Modulation of sensitivity to gaseous signaling by sterol-regulatory hypoxic transcription factors in Aspergillus nidulans biofilm cells.

Fungal biofilm founder cells experience self-generated hypoxia leading to dramatic changes in their cell biology. For example, during Aspergillus nidulans biofilm formation microtubule (MT) disassembly is triggered causing dispersal of EB1 from MT tips. This process is dependent on SrbA, a sterol regulatory element-binding transcription factor required for adaptation to hypoxia. We show that SrbA, an ER resident protein prior to activation, is proteolytically activated during early stages of biofilm formation and that, like SrbA itself, its activating proteases are also required for normal biofilm MT disassembly. In addition to SrbA, the AtrR transcription factor is also found to be required to modulate cellular responses to gaseous signaling during biofilm development. Using co-cultures, we further show that cells lacking srbA or atrR are capable of responding to biofilm generated gaseous microenvironments but are actually more sensitive to this signal than wild type cells. SrbA is a regulator of ergosterol biosynthetic genes and we find that the levels of seven GFP-tagged Erg proteins differentially accumulate during biofilm formation with various dependencies on SrbA for their accumulation. This uncovers a complex pattern of regulation with biofilm accumulation of only some Erg proteins being dependent on SrbA with others accumulating to higher levels in its absence. Because different membrane sterols are known to influence cell permeability to gaseous molecules, including oxygen, we propose that differential regulation of ergosterol biosynthetic proteins by SrbA potentially calibrates the cell's responsiveness to gaseous signaling which in turn modifies the cell biology of developing biofilm cells.

Aspergillus nidulans biofilm formation modifies cellular architecture and enables light-activated autophagy.

After growing on surfaces, including those of medical and industrial importance, fungal biofilms self-generate internal microenvironments. We previously reported that gaseous microenvironments around founder Aspergillus nidulans cells change during biofilm formation causing microtubules to disassemble under control of the hypoxic transcription factor SrbA. Here we investigate if biofilm formation might also promote changes to structures involved in exocytosis and endocytosis. During biofilm formation, the endoplasmic reticulum (ER) remained intact but ER exit sites and the Golgi apparatus were modified as were endocytic actin patches. The biofilm-driven changes required the SrbA hypoxic transcription factor and could be triggered by nitric oxide, further implicating gaseous regulation of biofilm cellular architecture. By tracking green fluorescent protein (GFP)-Atg8 dynamics, biofilm founder cells were also observed to undergo autophagy. Most notably, biofilm cells that had undergone autophagy were triggered into further autophagy by spinning disk confocal light. Our findings indicate that fungal biofilm formation modifies the secretory and endocytic apparatus and show that biofilm cells can also undergo autophagy that is reactivated by light. The findings provide new insights into the changes occurring in fungal biofilm cell biology that potentially impact their unique characteristics, including antifungal drug resistance.

The spindle pole-body localization of activated cytoplasmic dynein is cell cycle-dependent in Aspergillus nidulans.

Cytoplasmic dynein is a minus end-directed microtubule motor that can be activated by cargo adapters. In Aspergillus nidulans, overexpression of ΔC-HookA, the early endosomal adapter HookA missing its cargo-binding site, causes activated dynein to accumulate at septa and spindle pole bodies (SPBs) where the microtubule-organizing centers are located. Intriguingly, only some interphase nuclei show SPB signals of dynein. Here we present data demonstrating that localization of the activated dynein at SPBs is cell cycle-dependent: SPB dynein signals are seen to associate with nuclei at early G1 but disappear at about the G1-S boundary.

SUMOlock reveals a more complete Aspergillus nidulans SUMOylome.

SUMOylation, covalent attachment of the small ubiquitin-like modifier protein SUMO to proteins, regulates protein interactions and activity and plays a crucial role in the regulation of many key cellular processes. Understanding the roles of SUMO in these processes ultimately requires identification of the proteins that are SUMOylated in the organism under study. The filamentous fungus Aspergillus nidulans serves as an excellent model for many aspects of fungal biology, and it would be of great value to determine the proteins that are SUMOylated in this organism (i.e. its SUMOylome). We have developed a new and effective approach for identifying SUMOylated proteins in this organism in which we lock proteins in their SUMOylated state, affinity purify SUMOylated proteins using the high affinity S-tag, and identify them using sensitive Orbitrap mass spectroscopy. This approach allows us to distinguish proteins that are SUMOylated from proteins that are binding partners of SUMOylated proteins or are bound non-covalently to SUMO. This approach has allowed us to identify 149 proteins that are SUMOylated in A. nidulans. Of these, 67 are predicted to be involved in transcription and particularly in the regulation of transcription, 21 are predicted to be involved in RNA processing and 16 are predicted to function in DNA replication or repair.

Nup2 performs diverse interphase functions in Aspergillus nidulans.

The nuclear pore complex (NPC) protein Nup2 plays interphase nuclear transport roles and in Aspergillus nidulans also functions to bridge NPCs at mitotic chromatin for their faithful coinheritance to daughter G1 nuclei. In this study, we further investigate the interphase functions of Nup2 in A. nidulans. Although Nup2 is not required for nuclear import of all nuclear proteins after mitosis, it is required for normal G1 nuclear accumulation of the NPC nuclear basket-associated components Mad2 and Mlp1 as well as the THO complex protein Tho2. Targeting of Mlp1 to nuclei partially rescues the interphase delay seen in nup2 mutants indicating that some of the interphase defects in Nup2-deleted cells are due to Mlp1 mislocalization. Among the inner nuclear membrane proteins, Nup2 affects the localization of Ima1, orthologues of which are involved in nuclear movement. Interestingly, nup2 mutant G1 nuclei also exhibit an abnormally long period of extensive to-and-fro movement immediately after mitosis in a manner dependent on the microtubule cytoskeleton. This indicates that Nup2 is required to limit the transient postmitotic nuclear migration typical of many filamentous fungi. The findings reveal that Nup2 is a multifunctional protein that performs diverse functions during both interphase and mitosis in A. nidulans.

Tools for retargeting proteins within Aspergillus nidulans.

Endogenously tagging proteins with green fluorescent protein (GFP) enables the visualization of the tagged protein using live cell microscopy. GFP-tagging is widely utilized to study biological processes in model experimental organisms including filamentous fungi such as Aspergillus nidulans. Many strains of A. nidulans have therefore been generated with different proteins endogenously tagged with GFP. To further enhance experimental approaches based upon GFP-tagging, we have adapted the GFP Binding Protein (GBP) system for A. nidulans. GBP is a genetically encoded Llama single chain antibody against GFP which binds GFP with high affinity. Using gene replacement approaches, it is therefore possible to link GBP to anchor proteins, which will then retarget GFP-tagged proteins away from their normal location to the location of the anchor-GBP protein. To facilitate this approach in A. nidulans, we made four base plasmid cassettes that can be used to generate gene replacement GBP-tagging constructs by utilizing fusion PCR. Using these base cassettes, fusion PCR, and gene targeting approaches, we generated strains with SPA10-GBP and Tom20-GBP gene replacements. These strains enabled test targeting of GFP-tagged proteins to septa or to the surface of mitochondria respectively. SPA10-GBP is shown to effectively target GFP-tagged proteins to both forming and mature septa. Tom20-GBP has a higher capacity to retarget GFP-tagged proteins being able to relocate all Nup49-GFP from its location within nuclear pore complexes (NPCs) to the cytoplasm in association with mitochondria. Notably, removal of Nup49-GFP from NPCs causes cold sensitivity as does deletion of the nup49 gene. The cassette constructs described facilitate experimental approaches to generate precise protein-protein linkages in fungi. The A. nidulans SPA10-GBP and Tom20-GBP strains can be utilized to modulate other GFP-tagged proteins of interest.

Microtubule-organizing centers of Aspergillus nidulans are anchored at septa by a disordered protein.

Microtubule-organizing centers (MTOCs) are large, multi-subunit protein complexes. Schizosaccharomyces pombe harbors MTOCs at spindle pole bodies, transient MTOCs in the division plane (eMTOCs) and nuclear-envelope associated MTOCs in interphase cells (iMTOCs). In the filamentous fungus Aspergillus nidulans SPBs and septum-associated MTOCs were described. Although comparable to S. pombe eMTOCs, A. nidulans sMTOCS are permanent septum-associated structures. The composition of sMTOCs is poorly understood and how they are targeted to septa was unknown. Here, we show that in A. nidulans several SPB outer plaque proteins also locate to sMTOCs while other SPB proteins do not, including SfiA, a protein required for SPB duplication in Saccharomyces cerevisiae and S. pombe and PcpA, the anchor for γ-TuSCs at the SPB inner plaque. The A. nidulans disordered protein Spa18Mto2 and the centrosomin-domain containing protein ApsBMto1 were required for recruiting the γ-TuRC component GcpC to sMTOCs and for seeding MT formation from septa. Testing different septum-associated proteins for a role in sMTOC function, Spa10 was identified. It forms a septal pore disc structure, recruits Spa18 and ApsB to septa and is required for sMTOC activity. This is the first evidence for a septum-specific protein, Spa10, as anchor for a specific class of MTOCs.

Mitotic nuclear pore complex segregation involves Nup2 in Aspergillus nidulans.

Transport through nuclear pore complexes (NPCs) during interphase is facilitated by the nucleoporin Nup2 via its importin α- and Ran-binding domains. However, Aspergillus nidulans and vertebrate Nup2 also locate to chromatin during mitosis, suggestive of mitotic functions. In this study, we report that Nup2 is required for mitotic NPC inheritance in A. nidulans Interestingly, the role of Nup2 during mitotic NPC segregation is independent of its importin α- and Ran-binding domains but relies on a central targeting domain that is necessary for localization and viability. To test whether mitotic chromatin-associated Nup2 might function to bridge NPCs with chromatin during segregation, we provided an artificial link between NPCs and chromatin via Nup133 and histone H1. Using this approach, we bypassed the requirement of Nup2 for NPC segregation. This indicates that A. nidulans cells ensure accurate mitotic NPC segregation to daughter nuclei by linking mitotic DNA and NPC segregation via the mitotic specific chromatin association of Nup2.

Location and functional analysis of the Aspergillus nidulans Aurora kinase confirm mitotic functions and suggest non-mitotic roles.

Filamentous fungi have devastating negative impacts as pathogens and agents of food spoilage but also have critical ecological importance and are utilized for industrial applications. The characteristic multinucleate nature of filamentous fungi is facilitated by limiting if, when and where septation, the fungal equivalent of cytokinesis, occurs. In the model filamentous fungus Aspergillus nidulans septation does not occur immediately after mitosis and is an incomplete process resulting in the formation of a septal pore whose permeability is cell cycle regulated. How mitotic regulators, such as the Aurora kinase, contribute to the often unique biology of filamentous fungi is not well understood. The Aurora B kinase has not previously been investigated in any detail during hyphal growth. Here we demonstrate for the first time that Aurora displays cell cycle dependent locations to the region of forming septa, the septal pore and mature septa as well as the mitotic apparatus. To functionally analyze Aurora, we generated a temperature sensitive allele revealing essential mitotic and spindle assembly checkpoint functions consistent with its location to the kinetochore region and spindle midzone. Our analysis also reveals that cellular and kinetochore Aurora levels increase during a mitotic spindle assembly checkpoint arrest and we propose that this could be important for checkpoint inactivation when spindle formation is prevented. We demonstrate that Aurora accumulation at mature septa following mitotic entry does not require mitotic progression but is dependent upon a timing mechanism. Surprisingly we also find that Aurora inactivation leads to cellular swelling and lysis indicating an unexpected function for Aurora in fungal cell growth. Thus in addition to its conserved mitotic functions our data suggest that Aurora has the capacity to be an important regulator of septal biology and cell growth in filamentous fungi.

Microtubules are reversibly depolymerized in response to changing gaseous microenvironments within Aspergillus nidulans biofilms.

How microtubules (MTs) are regulated during fungal biofilm formation is unknown. By tracking MT +end-binding proteins (+TIPS) in Aspergillus nidulans, we find that MTs are regulated to depolymerize within forming fungal biofilms. During this process, EB1, dynein, and ClipA form transient fibrous and then bar-like structures, novel configurations for +TIPS. Cells also respond in an autonomous manner, with cells separated by a septum able to maintain different MT dynamics. Surprisingly, all cells with depolymerized MTs rapidly repolymerize their MTs after air exchange above the static culture medium of biofilms. Although the specific gasotransmitter for this biofilm response is not known, we find that addition of hydrogen sulfide gas to growing cells recapitulates all aspects of reversible MT depolymerization and transient formation of +TIPs bars. However, as biofilms mature, physical removal of part of the biofilm is required to promote MT repolymerization, which occurs at the new biofilm edge. We further show MT depolymerization within biofilms is regulated by the SrbA hypoxic transcription factor and that without SrbA, MTs are maintained as biofilms form. This reveals a new mode of MT regulation in response to changing gaseous biofilm microenvironments, which could contribute to the unique characteristics of fungal biofilms in medical and industrial settings.

A mitotic nuclear envelope tether for Gle1 also impacts nuclear and nucleolar architecture.

During Aspergillus nidulans mitosis peripheral nuclear pore complex (NPC) proteins (Nups) disperse from the core NPC structure. Unexpectedly, one predicted peripheral Nup, Gle1, remains at the mitotic NE via an unknown mechanism. Gle1 affinity purification identified MtgA ( M: itotic T: ether for G: le1), which tethers Gle1 to the NE during mitosis, but not during interphase when Gle1 is at NPCs. MtgA is the ortholog of the Schizosaccharomyces pombe telomere-anchoring inner nuclear membrane protein Bqt4. Like Bqt4, MtgA has meiotic roles but is functionally distinct from Bqt4 as MtgA is not required for tethering telomeres to the NE. Domain analyses revealed MtgA targeting to the NE requires its C-terminal transmembrane domain and a nuclear localization signal. Importantly, MtgA functions beyond Gle1 mitotic targeting and meiosis and impacts nuclear and nucleolar architecture when deleted or overexpressed. Deletion of MtgA generates small, round nuclei whereas overexpressing MtgA generates larger nuclei with altered nuclear compartmentalization resulting from NE expansion around the nucleolus. The accumulation of MtgA around the nucleolus promotes a similar accumulation of the endoplasmic reticulum (ER) protein Erg24 lowering its levels in the ER. This study extends the functions of Bqt4-like proteins to include mitotic Gle1 targeting and modulation of nuclear and nucleolar architecture.

The Inner Nuclear Membrane Protein Src1 Is Required for Stable Post-Mitotic Progression into G1 in Aspergillus nidulans.

How membranes and associated proteins of the nuclear envelope (NE) are assembled specifically and inclusively around segregated genomes during exit from mitosis is incompletely understood. Inner nuclear membrane (INM) proteins play key roles by providing links between DNA and the NE. In this study we have investigated the highly conserved INM protein Src1 in Aspergillus nidulans and have uncovered a novel cell cycle response during post mitotic formation of G1 nuclei. Live cell imaging indicates Src1 could have roles during mitotic exit as it preferentially locates to the NE abscission points during nucleokinesis and to the NE surrounding forming daughter G1 nuclei. Deletion analysis further supported this idea revealing that although Src1 is not required for interphase progression or mitosis it is required for stable post-mitotic G1 nuclear formation. This conclusion is based upon the observation that in the absence of Src1 newly formed G1 nuclei are structurally unstable and immediately undergo architectural modifications typical of mitosis. These changes include NPC modifications that stop nuclear transport as well as disassembly of nucleoli. More intriguingly, the newly generated G1 nuclei then cycle between mitotic- and interphase-like states. The findings indicate that defects in post-mitotic G1 nuclear formation caused by lack of Src1 promote repeated failed attempts to generate stable G1 nuclei. To explain this unexpected phenotype we suggest a type of regulation that promotes repetition of defective cell cycle transitions rather than preventing progression past the defective cell cycle transition. We suggest the term "reboot regulation" to define this mode of cell cycle regulation. The findings are discussed in relationship to recent studies showing the Cdk1 master oscillator can entrain subservient oscillators that when uncoupled cause cell cycle transitions to be repeated.

Nup2 requires a highly divergent partner, NupA, to fulfill functions at nuclear pore complexes and the mitotic chromatin region.

Chromatin and nuclear pore complexes (NPCs) undergo dramatic changes during mitosis, which in vertebrates and Aspergillus nidulans involves movement of Nup2 from NPCs to the chromatin region to fulfill unknown functions. This transition is shown to require the Cdk1 mitotic kinase and be promoted prematurely by ectopic expression of the NIMA kinase. Nup2 localizes with a copurifying partner termed NupA, a highly divergent yet essential NPC protein. NupA and Nup2 locate throughout the chromatin region during prophase but during anaphase move to surround segregating DNA. NupA function is shown to involve targeting Nup2 to its interphase and mitotic locations. Deletion of either Nup2 or NupA causes identical mitotic defects that initiate a spindle assembly checkpoint (SAC)-dependent mitotic delay and also cause defects in karyokinesis. These mitotic problems are not caused by overall defects in mitotic NPC disassembly-reassembly or general nuclear import. However, without Nup2 or NupA, although the SAC protein Mad1 locates to its mitotic locations, it fails to locate to NPCs normally in G1 after mitosis. Collectively the study provides new insight into the roles of Nup2 and NupA during mitosis and in a surveillance mechanism that regulates nucleokinesis when mitotic defects occur after SAC fulfillment.

The Set1/COMPASS histone H3 methyltransferase helps regulate mitosis with the CDK1 and NIMA mitotic kinases in Aspergillus nidulans.

Mitosis is promoted and regulated by reversible protein phosphorylation catalyzed by the essential NIMA and CDK1 kinases in the model filamentous fungus Aspergillus nidulans. Protein methylation mediated by the Set1/COMPASS methyltransferase complex has also been shown to regulate mitosis in budding yeast with the Aurora mitotic kinase. We uncover a genetic interaction between An-swd1, which encodes a subunit of the Set1 protein methyltransferase complex, with NIMA as partial inactivation of nimA is poorly tolerated in the absence of swd1. This genetic interaction is additionally seen without the Set1 methyltransferase catalytic subunit. Importantly partial inactivation of NIMT, a mitotic activator of the CDK1 kinase, also causes lethality in the absence of Set1 function, revealing a functional relationship between the Set1 complex and two pivotal mitotic kinases. The main target for Set1-mediated methylation is histone H3K4. Mutational analysis of histone H3 revealed that modifying the H3K4 target residue of Set1 methyltransferase activity phenocopied the lethality seen when either NIMA or CDK1 are partially functional. We probed the mechanistic basis of these genetic interactions and find that the Set1 complex performs functions with CDK1 for initiating mitosis and with NIMA during progression through mitosis. The studies uncover a joint requirement for the Set1 methyltransferase complex with the CDK1 and NIMA kinases for successful mitosis. The findings extend the roles of the Set1 complex to include the initiation of mitosis with CDK1 and mitotic progression with NIMA in addition to its previously identified interactions with Aurora and type 1 phosphatase in budding yeast.

Application of a new dual localization-affinity purification tag reveals novel aspects of protein kinase biology in Aspergillus nidulans.

Filamentous fungi occupy critical environmental niches and have numerous beneficial industrial applications but devastating effects as pathogens and agents of food spoilage. As regulators of essentially all biological processes protein kinases have been intensively studied but how they regulate the often unique biology of filamentous fungi is not completely understood. Significant understanding of filamentous fungal biology has come from the study of the model organism Aspergillus nidulans using a combination of molecular genetics, biochemistry, cell biology and genomic approaches. Here we describe dual localization-affinity purification (DLAP) tags enabling endogenous N or C-terminal protein tagging for localization and biochemical studies in A. nidulans. To establish DLAP tag utility we endogenously tagged 17 protein kinases for analysis by live cell imaging and affinity purification. Proteomic analysis of purifications by mass spectrometry confirmed association of the CotA and NimXCdk1 kinases with known binding partners and verified a predicted interaction of the SldABub1/R1 spindle assembly checkpoint kinase with SldBBub3. We demonstrate that the single TOR kinase of A. nidulans locates to vacuoles and vesicles, suggesting that the function of endomembranes as major TOR cellular hubs is conserved in filamentous fungi. Comparative analysis revealed 7 kinases with mitotic specific locations including An-Cdc7 which unexpectedly located to mitotic spindle pole bodies (SPBs), the first such localization described for this family of DNA replication kinases. We show that the SepH septation kinase locates to SPBs specifically in the basal region of apical cells in a biphasic manner during mitosis and again during septation. This results in gradients of SepH between G1 SPBs which shift along hyphae as each septum forms. We propose that SepH regulates the septation initiation network (SIN) specifically at SPBs in the basal region of G1 cells and that localized gradients of SIN activity promote asymmetric septation.

Mitotic regulation of fungal cell-to-cell connectivity through septal pores involves the NIMA kinase.

Intercellular bridges are a conserved feature of multicellular organisms. In multicellular fungi, cells are connected directly via intercellular bridges called septal pores. Using Aspergillus nidulans, we demonstrate for the first time that septal pores are regulated to be opened during interphase but closed during mitosis. Septal pore-associated proteins display dynamic cell cycle-regulated locations at mature septa. Of importance, the mitotic NIMA kinase locates to forming septa and surprisingly then remains at septa throughout interphase. However, during mitosis, when NIMA transiently locates to nuclei to promote mitosis, its levels at septa drop. A model is proposed in which NIMA helps keep septal pores open during interphase and then closed when it is removed from them during mitosis. In support of this hypothesis, NIMA inactivation is shown to promote interphase septal pore closing. Because NIMA triggers nuclear pore complex opening during mitosis, our findings suggest that common cell cycle regulatory mechanisms might control septal pores and nuclear pores such that they are opened and closed out of phase to each other during cell cycle progression. The study provides insights into how and why cytoplasmically connected Aspergillus cells maintain mitotic autonomy.

Functional analysis of the Aspergillus nidulans kinome.

The filamentous fungi are an ecologically important group of organisms which also have important industrial applications but devastating effects as pathogens and agents of food spoilage. Protein kinases have been implicated in the regulation of virtually all biological processes but how they regulate filamentous fungal specific processes is not understood. The filamentous fungus Aspergillus nidulans has long been utilized as a powerful molecular genetic system and recent technical advances have made systematic approaches to study large gene sets possible. To enhance A. nidulans functional genomics we have created gene deletion constructs for 9851 genes representing 93.3% of the encoding genome. To illustrate the utility of these constructs, and advance the understanding of fungal kinases, we have systematically generated deletion strains for 128 A. nidulans kinases including expanded groups of 15 histidine kinases, 7 SRPK (serine-arginine protein kinases) kinases and an interesting group of 11 filamentous fungal specific kinases. We defined the terminal phenotype of 23 of the 25 essential kinases by heterokaryon rescue and identified phenotypes for 43 of the 103 non-essential kinases. Uncovered phenotypes ranged from almost no growth for a small number of essential kinases implicated in processes such as ribosomal biosynthesis, to conditional defects in response to cellular stresses. The data provide experimental evidence that previously uncharacterized kinases function in the septation initiation network, the cell wall integrity and the morphogenesis Orb6 kinase signaling pathways, as well as in pathways regulating vesicular trafficking, sexual development and secondary metabolism. Finally, we identify ChkC as a third effector kinase functioning in the cellular response to genotoxic stress. The identification of many previously unknown functions for kinases through the functional analysis of the A. nidulans kinome illustrates the utility of the A. nidulans gene deletion constructs.

Functional characterization of a new member of the Cdk9 family in Aspergillus nidulans.

Cdk9-like kinases in complex with T-type cyclins are essential components of the eukaryotic transcription elongation machinery. The full spectrum of Cdk9/cyclin T targets, as well as the specific consequences of phosphorylations, is still largely undefined. We identify and characterize here a Cdk9 kinase (PtkA) in the filamentous ascomycete Aspergillus nidulans. Deletion of ptkA had a lethal effect in later stages of vegetative growth and completely impeded asexual development. Overexpression of ptkA affected directionality of polarized growth and the initiation of new branching sites. A green fluorescent protein-tagged PtkA version localized inside the nucleus during interphase, supporting a role of PtkA in transcription elongation, as observed in other organisms. We also identified a putative cyclin T homolog, PchA, in the A. nidulans genome and confirmed its interaction with PtkA in vivo. Surprisingly, the Pcl-like cyclin PclA, previously described to be involved in asexual development, was also found to interact with PtkA, indicating a possible role of PtkA in linking transcriptional activity with development and/or morphogenesis in A. nidulans. This is the first report of a Cdk9 kinase interacting with a Pcl-like cyclin, revealing interesting new aspects about the involvement of this Cdk-subfamily in differential gene expression.

Single-step affinity purification for fungal proteomics.

A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

The three fungal transmembrane nuclear pore complex proteins of Aspergillus nidulans are dispensable in the presence of an intact An-Nup84-120 complex.

In Aspergillus nidulans nuclear pore complexes (NPCs) undergo partial mitotic disassembly such that 12 NPC proteins (Nups) form a core structure anchored across the nuclear envelope (NE). To investigate how the NPC core is maintained, we affinity purified the major core An-Nup84-120 complex and identified two new fungal Nups, An-Nup37 and An-ELYS, previously thought to be vertebrate specific. During mitosis the An-Nup84-120 complex locates to the NE and spindle pole bodies but, unlike vertebrate cells, does not concentrate at kinetochores. We find that mutants lacking individual An-Nup84-120 components are sensitive to the membrane destabilizer benzyl alcohol (BA) and high temperature. Although such mutants display no defects in mitotic spindle formation, they undergo mitotic specific disassembly of the NPC core and transient aggregation of the mitotic NE, suggesting the An-Nup84-120 complex might function with membrane. Supporting this, we show cells devoid of all known fungal transmembrane Nups (An-Ndc1, An-Pom152, and An-Pom34) are viable but that An-ndc1 deletion combined with deletion of individual An-Nup84-120 components is either lethal or causes sensitivity to treatments expected to destabilize membrane. Therefore, the An-Nup84-120 complex performs roles, perhaps at the NPC membrane as proposed previously, that become essential without the An-Ndc1 transmembrane Nup.