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Characterization of a novel interaction of the Nup159 nucleoporin with asymmetrically localized spindle pole body proteins and its link with autophagy [1]

['Inés García De Oya', 'Centro Andaluz De Biología Molecular Y Medicina Regenerativa', 'Cabimer', 'Spanish National Research Council', 'Csic', '- University Of Seville - University Pablo De Olavide', 'Sevilla', 'Javier Manzano-López', 'Alejandra Álvarez-Llamas', 'María De La Paz Vázquez-Aroca']

Date: 2023-08

Both the spindle microtubule-organizing centers and the nuclear pore complexes (NPCs) are convoluted structures where many signaling pathways converge to coordinate key events during cell division. Interestingly, despite their distinct molecular conformation and overall functions, these structures share common components and collaborate in the regulation of essential processes. We have established a new link between microtubule-organizing centers and nuclear pores in budding yeast by unveiling an interaction between the Bfa1/Bub2 complex, a mitotic exit inhibitor that localizes on the spindle pole bodies, and the Nup159 nucleoporin. Bfa1/Bub2 association with Nup159 is reduced in metaphase to not interfere with proper spindle positioning. However, their interaction is stimulated in anaphase and assists the Nup159-dependent autophagy pathway. The asymmetric localization of Bfa1/Bub2 during mitosis raises the possibility that its interaction with Nup159 could differentially promote Nup159-mediated autophagic processes, which might be relevant for the maintenance of the replicative lifespan.

Intriguingly, several connections have been established between the spindle MTOCs and the NPCs, which even share common components, suggesting that these structures collaborate in the regulation of key cellular processes [ 19 ]. Our results reveal a new link between proteins located on the SPBs and the NPCs. Specifically, we show that the Bfa1/Bub2 complex associates with the Nup159 nucleoporin. This interaction is cell cycle-regulated and requires Bfa1/Bub2 localization to the SPBs. Furthermore, we demonstrate that Bfa1/Bub2 association with Nup159 is prevented during the initial stages of spindle positioning but it is then promoted in anaphase to facilitate the activity of the Nup159-dependent autophagic pathway. The asymmetric localization of Bfa1/Bub2, which exclusively loads on the SPB that is delivered to the daughter cell [ 3 ], raises the interesting possibility that this novel connection between MEN components and Nup159 could mediate a differential regulation of the autophagic degradation of nucleoporins or other cellular components in the mother and daughter cells that might be important for the maintenance of the replicative lifespan in S. cerevisiae.

S. cerevisiae displays a closed mitosis and the SBPs are embedded in the nuclear envelope, which remains intact during the whole process of cell division [ 9 ]. Bfa1 and Bub2, as well as the rest of SPB-associated MEN components, reside on the cytoplasmic side of the SPBs. However, signals that activate the GAP complex are also generated within the nucleus. As such, Bfa1/Bub2 activity is required to maintain the functionality of the DNA damage checkpoint (DDC) and the spindle assembly checkpoint (SAC), 2 surveillance mechanisms that are respectively triggered by DNA lesions and the incorrect attachment of chromosomes to the mitotic spindle [ 4 , 10 , 11 ]. Hence, nucleocytoplasmic transport plays an important role in the regulation of the MEN. Transport across the nuclear envelope is mediated by nuclear pore complexes (NPCs), which are convoluted structures inserted in the nuclear membrane and organized into different subcomplexes of proteins named nucleoporins [ 12 ]. One of the modules that constitute the NPC in S. cerevisiae is the Nup82 complex, located at the cytoplasmic side and formed by the association of the Nup159, Nsp1, and Nup82 nucleoporins, which collaborate with Nup116, Nup42, Gle1, and Nup100 to facilitate nuclear mRNA export [ 12 – 14 ]. Notably, Nup159 has been identified as one of the proteins recognized by Atg8 to promote the autophagic degradation of NPC components [ 15 , 16 ]. Furthermore, the dynein light chain Dyn2 was recently identified as a novel constituent of the Nup82 complex that is recruited by Nup159 to the nuclear pores, which suggests that NPCs might also be important for the correct alignment of the mitotic spindle [ 17 , 18 ]. Therefore, similar to the MTOCs, NPCs are crucial elements in the regulation of many cellular processes besides their main function in nucleocytoplasmic transport.

The microtubules that constitute the mitotic spindle, position this structure within the cell, and enable its function in chromosome segregation, emanate from microtubule-organizing centers (MTOCs) located at both spindle poles [ 1 ]. The MTOCs, named centrosomes in mammalian cells and spindle pole bodies (SPBs) in the budding yeast Saccharomyces cerevisiae, are fundamental players in the regulation of cell division [ 1 ]. Besides their essential role in genome distribution, centrosomes and SPBs are platforms where many cell signaling pathways converge to regulate different aspects of mitotic progression [ 2 ]. In this way, most constituents of the mitotic exit network (MEN), a signaling cascade that triggers exit from mitosis in S. cerevisiae, are localized to the SPBs [ 3 ]. This is the case of Bfa1/Bub2, a two-component GTPase-activating protein (GAP) that inhibits MEN signaling and constitutes a central target of the main cell cycle checkpoints [ 4 – 6 ]. Bfa1 and Bub2 integrate signals from multiple sources in order to coordinate mitotic exit with the successful completion of key cellular events. To this end, the GAP complex is regulated by different kinases that control its activity and/or localization, such as the Polo-like kinase Cdc5, which phosphorylates Bfa1/Bub2 in anaphase to restrain its inhibitory action on the MEN [ 6 ]. Additionally, when the spindle position checkpoint (SPOC) is triggered as a consequence of spindle misalignment, Bfa1/Bub2 phosphorylation by the Kin4 kinase prevents the inhibitory action of Cdc5 on Bfa1/Bub2, thereby impeding mitotic exit until the spindle is finally correctly positioned along the mother-daughter cell axis [ 7 , 8 ]. Despite a lot of work has been put into understanding the mechanisms that control the activity and localization of Bfa1/Bub2, many aspects of their regulation are nonetheless still unknown.

Results

A global screening reveals a novel interaction between nuclear pore components and the mitotic exit inhibitor Bfa1 In order to uncover yet undescribed proteins that could interact with Bfa1/Bub2 and regulate their function, we carried out a global screening using a two-hybrid assay and Bfa1 as the bait [20]. Both Bub2 and the Cdc5 kinase, which phosphorylates and inactivates the Bfa1/Bub2 complex during anaphase [6], were identified among the proteins that associated with Bfa1 in our screening, demonstrating the validity of the approach. Interestingly, Nup159 and Nup42 were also found to interact with Bfa1 in the two-hybrid assay. These 2 FG-nucleoporins, characterized by phenylalanine- and glycine-rich sequences, localize to the cytoplasmic side of the nuclear pore and contribute to the formation of the filaments that project from this structure [12–14]. The cytoplasmic localization of Nup159 and Nup42 is in agreement with their potential interaction with the SPB-associated Bfa1/Bub2 complex. Moreover, since many of the signals that are transmitted to the GAP in order to prevent mitotic exit are generated within the nucleus, the interaction of these nucleoporins with Bfa1/Bub2 might represent a potential step mediating the communication between the nuclear compartment and the MEN inhibitors at the SPBs. In order to verify the interaction between Bfa1 and the nucleoporins, we used co-immunoprecipitation assays. Indeed, 3HA-tagged Bfa1 was clearly pulled down together with green fluorescent protein (GFP)-labeled Nup159 in exponentially growing cells expressing both protein fusions, despite a residual background signal could sometimes be observed in control cells only expressing 3HA-Bfa1 due to unspecific binding of this protein to the magnetic beads used in the assay (Fig 1A). We also noticed that, independently of the epitope used for tagging, Nup159 is prone to degradation in protein extracts, which gives rise to several faster migrating bands in PAGE gels besides that of the full-length protein (Fig 1A). In contrast to what observed for Nup159, we could not co-immunoprecipitate Bfa1 together with Nup42 (S1A Fig), thus being unable to confirm their association with this assay. We also evaluated whether the confirmed Nup159 interaction with the GAP complex could depend on Nup42 expression. Deletion of the NUP42 gene, however, did not impair the capacity of Nup159-GFP to pull down 3HA-Bfa1 in our assays (S1B Fig). Hence, we decided to not pursue the study of the possible Bfa1-Nup42 association any further. PPT PowerPoint slide

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TIFF original image Download: Fig 1. The nucleoporin Nup159 is a novel Bfa1 interactor. (A–D) Stationary phase cultures in YPAD were diluted to OD 600 = 0.2 in fresh medium and then grown for 6 h at 26°C. (A, B) Co-immunoprecipitation analysis in cells simultaneously expressing Nup159-GFP and either 3HA-Bfa1 or Bub2-3HA in different genetic backgrounds. Cells exclusively expressing 3HA-Bfa1, Bub2-3HA, or Nup159-GFP were included as controls. Western blot gel images for 3HA-Bfa1, Bub2-3HA, and Nup159-GFP are shown for both the input (INPUT) and the immunoprecipitated (Co-IP) samples. The Co-IP efficiency for 3HA-Bfa1 relative to the corresponding control with untagged Nup159 (-) and referred to the strain used as a reference (×1.00) in (A) is indicated in each case. Each sample in (A) was separated from the rest with an empty well to discard any residual transfer between lanes. Experiments were carried out thrice (n = 3) and a representative image is shown. (C, D) BiFC analysis of Bfa1-VC interaction with VN-tagged nucleoporins. (C) Illustrative image displaying a positive BiFC interaction (Bfa1-VC/Nup159-VN, in green and marked with an arrow) and SPB localization (Spc42-mCherry, in red). Nuclear morphology (DAPI, in blue), PhC, and merged images are also shown. (D) Quantification of the percentage of cells displaying positive BiFC interaction. Data are the average of 3 samples (n = 3; 100 cells/each) and are available in S1 Data. Error bars represent SD. BiFC, bimolecular fluorescence complementation; GFP, green fluorescent protein; PhC, phase-contrast; SPB, spindle pole body. https://doi.org/10.1371/journal.pbio.3002224.g001 The localization of Bfa1 and Bub2 to the SPBs is interdependent and the lack of Bub2 prevents Bfa1 phosphorylation, which likely takes place at this location [5,21]. Therefore, we next analyzed whether the Bfa1-Nup159 interaction was dependent on the integrity of the Bfa1/Bub2 complex. Remarkably, despite showing similar levels of total protein in the initial extract, 3HA-Bfa1 did not efficiently co-immunoprecipitate with Nup159-GFP in bub2Δ cells (Fig 1A). In addition, Bub2-3HA could also be pulled down together with Nup159-GFP in co-immunoprecipitation assays, which indicates that this nucleoporin can associate with the whole GAP complex but not necessarily directly interact with both its components (Fig 1B). These results thus demonstrate that both Bfa1 and Bub2 associate with Nup159 and that an intact Bfa1/Bub2 complex is necessary for this interaction. To provide further support to our observations, we also evaluated whether Nup159 and Bfa1 interacted in a bimolecular fluorescence complementation (BiFC) assay [22], which not only allows to detect the in vivo association between 2 proteins but also to determine where their interaction takes place. The BiFC is based in the reconstitution of the Venus yellow fluorescent protein by means of the association of 2 proteins that have been respectively fused to the N-terminal (VN) and C-terminal (VC) halves of this molecule [22]. Corroborating our prior results, Bfa1-VC interacted with Nup159-VN in the BiFC assay (Fig 1C and S1C Fig). Remarkably, despite NPCs spreading all over the nuclear envelope, the association between Bfa1-VC and Nup159-VN was mainly restricted to the context of the SPBs, as demonstrated by colocalization of the BiFC signal with that of an mCherry-tagged version of the SPB component Spc42 (Fig 1C). The BiFC signal was faint and not detected in every cell (Fig 1C and 1D). Interestingly, Bfa1-VC also showed positive BiFC interaction with Dyn2-VN, another component from the Nup82 complex [13], and a limited association to Nup100-VN, which collaborates with Nup159-Nup82 [13] (Fig 1D). However, Bfa1-VC did not interact with Gle1-VN, a nucleoporin that more externally localizes in the cytoplasmic side of the NPC [12], or Nup42-VN (Fig 1D), in agreement with our previous observations (S1A Fig). Overall, these results demonstrate the interaction between Nup159 and Bfa1/Bub2 and support that it likely takes place in the context of the SPBs.

The association of Bfa1 and Nup159 is cell cycle regulated The Bfa1/Bub2 complex is posttranslationally modified and subjected to changes in its localization both as cells progress through the cell cycle and after activation of the mitotic checkpoints [5,6,21,23,24]. Therefore, we next analyzed whether the association of Nup159 with Bfa1/Bub2 was modulated in a cell cycle-dependent manner. To this end, we synchronized cells in G1 with the α-factor pheromone and in metaphase or anaphase by means of the conditional inactivation of the thermosensitive cdc13-1 or cdc15-2 alleles, respectively [25–28]. Notably, the amount of 3HA-Bfa1 protein that was pulled down with Nup159-GFP in co-immunoprecipitation assays was particularly reduced in metaphase-arrested cdc13-1 cells, especially when compared to anaphase-blocked cdc15-2 cells (Fig 2A). The efficiency of the cell cycle arrest, which was confirmed in each case (S2A Fig), could be also easily verified by assessing the electrophoretic mobility of Bfa1, a protein that is unphosphorylated in G1 and gets progressively phosphorylated as cells go through mitosis, reaching its maximal phosphorylation level during anaphase [5]. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Nup159-Bfa1 interaction is cell cycle regulated. (A, B) Co-immunoprecipitation analysis in cells simultaneously expressing Nup159-GFP and 3HA-Bfa1 in the indicated genetic backgrounds. In each case, cells that only expressed 3HA-Bfa1 were included as controls. Western blot gel images for 3HA-Bfa1 and Nup159-GFP are shown for both the input (INPUT) and the immunoprecipitated (Co-IP) samples. The Co-IP efficiency for 3HA-Bfa1 relative to the corresponding control with untagged Nup159 (-) and referred to the strain or condition used as a reference (×1.00) is indicated in each case. (A) Stationary phase cells in YPAD were diluted to OD 600 = 0.2 in fresh medium and then grown for 6 h at 26°C in YPAD (asynchronous culture) or arrested in G1 with 5 μg/ml α-factor in YPAD at 26°C (G1 arrest) and, in the case of cdc13-1 (Metaphase) and cdc15-2 (Anaphase) cells, subsequently released for 2 h in YPAD at 34°C. Experiment was carried out thrice (n = 3) and a representative image is shown. (B) Stationary phase cells in YPAD were diluted to OD 600 = 0.2 in fresh medium, arrested in G1 with 5 μg/ml α-factor and released for 2 h either in YPAD at 34°C (cdc13-1, cdc20-3, and cdc15-2 cells) or in YPAD with 500 μm IAA at 26°C (cdc20-AID cells). (C, D) BiFC analysis of Bfa1 and Nup159 interaction. (C) Illustrative image displaying a positive BiFC interaction (Bfa1-VC/Nup159-VN, in green and indicated with an arrow) and SPB localization (Spc42-mCherry, in red). Nuclear morphology (DAPI, in blue), PhC, and merged images are also shown. (D) Quantification of the percentage of cells displaying positive BiFC interaction. Data are the average of 3 samples (n = 3; 100 cells/each) and are available in S1 Data. Error bars represent SD. BiFC, bimolecular fluorescence complementation; GFP, green fluorescent protein; PhC, phase-contrast; SPB, spindle pole body. https://doi.org/10.1371/journal.pbio.3002224.g002 In contrast to pheromone addition or inactivation of cdc15-2, expression of cdc13-1 at the restrictive temperature restrains cell cycle progression due to the activation of a cell cycle checkpoint. Cdc13 is required to protect telomeres from degradation and, in its absence, cells accumulate single-stranded DNA at the chromosome ends, a signal that triggers a DDC-dependent metaphase arrest [25,26]. The reduced Nup159-Bfa1 association in cdc13-1 cells at the restrictive temperature could thus be reliant either on cell cycle stage or on checkpoint activation. To discern between these 2 possibilities, we generated cells that expressed either the thermosensitive cdc20-3 allele or, alternatively, an auxin-inducible degron of the anaphase-promoting complex (APC/C) cofactor Cdc20 (Cdc20-AID-9Myc) [29,30]. APC/CCdc20 elicits the metaphase-to-anaphase transition by promoting the proteasome-dependent degradation of both securin and the mitotic cyclins [31]. Hence, inactivation of cdc20-3 or degradation of Cdc20-AID-9Myc impairs APC/CCdc20 activity and, consequently, blocks mitotic progression in metaphase without triggering any checkpoint [29,31]. Despite 3HA-Bfa1 co-immunoprecipitated more efficiently with Nup159-GFP in anaphase-arrested cdc15-2 cells than after DDC activation in cdc13-1 cells, their interaction was similarly reduced both in cdc20-3 cells at the restrictive temperature and in Cdc20-AID-9Myc cells after auxin addition (Fig 2B and S2B Fig). Hence, the decreased Nup159-Bfa1 association is due to the cell cycle stage and not to checkpoint activation. Accordingly, the BiFC interaction of Bfa1-VC and Nup159-VN also showed a cell cycle dependence, being less frequently observed in metaphase-arrested cdc13-1 or cdc20-3 mutants than in cdc15-2 mutants blocked in anaphase (Fig 2C and 2D). Our results thus demonstrate that the interaction of Bfa1 and Nup159 is cell cycle-modulated, being their association specifically prevented during metaphase and strongly stimulated during anaphase.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002224

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