New Results

Exportin-mediated nucleocytoplasmic transport maintains Pch2 homeostasis during meiosis

Esther Herruzo, Estefanía Sánchez-Díaz, Sara González-Arranz, Beatriz Santos, Jesús A. Carballo, Pedro A. San-Segundo
doi: https://doi.org/10.1101/2023.07.10.548332

Abstract

The meiotic recombination checkpoint reinforces the order of events during meiotic prophase I, ensuring the accurate distribution of chromosomes to the gametes. The AAA+ ATPase Pch2 remodels the Hop1 axial protein enabling adequate levels of Hop1-T318 phosphorylation to support the ensuing checkpoint response. While these events are focalized at chromosome axes, the checkpoint activating function of Pch2 relies on its cytoplasmic population. In contrast, forced nuclear accumulation of Pch2 leads to checkpoint inactivation. Here, we reveal the mechanism by which Pch2 travels from the cell nucleus to the cytoplasm to maintain Pch2 cellular homeostasis. Leptomycin B treatment provokes the nuclear accumulation of Pch2, indicating that its nucleocytoplasmic transport is mediated by the Crm1 exportin recognizing proteins containing Nuclear Export Signals (NESs). Consistently, leptomycin B leads to checkpoint inactivation and impaired Hop1 axial localization. Pch2 nucleocytoplasmic traffic is independent of its association with Zip1 and Orc1. We also identify a conserved functional NES in the non-catalytic N-terminal domain of Pch2 that is required for its nucleocytoplasmic traffic and proper checkpoint activity. In sum, we unveil another layer of control of Pch2 function during meiosis involving the nuclear export via the exportin pathway that is crucial to maintain the critical balance of Pch2 distribution among different cellular compartments.

INTRODUCTION

In sexually-reproducing organisms, chromosome distribution to the gametes relies on a specialized type of cell division, called meiosis, which reduces the number of chromosomes to the half prior to fertilization (Bolcun-Filas & Handel, 2018). During the long meiotic prophase I, chromosomes undergo several elaborate and carefully-regulated processes: pairing, synapsis and recombination. Recombination involves the formation of developmentally programmed DNA double-strand breaks (DSBs) by the conserved Spo11 protein and its partners (Keeney et al, 2014). The interhomolog repair of a subset of these DSBs as crossovers (COs) is essential for accurate chromosome segregation (Hunter, 2015; Zickler & Kleckner, 2015; San-Segundo & Clemente-Blanco, 2020). This process requires chromosomes to undergo intimate interactions during meiotic prophase that are facilitated by active chromosome movements coordinated by cytoskeletal forces and the widely conserved LINC complex (Zeng et al, 2017; González-Arranz et al, 2020; Kim et al, 2022). Once each chromosome has paired with its homologous partner, a tripartite structure called the synaptonemal complex (SC) is assembled along the length of each chromosome pair to maintain a stable association between paired homologs. In Saccharomyces cerevisiae, Hop1, Red1 and Rec8 are structural components of the lateral elements of the SC (Hollingsworth et al, 1990; Smith & Roeder, 1997; Klein et al, 1999; Láscarez-Lagunas et al, 2020), and the transverse filament Zip1 protein composes the central region which holds together the axes (Sym et al, 1993). The central region also includes the central element made of the Ecm11 and Gmc2 proteins (Humphryes et al, 2013).

The fidelity of meiotic divisions is ensured by the meiotic recombination checkpoint, which blocks cell cycle progression at the end of prophase I while recombination intermediates (i.e., unrepaired DSBs) persist (Subramanian & Hochwagen, 2014). Briefly, the Mec1-Ddc2 sensor kinase is recruited to resected DSBs (Refolio et al, 2011) and phosphorylates Hop1 at various consensus S/T-Q sites in a Red1-dependent manner. Among the multiple S/T-Q sites in Hop1, phosphorylation of T318 (hereafter, Hop1-T318ph) is critical for checkpoint activation (Carballo et al, 2008; Lo et al, 2014; Penedos et al, 2015). The Pch2 AAA+ ATPase sustains adequate levels of Hop1-T318ph on chromosomes to signal checkpoint activity in zip1Δ (Herruzo et al, 2016). Hop1-T318ph, in turn, promotes the activation of Mek1 and its recruitment to chromosomes (Niu et al, 2005; Ontoso et al, 2013; Hollingsworth & Gaglione, 2019). Finally, the Mek1 effector kinase phosphorylates and inhibits the Ndt80 transcription factor leading to pachytene arrest (Chen et al, 2018). In addition, Mek1 acts on Rad54 and Hed1 helping to prevent intersister recombination (Niu et al, 2009; Callender et al, 2016).

Pch2 (known as TRIP13 in mammals) is a member of the AAA+ ATPase family; these AAA+ proteins use the energy provided by ATP hydrolysis to provoke conformational changes on their substrates (Hanson & Whiteheart, 2005; Puchades et al, 2020). The conserved Pch2 protein was initially identified in a screen for meiotic recombination checkpoint mutants in budding yeast (San-Segundo & Roeder, 1999), but in addition to the checkpoint it also participates in multiple meiotic processes regulating several aspects of CO recombination and chromosome morphogenesis both in yeast and other organisms (Bhalla & Dernburg, 2005; Li & Schimenti, 2007; Joshi et al, 2009; Joyce & McKim, 2009; Zanders & Alani, 2009; Farmer et al, 2012; Joshi et al, 2015; Lambing et al, 2015). Despite the varying effects resulting from the absence of Pch2 in different organisms, it has been proposed that the common meiotic function of Pch2 is the coordination of recombination with chromosome synapsis to ensure proper CO number and distribution (Bhalla, 2023).

The meiotic roles of Pch2 are exerted through its action on its preferred client: the HORMAD Protein Hop1 (Vader, 2015; Prince & Martinez-Perez, 2022). As a member of the HORMAD family, Hop1 contains a flexible safety belt in its HORMA domain, which enables it to adopt an open/unbuckled or closed conformation (Ye et al, 2017; West et al, 2018). The transition from closed-Hop1 to unbuckled-Hop1 is thought to be accomplished by Pch2 ATPase activity, poising Hop1 for binding to a closure motif in Hop1 itself, or in other proteins that interact with Hop1, like Red1 (Chen et al, 2014; Ye et al., 2017; Alfieri et al, 2018; Yang et al, 2020). In other organisms, Pch2TRIP13 requires the p31(COMET) adaptor, but no cofactor has yet been described for Pch2 action in yeast (Eytan et al, 2014; Ye et al, 2015; Balboni et al, 2020; Giacopazzi et al, 2020).

Pch2 localization studies feature a complicated scenario. Pch2 is highly enriched in the unsynapsed ribosomal DNA (rDNA) region. This rDNA-specific recruitment requires Orc1, which collaborates with Pch2 to exclude Hop1 from the nucleolus thus limiting meiotic DSB formation at the repetitive rDNA array (San-Segundo & Roeder, 1999; Vader et al, 2011). Pch2 is also detected as individual foci colocalizing with Zip1 on synapsed chromosomes (San-Segundo & Roeder, 1999; Joshi et al., 2009). Targeting of Pch2 to the SC depends on Zip1, but other factors such as RNAPII-dependent transcription (Cardoso da Silva et al, 2020), Top2 (Heldrich et al, 2020), Nup2 (Subramanian et al, 2019) or chromatin modifications driven by Sir2 and Dot1 (San-Segundo & Roeder, 2000; Ontoso et al., 2013; Cavero et al, 2016), also influence Pch2 chromosomal distribution. Pch2 recruitment to chromosomes removes Hop1 from the axes (Borner et al, 2008; Herruzo et al., 2016; Subramanian et al, 2016), likely by disrupting Hop1-Red1 interaction via its remodeling activity towards the HORMA domain (West et al., 2018). Hop1 removal downregulates DSB formation on chromosomes that have successfully identified their homologs and formed COs, and silences the meiotic recombination checkpoint (Raina & Vader, 2020; Herruzo et al, 2021). In addition to the nucleolar and chromosomal population, a cytoplasmic supply of Pch2 also exists; this cytoplasmic pool is necessary and sufficient to sustain meiotic checkpoint activation (Herruzo et al, 2019; Herruzo et al., 2021). Based on all these observations, and the demonstrated role of Pch2 in promoting Hop1 chromosomal incorporation and T318 phosphorylation in zip1Δ (Herruzo et al., 2016), a model emerges in which Pch2, from the cytoplasm, structurally remodels Hop1 by using its ATPase activity, ensuring that unbuckled Hop1 is available to associate with Red1 via the closure motif determining its axial incorporation and achieving proper phosphorylation levels at T318 to support checkpoint activity (Herruzo et al., 2021).

The importance of a precise localization of Pch2 for an accurate meiotic checkpoint response argues that its distribution between the different cellular compartments must be finely regulated. Here, we combine detailed cytological, molecular and genetics studies to provide novel insights into how Pch2 travels from the nucleus to the cytoplasm, thereby contributing to delineate the regulatory network governing Pch2 subcellular distribution and function.

RESULTS and DISCUSSION

Pch2 nuclear export is mediated by the Crm1 exportin

The existence of at least three different subpopulations of Pch2 residing in the nucleolus, chromosomes and cytoplasm, together with the recent observation that an exquisite balance of Pch2 subcellular distribution is crucial to maintain a proper meiotic recombination checkpoint response (Herruzo et al., 2021), suggest that the nucleocytoplasmic transport of Pch2 must be tightly controlled. As an initial approach to explore this mechanism, we analyzed if Pch2 travels from the nucleus to the cytoplasm via Crm1, which is the main exportin mediating the nuclear export of proteins containing a Nuclear Export Signal (NES) (Stade et al, 1997). The drug leptomycin B (LMB) is a powerful tool to establish whether the nuclear export of a NES containing protein is mediated by the CRM1/XPO1/KAP124 exportin pathway. LMB binds to CRM1 disrupting the formation of the trimeric NES-CRM1-RanGTP export complex required for the transport from the nucleus to the cytoplasm (Sun et al, 2013). Thus, LMB treatment leads to the nuclear accumulation of NES-containing proteins. In Schizosaccharomyces pombe and mammalian cells, LMB is capable of binding directly to CRM1 inhibiting nuclear export, but S. cerevisiae cells are resistant to LMB because the wild-type budding yeast Crm1 does not bind to the drug. However, the Crm1-T539C mutant version is capable of binding LMB with high affinity and renders S. cerevisiae cells sensitive to LMB (Neville & Rosbash, 1999). Thus, to determine whether Pch2 travels from the nucleus to the cytoplasm via the Crm1 pathway, we analyzed Pch2 subcellular localization after LMB treatment in a crm1-T539C mutant background (Figure 1A-D). First, we confirmed that control crm1-T539C diploid cells, in the absence of LMB, completed meiosis and sporulation with fairly normal kinetics and efficiency (Figure S1A, S1B); however, addition of LMB during prophase I (15h) blocked meiotic progression (Figure S1B), indicating that, as expected, nuclear export is required for completion of meiosis and sporulation. More important, we also checked that the untreated zip1Δ crm1-T539C mutant showed a strong meiotic arrest (Figure S1A, S1B), indicating that the crm1-T539C mutation itself, in the absence of LMB, does not alter the zip1Δ-induced checkpoint response. Thus, the crm1-T539C mutant is a valid tool to explore the implication of nuclear export in Pch2 subcellular distribution in S. cerevisiae.

Figure 1.
Figure 1. Nuclear export of Pch2 is blocked by leptomycin B (LMB).

(A) Schematic representation of protein nuclear export via nuclear pore complexes mediated by the interaction of a Nuclear Export Sequence (NES) with the Crm1 exportin (see text for details). LMB blocks NES binding to Crm1 preventing nuclear export. The presence of a putative NES in Pch2 is depicted. For simplicity, the NES is drawn only in one subunit of the Pch2 hexamer. BioRender.com was used to create this figure.

(B, C) Fluorescence microscopy images of GFP-Pch2 localization in ZIP1 and zip1Δ cells, mock-treated (B), or treated with 500 ng/ml LMB (C), 15 h after meiotic induction. Images were taken at 24 h. Two representative individual cells for each condition are shown. Localization of GFP-Pch2 in the nucleolus (nlo), chromosomes (chr), and cytoplasm (cyt) is indicated. Scale bar, 2 μm. Strains are: DP1927 (ndt80Δ crm1-T539C GFP-PCH2) and DP1837 (zip1Δ ndt80Δ crm1-T539C GFP-PCH2).

(D) Quantification of the ratio of nuclear (including nucleolar) to cytoplasmic GFP fluorescent signal for the experiment shown in (B, C). Error bars, SD.

(E) Western blot analysis of GFP-Pch2 production (using anti-Pch2 antibodies) in the absence (mock) or presence of LMB. Pgk1 was used as loading control. The graph shows the quantification of GFP-Pch2 relative levels.

(F, G) Immunofluorescence of spread meiotic chromosomes at pachytene stained with anti-Pch2 antibodies (to detect GFP-Pch2; green), anti-Nsr1 antibodies (red) and DAPI (blue). Representative ZIP1 (F) and zip1Δ (G) nuclei, mock-treated, or treated with 500 ng/ml LMB 15 h after meiotic induction are shown. Arrows point to the rDNA region. Spreads were prepared at 19 h. Scale bar, 2 μm. Strains are: DP1717 (crm1-T539C GFP-PCH2) and DP1721 (zip1Δ crm1-T539C GFP-PCH2).

(H) Quantification of the GFP-Pch2 total signal for the experiment shown in (F, G). Error bars, SD; a.u., arbitrary units.

(I-J) Quantification of the nucleolar (I) and chromosomal (J) GFP-Pch2 signal for the experiment shown in (F). Error bars, SD; a.u., arbitrary units.

Pch2 localization was assessed by fluorescence microscopy analysis of a functional version of GFP-tagged Pch2 expressed from the previously described PHOP1-GFP-PCH2 construct (Herruzo et al., 2019; Herruzo et al., 2021), referred to as GFP-PCH2 throughout the article for simplicity. To avoid side effects resulting from differences in meiotic progression, GFP-Pch2 subcellular distribution was studied in prophase I-arrested ndt80Δ crm1-T539C live meiotic cells in otherwise wild-type (ZIP1) and zip1Δ backgrounds. Cells were treated with LMB, or mock-treated with the solvent ethanol, as control. As previously described, in mock treated wild-type ZIP1 cells, Pch2 localized to one side of the nucleus in a region corresponding to the nucleolus, it was also detected as discrete faint nuclear foci corresponding to the synapsed chromosomes, and it showed diffuse homogenous cytoplasmic signal as well (Figure 1B, top panels). Addition of LMB led to a strong accumulation of Pch2 in the nucleus displaying a much more intense signal especially in the presumed nucleolus, but also on the chromosomes and even in the nucleoplasm (Figure 1C, top panels). On the other hand, in the zip1Δ untreated control, Pch2 was only present in the nucleolus and the cytoplasm (Figure 1B, bottom panels), but LMB addition resulted in a prominent nucleolar accumulation and nucleoplasmic distribution, concomitant with reduced cytoplasmic localization (Figure 1C, bottom panels). Accordingly, the ratio between nuclear (including nucleolus) and cytoplasmic GFP signal increased after LMB treatment both in ZIP1 or zip1Δ strains (Figure 1D). Despite the altered subcellular distribution, total GFP-Pch2 protein levels remained unchanged in the presence or absence of LMB (Figure 1E).

To obtain more detailed information, we also analyzed the localization of GFP-Pch2 by immunofluorescence on pachytene chromosome spreads after LMB treatment. We first describe the distribution pattern in ZIP1 strains. In the absence of LMB, and consistent with the well-characterized localization of Pch2 (San-Segundo & Roeder, 1999; Joshi et al., 2009; Herruzo et al., 2016) and the observations in live meiotic cells (Figure 1B), the GFP-Pch2 protein localized mainly in the rDNA region, marked by the nucleolar protein Nsr1 (Figure 1F). The chromosome-associated population of Pch2, which is only present in synapsis-proficient strains, was not easily detectable with this technique in the BR strain background, as previously reported (Herruzo et al., 2016; Herruzo et al., 2019) (Figure 1F). However, the addition of LMB led to a general increase of GFP-Pch2 amount on the spread nuclei (Figure 1H), affecting both the nucleolar (Figure 1I) and the chromosomal (Figure 1J) populations. Moreover, in LMB treated ZIP1 cells, the accumulation of Pch2 in the nucleus was also associated to the increased formation of polycomplexes, which are organized extrachromosomal assemblies of SC components (Dong & Roeder, 2000), in 29.4% of nuclei (Figure S2A, S2C). On the other hand, on spread chromosomes of the zip1Δ mutant, Pch2 is only associated to the rDNA region; the addition of LMB led to an increased GFP-Pch2 signal restricted to the nucleolar area (Figure 1G, 1H).

In sum, these results demonstrate that inhibition of the Crm1 exportin pathway leads to Pch2 nuclear accumulation indicating that Pch2 travels from the nucleus to the cytoplasm using this pathway.

Effect of Crm1-mediated export inhibition on checkpoint function

We have previously described that the main role of Pch2 in the zip1Δ-induced checkpoint is to promote Hop1 association to unsynapsed chromosome axes supporting high levels of Hop1 phosphorylation at T318, which in turn sustain Mek1 activation (Herruzo et al., 2016). Artificial redirection of Pch2 to different subcellular compartments (i.e., forced nuclear accumulation) impairs these functions (Herruzo et al., 2021). To determine whether inhibition of the Crm1 nuclear export pathway, which alters Pch2 subcellular distribution, affects checkpoint function in zip1Δ, we examined the impact on Hop1 incorporation onto chromosome axes as well as the checkpoint status using Hop1-T318 phosphorylation as marker for Pch2-dependent checkpoint activity (Carballo et al., 2008; Penedos et al., 2015; Herruzo et al., 2016).

Analysis of Hop1 localization by immunofluorescence of zip1Δ spread nuclei in the absence of LMB revealed its characteristic intense and continuous signal along the unsynapsed axes, and its exclusion from the rDNA region containing Pch2 (Figure 2A). In contrast, after LMB treatment, Hop1 localization was compromised, displaying reduced intensity and a less continuous axial pattern (Figure 2A, 2B). To elude the possible effect of the different kinetics of meiotic progression, emphasized by the meiotic block conferred by the addition of LMB (Figure S1), we used prophase I-arrested ndt80Δ strains for an accurate analysis of Hop1-T318 phosphorylation levels without interference from cell cycle progression. As observed in Figure 2C, in the absence of LMB, the checkpoint was active in zip1Δ cells, as manifested by the high levels of Hop1-T318ph, compared to the wild type (ZIP1). However, phosphorylation levels of this checkpoint marker were notably reduced by the addition of LMB in zip1Δ cells (Figure 2C).

Figure 2.
Figure 2. LMB treatment impairs the meiotic recombination checkpoint

(A) Immunofluorescence of zip1Δ spread meiotic chromosomes stained with anti-Hop1 (red) and anti-GFP (to detect GFP-Pch2; green) antibodies. Representative nuclei, either mock-treated, or treated with 500 ng/ml LMB 15 h after meiotic induction, are shown. Arrows point to the rDNA region. Spreads were prepared at 19 h. Scale bar, 2 μm. The strain is DP1721 (zip1Δ crm1-T539C GFP-PCH2).

(B) Quantification of Pch2 (top graph) and Hop1 (bottom graph) signal for the experiment shown in (A). Error bars, SD; a.u., arbitrary units.

(C) Western blot analysis of Hop1-T318 phosphorylation (ph) in the absence (mock) or presence of LMB added at 15 h after meiotic induction (arrows). Pgk1 was used as loading control. The graph shows the quantification of Hop1-T318ph relative levels. Strains are: DP1927 (ndt80Δ crm1-T539C GFP-PCH2) and DP1837 (zip1Δ ndt80Δ crm1-T539C GFP-PCH2).

Thus, the accumulation of Pch2 in the nucleus of zip1Δ cells imposed by inhibition of Crm1 correlates with defects in Hop1 localization and phosphorylation, and inactivation of the meiotic recombination checkpoint. We cannot rule out that the nuclear accumulation of other factors involved in the checkpoint besides Pch2 could be responsible for the defective response observed after LMB treatment. For example, nucleocytoplasmic transport also appears to be involved in Ndt80 regulation (Wang et al, 2011). Nonetheless, we point out that the forced nuclear accumulation of Pch2 alone by fusion to a strong NLS leads to checkpoint downregulation (Herruzo et al., 2021), arguing that the simply block of Pch2 nuclear export by itself may account for the impaired checkpoint activity observed in LMB-treated zip1Δ cells.

Pch2 nucleocytoplasmic traffic is independent of Zip1 and Orc1

Since Orc1 is required for Pch2 nucleolar targeting (Vader et al., 2011), to further delineate the requirements for Pch2 nucleocytoplasmic transport we analyzed the effect of depleting Orc1 under LMB treatment conditions. That is, we explored if Pch2 nucleocytoplasmic traffic involves its transit through the rDNA region. To this end, we used the orc1-3mAID degron allele previously described; in this mutant, Pch2 does not localize to the nucleolus because of Orc1 degradation induced by auxin (Herruzo et al., 2019). We studied the localization of GFP-Pch2 in orc1-3mAID meiotic live cells and on chromosome spreads of both ZIP1 and zip1Δ strains. All the experiments involving orc1-3mAID were performed with auxin added at 12 h after meiotic induction to induce Orc1 depletion during prophase I. For clarity, we first describe the localization patterns in ZIP1 cells. In most mock-treated cells of the orc1-3mAID mutant, GFP-Pch2 was only detected in the cytoplasm and on nuclear foci or lines of different sizes likely corresponding to the chromosomes (Figure 3A, mock panel; Figure S2D). Indeed, immunofluorescence analyses of nuclear spreads confirmed the lack of Pch2 in the nucleolus (marked by Nsr1) and its presence on the chromosomes displaying a dotty-linear signal in the LMB-untreated control (Figure 3C, mock panels). In contrast, in the majority of LMB-treated ZIP1 cells, the cytoplasmic signal was reduced and GFP-Pch2 displayed a robust nuclear localization with accumulation in a strong large focus that could be combined with a dotty or linear pattern (Figure 3A, 3B; Figure S2D). In a small fraction of cells (16-20%), a diffuse nuclear signal could also be found in the presence or absence of LMB. This minor pattern appeared to be exclusive of orc1-3mAID crm1-T539C cells (Figure S2D). Analyses of nuclear spreads revealed that, in the presence of LMB, the GFP-Pch2 signal was much more intense, showing a more continuous linear chromosomal pattern (Figure 3C, left LMB panel; Figure 3E). Moreover, 45% of the spread nuclei showed a very intense Pch2 focus that did not correspond to the nucleolus (Figure 3C, right LMB panels), but colocalized with Zip1 at polycomplexes (Figure S2B, S2C). In these cases, the chromosomal staining of Zip1 and Pch2 was somewhat masked by the strong signal of the polycomplex. Thus, we conclude from these results that the block of nucleocytoplasmic traffic in Orc1-depleted cells, lacking nucleolar Pch2, leads to a stronger association of Pch2 with the SC or assemblies of SC components. Next, to determine whether the nuclear retention of Pch2 upon LMB treatment requires its interaction with Zip1, we analyzed Pch2 localization in the zip1Δ orc1-3mAID mutant. We observed that GFP-Pch2 localized exclusively in the cytoplasm in the absence of LMB (Figure 3A, mock panel) but, upon LMB treatment, Pch2 was retained in the nucleus displaying a diffuse nucleoplasmic distribution (Figure 3A, LMB panel, Figure 3B). There was no chromatin-associated Pch2 signal in either untreated or LMB-treated nuclei (Figure 3D, 3E). In sum, these observations suggest that shuttling of Pch2 between the nucleus and the cytoplasm involves neither the SC (Zip1-dependent) nor the nucleolar (Orc1-dependent) association of Pch2.

Figure 3.
Figure 3. Pch2 nucleocytoplasmic traffic does not involve its association with Orc1 and Zip1

(A) Fluorescence microscopy images of GFP-Pch2 localization in ZIP1 orc1-3mAID and zip1Δ orc1-3mAID cells, mock-treated, or treated with 500 ng/ml LMB, 15 h after meiotic induction. Auxin (500μM) was added to the cultures 12 h after meiotic induction to induce Orc1 degradation. Images were taken at 19 h. Cells representing the predominant GFP-Pch2 localization pattern for each condition are shown. Additional examples of GFP-Pch2 distribution are presented in Figure S2D. Scale bar, 2 μm. Strains are: DP1885 (orc1-3mAID crm1-T539C GFP-PCH2) and DP1886 (zip1Δ orc1-3mAID crm1-T539C GFP-PCH2).

(B) Quantification of the ratio of nuclear (including nucleolar) to cytoplasmic GFP fluorescent signal for the experiment shown in (A). Error bars, SD.

(C-D) Immunofluorescence of ZIP1 orc1-3mAID (C) and zip1Δ orc1-3mAID (D) spread meiotic chromosomes stained with anti-Pch2 (green) and anti-Nsr1 (red) antibodies, and DAPI (blue). Representative nuclei, either mock-treated, or treated with 500 ng/ml LMB 15 h after meiotic induction, are shown. Auxin (500 μM) was added to the cultures 12 h after meiotic induction to induce Orc1 degradation. Spreads were prepared at 19 h. Arrows point to the nucleolar region (nlo) and the Polycomplex (PC). Scale bar, 2 μm. The strains are the same used in (A).

(D) Quantification of Pch2 signal for the experiment shown in (C-D). Error bars, SD; a.u., arbitrary units.

A NES sequence in the NTD of Pch2 drives its export out of the nucleus

It is well known that the Crm1 exportin binds proteins possessing Nuclear Export Signals (NESs) for its transport out of the nucleus (Fung et al, 2021). We have observed that Pch2 export depends on Crm1 but, in principle, Pch2 could travel bound to putative partner(s) containing NESs or via NES(s) present in Pch2 itself. Thus, we searched for consensus NESs in the Pch2 sequence using the LocNES prediction tool (Xu et al, 2015) combined with visual matching to known patterns for hydrophobic residues distribution in NESs (Fung et al, 2017). Three putative NESs with high score probability were predicted; all of them present in the N-terminal domain (NTD) of Pch2 at amino acid positions 98-107, 127-136 and 205-214. To determine whether these putative NESs are functionally relevant for Pch2 nucleocytoplasmic traffic, we generated three GFP-tagged Pch2 versions harboring substitutions of hydrophobic residues (Leu, Ile, Val or Phe) present in each predicted putative NES to alanine (pch2-ntd98- 107-6A, pch2-ntd127-136-5A and pch2-ntd205-214-4A) (Figure S3A). Centromeric plasmids containing these constructs were transformed into a zip1Δ pch2Δ strain and the localization of GFP-Pch2 was examined in live meiotic cells. The pch2-ntd98-107-6A and pch2-ntd127-136-5A mutations both abolished nucleolar localization and led to an exclusively cytoplasmic distribution of Pch2 (Figure S3A, S3B). These mutations lie in the extended region of the NTD required for Orc1 interaction (Villar-Fernández et al, 2020), thus explaining the defective nucleolar targeting; therefore, they were discarded for further analyses. However, the GFP-pch2-ntd205-214-4A version showed a strong accumulation inside the nucleus as expected if a functional NES is disrupted (Figure S3A, S3B). Therefore, we generated strains harboring the GFP-pch2-ntd205-214-4A mutation (hereafter named as pch2-nes4A) integrated at the PCH2 genomic locus for more detailed analyses. We confirmed the significant increase in nuclear localization both in the nucleolus and nucleoplasm of the Pch2-nes4A version (Figure 4A, 4B). Consistent with the fact that Pch2 nuclear buildup in zip1Δ cells is deleterious for the checkpoint (Herruzo et al., 2021), the GFP-pch2-nes4A mutant suppressed the meiotic block of zip1Δ though to a lesser extent than the zip1Δ pch2Δ mutant did (Figure 4C). Furthermore, the levels of Hop1-T318 phosphorylation were reduced in the zip1Δ GFP-pch2-nes4A double mutant compared to zip1Δ (Figure 4D) reflecting impaired checkpoint activity. To confirm that these checkpoint defects stem from the impaired Pch2 nucleocytoplasmic transport resulting from a defective NES and not from other possible alterations in Pch2 activity and/or structure, we added an ectopic bona-fide strong NES from the PKI protein (Wen et al, 1995) generating a GFP-NESPKI-pch2-nes4A version. Importantly, the GFP-NESPKI-pch2-nes4A construct alleviated the nuclear accumulation of GFP-Pch2-nes4A (Figure 4A, 4B) and reestablished checkpoint function, as revealed by the restoration of the meiotic block (Figure 4C) and higher Hop1-T318 phosphorylation levels especially at the later time point (48 h) (Figure 4D). Thus, we conclude that the 205LSTEFDKIDL214 sequence in the Pch2 NTD domain (designated as NESPch2) behaves as a functional NES matching the Class 1a signature (Fung et al., 2017). We demonstrate that NESPch2 drives the nuclear export of Pch2, and it is critical for maintaining an exquisite balance of Pch2 subcellular distribution essential for a precise meiotic recombination checkpoint response. These findings also indicate that Pch2 does not need additional partners, besides the nuclear export machinery, to travel from the nucleus to the cytoplasm where it exerts its essential checkpoint activating function.

Figure 4.
Figure 4. A conserved functional NES in the NTD of Pch2 drives its nuclear export

(A) Fluorescence microscopy images of the localization of GFP-Pch2, GFP-Pch2-nes4 and GFP-NESPKI-Pch2-nes4A in zip1Δ cells. Images were taken 15 h after meiotic induction. Representative cells are shown. A schematic representation of the different GFP-Pch2 versions is also shown on top. Scale bar, 2 μm. Strains are: DP1625 (zip1Δ GFP-PCH2), DP1986 (zip1Δ GFP-pch2-nes4) and DP1992 (zip1Δ GFP-NESPKI-pch2-nes4A).

(B) Quantification of the ratio of nuclear (including nucleolar) to cytoplasmic GFP fluorescent signal for the experiment shown in (A). Error bars, SD.

(C) Time course analysis of meiotic nuclear divisions; the percentage of cells containing two or more nuclei is represented. Error bars: SD; n=3. At least 300 cells were scored for each strain at every time point. Strains are: DP1624 (GFP-PCH2), DP1625 (zip1Δ GFP-PCH2), DP1029 (zip1Δ pch2Δ), DP1986 (zip1Δ GFP-pch2-nes4A) and DP1992 (zip1Δ GFP-NESPKI-pch2-nes4A).

(D) Western blot analysis of Hop1-T318 phosphorylation in the strains analyzed in (C) at the indicated time points in meiosis. Pgk1 was used as a loading control. The graph shows the quantification of Hop1-T318ph relative levels.

(E) Schematic representation of the S. cerevisiae Pch2 protein (ScPch2) and the and human ortholog (HsTRIP13) indicating the characteristic AAA+ ATPase motifs, the conserved N-terminal domain (NTD) and the extended N-terminal domain exclusive of yeast Pch2 (NTDEXT). The positions and sequences of the NESs identified in this work are depicted (purple boxes).

(F) Fluorescence microscopy images of the localization of GFP-Pch2, GFP-Pch2-nes4A, GFP-NESTRIP13-Pch2-nes4A, and GFP-nes7ATRIP13-Pch2-nes4A in zip1Δ cells. Images were taken at 16 h after meiotic induction. Representative cells are shown. A schematic representation of the different GFP-Pch2 versions is also shown on top. Scale bar, 2 μm. The strain is DP1405 (zip1Δ pch2Δ) transformed with the centromeric plasmids pSS393 (GFP-PCH2), pSS459 (GFP-pch2-nes4A), pSS472 (GFP-NESTRIP13-pch2-nes4A) and pSS474 (GFP-nes7ATRIP13-pch2-nes4A).

(G) Quantification of the ratio of nuclear (including nucleolar) to cytoplasmic GFP fluorescent signal for the experiment shown in (F). Error bars, SD.

(H) Time course analysis of meiotic nuclear divisions. The percentage of cells containing two or more nuclei is represented. Error bars: SD; n=3. At least 300 cells were scored for each strain at every time point. Strains are: DP1625 (zip1Δ GFP-PCH2), DP1986 (zip1Δ GFP-pch2-nes4), DP2025 (zip1Δ GFP-NESTRIP13-pch2-nes4A) and DP2033 (zip1Δ GFP-nes7ATRIP13-pch2-nes4A).

(I) Western blot analysis of Hop1-T318 phosphorylation in the strains analyzed in (H) at the indicated time points in meiosis. Pgk1 was used as a loading control. The graph shows the quantification of Hop1-T318ph relative levels.

Evolutionary conservation of Pch2/TRIP13 nuclear export mechanism

Although the budding yeast Pch2 is meiosis specific, the mammalian homolog TRIP13 is also expressed in somatic cells besides the germline. TRIP13 has been reported to be localized both in the nucleus and the cytoplasm in mammalian somatic cells (Tipton et al, 2012; Wang et al, 2014; Thul et al, 2017). The Pch2/TRIP13 protein family possesses a highly conserved C-terminal AAA+ ATPase domain and a non-catalytic NTD with a lower similarity degree. In addition, yeast Pch2 contains a non-conserved extension at the very beginning of the protein (Figure 4E, Figure S4) that may be involved in the exclusive Pch2 nucleolar localization found in budding yeast via Orc1 interaction (Herruzo et al., 2019; Villar-Fernández et al., 2020). Using the LocNES prediction tool to analyze the human TRIP13 NTD, we detected a presumptive NES (at positions 65-80) close to the region corresponding to NESPch2 (Figure 4E, S4). To determine whether this putative NESTRIP13 is functionally active, we fused it to the NESPch2-deficient pch2-nes4A mutant generating a GFP-NESTRIP13-pch2-nes4A construct. Remarkably, addition of the proposed NESTRIP13 partially restored cytoplasmic localization of Pch2-nes4 (Figure 4F, 4G). Likewise, the zip1Δ GFP-NESTRIP13-pch2-nes4A double mutant displayed a notable meiotic block (Figure 4H) and increased levels of Hop1-T318ph at the 48-h time point (Figure 4I), consistent with a restoration of checkpoint activity. Furthermore, mutation to alanine of all NESTRIP13 hydrophobic residues (nes7ATRIP13) fail to reinstate cytoplasmic localization and checkpoint activity in pch2-nes4A (Figure 4F-4I). In sum, these observations strongly suggest that, indeed, the 65-80 amino acid fragment of TRIP13 NTD (FLTRNVQSVSIIDTEL) contains a functional NES capable of substituting the requirement for NESPch2 to implement a balanced localization of Pch2 in the different subcellular compartments during yeast meiosis.

Concluding remarks

Homeostatic control of Pch2 subcellular localization is crucial for a proper meiotic recombination checkpoint response (Raina & Vader, 2020; Herruzo et al., 2021). We unveil here another layer of control of Pch2 function during meiosis involving the nuclear export via the exportin pathway that is essential to maintain the critical balance of Pch2 distribution among different compartments (Figure 5). Functions for Pch2 outside the nucleus are not exclusive of yeast meiosis; in plants, PCH2 also operates in the cytoplasm promoting the nuclear targeting of the HORMA protein ASY1 (Hop1 homolog), in addition to its nuclear action in meiotic chromosome axis remodeling (Balboni et al., 2020; Yang et al., 2020). We provide evidence here for a possible evolutionary conservation of the nuclear export mechanism for Pch2/TRIP13 from yeast to mammals. In mice, TRIP13 is required for proper completion of meiotic chromosome synapsis and recombination, and Trip13-deficient mice are sterile (Li & Schimenti, 2007; Roig et al, 2010). Although the localization of TRIP13 during mammalian meiosis has not been reported, it can be detected both in the cytoplasm and nucleus of mouse spermatocytes (I. Roig, personal communication), suggesting that the meiotic role of mammalian TRIP13 may also be modulated by nucleocytoplasmic transport. In humans, biallelic mutations in the Trip13 gene are associated with female infertility and Wilms tumor (Yost et al, 2017; Zhang et al, 2020; Hu et al, 2022). Interestingly, the CRM1/XPO1 exportin has been recently identified in a screen for potential druggable targets involving TRIP13 function in Wilms tumor derived lines. Furthermore, the FDA-approved drug selinexor (KPT-330), which inhibits nuclear export, leads to suppression of TRIP13 function in these cell lines and it has been proposed as a potential therapeutic strategy for Wilms tumor patients (Mittal et al, 2022). In sum, all these observations underscore the importance of Pch2/TRIP13 nucleocytoplasmic traffic in the multiple biological processes impacted by this protein family.

Figure 5.
Figure 5. NES-mediated nuclear export of Pch2 is essential for meiotic recombination checkpoint function.

The nucleocytoplasmic transport of Pch2 via the exportin pathway maintains Pch2 homeostasis and supports proper checkpoint activity. In the NES-deficient pch2-nes4A mutant, the accumulation of Pch2 in the nucleus leads to checkpoint inactivation. The addition of the putative NES of the human TRIP13 ortholog to Pch2-nes4A restores the balanced subcellular distribution of Pch2 and restores checkpoint function. For simplicity, the NES is drawn only in one subunit of the Pch2 hexamer. BioRender.com was used to create this figure.

MATERIALS AND METHODS

Yeast strains

The genotypes of yeast strains are listed in Table S1. All strains are in the BR1919 background (Rockmill and Roeder, 1990). The zip1Δ::LEU2, zip1Δ::LYS2, ndt80Δ::kanMX6, pch2Δ::TRP1 and pch2Δ::URA3 gene deletions were previously described (Sym et al., 1993; Sym & Roeder, 1994; San-Segundo & Roeder, 1999; Herruzo et al., 2016). The orc1-3mAID and PHOP1-GFP-PCH2 constructs have been previously described (Herruzo et al., 2019; Herruzo et al., 2021). For the experiments involving the inactivation of the Crm1 exportin with LMB we employed diploid strains with both copies of the CRM1 essential gene deleted (crm1Δ::hphMX4 or crm1::natMX4) using a PCR-based approach (Goldstein & McCusker, 1999) carrying a centromeric plasmid expressing the crm1-T539C allele (pSS416) as the only source of exportin in the cell. The PHOP1-GFP-pch2-nes4A, PHOP1-GFP-NESPKI-pch2-nes4A, PHOP1-GFP-NESTRIP13-pch2-nes4A, and PHOP1-GFP-nes7ATRIP13-pch2-nes4A constructs were introduced into the genomic locus of PCH2 using an adaptation of the delitto perfetto technique (Stuckey et al, 2011). Basically, PCR fragments flanked by the appropriate sequences containing the HOP1 promoter followed by the GFP-pch2-nes4A, GFP-NESPKI-pch2-nes4A, GFP-NESTRIP13-pch2-nes4A or GFP-nes7ATRIP13-pch2-nes4A sequences, and a five Gly-Ala repeat linker before the second codon of PCH2, were amplified from pSS459, pSS462, pSS472 and pSS474, respectively (see below). These fragments were transformed into a strain carrying the CORE cassette (kanMX4-URA3) inserted close to the 5’ end of PCH2. G418-sensitive and 5-FOA resistant clones containing the correct integrated construct, which results in the elimination of 91 nt of the PCH2 promoter, were selected. All constructions and mutations were verified by PCR analysis and/or sequencing. The sequences of all primers used in strain construction are available upon request. All strains were made by direct transformation of haploid parents or by genetic crosses always in an isogenic background. Diploids were made by mating the corresponding haploid parents and isolation of zygotes by micromanipulation.

Plasmids

The plasmids used are listed in Table S2. The pSS393 centromeric plasmid expressing PHOP1-GFP-PCH2 was previously described (Herruzo et al., 2019). The pSS416 plasmid containing crm1-T539C was kindly provided by M. Dosil (CIC, Salamanca). The pSS448 and pSS451 plasmids, driving the expression of PHOP1-GFP-pch2(98-107)-6A and PHOP1-GFP-pch2(127-136)-5A, respectively, were made by non-overlapping mutagenesis following the procedure described in the Q5 site-directed mutagenesis kit (New England Biolabs), using the pSS393 as template and divergent primers. The forward primers carried the sequence encoding the mutated 98-107 (LIRSLAKVLL to AARSAAKAAA) or 127-136 (LFLSLFVKKI to AFASAFAKKA) regions in the Pch2 NTD. The pSS459 plasmid driving the expression of PHOP1-GFP-pch2-nes4A was derived from pSS393 by using the NEBuilder assembly kit (New England Biolabs) and a synthesized gBlock fragment (IDT) containing the mutated sequence corresponding to the 205-214 region (LSTEFDKIDL to ASTEADKADA). To construct the pSS462 plasmid containing PHOP1-GFP-NESPKI-pch2-nes4A, a 1.8-kb fragment was amplified from pSS459 (PHOP1-GFP-pch2-nes4A), using a forward primer containing the sequence encoding the Nuclear Export Signal (NES) from the PKI protein (LALKLAGLDI) (Wen et al., 1995) preceded by a NotI site at the 5’ end and a reverse primer within the PCH2 coding sequence downstream of the endogenous BlpI site. The fragment was digested with NotI-BlpI and cloned into the same sites of pSS393. To generate the pSS472 plasmid that contains PHOP1-GFP-NESTRIP13-pch2-nes4A, a 418-bp fragment was amplified from pSS393 (PHOP1-GFP-PCH2) using a forward primer containing the presumptive NES of TRIP13 (at positions 65-80) preceded by a NotI site at its 5’end, and a reverse primer within the PCH2 coding sequence downstream of the endogenous BamHI site. The fragment was digested with NotI-BamHI and cloned into the same sites of pSS459. To make the pSS474 plasmid containing PHOP1-GFP-nes7ATRIP13-pch2-nes4A, a 418pb fragment was amplified from pSS393, using a forward primer containing the mutation to alanine of all NESTRIP13 hydrophobic residues (FLTRNVQSVSIIDTEL to AATRNAQSASAADTEA), preceded by a NotI site at its 5’end, and a reverse primer within the PCH2 coding sequence downstream of the endogenous BamHI site. The fragment was digested with NotI-BamHI and cloned into the same sites of pSS459.

Meiotic cultures and meiotic time courses

To induce meiosis and sporulation, BR strains were grown in 3.5 ml of synthetic complete medium (2% glucose, 0.7% yeast nitrogen base without amino acids, 0.05% adenine, and complete supplement mixture from Formedium at twice the particular concentration indicated by the manufacturer) for 20–24 h, then transferred to 2.5 ml of YPDA (1% yeast extract, 2% peptone, 2% glucose, and 0.02% adenine) and incubated to saturation for an additional 8 h. Cells were harvested, washed with 2% potassium acetate (KAc), resuspended into 2% KAc (10 ml), and incubated at 30°C with vigorous shaking to induce meiosis. Both YPDA and 2% KAc were supplemented with 20 mM adenine and 10 mM uracil. The culture volumes were scaled up when needed. To inhibit Crm1-T539C, cultures were treated with 500 ng/ml of leptomycin B (LMB) at 15h in meiosis for the indicated periods of time. To induce Orc1-3mAID degradation, auxin (500μM) was added to the cultures 12 h after meiotic induction. To score meiotic nuclear divisions, samples from meiotic cultures were taken at different time points, fixed in 70% ethanol, washed in phosphate-buffered saline (PBS) and stained with 1 μg/μl 4′,6-diamidino-2-phenylindole (DAPI) for 15 min. At least 300 cells were counted at each time point. Meiotic time courses were repeated several times; averages and error bars from at least three replicates are shown.

Western blotting

Total cell extracts for Western blot analysis were prepared by trichloroacetic acid (TCA) precipitation from 3-ml aliquots of sporulation cultures, as previously described (Acosta et al, 2011). The antibodies used are listed in Table S3. The Pierce ECL Plus reagents (ThermoFisher Scientific) were used for detection. The signal was captured with a Fusion FX6 system (Vilber) and quantified with the Evolution-Capt software (Vilber).

Cytology

Immunofluorescence of chromosome spreads was performed essentially as described (Rockmill, 2009). The antibodies used are listed in Table S3. Images of spreads were captured with a Nikon Eclipse 90i fluorescence microscope controlled with MetaMorph software (Molecular Devices) and equipped with a Hammamatsu Orca-AG charge-coupled device (CCD) camera and a PlanApo VC 100x 1.4 NA objective. To measure Pch2 and Hop1 intensity on chromosome spreads, a region containing DAPI-stained chromatin was defined and the Raw Integrated Density values were measured. Background values were subtracted using the rolling ball algorithm from Fiji setting the radius to 50 pixels. Images of whole live cells expressing GFP-PCH2 were captured with an Olympus IX71 fluorescence microscope equipped with a personal DeltaVision system, a CoolSnap HQ2 (Photometrics) camera, and 100x UPLSAPO 1.4 NA objective. Stacks of 7 planes at 0.8-μm intervals were collected. Maximum intensity projections of 3 planes containing GFP-Pch2 are shown for ZIP1 cells and single planes for zip1Δ cells. To determine the nuclear/cytoplasm GFP fluorescence ratio shown in Figures 1D, 3B, 4B, 4G and S3B, the ROI manager tool of Fiji software (Schindelin et al, 2012) was used to define the cytoplasm and nuclear (including the nucleolus) areas; the mean intensity values were measured and subjected to background subtraction. In the specific case of Figure 4G, due to the difficulty in the discrimination of the nuclei in the strain expressing GFP-NESTRIP13-pch2-nes4A, we measured the average area of more than 50 nuclei from each one of the other strains analyzed in that Figure in which the contour of the nucleus was visible, resulting in a value of 7.02 μm2. We then applied this value to draw a circle containing the nucleolus in all the cells to define the nucleus and measured the nuclear/cytoplasmic fluorescence intensity ratio.

Sporulation efficiency

Sporulation efficiency was quantitated by microscopic examination of asci formation after 3 days on sporulation plates. Both mature and immature asci were scored. At least 300 cells were counted for every strain.

Statistics

To determine the statistical significance of differences, a two-tailed Student t-test was used. P-values were calculated with the GraphPad Prism 9.0 software. P<0.05 (*); P<0.01 (**); P<0.001 (***); P<0.0001 (****). The nature of the error bars in the graphical representations and the number of biological replicates are indicated in the corresponding figure legend.

AUTHOR CONTRIBUTIONS

EH: conceptualization, investigation, formal analysis, visualization. ESD: conceptualization, investigation, formal analysis, visualization. SGA: investigation, formal analysis.

BS: conceptualization, supervision, project administration. JAC: conceptualization, resources, funding acquisition

PSS: conceptualization, investigation, supervision, funding acquisition, visualization, writing original draft.

All authors revised, commented, and approved the manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplemental Figure Legends

Figure S1.
Figure S1. The crm1-T539C mutation does not substantially impair meiosis or checkpoint function in the absence of LMB

(A) Sporulation efficiency was examined after 3 days on sporulation plates. Error bars, SD; n=3. At least 300 cells were counted for each strain. Strains are: DP421 (wild type), DP1717 (crm1-T539C GFP-PCH2), DP422 (zip1Δ), and DP1721 (zip1Δ crm1-T539C GFP-PCH2).

(B) Time course analysis of meiotic nuclear divisions. The percentage of cells containing two or more nuclei is represented. Ethanol (Mock) or Leptomycin B (LMB) were added 15 h after meiotic induction (arrow). Error bars: SD; n=3. At least 300 cells were scored for each strain at every time point. Strains are: DP1717 (crm1-T539C GFP-PCH2) and DP1721 (zip1Δ crm1-T539C GFP-PCH2).

Figure S2.
Figure S2. Nuclear accumulation of Pch2 is linked to increased association of Pch2 with the SC and SC assemblies in ZIP1+ cells

(A-B) Immunofluorescence of spread meiotic chromosomes at pachytene stained with anti-GFP antibodies (to detect GFP-Pch2; green), anti-Zip1 antibodies (red) and DAPI (blue). Representative nuclei are shown. In both, (A) and (B), cultures were mock-treated, or treated with 500 ng/ml LMB 15 h after meiotic induction. In (B), Auxin (500μM) was also added 12 h after meiotic induction to degrade Orc1. Spreads were prepared at 19 h. Arrows point to the rDNA region (nlo) and Polycomplex (PC). Scale bar, 2 μm. The strain in (A) is: DP1717 (crm1-T539C GFP-PCH2). The strain in (B) is: DP1885 (orc1-3mAID crm1-T539C GFP-PCH2).

(C) Percentage of nuclei containing polycomplexes in the experiments shown in A and B. Between 10 to 20 nuclei were counted for each strain and condition.

(D) Quantification of the different patterns of Pch2 localization in the experiment presented in Figure 3A-3B. Representative cells are shown.

Figure S3.
Figure S3. Identification of NES sequences in Pch2

(A) Schematic representation of the S. cerevisiae Pch2 protein. The position and sequence of the three putative NESs predicted by LocNES in the non-catalytic N-terminal domain of Pch2 are depicted, as well as the corresponding mutants generated. The images show representative zip1Δ pch2Δ cells transformed with centromeric plasmids expressing wild-type GFP-PCH2 or the different mutated versions of the predicted NESs, as indicated. Note that only the mutation of the 205-214 region (boxed) leads to Pch2 accumulation in the nucleus. Images were taken 15 h after meiotic induction. The strain is DP1405 (zip1Δ pch2Δ) transformed with the centromeric plasmids pSS393 (GFP-PCH2), pSS448 (GFP-pch2-ntd98-107-6A), pSS451 (GFP-pch2-ntd127-136-5A) and pSS459 (GFP-pch2-ntd205-214-4A).

(B) Quantification of the ratio of nuclear (including nucleolar) to cytoplasmic GFP fluorescent signal for the experiment shown in (A). Error bars, SD.

Figure S4.
Figure S4. Conserved position of the NES in Pch2 and TRIP13

ClustalW alignment of the protein sequences of Pch2 orthologs from S. cerevisiae (ScPch2) and human (HsTRIP13). The characteristic AAA+ ATPase features are boxed in blue. The identified NESs are boxed in red. The color code for amino acids is the following: AVFPMILW (small + hydrophobic -Y): red. DE (acidic): blue. RK (basic -H): magenta. STYHCNGQ (hydroxyl +sulfhydryl + amine + G): green. Alignment was performed at https://www.ebi.ac.uk/Tools/msa/clustalo/.

View this table:
Table S1. Saccharomyces cerevisiae strains
View this table:
Table S2. Plasmids
View this table:
Table S3. Primary antibodies

ACKNOWLEDGEMENTS

We are grateful to Andrés Clemente for helpful comments and discussions and to Ignasi Roig for sharing unpublished results. We also thank Mercedes Dosil and Yolanda Sánchez for reagents, Jesús Pinto, Carmen Castro and Carlos R. Vázquez for advice on microscopy analysis, Isabel Acosta for technical support, and Ana Lago-Maciel for help in strains construction. This work was supported by the grant PID2021-125830NB-I00 from Ministry of Science and Innovation (MCIN/AEI/FEDER, EU) of Spain to PSS and JAC. EH was partially supported by the grant CSI259P20 from the “Junta de Castilla y León” (FEDER, EU). ESD is supported by a predoctoral contract from the “Junta de Castilla y León” (co-funded by the Education Department and the European Social Fund FSE+).

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Posted July 10, 2023.
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Exportin-mediated nucleocytoplasmic transport maintains Pch2 homeostasis during meiosis
Esther Herruzo, Estefanía Sánchez-Díaz, Sara González-Arranz, Beatriz Santos, Jesús A. Carballo, Pedro A. San-Segundo
bioRxiv 2023.07.10.548332; doi: https://doi.org/10.1101/2023.07.10.548332
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