the absence (YAG), of uridine and uracil (see text). (B) Examination of spores from the same heterokaryon germinated in liquid medium without uridine and uracil (YG) for 18.5 hr at 32° by DAPI staining for DNA. Gross nuclear abnormalities are apparent in the germling (sonB disruption; pyr4+) containing the disrupted sonB allele. Arrowhead indicates a wild-type (sonB+; pyr4-) spore from the heterokaryon, which cannot germinate without uridine and uracil and arrests with a single nucleus. Bar, 3 µm. (C) Cells (CDS91) containing sonB under control of the regulatable alcA promotor grow under repressing (glucose) but not inducing (ethanol) conditions. (D and E) Examination of wild-type (D, R153) and alcA::sonB (E, CDS91) strains germinated for 15 hr at 32° under inducing conditions (minimal medium + 40 mM threonine) after DAPI staining for DNA. Bar, 5 µm. (F) Quantitation of the average number of nuclei per germling under the same conditions as in D and E.

Overexpression studies also suggested that SONB^NUP98/NUP96 has important functions. Strains containing extra copies of sonB under control of the alcA promotor were highly sensitive to overexpression of SONB^NUP98/NUP96 (Fig 3C). This sensitivity was not due to the presence of 208-kD SONB^NUP98/NUP96 as SONB^NUP98 and SONB^NUP96 were rapidly generated (Fig 2D; data not shown). Analysis of germlings grown under inducing conditions indicated that they arrested with a single interphase nucleus characteristic of a nim phenotype (Fig 3E and Fig F) while wild-type strains carried out nuclear division as normal (Fig 3D and Fig F).

sonB1 mutants display sensitivity to hydroxyurea:
sonB1 mutants also displayed significant sensitivity to low concentrations of the ribonucleotide reductase inhibitor HU, which slows progression through S-phase (Fig 4A). The slowed S-phase checkpoint over mitosis in A. nidulans ensures that S-phase is completed before mitotic entry (YE et al. 1996 ). Given that the sonB1 mutation allows nimA1 mutants to enter mitosis under conditions in which they normally arrest in G₂, one explanation for sonB1 HU sensitivity is that such mutants enter mitosis inappropriately during a slowed S-phase. We thus germinated wild-type and sonB1 strains in the presence or absence of 6 mM HU and followed entry into mitosis by assessing DNA morphology (Fig 4B). Wild-type and sonB1 mutants both entered mitosis with similar kinetics in either the presence or the absence of 6 mM HU although both delayed mitotic entry by 1 hr in the presence of 6 mM HU (Fig 4B). Thus sonB1 mutants do not have a defect in the checkpoint that assures completion of S-phase before entry into mitosis.

View larger version (34K): In this window In a new window Download PPT slide   Figure 4. sonB1 HU sensitivity is not due to a defect in the slowed S-phase checkpoint preventing mitosis before completion of DNA replication. (A) Colony-growth phenotypes of a wild-type (R153), sonB1 (CDS40), and sonB1 strain containing an extra copy of histone H2A-H2B (CDS38) at 32° in the presence or absence of 6 mM HU and at 20°. (B) Mitotic entry of a wild-type and sonB1 strain in the presence or absence of 6 mM HU showing that sonB1 mutants do not enter mitosis early during a slowed S-phase. Spores were germinated in the presence of 5 µg/ml nocodazole to trap cells in pseudometaphase arrest and the chromosome mitotic index was accessed after DAPI staining to visualize DNA.

The histone H2A-H2B gene pair act as a copy-number suppressor of sonB1 cold sensitivity and HU sensitivity:
While cloning sonB1 we also isolated transformants, which were partially able to complement sonB1 cold sensitivity. Sequencing of recovered plasmids indicated that they all contained the histone H2A-H2B gene pair. We constructed a plasmid containing just the histone H2A-H2B gene pair and found that this was sufficient to suppress both sonB1 cold sensitivity and sonB1 HU sensitivity (Fig 4A). This suppression required histone H2A and histone H2B in combination, as plasmids containing either one of these genes alone were not able to suppress the sonB1 phenotypes (data not shown). Interestingly, extra copies of the histone H2A-H2B gene pair were not able to suppress the cold sensitivity of the sonA1 allele that was also isolated as an extragenic suppressor of nimA1 (WU et al. 1998 ; data not shown).

NIMA1 is cytoplasmic at the nimA1 arrest point:
We have previously demonstrated that at nimA5 arrest, NIMA5 has yet to accumulate in the nucleus and is predominantly cytoplasmic (DE SOUZA et al. 2000 ). Given that we have now isolated mutations in two nucleoporins that suppress the nimA1 mutation, we wished to determine the localization of NIMA1 during nimA1 arrest. We therefore generated a strain containing a single copy of NIMA1 C-terminally tagged with four copies of the HA epitope. At the nimA1 arrest point, NIMA1 was localized mainly to the cytoplasm (Fig 5A), but when cells were released into mitosis, NIMA1 became enriched in the nucleus (Fig 5B). These results are consistent with what we have observed previously for wild-type NIMA localization in G₂ and mitosis (DE SOUZA et al. 2000 ). For sonB1 to suppress the nimA1 temperature sensitivity, we assumed NIMA1 must be able to enter the nucleus. To determine if this were the case, we examined the localization of NIMA1 in a sonB1 + nimA1 strain containing a single HA-tagged version of NIMA1 at the restrictive temperature for nimA1. We found that cells containing mitotic nuclei did contain nuclear NIMA1 as predicted (data not shown), indicating that the sonB1 NPC mutant allows NIMA1 into the nucleus, thus relieving the G₂ arrest.

View larger version (24K): In this window In a new window Download PPT slide   Figure 5. NIMA1 is cytoplasmic during nimA1 arrest. CDS70 containing HA-tagged nimA1 as its only version of nimA was arrested at the restrictive temperature of 42° and released into mitosis by temperature shift to 32°. Staining is for NIMA1-HA, tubulin, and DNA as labeled. Note nuclear accumulation of NIMA1-HA at prophase. Bar, 3 µm.

GFP-SONB^NUP98 localizes to the nuclear pore complex:
To investigate the localization of SONB^NUP98, we constructed a strain containing single-copy SONB^NUP98/NUP96 N-terminally tagged with GFP and NLS-DsRed. NLS-DsRed is made up of the C terminus of A. nidulans STUA, including its NLS (SUELMANN et al. 1997 ) fused to Discosoma red fluorescent protein (DsRedT4, a kind gift from Reinhard Fischer; BEVIS and GLICK 2002 ). GFP-SONB^NUP98/NUP96 was cleaved to generate GFP-SONB^NUP98 as expected (data not shown). Confocal imaging indicated a characteristic NPC localization for GFP-SONB^NUP98 at the nuclear periphery surrounding the NLS-DsRed within the nucleus, confirming that SONB^NUP98 is a nucleoporin (Fig 6).

View larger version (111K): In this window In a new window Download PPT slide   Figure 6. Localization of GFP-SONBNUP98 to the NPC. A single confocal slice through a germling (CDS170) expressing GFP-SONBNUP98/NUP96 off its own promotor and the nuclear marker NLS-DsRed. (A) The localization of GFP-SONBNUP98 showing a characteristic NPC localization at the nuclear periphery. (B) NLS-DsRed is localized within the nucleus. (C) A differential interference contrast image of the same cell in A and B. Bar, 5 µm.

SONB^NUP98 and SONA^GLE2 are present in a complex in A. nidulans:
The presence of a GLEBS domain in SONB^NUP98 (Fig 2C) suggests that SONB^NUP98 and SONA^GLE2 physically interact. As our peptide-specific antibodies failed to immunoprecipitate SONB^NUP98, we used a strain containing an extra HA-tagged copy of SONA^GLE2 to immunoprecipitate SONA^GLE2-HA with antibodies specific for the HA epitope. If the GLEBS domain in SONB^NUP98 is functional, we should be able to detect endogenous SONB^NUP98 in immunoprecipitates of SONA^GLE2-HA. We were able to specifically immunoprecipitate SONA^GLE2-HA to near completion and these immunoprecipitates also contained a 104-kD doublet recognized by the SONB^NUP98 antibodies, confirming that these nucleoporins physically interact (Fig 7A). This doublet was of identical mobility to that of SONB^NUP98 found in extracts containing ectopically expressed SONB^NUP98/NUP96 (Fig 2D) and was not present when immunoprecipitations were performed using control extracts not containing SONA^GLE2-HA (Fig 7A). Although we were unable to detect the C-terminal cleavage product SONB^NUP96 in SONA^GLE2-HA immunoprecipitates from 3 mg of extract, other NPC proteins were present because the Mab414 antibody (DAVIS and BLOBEL 1986 ) raised against NPC proteins identified several bands present in the SONA^GLE2-HA but not control immunoprecipitates (data not shown).

View larger version (48K): In this window In a new window Download PPT slide   Figure 7. The sonB1 allele is defective for SONAGLE2 binding. (A) SONAGLE2-HA immunoprecipitates from log-phase cultures of a strain containing extra copies of SONAGLE2-HA (LPW42). Protein lysates (Input) and SONAGLE2-HA immunoprecipitates (SONA-HA IP) were tested for SONAGLE2 and SONAGLE2-HA using an anti-SONAGLE2 antibody and for SONBNUP98 using anti-SONBNUP98 Ab-B2. Immunoprecipitates from a strain not containing SONAGLE2-HA (Control IP) were used as a control for specificity. Immunoprecipitations were performed from 3 mg total protein extract and the input represents one-thirtieth of this amount of protein. (B) Alignment (ClustalW, Biology Workbench; http://workbench.sdsc.edu/) of the GLEBS domains from the indicated National Center for Biotechnology Information PubMed sequences from A. nidulans (SONBNUP98/NUP96; AY223678), an uncharacterized gene in N. crassa (EAA31174), S. pombe (NUP189; SPAC1486.05), S. cerevisiae (NUP116; NP_013762), human (NUP98/NUP96 precursor; AF071076.1), and C. elegans (T29008). Human and rat (NP_112336.1) NUP98/NUP96 are identical over this region. Identical (*), conserved strong groups (:), and conserved weak groups (.) are indicated. The glutamic acid mutated to lysine in sonB1 is indicated. (C) Lysates prepared from strains containing an extra copy of SONAGLE2-HA in a wild-type (CDS108) or a sonB1 (CDS62) background were tested for SONBNUP98 or SONB1NUP98 association. "Input" indicates the relative amounts of SONBNUP98/SONB1NUP98 present in 250 µg of lysate or of SONAGLE2 and SONAGLE2-HA in 100 µg lysate. "SONA-HA IP" indicates the relative amounts of SONBNUP98/SONB1NUP98 present in SONAGLE2-HA immunoprecipitates from a 3-mg extract.

sonB1 mutants are defective in binding SONA^GLE2:
As a step toward understanding SONB^NUP98/NUP96 function, we isolated genomic DNA from a sonB1 mutant and determined the sequence of the mutant allele. Analysis indicated that sonB1 contains a single point mutation (GAG AAG at amino acid 193), resulting in a substitution of lysine for a glutamic acid conserved in the GLEBS domains of N. crassa, S. pombe, and S. cerevisiae, although not present in the GLEBS domains of C. elegans or mammalian proteins (Fig 7B). This substitutes a basic for an acidic amino acid within the GLEBS domain, suggesting that the primary defect in sonB1 mutants may be due to a weakened binding of SONB1^NUP98 to SONA^GLE2. To test this, we constructed strains containing an extra copy of SONA^GLE2-HA in either a wild-type or a sonB1 background and tested for the presence of SONB^NUP98 in SONA^GLE2-HA immunoprecipitates. Prior to immunoprecipitation, both strains contained a comparable amount of both SONA^GLE2 and SONA^GLE2-HA (Fig 7C). Although gels had to be overloaded to detect SONB^NUP98 and SONB1^NUP98, both were present at similar levels (Fig 7C). Similar levels of SONA^GLE2-HA were immunoprecipitated from wild-type and sonB1 lysates. In lysate from the wild-type strain, SONB^NUP98 was associated with SONA^GLE2-HA (Fig 7C). In contrast, in three independent experiments there was a pronounced reduction in the level of SONB1^NUP98 in the SONA^GLE2-HA immunoprecipitate from the sonB1 lysate (Fig 7C). This indicates that the mutation in the sonB1 allele compromises the ability of SONB1^NUP98 and SONA^GLE2 to associate. Interestingly, similar experiments in a sonA1 strain containing an extra copy of SONA1^GLE2-HA indicated that SONA1^GLE2-HA and SONB^NUP98 still associated normally (data not shown).

Thus the two genes we have isolated as suppressors of the nimA1 mutation are both nucleoporins and these nucleoporins physically interact. What is more, the point mutation in sonB1 weakens the association of these two nucleoporins.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Nuclear pore complex function at G₂/M:
In screening for mutants that allow mitotic entry from the G₂ nimA1 arrest point, we have isolated two mutations in NPC proteins. The fact that mutations in no other NPC proteins were isolated in the nimA1 suppressor screen and that SONA^GLE2 and SONB^NUP98 physically interact suggests high specificity of the suppression of nimA1 rather than any global defect in NPC architecture. In particular, the fact that the only allele of sonB isolated contains a single point mutation within the GLEBS domain and affects binding of SONA^GLE2 to SONB1^NUP98 further suggests that interaction of these two proteins is specifically important for relieving the G₂ nimA1 arrest. Our data suggest that the sonA1 and sonB1 mutations relieve nimA1 G₂ arrest in part by allowing NIMA1 into the nucleus where it carries out its essential mitotic functions. We previously have shown that the sonA1 mutation allows NIMX^cdc2/NIME^cyclinB into the nucleus, thus suppressing the temperature sensitivity of nimA1 (WU et al. 1998 ). The collective data suggest a specific role for the NPC in the G₂/M transition. Supporting this, overexpression of SONB^NUP98/NUP96 is lethal and leads to a cell-cycle-specific nim phenotype. Given these data, it is of interest that mutants of the fission yeast SONA^GLE2 homolog Rae1 arrest the cell cycle in G₂ with high Cdc2 kinase activity (WHALEN et al. 1997 ), strongly implicating a G₂/M function for this nucleoporin in another system. It is unlikely that the essential function of SONB^NUP98/NUP96 in A. nidulans is its role in the G₂/M transition because the phenotype of the disrupted allele profoundly affects nuclear structure and may reflect other roles of SONB^NUP98/NUP96 homologs in nuclear transport (RADU et al. 1995 ; POWERS et al. 1997 ). Recently, a fission yeast gene encoding a NUP98/NUP96-like protein has also been demonstrated to have an essential function (TANGE et al. 2002 ).

The nimA1 allele is unique in that it contains a point mutation within the noncatalytic domain of NIMA and has a basal level of activity at restrictive temperature (PU et al. 1995 ; WU et al. 1998 ). In contrast, the nimA5 and nimA7 alleles contain point mutations in the catalytic domain and are catalytically inactive at restrictive temperature (PU et al. 1995 ; YE et al. 1995 ). In the nimA1/sonB1 interaction, nuclear entry of catalytically active NIMA1 would likely allow mitotic progression as we have documented. In contrast, in the nimA5/sonB1 or nimA7/sonB1 interactions, entry of inactive NIMA5/NIMA7 to the nucleus is likely to cause defects and the synthetic lethality we have observed.

While our data suggest that sonA1 and sonB1 NPC mutants allow mitotic entry in a process involving NIMA, more experiments are needed to establish a mechanism. Given that many aspects of SONB^NUP98/NUP96 and SONA^GLE2 regulation and function are conserved through evolution, it will be of interest to see if such a mechanism is conserved in higher eukaryotes. However, in contrast to higher eukaryotes, many simple eukaryotes, including S. pombe, S. cerevisiae, and A. nidulans undergo a closed mitosis in which the nuclear envelope remains intact. Our data linking the mitosis-promoting functions of the NIMA kinase with two nucleoporins has begun to address the issue of how nuclear transport occurs during mitosis in organisms that carry out a closed mitosis. Both SONA^GLE2 and SONB^NUP98 are likely to be key elements in this regulation, as is NIMA.

A potential role for SONB^NUP98/NUP96 in S-phase:
The sensitivity of sonB1 mutants to HU suggests a role in S-phase regulation. Although sonB1 can promote mitotic entry from a nimA1 arrest, sonB1 mutants do not enter mitosis early in the presence of HU, indicating that they have an intact slowed S-phase checkpoint ensuring mitosis does not occur before completion of DNA replication. Thus another explanation is needed to explain sensitivity to HU. A clue to this may come from our observation that the histone H2A-H2B gene pair acts as a copy-number suppressor of sonB1 HU sensitivity. The NPC is associated with telomeric chromatin (GALY et al. 2000 ) and recently histones were isolated and identified by mass spectrometry in purified NPC fractions (CRONSHAW et al. 2002 ). Moreover, a residue conserved in A. nidulans histone H2A is phosphorylated on histone H2AX in response to low concentrations of HU (WARD and CHEN 2001 ) and this could potentially be involved in the suppression of sonB1 HU sensitivity by histone H2A-H2B. Experiments are in progress to further understand these intriguing interactions.

NIMA-related kinases and the nuclear envelope in higher eukaryotes:
Our data suggest that NIMA is required for the nuclear entry of NIMA and NIMX^cdc2/NIME^cyclinB in a process involving SONA^GLE2 and SONB^NUP98/NUP96, and we are investigating how NIMA may regulate NPC function at mitosis. One explanation is that phosphorylation of SONB^NUP98/NUP96 and/or SONA^GLE2 by NIMA is required for mitotic NPC regulation. Interestingly, SONB^NUP98 contains 29 NIMA phosphorylation sites corresponding to the consensus sequence Phe-Xaa-Xaa-Ser/Thr (LU et al. 1994 ) while SONA^GLE2 contains 3 NIMA phosphorylation sites. In this regard, mitotic phosphorylation of Xenopus NUP98 has been demonstrated (MACAULAY et al. 1995 ; MILLER et al. 1999 ) and NUP98 proteins have NIMA consensus phosphorylation sites. Mitotic phosphorylation of other nucleoporins has been demonstrated and suggested to be important for the dispersal of NPC components at mitosis (FAVREAU et al. 1996 ). NIMA-related kinases may be important for this because overexpression of NIMA in HeLa cells leads to dispersal of the p62 nucleoporin and nuclear membrane breakdown (LU and HUNTER 1995 ). Evidence of another NIMA-related kinase having a role in nuclear envelope regulation comes from fission yeast that display marked nuclear envelope abnormalities if the NIMA-related kinase fin1 is deleted (KREIN et al. 2002 ). Interestingly, fin1 nulls also display synthetic lethality with pim1-d1, a mutant of the RCC1 homolog pim1 involved in nuclear transport (KREIN et al. 1998 , KREIN et al. 2002 ). In addition, fin1 nulls are not viable in combination with temperature-sensitive alleles of cut11 whose gene product colocalizes to the NPC in interphase and mitotic spindle pole bodies (WEST et al. 1998 ; KREIN et al. 2002 ). It will be of interest to see whether any of the 11 NIMA-related kinases identified in humans plays a role in nuclear transport and/or NPC function during mitosis.

Finally, chromosomal rearrangements in many human hematologic malignancies involve NUP98, resulting in NUP98 chimeric proteins that are likely oncogenic (for review see LAM and APLAN 2001 ). Interestingly, all of these fusions contain the FG repeat region of NUP98 that contains many consensus NIMA phosphorylation sites. The data presented here implicate a role for SONB^NUP98 in mitotic progression, and any potential roles that human NUP98 may have in mitotic regulation, or response to delayed DNA replication, may contribute to the oncogenic properties of these NUP98 chimeric proteins.

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession no. AY223678.
¹ Present address: Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106.
² Present address: Weis Center for Research, Geisinger Clinic, Danville, PA 17822.

	ACKNOWLEDGMENTS

We thank all members of the Osmani laboratory and Xiang Ye (Lilly Research Laboratories, Eli Lilly, Indianapolis) for their interest and input into this work. We also thank Greg May and Nir Osherov (The University of Texas, M. D. Anderson Cancer Center) for their gift of the pRG3-AMA-1 library and technical advice, John Donnan (John Innes Centre, Norwich, UK) for the plant adapted-GFP plasmid, and Reinhard Fischer (Max-Planck-Institut, Marburg, Germany) for the pJH19 plasmid. This work was supported by National Institutes of Health grant GM-42564.

Manuscript received May 14, 2003; Accepted for publication June 27, 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ALLEN, N. P., L. HUANG, A. BURLINGAME, and M. REXACH, 2001  Proteomic analysis of nucleoporin interacting proteins. J. Biol. Chem. 276:29268-29274.[Abstract/Free Full Text]

BAILER, S. M., S. SINIOSSOGLOU, A. PODTELEJNIKOV, A. HELLWIG, and M. MANN et al., 1998  Nup116p and nup100p are interchangeable through a conserved motif which constitutes a docking site for the mRNA transport factor gle2p. EMBO J. 17:1107-1119.[Medline]

BEVIS, B. J. and B. S. GLICK, 2002  Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol. 20:83-87.[Medline]

BROWN, J. A., A. BHARATHI, A. GHOSH, W. WHALEN, and E. FITZGERALD et al., 1995  A mutation in the Schizosaccharomyces pombe rae1 gene causes defects in poly(A)+ RNA export and in the cytoskeleton. J. Biol. Chem. 270:7411-7419.[Abstract/Free Full Text]

CHOOK, Y. M. and G. BLOBEL, 2001  Karyopherins and nuclear import. Curr. Opin. Struct. Biol. 11:703-715.[Medline]

CRONSHAW, J. M., A. N. KRUTCHINSKY, W. ZHANG, B. T. CHAIT, and M. J. MATUNIS, 2002  Proteomic analysis of the mammalian nuclear pore complex. J. Cell Biol. 158:915-927.[Abstract/Free Full Text]

DASSO, M., 2002  The Ran GTPase: theme and variations. Curr. Biol. 12:R502.[Medline]

DAVIS, L. I. and G. BLOBEL, 1986  Identification and characterization of a nuclear pore complex protein. Cell 45:699-709.[Medline]

DE SOUZA, C. P., X. S. YE, and S. A. OSMANI, 1999  Checkpoint defects leading to premature mitosis also cause endoreplication of DNA in Aspergillus nidulans.. Mol. Biol. Cell 10:3661-3674.[Abstract/Free Full Text]

DE SOUZA, C. P., A. H. OSMANI, L. P. WU, J. L. SPOTTS, and S. A. OSMANI, 2000  Mitotic histone H3 phosphorylation by the NIMA kinase in Aspergillus nidulans.. Cell 102:293-302.[Medline]

DOONAN, J. H., C. MACKINTOSH, P. COHEN, S. A. OSMANI, and E. LEE et al., 1991  A cDNA encoding rabbit muscle protein phosphatase 1 complements the Aspergillus cell cycle mutation, bimG11. J. Biol. Chem. 266:18889-18894.[Abstract/Free Full Text]

FAVREAU, C., H. J. WORMAN, R. W. WOZNIAK, T. FRAPPIER, and J. C. COURVALIN, 1996  Cell cycle-dependent phosphorylation of nucleoporins and nuclear pore membrane protein Gp210. Biochemistry 35:8035-8044.[Medline]

FERNANDEZ-ABALOS, J. M., H. FOX, C. PITT, B. WELLS, and J. H. DOONAN, 1998  Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans.. Mol. Microbiol. 27:121-130.[Medline]

FONTOURA, B. M., G. BLOBEL, and M. J. MATUNIS, 1999  A conserved biogenesis pathway for nucleoporins: proteolytic processing of a 186-kilodalton precursor generates Nup98 and the novel nucleoporin, Nup96. J. Cell Biol. 144:1097-1112.[Abstract/Free Full Text]

FONTOURA, B. M., S. DALES, G. BLOBEL, and H. ZHONG, 2001  The nucleoporin Nup98 associates with the intranuclear filamentous protein network of TPR. Proc. Natl. Acad. Sci. USA 98:3208-3213.[Abstract/Free Full Text]

GALY, V., J.-C. OLIVO-MARIN, H. SCHERTHAN, V. DOYE, and N. RASCALOU et al., 2000  Nuclear pore complexes in the organization of silent telomeric chromatin. Nature 403:108-112.[Medline]

KREIN, M. J., S. J. BUGG, M. PALATSIDES, G. ASOULINE, and M. MORIMYO et al., 1998  A NIMA homologue promotes chromatin condensation in fission yeast. J. Cell Sci. 111:967-976.[Abstract]

KREIN, M. J., R. R. WEST, U. P. JOHN, K. KONIARAS, and J. R. MCINTOSH et al., 2002  The fission yeast NIMA kinase Fin1p is required for spindle function and nuclear envelope integrity. EMBO J. 21:1713-1722.[Medline]

LAM, D. H. and P. D. APLAN, 2001  NUP98 gene fusions in hematologic malignancies. Leukemia 15:1689-1695.[Medline]

LU, K. P. and T. HUNTER, 1995  Evidence for a NIMA-like mitotic pathway in vertebrate cells. Cell 81:413-424.[Medline]

LU, K. P., B. E. KEMP, and A. R. MEANS, 1994  Identification of substrate specificity determinants for the cell cycle-regulated NIMA protein kinase. J. Biol. Chem. 269:6603-6607.[Abstract/Free Full Text]

MACAULAY, C., E. MEIER, and D. J. FORBES, 1995  Differential mitotic phosphorylation of proteins of the nuclear pore complex. J. Biol. Chem. 270:254-262.[Abstract/Free Full Text]

MILLER, M. W., M. R. CARACCIOLO, W. K. BERLIN, and J. A. HANOVER, 1999  Phosphorylation and glycosylation of nucleoporins. Arch. Biochem. Biophys. 367:51-60.[Medline]

MURPHY, R., J. L. WATKINS, and S. R. WENTE, 1996  GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function. Mol. Biol. Cell 7:1921-1937.[Abstract]

OAKLEY, B. R., and S. A. OSMANI, 1993 Cell-cycle analysis using the filamentous fungus Aspergillus nidulans, pp. 127–142 in The Cell Cycle: A Practical Approach, edited by P. FANTES and R. BROOKS. IRL Press/Oxford University Press, Oxford/New York.

OSHEROV, N. and G. S. MAY, 2000  Conidial germination in Aspergillus nidulans requires RAS signaling and protein synthesis. Genetics 155:647-656.[Abstract/Free Full Text]

OSMANI, A. H., S. L. MCGUIRE, and S. A. OSMANI, 1991  Parallel activation of the NIMA and p34^cdc2 cell cycle-regulated protein kinases is required to initiate mitosis in A. nidulans. Cell 67:283-291.[Medline]

OSMANI, A. H., N. VAN PEIJ, M. MISCHKE, M. J. O'CONNELL, and S. A. OSMANI, 1994  A single p34^cdc2 protein kinase (nimX^cdc2) is required at G1 and G2 in Aspergillus nidulans.. J. Cell Sci. 107:1519-1528.[Abstract]

OSMANI, S. A., G. S. MAY, and N. R. MORRIS, 1987  Regulation of the mRNA levels of nimA, a gene required for the G2-M transition in Aspergillus nidulans.. J. Cell Biol. 104:1495-1504.[Abstract/Free Full Text]

POWERS, M. A., D. J. FORBES, J. E. DAHLBERG, and E. LUND, 1997  The vertebrate GLFG nucleoporin, Nup98 is an essential component of multiple RNA export pathways. J. Cell Biol. 136:241-250.[Abstract/Free Full Text]

PRITCHARD, C. E., M. FORNEROD, L. H. KASPER, and J. M. VAN DEURSEN, 1999  RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains. J. Cell Biol. 145:237-254.[Abstract/Free Full Text]

PU, R. T., G. XU, L. WU, J. VIERULA, and K. O'DONNELL et al., 1995  Isolation of a functional homolog of the cell cycle specific NIMA protein kinase and functional analysis of conserved residues. J. Biol. Chem. 271:18110-18116.

RADU, A., M. S. MOORE, and G. BLOBEL, 1995  The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex. Cell 81:215-222.[Medline]

REXACH, M. and G. BLOBEL, 1995  Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell 83:683-692.[Medline]

ROSENBLUM, J. S. and G. BLOBEL, 1999  Autoproteolysis in nucleoporin biogenesis. Proc. Natl. Acad. Sci. USA 96:11370-11375.[Abstract/Free Full Text]

ROUT, M. P. and J. D. AITCHISON, 2001  The nuclear pore complex as a transport machine. J. Biol. Chem. 276:16593-16596.[Free Full Text]

ROUT, M. P., J. D. AITCHISON, A. SUPRAPTO, K. HJERTAAS, and Y. ZHAO et al., 2000  The yeast nuclear pore complex: composition, architecture, and transport mechanism. J. Cell Biol. 148:635-651.[Abstract/Free Full Text]

RYAN, K. J. and S. R. WENTE, 2000  The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm. Curr. Opin. Cell Biol. 12:361-371.[Medline]

SUELMANN, R., N. SIEVERS, and R. FISCHER, 1997  Nuclear traffic in fungal hyphae: in vivo study of nuclear migration and positioning in Aspergillus nidulans.. Mol. Microbiol. 25:757-769.[Medline]

TANGE, Y, A. HIRATA, and O. NIWA, 2002  An evolutionarily conserved fission yeast protein, Ned1, implicated in normal nuclear morphology and chromosome stability, interacts with Dis3, Pim1/RCC1 and an essential nucleoporin. J. Cell Sci. 115:4375-4385.[Abstract/Free Full Text]

TEIXEIRA, M. T., E. FABRE, and B. DUJON, 1999  Self-catalyzed cleavage of the yeast nucleoporin Nup145p precursor. J. Biol. Chem. 274:32439-32444.[Abstract/Free Full Text]

WARD, I. M. and J. CHEN, 2001  Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J. Biol. Chem. 276:47759-47762.[Abstract/Free Full Text]

WARING, R. B., G. S. MAY, and N. R. MORRIS, 1989  Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes. Gene 79:119-130.[Medline]

WENTE, S. R., 2000  Gatekeepers of the nucleus. Science 288:1374-1377.[Abstract/Free Full Text]

WEST, R. R., E. V. VAISBERG, R. DING, P. NURSE, and J. R. MCINTOSH, 1998  cut11(+): a gene required for cell cycle-dependent spindle pole body anchoring in the nuclear envelope and bipolar spindle formation in Schizosaccharomyces pombe.. Mol. Biol. Cell 9:2839-2855.[Abstract/Free Full Text]

WHALEN, W. A., A. BHARATHI, D. DANIELEWICZ, and R. DHAR, 1997  Advancement through mitosis requires rae1 gene function in fission yeast. Yeast 13:1167-1179.[Medline]

WU, L., S. A. OSMANI, and P. M. MIRABITO, 1998  A role for NIMA in the nuclear localization of cyclin B in Aspergillus nidulans.. J. Cell Biol. 141:1575-1587.[Abstract/Free Full Text]

YE, X. S., G. XU, P. T. PU, R. R. FINCHER, and S. L. MCGUIRE et al., 1995  The NIMA protein kinase is hyperphosphorylated and activated downstream of p34^cdc2/cyclin B: coordination of two mitosis promoting kinases. EMBO J. 14:986-994.[Medline]

YE, X. S., R. R. FINCHER, A. TANG, K. O'DONNELL, and S. A. OSMANI, 1996  Two S-phase checkpoint systems, one involving the function of both BIME and Tyr15 phosphorylation of p34^cdc2, inhibit NIMA and prevent premature mitosis. EMBO J. 15:3599-3610.[Medline]

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