Downregulation of the Tem1 GTPase by Amn1 after cytokinesis involves both nuclear import and SCF-mediated degradation

The mitotic exit network (MEN) is an essential kinase cascade that promotes mitotic exit and cytokinesis in budding yeast (Bardin and Amon, 2001; Simanis, 2003). The MEN has an organisation similar to the septation initiation network in fission yeast and the Hippo pathway in metazoans, in that it includes a Ste20-like protein kinase (Cdc15) that activates a LATS-NDR kinase (Mob1–Dbf2 or Mob1b–Dbf20, which share overlapping functions) (Hergovich and Hemmings, 2012). A major function of the MEN is to promote the release from the nucleolus and the activation of the Cdc14 phosphatase (Mohl et al., 2009; Shou et al., 1999; Visintin et al., 1999), which is the main CDK-counteracting phosphatase in budding yeast (Bouchoux and Uhlmann, 2011; Gray et al., 2003; Visintin et al., 1998).

MEN signalling is triggered by the Tem1 GTPase, which is activated at microtubule-organising centres or spindle pole bodies (SPBs) (reviewed in Scarfone and Piatti, 2015). During metaphase, Tem1 is present on both SPBs, albeit with a slight preference for the bud-proximal SPB. In anaphase it becomes markedly asymmetric, becoming progressively concentrated on the bud-directed SPB (Bardin et al., 2000; Campbell et al., 2020; Caydasi and Pereira, 2009; Fraschini et al., 2006; Molk et al., 2004; Monje-Casas and Amon, 2009; Pereira et al., 2000; Scarfone et al., 2015). In spite of this asymmetry, both SPB-bound pools of Tem1 seem to contribute to mitotic exit (Campbell et al., 2020).

Once active, Tem1 recruits to SPBs and activates the MEN kinase Cdc15, which in turn phosphorylates the MEN scaffold Nud1, thereby creating a phospho-docking motif for the Mob1–Dbf2 kinase. At SPBs, Cdc15 activates Mob1–Dbf2, ultimately allowing Cdc14 nucleolar release and activation (Mah et al., 2001; Mohl et al., 2009; Rock and Amon, 2011; Rock et al., 2013; Scarfone et al., 2015; Valerio-Santiago and Monje-Casas, 2011; Visintin and Amon, 2001).

How MEN signalling is extinguished after cytokinesis is an important, yet unanswered, question. Indeed, unscheduled Cdc14 phosphatase activity in G1 interferes with DNA replication (Bloom and Cross, 2007). Furthermore, Cdc14-independent MEN functions (Hotz et al., 2012; Meitinger et al., 2011, 2013; Oh et al., 2012) might also need to be turned off.

One mechanism that contributes to MEN downregulation after mitotic exit is the re-entrapment of Cdc14 in the nucleolus prompted by Cdc14 itself (Lu and Cross, 2010; Manzoni et al., 2010; Visintin et al., 2008).

Another actor that contributes to turn down MEN signalling after mitotic exit is the MEN antagonist Amn1. Amn1 is expressed from mitotic exit to late G1 and accumulates specifically in the nucleus of the bud-derived daughter cell (Wang et al., 2003). Asymmetric, cell cycle-dependent regulation of Amn1 expression requires the daughter-specific transcription factor Ace2 (Colman-Lerner et al., 2001) and Amn1 degradation in late G1 (Wang et al., 2003). In turn, Ace2 activation depends on the MEN and Cdc14 (Brace et al., 2011; Sanchez-Diaz et al., 2012; Weiss et al., 2002). Thus, besides promoting its own re-entrapment, Cdc14 also triggers Amn1-dependent downregulation of the MEN.

Exactly how Amn1 antagonises MEN signalling remains to be elucidated. Amn1 interacts physically with Tem1 and competes with Cdc15 for Tem1 binding (Wang et al., 2003). However, higher levels of Tem1 are present in amn1Δ cells (Wang et al., 2003), suggesting that Amn1 could also inhibit Tem1 function by promoting its turnover. Accordingly, in G1, Tem1 is present at low levels on SPBs (Campbell et al., 2020; Molk et al., 2004). Furthermore, Amn1 is an atypical F-box protein part of the Skp, Cullin and F-box-containing (SCF) ubiquitin ligase, driving proteolysis of Ace2 in most, but not all, laboratory strains (Fang et al., 2018).

Here, we show that Amn1 has a dual function in Tem1 downregulation after mitotic exit. On one side, it promotes Tem1 degradation as part of an SCF^Amn1 complex, while on the other it displaces Tem1 from SPBs and escorts it into the nucleus independently of SCF.

To study Tem1 localisation, we used a strain expressing the endogenous TEM1 gene tagged at the C terminus with yeast-enhanced GFP (yeGFP). This strain has a doubling time indistinguishable from that of the isogenic untagged strain, suggesting that the tag does not perturb Tem1 activity (Fig. S1A). By live-cell imaging, we observed a transient and asymmetric relocalisation of Tem1–yeGFP into the nucleus of the bud compartment, which was visualised using the nuclear marker mCherry–Pus1 (98/98 cells; Fig. 1A). Tem1 nuclear translocation occurred about 20 min after anaphase and was accompanied by a decrease in Tem1 levels at the daughter-bound SPB. To better resolve the timing of Tem1 nuclear import, we filmed cells co-expressing Tem1–yeGFP and mCherry-tagged septin Shs1. Tem1–yeGFP appeared in the nucleus of the bud 4 min (±3 min, s.d.; n=45) after septin ring disassembly, which marks the onset of cytokinesis (Lippincott et al., 2001; Tamborrini et al., 2018), and persisted in the nucleus throughout the following G1 phase until appearance of a new septin ring (Fig. S1B). Thus, Tem1 undergoes an asymmetric accumulation in the nucleus of the daughter cell after cytokinesis until late G1.

Next, we investigated the molecular basis of Tem1 nuclear import. We reasoned that Amn1 could be implicated in this process since it interacts with Tem1 and is concentrated in the daughter cell nucleus after mitotic exit (Wang et al., 2003). In agreement with our prediction, Tem1–yeGFP nuclear import was completely abolished in amn1Δ cells (93/93 cells), and the protein persisted at both SPBs throughout the cell cycle (Fig. 1B). Furthermore, AMN1 deletion increased the levels of Tem1 at the daughter cell SPB from cytokinesis to the following G1 without affecting Tem1 levels at the mother cell SPB (Fig. 1C).

Consistent with a role of Amn1 in downregulating Tem1 levels at SPBs specifically in G1, Tem1 levels were increased by more than twofold at SPBs of G1-arrested amn1Δ cells relative to levels in their wild-type counterparts (Fig. S1C).

Importantly, live-cell imaging of cells co-expressing Amn1–superfolder GFP (sfGFP) and Tem1–mScarlet-I showed that the two proteins concomitantly concentrate in the nucleus (104/109 cells; Fig. 1D). These data suggest that Amn1 could escort Tem1 into the nucleus of the daughter cell to reduce its active pool at the daughter-bound SPB after cytokinesis, once Tem1 has accomplished its essential functions.

Amn1 is an atypical F-box protein that has recently been shown to be part of an SCF complex (Fang et al., 2018). In agreement with these data, we confirmed, using co-immunoprecipitation experiments, that Flag-tagged Amn1 (Amn1–3Flag) interacts with HA-tagged cullin Cdc53 (Cdc53–6HA) and HA-tagged Skp1 (Skp1–6HA) in our strain background (W303) (Fig. 2A,B, lane 3).

We then asked whether Tem1 is associated with the SCF^Amn1 complex. First, we confirmed the association between HA-tagged Tem1 (Tem1–3HA) and Amn1–3Flag using a co-immunoprecipitation assay (Fig. 2C, lane 2). Second, we asked whether Cdc53–6HA could be co-immunoprecipitated with Flag-tagged Tem1 (Tem1–3Flag) from cells endogenously expressing both proteins. Results showed that indeed Tem1 binds to Cdc53 in wild-type cells, whereas the interaction is severely impaired in amn1Δ cells (Fig. 2D, lanes 2 and 3), suggesting that Tem1 binding to SCF is mediated by Amn1.

Tem1 interaction with SCF^Amn1 suggests that Tem1 might be targeted for ubiquitin-mediated degradation and/or ubiquitin-dependent nuclear import. Therefore, we assessed whether Tem1 ubiquitylation (Cassani et al., 2013; Tamborrini et al., 2018) is regulated during the cell cycle and is dependent on Amn1. To this end, we carried out Ni-NTA pulldowns of ubiquitylated proteins from synchronised cells overexpressing untagged or His-tagged ubiquitin, followed by western blotting to detect Tem1–3HA. Since Amn1 is expressed after mitotic exit, cells were synchronised in late mitosis using the temperature-sensitive cdc15-2 mutation and released at permissive temperature. Under these conditions, we could detect mono- and poly-ubiquitylated forms of Tem1, some of which were periodic during the cell cycle (Fig. 2E). Remarkably, cell cycle-regulated Tem1 ubiquitylation peaked at 30 min after the late mitotic release, that is, when Amn1–3Flag levels rose (Fig. 2E), and was drastically impaired by AMN1 deletion, suggesting that it is greatly facilitated by Amn1, presumably bound to SCF.

Finally, we analysed the impact of Amn1-dependent ubiquitylation on Tem1 turnover. To this end, amn1Δ and GAL1-AMN1 cells carrying an extra copy of TEM1 under the control of the GAL1 promoter were synchronised in G1 and subjected to a short pulse (30 min) of galactose induction, followed by glucose-mediated repression. Tem1 decay was accelerated by the pulse of Amn1 (Fig. 2F), suggesting that Tem1 ubiquitylation by SCF^Amn1 mildly stimulates Tem1 proteolysis. Consistently, AMN1 deletion led to a two- to three-fold increase in Tem1 steady-state levels (Fig. 3F), in agreement with a previous report (Wang et al., 2003). Taken together, these data suggest that Tem1 interaction with and ubiquitylation by SCF^Amn1 from cytokinesis to late G1 stimulates Tem1 degradation.

To assess whether Amn1-bound SCF is involved in Tem1 nuclear import, besides its proteolysis, we deleted the F-box domain of Amn1 (amino acids 166–263; Fig. 3A). F-box motifs mediate the binding of F-box proteins to SCF (Bai et al., 1996). As expected, Amn1-ΔFbox no longer associated with Skp1 (Fig. 2B, lane 4). In addition, F-box deletion in Amn1 abolished the binding of Tem1 to Cdc53 without affecting the Tem1–Amn1 interaction (Fig. 2C,D, lane 4). Thus, the F-box motif of Amn1 is necessary for Amn1 and Tem1 interaction with SCF, but not for Amn1 binding to Tem1. Interestingly, F-box deletion led to higher levels of Amn1–3Flag, as assessed by western blotting (Fig. S2), consistent with the finding that Amn1 proteolysis in G1 requires SCF (Wang et al., 2003). It should be noticed, however, that Amn1-ΔFbox protein levels nevertheless oscillated during the cell cycle with similar kinetics to full-length Amn1, suggesting that additional ubiquitin ligases control its degradation.

Live-cell imaging of Tem1–GFP localisation in amn1-ΔFbox cells showed that Tem1 nuclear import was not affected (102/102 cells; Fig. 3B), while its levels at the daughter SPB in G1 were moderately increased (Fig. 3C). Therefore, Amn1 promotes Tem1 nuclear import independently of SCF.

We then wondered which features of Amn1 could be key for mediating Tem1 nuclear import. Using the cNLS-Mapper prediction software (Kosugi et al., 2009), we found a putative bipartite nuclear localisation signal (NLS) in the primary sequence of Amn1 (amino acids 13–31; Fig. 3A). GFP-tagged Amn1 was readily detected in the nucleus of daughter cells after cytokinesis (59/60 cells; Fig. 3D, left), as previously shown (Wang et al., 2003). Mutating pairs of basic residues in the NLS into alanine (amino acids 13–14 and 30–31), either together (Amn1-4A; Fig. 3D, right) or separately (Amn1-A₁₃A₁₄ and Amn1-A₃₀A₃₁; Fig. S3), prevented Amn1 nuclear translocation (55/56 amn1-4A cells, 1/56 cells with no fluorescence), while the protein accumulated asymmetrically in the cytoplasm of the daughter cell. Thus, the identified NLS is crucial for Amn1 nuclear localisation. The lack of Amn1 diffusion between mother and bud compartment is consistent with the notion that Amn1 synthesis occurs after cytokinesis (i.e. after complete separation of mother and bud cytoplasm). Importantly, the amn1-4A mutant allele affected neither Amn1 binding to Skp1 (Fig. 2B, lane 5) nor Tem1 interaction with Amn1 and Cdc53 (Fig. 2C, lane 3; Fig. 2D, lane 5).

In agreement with an escorting function of Amn1 in Tem1 nuclear import, Tem1 was not translocated into the nucleus of amn1-4A cells at any cell cycle stage (70/70 cells; Fig. 3E; Fig. S4). Moreover, Tem1 completely disappeared from the daughter cell SPB during G1 (i.e. around the time of cytoplasmic accumulation of Amn1; Fig. 3E; Fig. S4), indicating that binding to Amn1 evicts Tem1 from SPBs even in the absence of nuclear translocation. Remarkably, amn1-4A cells displayed even lower levels of Tem1 at the daughter SPB in G1 than wild-type cells (Fig. 3C). This is likely a consequence of Amn1 failure to enter the nucleus, which increases its window of opportunity for dislodging Tem1 from the SPB.

We took advantage of the amn1-4A mutant to assess the possible contribution of Tem1 nuclear import to its proteolysis. By analysing Tem1 protein levels using western blotting, we found that F-box deletion or NLS mutation individually caused a mild stabilisation of Tem1 in G1, whereas deletion of both motifs together led to an additive increase in Tem1 levels, albeit not as pronounced as in amn1Δ cells (Fig. 3F). Conversely, AMN1 overexpression from the galactose-inducible GAL1 promoter (GAL1-AMN1) dramatically decreased Tem1 protein levels in an F-box-dependent manner (Fig. 3G). Thus, nuclear import and binding to SCF both contribute to Tem1 proteolysis.

In summary, Amn1 inhibits Tem1 at SPBs by two different mechanisms: SCF-mediated protein degradation and SCF-independent nuclear translocation (Fig. 4). Whether Tem1 has functions independent of the MEN inside the nucleus is an intriguing possibility that deserves further investigation. Given that Tem1 becomes activated at SPBs, the dual mode of action of Amn1 may ensure rapid Tem1 inactivation after cytokinesis. Competition between Amn1 and Cdc15 for association with Tem1 (Wang et al., 2003) could provide an additional mode of Tem1 downregulation. Because AMN1 expression requires the transcription factor Ace2, which in turn enters the nucleus upon MEN-dependent activation of Cdc14, Tem1 eviction from the daughter SPB and degradation are only possible after Tem1 has fulfilled its MEN-promoting functions. On the other hand, because Amn1 itself and Ace2 are targets of SCF^Amn1, their clearance in G1 re-sets the stage for Tem1 re-accumulation in the following S and M phases. Thus, this sophisticated cell cycle circuit contributes to the ordered relay of MEN-regulated processes.

All yeast strains (Table S1) are congenic to or at least four times backcrossed to W303 (ade2-1, trp1-1, leu2-3,112, his3-11,15, ura3). One-step tagging techniques were used to generate 3HA-, yeGFP-, sfGFP-, mScarlet-I- and mCherry-tagged proteins at the C terminus. The Tem1–yeGFP construct had been previously described (Scarfone et al., 2015). A Yiplac211 plasmid bearing mCherry-PUS1 (gift from Maria Moriel Carretero, CRBM, Montpellier) was integrated at the ura3 locus after cutting with ApaI. The shs1-GFP-bearing BYP6904 plasmid was obtained from the National BioResource Project (Yeast) (https://yeast.nig.ac.jp/yeast) and was used to generate the shs1::LEU2::SHS1-GFP strain after cutting with BglII and integration at the SHS1 locus. GAL1-TEM1 and GAL1-AMN1 strains were generated by inserting a natNT2-GAL1 cassette upstream the ATG at the TEM1-3HA and AMN1-3Flag locus, respectively. Since TEM1 is an essential gene, the natNT2-GAL1 cassette was integrated into a strain that carried an extra copy of TEM1 on a centromeric plasmid. Strains deleted of the entire AMN1 coding sequence were obtained by insertion of the KanMX, K.l.URA3 or C.a.URA3 cassettes by one-step transformation. A pRS313-AMN1 plasmid (pSP1402) was constructed by inserting a DNA fragment covering the entire coding sequence of AMN1 with 500 bp of 5′ UTR and 178 bp of 3′ UTR, amplified by PCR from genomic DNA of a wild-type strain. Lys13 and Arg14 of Amn1 were both mutated to Ala by inserting a synthetic DNA duplex made of oligonucleotides MP902 and MP903 between the MluI and EcoRI sites in the AMN1 gene. Lys30 and Lys31 of Amn1 were both mutated to Ala by inserting a synthetic DNA duplex carrying these mutations (Genecust) between the EcoRV and EcoRI sites in the AMN1 gene. The deletion of the F-box domain of Amn1 (amino acids 163 to 263) was obtained by inverse PCR using oligonucleotides MP1037 and MP1038. Mutated AMN1 alleles were transplaced at the AMN1 locus by replacing the URA3 marker in amn1::K.l.URA3 or amn1::C.a.URA3 strains followed by counter-selection on FoA-containing plates. The pRS413-TEM1 plasmid (pSP526) contains the entire coding sequence for TEM1 and 524 bp of 5′ UTR. Details of oligonucleotides used are provided in Table S2.

Yeast cultures were grown at 25–30°C, in either SD medium (6.7 g/L yeast nitrogen base without amino acids), supplemented with the appropriate nutrients, or YEP (1% yeast extract, 2% bactopeptone, 50 mg/l adenine) medium. Raffinose was supplemented to 2% (SD-raffinose or YEPR), glucose to 2% (SD-glucose or YEPD), and galactose to 1% (SD-raffinose/galactose or YEPRG). Cells were synchronised in G1 by alpha factor (4 µg/ml; Genscript) in YEP medium containing the appropriate sugar at 25°C. G1 arrest was monitored under a transmitted light microscope and cells were released in fresh medium (typically after 120–135 min of alpha factor treatment) at 30°C, unless otherwise specified, after being collected by centrifugation at 2000 g followed by one wash with YEP containing the appropriate sugar.

Analysis of 6His-tagged ubiquitin Tem1 conjugates was performed as previously described (Tamborrini et al., 2018).

For fluorescence-activated cell sorting (FACS) analysis, yeast cells were collected and treated as described previously (Benzi et al., 2020) and were analysed on a ACEA NovoCyte cytometer.

For trichloroacetic acid (TCA) protein extracts, 10–15 ml of cell culture in logarithmic phase (OD600=0.5–1) was collected by centrifugation at 2000 g, washed with 1 ml of 20% TCA and resuspended in 100 µl of 20% TCA before breakage of cells with glass beads (diameter 0.5–0.75 mm) on a Vibrax VXR (IKA). After addition of 400 µl of 5% TCA, lysates were centrifuged for 10 min at 845 g. Protein precipitates were resuspended in 100 µl of 3× SDS sample buffer (240 mM Tris-HCl pH6.8, 6% SDS, 30% glycerol, 2.28 M β-mercaptoethanol and 0.06% Bromophenol Blue), denatured at 99°C for 3 min and loaded on SDS–PAGE after elimination of cellular debris by centrifugation (5 min at 20,000 g).

Native yeast protein extracts for immunoprecipitations were obtained from cell pellets (equivalent to ∼50 OD₆₀₀), after centrifugation at 2000 g and washing with 1 ml of cold 10 mM Tris-HCl pH 7.5. Cells were then lysed using glass beads in a Bullet Blender in 25 mM Tris-HCl pH 7.4, 100 mM NaCl, 2 mM EDTA, 1 mM DTT and 0.1% IGEPAL, containing a cocktail of protease inhibitors (Complete EDTA-free, Roche) and phosphatase inhibitors (PhosSTOP, Roche). Lysates were cleared at 20,000 g for 10 min at 4°C and incubated on a nutator for 2 h at 4°C with 15 µl of Protein G Dynabeads (Invitrogen) and anti-Flag-M2 antibodies (F1804 Sigma-Aldrich; 1 μg/IP). After washing the beads three times, proteins were eluted in 50 mM Tris-HCl pH 8.3, 1 mM EDTA and 0.1% SDS containing 0.5 mg/ml of Flag peptide (Genscript).

For western blotting, proteins were wet-transferred on Protran membranes (Schleicher and Schuell). Total proteins were revealed by Amido Black staining and quantified with ImageJ (NIH, Bethesda, MD) after scanning the membranes. Specific proteins were detected with monoclonal anti-HA 12CA5 (AgroBio, 1:5000) or monoclonal anti-Flag M2 (Sigma F3165, 1:5000). Antibodies were diluted in 5% low-fat milk (Regilait) dissolved in TBST (25 mM Tris, 137 mM NaCl, 2.68 mM KCl, 0.1% Tween-20). Secondary antibodies were purchased from GE Healthcare, and proteins were detected by a luminol/p-coumaric acid enhanced chemiluminescence system. Membranes were imaged with an Amersham Imager 600, and protein bands were quantified using ImageJ.

Imaging of Tem1 at SPBs in cells arrested in G1 with alpha factor was performed after fixation in 4% paraformaldehyde (PFA). Still digital images were taken with an oil immersion 100×1.4 HCX PlanApochromat objective (Zeiss) with a Coolsnap-HQ2 CDD camera (Photometrics) mounted on a Zeiss AxioimagerZ1 fluorescence microscope and controlled by the MetaMorph imaging system software (Molecular Devices). Z stacks containing 18 planes were acquired with a step size of 0.3 µm and were maximum projected using ImageJ. Fluorescence intensities were measured using ImageJ. SPB particles were identified on the Spc42–mCherry images using the ImageJ Analyze Particles tool after applying a threshold. Maximum pixel values present in these SPB particles were extracted in both Spc42–mCherry and Tem1–yeGFP images. Background values for both channels were determined as the mean pixel value of several regions outside SPB particles. The log₂ of Tem1 signals was finally plotted according to the following equation: log₂[(SPB^MAX−Bkgnd^GFP)/(SPB^MAX−Bkgnd^mCherry)] (Fig. 3C; Fig. S1B). For time-lapse video microscopy, cells were mounted on 0.8% agarose pads in SD medium on Fluorodishes (FD35-100 World Precision Instruments) and filmed at controlled temperature (30°C) with a 100×1.49 NA oil immersion objective mounted on a Nikon Eclipse Ti microscope equipped with an EMCCD Evolve-512 Camera (Photometrics) and iLAS2 module (Roper Scientific), which was controlled using Metamorph. Z stacks of 13 planes were acquired every 3–4 min with a step size of 0.4 µm in HILO mode. Z stacks were maximum projected using ImageJ. Alternatively, time-lapse video microscopy was performed using a 100× Plan Apo lambda 1.45 NA objective mounted on a Dragonfly Andor spinning-disk equipped with dual camera for two-channel simultaneous acquisition and coupled to a Ti2 Nikon microscope (Fig. 3C). Tem1 signals at SPBs were quantified as above, except that the background was calculated for each individual SPB as the mean pixel value in the vicinity of the SPB.

We are grateful to Maria Moriel-Carretero for the mCherry-PUS1 plasmid, to Marco Geymonat and all members of Piatti's lab for useful discussions. We acknowledge the imaging core facility MRI, member of the national infrastructure France-BioImaging, supported by the French National Research Agency (ANR-10-INBS-04, ‘Investments for the future’).

The peer review history is available online at https://journals.biologists.com/jcs/article-lookup/doi/10.1242/jcs.258972