Molecular Mechanisms that Restrict Yeast Centrosome Duplication to One Event per Cell Cycle

Please cite this article in press as: Elserafy et al., Molecular Mechanisms that Restrict Yeast Centrosome Duplication to One Event per Cell Cycle, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.05.032 Current Biology 24, 1–11, July 7, 2014 ª2014 Elsevier Ltd All rights reserved

http://dx.doi.org/10.1016/j.cub.2014.05.032

Article Molecular Mechanisms that Restrict Yeast Centrosome Duplication to One Event per Cell Cycle  c ,1,2 Annett Neuner,1,2 Menattallah Elserafy,1,2 Mirela Sari Tien-chen Lin,1 Wanlu Zhang,1 Christian Seybold,1 Lavanya Sivashanmugam,1 and Elmar Schiebel1,* 1Zentrum fu ¨ r Molekulare Biologie der Universita¨t Heidelberg, DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany

Summary Background: The spindle pole body (SPB) of budding yeast is the functional equivalent of the mammalian centrosome. Like the centrosome, the SPB duplicates once per cell cycle. The new SPB assembles adjacent to the mother SPB at a substructure called the bridge. The half-bridge, the bridge precursor, is a one-sided extension of the SPB central plaque layered on both sides of the nuclear envelope. Parallel Sfi1 molecules longitudinally span the half-bridge with their N termini embedded in the SPB central plaque, whereas their C termini mark the half-bridge distal end. In early G1, half-bridge elongation by antiparallel C-to-C dimerization of Sfi1 exposes free N-Sfi1 where the new SPB assembles. After SPB duplication, the dimerized Sfi1 is severed to allow spindle formation and SPB reduplication. Results: We show that Sfi1 C-terminal domain harbors phosphorylation sites for Cdk1 and the polo-like kinase Cdc5. Cdk1 and, to a lesser extent, Cdc5 inhibit SPB duplication as phosphomimetic sfi1 mutations lead to metaphase cells with a single SPB. In contrast, phosphoinhibitory sfi1 mutations in Cdk1 sites are lethal because cells fail to sever the bridge after SPB duplication. Moreover, Cdc14 dephosphorylates C-Sfi1 to prepare it for a new round of duplication, and the kinase Mps1 promotes Sfi1 extension in G1. Conclusions: Positive (Cdc14) and negative (Cdk1 and Cdc5) SPB duplication signals are integrated at the level of the halfbridge component Sfi1. In addition, Mps1 activates Sfi1 duplication. Fluctuating activities of these regulators ensure one SPB duplication event per cell cycle. Introduction The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. Both organize microtubules, and both are duplicated in a cell-cycle-dependent manner only once per cell cycle [1–3]. Misregulation of centrosome duplication in human cells leads to chromosome segregation defects and eventually to cancer [4]. The SPB is a multilayered structure that is embedded in the nuclear envelope (NE) throughout the cell cycle [5]. SPB duplication takes place at the half-bridge, an electron-dense, onesided extension of the central SPB plaque that is layered on the nuclear and cytoplasmic sides of the NE [5, 6] (Figure 1A). SPB duplication starts in early G1 phase with the elongation of the half-bridge and the formation of the bridge structure,

2Co-first

author *Correspondence: [email protected]

which, at its distal end, serves as an assembly platform for the formation of the new SPB [5]. In late S phase, the two side-by-side SPBs are separated by medial fission of the bridge to allow spindle formation by the two SPBs each bearing a half-bridge. Genetic data suggest that the kinesin5 motor proteins Cin8 and Kip1, at least partly, drive this separation by creating a force that pulls both SPBs apart [12–14]. The half-bridge is composed of four proteins: the yeast centrin Cdc31 and its binding partner Sfi1, the tail-anchored protein Kar1 that also interacts with Cdc31, and the SUN domain protein Mps3 [15–22] (Figure 1A). Sfi1 plays a major role in halfbridge elongation. Sfi1 is a long filamentous protein that spans the length of the half-bridge on the cytoplasmic side of the NE. The N terminus of Sfi1 is close to the SPB central plaque, whereas the C terminus defines the distal end of the halfbridge [20, 21]. The central part of Sfi1 contains 20 centrin binding sites. Half-bridge extension is probably driven by C-to-C dimerization of Sfi1 molecules (Figure 1A). This leads to free Sfi1 N termini at the distal end of the bridge. It was suggested that in G1, Sfi1 N termini initiate the assembly of a miniature version of the SPB that is known as the ‘‘satellite.’’ The satellite proceeds in the formation of the complete SPB after START of the cell cycle [5, 20] (Figure 1A). SPB duplication is regulated by phosphorylation and dephosphorylation events. Cyclin-dependent kinase (Cdk1) activity either promotes or inhibits SPB duplication depending on the cell-cycle phase in which it acts [23]. Cdk1 in complex with G1 cyclins Cln1–Cln3 stimulates the formation of the new SPB by expanding the satellite into a full SPB [24]. S phase Cdk1 (Clb5 and Clb6) activity promotes separation of the two side-by-side SPBs by inhibiting APCCdh1 in order to protect the motor proteins Cin8 and Kip1 from degradation [12–14, 25, 26]. Later in the cell cycle, mitotic Cdk1 (Clb1–Clb4) inhibits SPB reduplication through a mechanism that is currently unclear [23, 26, 27]. In addition, the kinase Mps1 is essential for a number of SPB duplication steps [28, 29]. Here, we show that SPB duplication in budding yeast is regulated through the phosphorylation and dephosphorylation of the C terminus of Sfi1 by the kinases Cdk1 and polo-like Cdc5 and the phosphatase Cdc14. Cdc14 drives mitotic exit by dephosphorylating a subset of Cdk1 substrates in anaphase [30]. Cdc14 and Mps1 kinase promote C-to-C dimerization of Sfi1 in G1. Phosphorylation of Sfi1 by Cdk1 and Cdc5 prevents this dimerization step in mitosis. After SPB duplication, phosphorylation of Sfi1 by Cdk1 is important for the separation of the two adjacent SPBs. Thus, we describe how phosphoregulation of Sfi1 restricts SPB duplication to one event per cell cycle. Results Cdk1 Phosphorylation Regulates Sfi1 The molecular basis by which cell-cycle regulators control the initiation of SPB duplication has yet to be elucidated. A potential target of Cdk1 is the half-bridge component Sfi1. Cells bearing a mutation in the SFI1 gene that impairs phosphorylation of the Cdk1 phosphosite S855 are unable to grow in a background deficient in spindle assembly checkpoint (SAC;

Please cite this article in press as: Elserafy et al., Molecular Mechanisms that Restrict Yeast Centrosome Duplication to One Event per Cell Cycle, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.05.032 Current Biology Vol 24 No 13 2

Figure 1. Cdk1 Phosphorylation Regulates Sfi1 (A) The SPB duplication model. Nuclear envelope (NE), N terminus of Sfi1 (N-Sfi1), C-terminal dimerization domain of Sfi1 (Sfi1 C-C), and Satellite (S) are shown here. (B) In vitro kinase reaction with purified Cdk1Clb2 and recombinant GST and GST-Sfi1. The kinase reaction was performed in the presence of g-32P-ATP. The experiment was analyzed by SDS-PAGE and autoradiography. (C) Mass spectrometry analysis of Sfi1 phosphorylated by Cdk1-Clb2 in vitro. Shown are the identified Cdk1 consensus sites in the C terminus of Sfi1. These sites were also mapped in vivo [7–9]. The plus symbol (+) in the third column indicates Cdk1 sites (SPxK/R) predicted to be dephosphorylated by Cdc14 [10]. (D) Plasmid shuffle experiment to test growth of yeast cells containing SFI1 alleles. The SFI1 shuffle strain (sfi1D::HIS3 pRS316-Sfi1) (left) and a SFI1 shuffle strain with mad2D (right) were transformed with the integration plasmid pRS305K (2), pRS305K-SFI1 (SFI1), or the indicated pRS305K-sfi1Cdk1mutant alleles. Cells were incubated on YPD and 5-fluoroorotic acid (5-FOA) plates for 3 days. Drop tests were performed with serial 10-fold dilutions starting with equivalent cell concentrations. (E) Dominancy of sfi1Cdk1-6A and sfi1Cdk1-6D alleles was tested by transforming high gene dosage pRS425 [11], pRS425-SFI1, pRS425sfi1Cdk1-6A, or pRS425-sfi1Cdk1-6D plasmids in wild-type yeast strain ESM356. Shown is the mean of two independent experiments using the same batch of frozen competent ESM356 cells. Error bars show mean 6 SD.

mad2D) because of an SPB separation defect [31]. To obtain a more-complete picture of how Cdk1 regulates Sfi1, we purified both proteins and performed an in vitro kinase reaction in the presence of g-32P-ATP. Sfi1 was phosphorylated by Cdk1Clb2, whereas glutathione S-transferase (GST) alone was not (Figure 1B). Mass spectrometric analysis of in vitro phosphorylated Sfi1 identified four phosphosites (T816, S855, S882, and S892) in the C terminus of Sfi1. These sites correspond to the Cdk1 consensus S/TPxK/R (Figure 1C) and are phosphorylated in vivo [8, 9]. In addition, the Cdk1 consensus sites S801 and S923 in C-Sfi1 are also known to be phosphorylated in vivo [7, 8] (Figure 1C). Thus, Cdk1 is able to phosphorylate several sites within the C-terminal region of Sfi1. To confirm that C-Sfi1 does not carry additional Cdk1 sites, we performed an in vitro kinase assay with C-Sfi1 and C-Sfi16A (serine and threonine residues of the six Cdk1 sites were changed to alanine) as substrates. We also tested whether the S phase cyclin Clb5 and the mitotic cyclin Clb2 have different specificity toward C-Sfi1. We coimmunoprecipitated Cdk1as1 in complex with either Clb5-TAP or Clb2-TAP from

yeast lysates [32]. cdk1as1 encodes a mutated allele of CDK1 that can be inhibited by the ATP analog 1NM-PP1 [33, 34]. The C terminus of Sfi1 was phosphorylated by both Cdk1as1-Clb2 and Cdk1as1-Clb5 (Figure S1A available online). However, Cdk1as1-Clb2 was clearly more efficient in phosphorylating C-Sfi1 than Cdk1as1-Clb5 was. In contrast, Cdk1as1-Clb5 phosphorylated a contaminating peptide more efficiently than Cdk1as1-Clb2. In the presence of the Cdk1as1 inhibitor 1NM-PP1, phosphorylation of C-Sfi1 could not be detected (Figure S1A). Importantly, Cdk1 did not phosphorylate the C terminus of Sfi1-6A, indicating the lack of additional Cdk1 phosphorylation sites in C-Sfi1. We next asked which Cdk1-cyclin complex may regulate C-Sfi1 in vivo. In the yeast two-hybrid system, the mitotic cyclins Clb2, Clb3, and Clb4 showed weak to strong interactions with C-Sfi1, respectively (Figure S1B). The B type cyclin Clb4 is especially interesting because the Cdk1-Clb4 complex already binds in early S to the cytoplasmic side of the SPB where it may also regulate Sfi1 [35, 36]. The Cdk1 sites in C-Sfi1 were mutated to investigate their function. The phosphoacceptor serine and threonine residues of each Cdk1 site were mutated to alanine (phosphoinhibiting) or aspartic acid (phosphomimetic). sfi1Cdk1-1A/1D (sfi1S855) and sfi1Cdk1-5D (sfi1-S801D T816D S882D S892D S923D) mutant cells grew at all temperatures tested, whereas sfi1Cdk1-5A (sfi1-S801A T816A S882A S892A S923A) cells

Please cite this article in press as: Elserafy et al., Molecular Mechanisms that Restrict Yeast Centrosome Duplication to One Event per Cell Cycle, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.05.032 Molecular Mechanisms of SPB Duplication 3

showed a conditional lethal growth defect at 37 C (Figure S1C). Inactivation of the SAC gene MAD2 was lethal for sfi1Cdk1-1A/ 1D and sfi1Cdk1-5A/5D cells even at 23 C, suggesting that the defect in these cells was compensated for by a SAC-dependent cell-cycle delay (Figure 1D, right). Importantly, phosphomimetic and phosphoinhibitory mutations in the six C-terminal Cdk1 sites (sfi1Cdk1-6A and sfi1Cdk1-6D) were lethal for cells even in the presence of a functional SAC (Figure 1D, left). In addition, we noticed that the sfi1Cdk1-6A or sfi1Cdk1-6D alleles integrated in the LEU2 locus of the shuffle strain (sfi1D pRS316-SFI1) inhibited cell growth, as indicated by the smaller colony size (Figure 1D, yeast peptone dextrose [YPD]). This suggests that both SFI1 alleles might be dominant lethal. Indeed, sfi1Cdk1-6A and sfi1Cdk1-6D on the high gene dosage plasmid pRS425 hardly gave transformants in wild-type cells, whereas the empty plasmid pRS425 or pRS425-SFI1 gave similar transformation efficiencies (Figure 1E). Together, phosphoregulation of C-Sfi1 by Cdk1 is important for cell viability. Sfi1 Phosphorylation by Cdk1 Is Important for SPB Separation and Prevents SPB Reduplication To analyze the phenotype of sfi1Cdk1-6A and sfi1Cdk1-6D in greater detail, we expressed the two alleles in an SFI1-degron strain (td-sfi1). In this strain, endogenous SFI1 was fused to a fragment coding for the DHFR-based temperature-inducible degron (td). The td-Sfi1 fusion protein was degraded upon shifting cells to 37 C and upon overexpression of the ubiquitin E3 ligase UBR1 [37]. Fluorescence measurements in yeGFPtd-sfi1 cells showed that w80% of the SPB pool of yeGFPtd-Sfi1 was degraded over a 3 hr incubation at restrictive temperature (Figures S1D and S1E). As reported for the conditional lethal sfi1-3 allele [20], temperature-induced degradation of td-Sfi1 blocked cell-cycle progression and induced an SPB duplication defect (Figures S1F and S1G). After 150 min at restrictive conditions, td-sfi1 cells carried a single Spc42mCherry fluorescent signal corresponding in intensity to the single SPB signal of G1 cells (Figure S1G). Spc42 is a component of the central plaque, and the Spc42-mCherry fluorescent signal at SPBs increases in intensity around 1.5 times from G1 (Figure 1A, SPB with satellite) to S phase (two side-by side SPBs) with SPB duplication (Figure S1G) [38, 39]. Introducing SFI1 alleles into the LEU2 locus of td-sfi1 cells allowed us to analyze the sfi1Cdk1-6A and sfi1Cdk1-6D phenotype after degradation of td-Sfi1. Consistent with the shuffle experiment (Figure 1D), sfi1Cdk1-6A and sfi1Cdk1-6D did not promote growth of td-sfi1 cells (Figure 2A). We used the tdsfi1 cells with SFI1, sfi1Cdk1-6A, or sfi1Cdk1-6D in a time course experiment to analyze the phenotype (Figure 2B). Cells were synchronized with a factor under permissive conditions. At 30 min before the release of the cell-cycle block, galactose was added to induce UBR1 expression. With the release of the cell-cycle block, cells were shifted to 37 C. Cells expressing wild-type SFI1 in the td-sfi1 background cycled through the cell cycle, as indicated by the appearance of budded cells with two separated SPBs after 30 min and by the migration of one of the two SPBs into the daughter cell in anaphase after 60 min (Figure 2C). The level of the mitotic cyclin Clb2 started to decline after 90 min, as the Cdk1-Clb inhibitor Sic1 accumulated during mitotic exit. In contrast, 40%–50% of sfi1Cdk1-6A (Figure 2D, 90 and 120 min) and >80% of sfi1Cdk1-6D cells (Figure 2E) arrested in the first cell cycle with a large bud and a single Spc42-mCherry SPB signal (Figures 2D and 2E, fluorescent images on the right). These data are indicative of either

an SPB duplication or a separation defect in sfi1Cdk1-6A and sfi1Cdk1-6D cells. To determine whether sfi1Cdk1-6A and sfi1Cdk1-6D cells were defective in either SPB duplication or separation, we quantified the Spc42-mCherry signal intensity (Figures 2C–2E, right). In wild-type cells, the intensity of the Spc42-Cherry signal showed a transient increase in signal intensity at the time of SPB duplication at 60 min (Figure 2C). This fluorescence increase was about 1.4-fold and was very close to the increase of the Spc42-mCherry SPB signal between G1 (a factor; SPB with satellite) and G1/S (side-by-side SPB) cells (Figure S1G). sfi1Cdk1-6A cells that arrested with a large bud and a single SPB signal showed an Spc42-mCherry signal that was 1.5fold of the corresponding sfi1Cdk1-6D cells or the single SPB signal of SFI1 wild-type cells at 120 and 150 min (Figures 2C–2E). This suggests that sfi1Cdk1-6A cells have duplicated but unseparated SPBs, whereas sfi1Cdk1-6D cells probably have an SPB duplication defect. To confirm the SPB defects, we analyzed sfi1Cdk1-6A, sfi1Cdk1-6D, and SFI1 cells by electron microscopy. td-sfi1 SFI1 (SFI1) cells separated their SPBs and assembled a bipolar spindle (Figure S1H). Consistent with the fluorescence measurements (Figure 2D), large budded sfi1Cdk1-6A cells harbored two side-by-side SPBs that were still connected by the bridge (Figures 2F and S1I). In contrast, sfi1Cdk1-6D cells arrested with a single SPB as large budded cells (Figures 2F and S1I). Taken together, the defect in SPB duplication that was observed in sfi1Cdk1-6D cells suggests that Cdk1 phosphorylation of Sfi1 blocks SPB reduplication in S phase and mitosis, whereas Cdk1 phosphorylation of C-Sfi1 is required for SPB separation, as indicated by the side-by-side SPB phenotype in sfi1Cdk1-6A cells. Cdc5 Assists Cdk1 in Phosphorylating Sfi1 to Inhibit SPB Duplication in Mitosis In higher eukaryotes, polo kinase promotes centrosome separation through promoting linker dissolution [40]. In addition, Shirk et al. [41] showed that Cdc5 ensures SPB separation in meiosis. These data raise the question of whether Cdc5 also regulates SPB duplication through Sfi1 phosphorylation. We first asked whether Sfi1 and Cdc5 are present in common complexes. The affinity-purified anti-Cdc5 antibodies detect Cdc5 in immunoblots (Figure S2A). Cdc5 and Sfi16HA were both found in anti-Cdc5 immunoprecipitations (Figure 3A). A control showed that Sfi1-6HA did not bind unspecifically to the protein G beads. Consistently, HA-tag-specific antibodies precipitated Cdc5 from Sfi1-6HA, but not with Sfi1 cells (Figure S2B). Thus, Cdc5 interacts with Sfi1. The C terminus of Sfi1 contains five Cdc5 kinase consensus sites (D/N/E-x-S/T) [42]. Two of these consensus sites, S801 and S882, are also Cdk1 phosphorylation sites. An in vitro kinase assay with recombinant GST-Sfi1 showed that purified Cdc5 phosphorylated Sfi1, but not GST (Figure 3B). Mass spectrometry of Cdc5-phosphorylated Sfi1 identified the Cdc5 consensus sites S826, T866, and T876 (Figure 3C). This indicates that Sfi1 is a bona fide Cdc5 substrate. To analyze the physiological significance of Sfi1 phosphorylation by Cdc5, we mutated S826, T866, and T876, as described above, for the Cdk1 sites. In a shuffle experiment, single SFI1 phosphomutants had no obvious growth defect at 23 C and 37 C (Figure S2C). Mutating two of the Cdc5 phosphorylation sites (S826D T876D and S826D T866D) impaired growth in a mad2D background at 23 C (Figure 3D). The triple sfi1Cdc5-3D mutant (S826D T866D T876D), however, failed to

Please cite this article in press as: Elserafy et al., Molecular Mechanisms that Restrict Yeast Centrosome Duplication to One Event per Cell Cycle, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.05.032 Current Biology Vol 24 No 13 4

Figure 2. Cdk1 Phosphorylation of C-Sfi1 Is Important for SPB Separation and Prevents SPB Reduplication (A) Drop test of pGal1-UBR1 td-sfi1 cells with pRS305K (2), pRS305K-SFI1 (SFI1), or the indicated pRS305K-sfi1Cdk1mutant alleles. Cells were grown on YPD and YPRaf/Gal plates at the indicated temperatures. Drop tests were performed with 10-fold serial dilutions. In this experiment, sfi1Cdk1-6A and sfi1Cdk1-6D were likely not dominant because td-sfi1 was overexpressed from the copper promoter [37], therefore outcompeting the mutant Sfi1 versions. (B) Outline of a time course experiment with td-sfi1 cells carrying SFI1, sfi1Cdk1-6A, or sfi1Cdk1-6D. Cells were grown in YPRaf media at 23 C. a factor was added for 4 hr to arrest cells in G1. At 30 min before a factor washout, galactose was added to induce UBR1 expression. After the release of the cell-cycle block, cells were shifted to 37 C to start td-Sfi1 degradation (t = 0). Samples were withdrawn every 30 min. (legend continued on next page)

Please cite this article in press as: Elserafy et al., Molecular Mechanisms that Restrict Yeast Centrosome Duplication to One Event per Cell Cycle, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.05.032 Molecular Mechanisms of SPB Duplication 5

support viability even when the SAC was functional (Figure 3D, bottom). In contrast, sfi1Cdc5-3A (S826A T866A T876A) cells grew in drop tests at the same rate as wild-type SFI1 cells. To characterize the phosphomimetic phenotype in greater depth, we expressed sfi1Cdc5-3D in the td-sfi1 strain. Time course analysis monitored cell-cycle progression and SPB duplication of SFI1 wild-type (Figure 3E), sfi1Cdc5-3A (Figure 3F), and sfi1Cdc5-3D (Figure 3G) cells. sfi1Cdc5-3A cells showed no obvious cell-cycle defect and behaved as td-sfi1 SFI1 control cells (Figures 3E and 3F). In contrast, w30% of sfi1Cdc5-3D cells arrested in the first cycle with a large bud and a single SPB signal (Figure 3G, red line). Analysis of cells by electron microscopy showed that these sfi1Cdc5-3D cells were defective in SPB duplication (Figures 3H and S2D). Thus, Cdc5 phosphorylation of Sfi1 is important to prevent SPB reduplication in S phase and mitosis. As outlined above, two of the five Cdc5 consensus sites are also Cdk1 sites (S801 and S882). We used sfi1Cdc5-5A and sfi1Cdc5-5D mutant alleles to test whether Cdc5 phosphorylation of Sfi1 potentially regulates SPB duplication in a similar strong manner as Cdk1. In a plasmid shuffle experiment, we could show that sfi1Cdc5-5A and sfi1Cdc5-5D cells grew as sfi1Cdc5-3A and sfi1Cdc5-3D cells at 23 C and 37 C (Figure S2E). Thus, based on the phosphorylation assay (Figure 3B) and the in vivo analysis (Figures 3E–3H and S2E), we can say that S826, T866, and T876 are functionally the major Cdc5 phosphorylation sites of Sfi1. Cdc14 Phosphatase Dephosphorylates Sfi1 During mitotic exit, the conserved phosphatase Cdc14 counteracts mitotic Cdk1 activity by inducing Clb2 degradation and dephosphorylation of a subset of Cdk1 substrates [10, 43]. Because Cdk1 phosphorylation of Sfi1 blocks SPB duplication (Figure 2), these phosphorylation modifications must be removed before cells enter G1. Dephosphorylation of Sfi1 by Cdc14 with mitotic exit would therefore be a mechanism that prepares Sfi1 for a new cycle of half-bridge elongation. In a global analysis, Bloom et al. identified Sfi1 as a binding partner and substrate of overexpressed Cdc14 [44]. Interestingly, four of the six Cdk1 sites in the C terminus of Sfi1 correspond to Cdc14 consensus dephosphorylation sites (SPxK/R) [10] (Figure 1C). We first confirmed the interaction between endogenous, nonoverexpressed Cdc14 and Sfi1. Cdc14-TAP was purified from yeast cells, and its binding partners were identified by mass spectrometry analysis. This analysis revealed an enrichment of SPB proteins, including Sfi1 (Figure S3A). Sfi1 was not found in the control purification using untagged CDC14 cells. These interactions are consistent with the binding of Cdc14 to anaphase SPBs [45]. Further characterization of the interaction between Sfi1 and Cdc14 by a yeast two-hybrid approach revealed binding of Cdc14 and the Cdc14 trap mutant protein Cdc14D253A [46] to the C terminus of Sfi1 (Figure 4A). To determine whether Cdc14 dephosphorylates Sfi1, we performed a phosphorylation

experiment followed by a dephosphorylation experiment [47]. First, recombinant GST-Sfi1 was incubated with g-32P-ATP in the presence of Cdk1-Clb2 to phosphorylate Sfi1. After inhibiting the Cdk1 kinase through the addition of EDTA, the phosphorylated Sfi1 was incubated with Cdc14WT or an inactive, phosphatase-dead version of Cdc14 (Cdc14C283A). This experiment revealed that Cdc14 was able to dephosphorylate Sfi1 (Figure 4B). We next studied SPB duplication in cells lacking Cdc14 activity. Mitotic progression in CDC14 wild-type and conditional lethal cdc14-2 cells was arrested at metaphase through the addition of the drug nocodazole at 37 C, the restrictive temperature of cdc14-2 cells. Nocodazole was then washed out to allow progression into anaphase. To overcome the mitotic exit defect of cdc14-2 cells [30], we overexpressed a stabilized version of the Cdk1 inhibitor SIC1 (sic1s) [48] (Figure 4C). Expression of sic1s inactivates mitotic Cdk1 activity and drives cdc14-2 cells into G1 phase, as indicated by rebudding. However, cytokinesis is disturbed in cdc14-2 sic1s cells because of a role of Cdc14 in this process [46]. We followed SPB duplication by using the Sfi1-yeGFP marker that doubles in signal intensity as soon as cells elongate the half-bridge into a bridge in early G1 (Figure 4D). In CDC14 wild-type cells, the Sfi1yeGFP signal doubled after 60 min of pGal1-sic1S induction, indicating half-bridge extension (Figure 4E). In contrast, the Sfi1-yeGFP signal of cdc14-2 cells increased only slowly and even after 120 min did not reach the CDC14 value of 60 min. Both CDC14 and cdc14-2 cells started to bud after 30 min, indicating that wild-type and mutant cells progressed at a similar rate through G1. Thus, Cdc14 promotes binding of Sfi1 to SPBs in G1 phase to achieve bridge extension. To confirm the delayed SPB duplication in cdc14-2 pGal1-sic1S, we analyzed cdc14-2 pGal1-sic1S and CDC14 pGal1-sic1S cells 60 min after Gal1-sic1S induction by electron microscopy. At t = 60 min, cdc14-2 pGal1-sic1S cells showed mainly one SPB (Figures 4F, S3B, and S3C), whereas most CDC14 pGal1-sic1S cells showed two SPBs (Figures 4F, S3B, and S3C). Thus, Cdc14 activity is needed for timely SPB duplication, most likely by removing the inhibitory Cdk1 phosphorylations at the C terminus of Sfi1. Mps1 Kinase Stimulates Sfi1 Binding to the SPB in G1 Mitotic Cdk1 activity phosphorylates the C terminus of Sfi1 and so prevents SPB reduplication during mitosis (Figure 2). Dephosphorylation of these Cdk1 sites by Cdc14 promotes half-bridge extension in G1 phase (Figures 4E and 4F). We therefore asked whether overexpression of CDC14 in metaphase would drive Sfi1 recruitment to SPBs. For this experiment, we used pMet3-CDC20 cdc26D cells in which expression of either the wild-type (pGal1-CDC14) or phosphatase-dead (pGal1-cdc14C283A) versions of Cdc14 was induced. Depletion of CDC20 arrested cells in metaphase, and the lack of CDC26 further inactivated the anaphase promoting complex (APC) at 37 C [49]. By using Sfi1-yeGFP, we followed halfbridge elongation over time. Although CDC14 was strongly

(C–E) The experiment was performed as described in (B). Cells from each time point were categorized according to bud morphology and the number of Spc42-mCherry signals. Note that we quantified the Spc42-mCherry fluorescent signal of only one SPB in case two separated SPBs were present per cell. The cell-cycle markers Sic1 and Clb2 were monitored over time. Tub2 (b-tubulin) was used as loading control. SPB duplication was analyzed by quantifying the signal intensity of Spc42-mCherry at the SPB. N = 50 cells per time point and strain. Error bars show mean 6 SD. Cells on the right are representative large budded cells. Scale bar of (C)–(E) represents 10 mm. (F) Electron microscopy of thin serial sections of cells from (D) and (E) after 120 min. The cartoons on the left summarize the SPB phenotype. Indicated are Bridge, cytoplasm (C), cytoplasmic microtubules (cMT), nucleus (N), nuclear envelope (NE), and nuclear microtubules (nMT). SPB1 and SPB2 indicate the two side-by-side SPBs of a sfi1Cdk1-6A cell. Scale bars of (F) represent 2 mm and 200 nm.

Please cite this article in press as: Elserafy et al., Molecular Mechanisms that Restrict Yeast Centrosome Duplication to One Event per Cell Cycle, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.05.032 Current Biology Vol 24 No 13 6

Figure 3. Cdc5 Phosphorylates Sfi1 and Prevents SPB Reduplication in Mitosis (A) Coimmunoprecipitation experiment. Cdc5 was pulled down with an anti-Cdc5 antibody bound to protein G Sepharose. Sfi1-6HA was detected with an anti-HA antibody. As control, the cell extract was incubated with protein G Sepharose only. (B) In vitro kinase assay with Cdc5 and recombinant GST-Sfi1. Reaction was performed in the presence of g-32P-ATP. Experiment was analyzed by SDS-PAGE and autoradiography. (legend continued on next page)

Please cite this article in press as: Elserafy et al., Molecular Mechanisms that Restrict Yeast Centrosome Duplication to One Event per Cell Cycle, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.05.032 Molecular Mechanisms of SPB Duplication 7

expressed and led to the dephosphorylation of Cdk1 substrates like Sli15 (data not shown) [50], the Sfi1-yeGFP signal did not increase upon CDC14 induction (Figures S3D and S3E). Thus, overexpression of CDC14 in mitosis did not promote Sfi1 accumulation at SPBs. One way to explain the outcome of the CDC14 overexpression experiment is that although CDC14 removes the blocking Cdk1 phosphorylations from Sfi1, a promoting activity that rises at the SPB in G1 is missing. The conserved Mps1 kinase has been shown to promote SPB duplication at the level of half-bridge extension and so could be a prime candidate for providing this activity [28, 51]. To address whether Mps1 activity is required for Sfi1 C-to-C dimerization in G1, we analyzed the Sfi1-yeGFP signal in the ATP-analog-sensitive mps1-as1 mutant [52] (Figures 5A–5C). mps1-as1 cells were arrested with a factor in G1, released by washing with YPD medium, and further cultivated until they entered metaphase. Then, the Mps1-as1 inhibitor 1NM-PP1 or DMSO were added together with a factor to rearrest the cells in G1 (Figure 5A). In the presence of 1NM-PP1, mps1-as1 cells failed to duplicate their SPBs and arrested with a single SPB signal [53] (Figure 5B). Quantification of the Sfi1-yeGFP signal revealed that Sfi1-yeGFP failed to incorporate into a bridge structure in the absence of Mps1 activity (Figure 5C). In contrast, mps1-as1 cells that were simply treated with DMSO doubled the Sfi1yeGFP signal. Thus, Mps1 activity is critical for half-bridge elongation in G1. Discussion Like their centrosome counterparts, SPB duplication is restricted to one event per cell cycle because overduplication leads to chromosome missegregation and aneuploidy. It has been previously established that Cdk1 activity has a dual role in both promoting and inhibiting SPB duplication [23]. It has also been clear that the activity of the conserved kinase Mps1 regulates multiple steps during the SPB duplication process [29, 53]. Here, we report that SPB duplication is also regulated by the polo-like kinase Cdc5 and the mitotic exit phosphatase Cdc14 (Figure 5D). Cdk1, Cdc5, and Cdc14 directly alter the phosphorylation status of the C-terminal portion of the half-bridge protein Sfi1. The action of Mps1 is broader because it phosphorylates at least three half-bridge and core SPB components: Cdc31, Spc29, and Kar1 [53, 54] (C.S., unpublished data). Our data define the following order of events during SPB duplication (Figure 5D). In early G1 phase of the cell cycle, the half-bridge consists of a parallel layer of Sfi1 proteins (Figure 1A). N-Sfi1 is embedded into the central core of the SPB and therefore not accessible to initiate a new round of SPB duplication. At this stage in the cell cycle, Mps1 kinase promotes C-to-C antiparallel interaction of Sfi1 that extends the half-bridge to form the bridge (Figure 5D, step 1) [20]. The

free N termini of Sfi1 that arose through Sfi1 tail-to-tail dimerization probably initiate the assembly of the satellite [3]. After passage through START, the new SPB fully assembles (Figure 5D, step 2). Phosphorylation of Spc42 by Cdk1-Cln1/2 activity is one of the driving forces for expansion of the satellite into the duplication plaque in G1 [24]. The two side-by-side SPBs are still connected by the bridge and reside next to one another in the NE. At the beginning of S phase, increasing Cdk1-Clb activity triggers SPB separation and spindle assembly by two parallel pathways (Figure 5D, step 3). Cdk1 inactivates the E3 enzyme APCCdh1, which leads to the accumulation of the two kinesin-5 motor proteins Cin8 and Kip1 [13, 14]. In addition, Cdk1 phosphorylates the C terminus of Sfi1 to promote the disassembly of the antiparallel Sfi1 oligomers. Yeast two-hybrid data indicate interaction of C-Sfi1 with Clb3 and Clb4 and a weaker interaction with Clb2 (Figure S1B). The interaction with Clb4 is interesting because the Cdk1-Clb4 complex associates with the cytoplasmic side of the SPB in early S phase [35]. Thus, Cdk1-Clb4 is in the right place at the right time to phosphorylate C-Sfi1 and to trigger SPB separation. The combined action of these two pathways results in the severing of the bridge and spindle assembly [13, 31]. In addition, Sfi1 phosphorylations in mitosis by Cdk1 and, to a lesser extent, also by Cdc5 block an additional round of Sfi1 C-to-C interaction and SPB reduplication (Figure 5D, step 4). However, Cdk1-Clb activity seems to be the predominant force that blocks SPB reduplication because the phosphoinhibitory sfi1Cdc5-3A cells have no obvious defect in SPB duplication, whereas the phosphomimetic sfi1Cdc5-3D cells only have a defect in about 30% of the cells (Figure 3G). Dephosphorylation of Sfi1 by Cdc14 at mitotic exit then prepares the SPB for duplication in G1 (Figure 5D, step 5). In summary, multiple mechanisms inhibit SPB overduplication in mitosis: phosphorylation of C-Sfi1 by Cdk1-Clb and Cdc5. In addition, Mps1 activity at SPBs is needed to promote SPB duplication. It is unclear whether the mitotic Mps1 that mainly functions at kinetochores is able to drive SPB duplication [55]. In any case, overexpression of yeast MPS1 in mitosis did not trigger half-bridge extension (M.E., unpublished data). It is therefore likely that it is mainly the residual Mps1 activity escaping the degradation by the anaphase-promoting complex at mitotic exit [56] that drives SPB duplication in G1. Thus, failure of one mechanism as seen in sfi1Cdk1-6A or sfi1Cdk1-6D cells (Figure 2) will unlikely lead to SPB overduplication. In human cells, the hierarchical assembly of a small number of proteins initially drives centrosome duplication [57]. Severing the connector that joins centrioles by separase and polo kinase Plk1 is the licensing step for a new round of duplication in S phase [40, 58]. Despite these similarities, the role of human Sfi1 at centrosomes remains unclear [20]. It will be interesting to determine whether its functions involve C-to-C dimerization accompanied by the exposure of free N termini, as is clearly the case in budding yeast.

(C) Mass spectrometry analysis of in vitro phosphorylated Sfi1 from (B). Shown are the Cdc5 consensus sites within the C terminus of Sfi1 that were phosphorylated by Cdc5. (D) Plasmid shuffle experiment to test the growth of yeast cells containing the indicated SFI1 alleles. SFI1 shuffle strains (sfi1D::HIS3 pRS316-SFI1 and sfi1D::HIS3 mad2D::natNT2 pRS316-SFI1) were transformed with pRS305K (2), pRS305K-SFI1 (SFI1), or pRS305K-sfi1Cdc5 mutant alleles. Cells were grown on YPD and on 5-FOA plates at 23 C. Drop tests were performed with serial 10-fold dilutions from equivalent cell concentrations. (E–G) Time course experiment of td-sfi1 cells with SFI1 (E), sfi1Cdc5-3A (F), or sfi1Cdc5-3D (G). Experiment was performed as in Figure 2B. Cells from each time point were categorized according to budding and SPB number. Cell-cycle progression was monitored by immunoblotting with antibodies against the cell-cycle markers Sic1 and Clb2. Tub2 was used as loading control. Scale bar of (E)–(G) represents 10 mm. (H) SPB duplication was analyzed by electron microscopy of serial thin sections. Sfi1Cdc5-3D cells were analyzed 120 min after a factor release. The cartoon on the left summarizes the SPB phenotype. Indicated are cytoplasmatic microtubules (cMT), cytoplasm (C), nucleus (N), nuclear envelope (NE), and nuclear microtubules (nMT). Scale bars of (H) represent 2 mm and 200 nm.

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Figure 4. Cdc14 Dephosphorylates Sfi1 (A) Yeast two-hybrid experiment with Cdc14 and the trap mutant Cdc14D253A fused to the activation domain (AD) of GAL4 and SFI11-187 and SFI1796-947 fused to DNA-binding domain (BD) of LexA. Interaction is indicated by blue color upon overlay with agarose containing X-Gal. (legend continued on next page)

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Figure 5. Sfi1 Oligomerization Is Promoted by the Activity of Mps1 Kinase (A) mps1-as1 SFI1-yeGFP SPC42-mCherry cells were synchronized with a factor at 23 C. Cells were released from the block by washing, and the Mps1-as1 inhibitor 1NM-PP1 or DMSO was added after spindle formation, as indicated. In addition, a factor was added again to arrest cells in G1. Cells were analyzed for Sfi1-yeGFP signal intensity at SPBs at the indicated time points. (B) Examples of a factor-arrested mps1-as cells that were incubated with DMSO or the Mps1-as1 inhibitor 1NM-PP1. Scale bar of (B) represents 5 mm. (C) Quantification of the Sfi1-yeGFP signal at SPBs of cells in (B). N = 50 cells were analyzed per time point and strain. Error bars show mean 6 SD. (D) Model for the action of Cdk1, Cdc5, Mps1, and Cdc14 on Sfi1. See Discussion for description.

Cells were washed twice with growth medium to remove a factor. 1NM-PP1 was purchased from Merck Biosciences. Yeast extracts were prepared using alkaline lyses and TCA precipitation [60]. Yeast Two-Hybrid Interactions SFI1 fragments and CDC14 were cloned into vectors pMM5 and pMM6 [50]. Yeast two-hybrid interactions were tested as described in [62].

Experimental Procedures Yeast Strains and Growth Conditions Yeast strains and plasmids are listed in Table S1. Deletion and epitope tagging of genes at their endogenous loci were constructed by PCR-based methods [59, 60]. Point mutations of SFI1 were introduced via site-directed mutagenesis using the QuikChange method (Stratagene). Single integration of several SFI1 alleles into the genome of yeast cells was achieved by using the single integration vector pRS305K [61]. For synchronization, yeast cells were grown in YPRaf (yeast extract, peptone, and raffinose) or SDRaf (synthetic defined and raffinose) medium and arrested in G1 by addition of 10 mg/ml a factor until about 95% of cells showed a mating projection.

In Vitro Kinase and In Vitro Dephosphorylation Assays In vitro kinase reactions were performed with 5 mCi of g-32P-ATP (0.05 nM) using the kinases Cdk1as1-Clb2-TAP, Cdk1as1-Clb5-TAP [33] or GST-Cdc5, and GST-Cdc5KD [63], all purified from yeast. Radioactivity was detected using a PhosphorImager (FLA-300; Fujifilm). For Cdc14 dephosphorylation, GST-Sfi1 was first phosphorylated with purified Cdk1-Clb2-TAP. The reaction was stopped by the addition of 20 mM EDTA. Phosphorylated GST-Sfi1 was incubated with recombinant Cdc14 and Cdc14C283A for 45 min at 30 C [64]. 1NM-PP1 (15 mM) was used as an inhibitor for cdk1-as allele. Electron Microscopy Cells were high pressure frozen, freeze substituted, sectioned, labeled, and stained for electron microscopy as described in [65]. Briefly, cells were collected onto a 0.45 mm polycarbonate filter (Millipore) using vacuum

(B) Recombinant GST-Sfi1 was incubated with Cdk1-Clb2 in the presence of g-32P-ATP to phosphorylate Sfi1 with (lane 2) and without (lanes 1, 3, and 4) EDTA at t = 0. EDTA inhibits the activity of Cdk1-Clb2. After the phosphorylation reaction, Cdk1-Clb2 activity was blocked by EDTA addition to samples in lanes 1, 3, and 4. Phosphorylated GST-Sfi1 (lane 1) was then incubated with Cdc14 (lane 3) or Cdc14C283A (lane 4). Shown is a SDS-PAGE and autoradiography. (C) Time line of the experiment in (E). (D) Quantification of the Sfi1-yeGFP signal at SPBs in G1 (no bud, one SPB) and anaphase cells (large budded cells with two separated SPBs). N = 40 cells. Error bars show mean 6 SD. (E) Cells with CDC14 wild-type (CDC14 SFI1-yeGFP pGal1-sic1S) and cdc14-2 (cdc14-2 SFI1-yeGFP pGal1-sic1S) were grown at 23 C in YPRaf medium and arrested in G1 with a factor. Cells were released and incubated with nocodazole at 37 C in order to promote metaphase arrest and inactivation of cdc14-2. At t = 0, galactose was added to express sic1S and to drive cells into G1 phase. In addition, nocodazole was washed out. Half-bridge elongation in G1 in the absence or in the presence of CDC14 activity was analyzed by quantifying Sfi1-yeGFP at SPBs (left). N = 50 cells per time point and strain. Error bars show mean 6 SD. Cell-cycle progression was monitored by budding and DAPI signals (middle). The level of Sic1 and Cdc14 was monitored by immunoblotting. Tub2 was used as loading control (right). (F) SPB duplication of cells in (E) was analyzed after 60 min by electron microscopy of serial thin sections. Cells were checked by phase contrast microscopy before preparing them for electron microscopy. The mutant cells showed the expected cell-separation defect [46]. However, during the freezing process many cells separated. The picture on the upper right is an enlargement of the SPB of the cdc14-2 pGal1-sic1S cell in the middle. Note that the SPB carries a half-bridge (about 70 nm long). The cartoon on the left summarizes the SPB phenotype. Indicated are Bridge, cytoplasm (C), nucleus (N), nuclear envelope (NE), and nuclear microtubules (nMT). SPB1 and SPB2 refer to the two side-by side SPBs in the CDC14 pGal1-sic1S cell. Scale bars of (F) represent 2 mm and 200 nm.

Please cite this article in press as: Elserafy et al., Molecular Mechanisms that Restrict Yeast Centrosome Duplication to One Event per Cell Cycle, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.05.032 Current Biology Vol 24 No 13 10

filtration and then cryoimmobilized by high-pressure freezing using an EM PACT2 machine (Leica Microsystems). Cells were freeze substituted using the EM-AFS2 device (Leica Microsystems; freeze substitution solution: 0.1% glutaraldehyde, 0.2% uranyl acetate, 1% water, all dissolved in anhydrous acetone) and stepwise infiltrated with Lowicryl HM20 (Polysciences), started by a low temperature of 290 C. For polymerization, the samples were exposed to UV light for 48 hr at 245 C and were gradually warmed up to 20 C. Embedded cells were serially sectioned by a Reichert Ultracut S Microtome (Leica Instruments) to a thickness of 60–70 nm. After staining with 2% uranyl acetate and lead citrate, sections were viewed in a CM120 electron microscope (Philips Electronics) operated at 120 kV. Images were captured with a CCD camera (Keen View, Soft Imaging systems) and viewed with DigitalMicrograph software. Mass Spectrometry Peptides were analyzed by LC-MS/MS (Orbitrap Elite, Thermo Fisher Scientific) and data were processed using Thermo Proteome Discoverer Software (version 1.4). Phosphorylation site localization was performed on the Mascot results using PhosphoRS. Protein Purifications and Antibodies Cdk1-Clb2-TAP, Cdk1-Clb5-TAP GST-Cdc5, and GST-Cdc5KD were purified from yeast cells [33, 66]. Tap-Cdc14 was purified from yeast cells, with rabbit-IgG coupled to epoxy-activated Dynabeads M-270 (Invitrogen). GST-Sfi1 and GST-Sfi1-C (wild-type and 6A mutant) were purified from E. coli using GST-Sepharose beads. For the GST-Sfi1-C (wild-type and 6A mutant), the GST tag was cleaved using PreScission Protease. Primary antibodies used were anti-HA (12CA5, Sigma), anti-Cdc5, anti-Clb2, anti-Tub2, and anti-Sic1. Antibodies were prepared in mice, rabbits, sheep, or guinea pigs against purified recombinant proteins [50]. Secondary antibodies were from Jackson ImmunoResearch Laboratories. Fluorescence Microscopy Images of cells were acquired through fluorescence microscopy with a DeltaVision RT system (Applied Precision, Olympus IX71 based). To quantify Spc42-mCherry or Sfi1-yeGFP signal intensity at SPBs, cells were analyzed without fixation. Pictures were processed with ImageJ 1.383 software. Supplemental Information Supplemental Information includes three figures and one table and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2014.05.032. Author Contributions M.E. performed experiments and analyzed data, finalized the manuscript, wrote list of strains and plasmids, handled the submission and revision.  designed the experiments, performed experiments and analyzed the M.S. data, and wrote a preliminary draft of the manuscript. A.N. handled all the electron microscopy imaging, analysis, and statistics. Acknowledgments This work was supported by a grant of the Deutsche Forschungsgemeinschaft Schi295/5-1. We thank S. Heinze and U. Ja¨kle for excellent technical support. We thank the ZMBH Mass Spectrometry Facility for analyzing phosphorylated proteins and the EMBL EM facility for providing their support. GST-Cdc5 and GST-Cdc5KD were provided by S. Sedgwick. We acknowledge H. Maekawa, D. Morgan, G. Pereira, and E. Schwob for providing plasmids. Received: March 26, 2014 Revised: May 11, 2014 Accepted: May 14, 2014 Published: June 19, 2014 References 1. Jaspersen, S.L., and Winey, M. (2004). The budding yeast spindle pole body: structure, duplication, and function. Annu. Rev. Cell Dev. Biol. 20, 1–28. 2. Pereira, G., and Schiebel, E. (2001). The role of the yeast spindle pole body and the mammalian centrosome in regulating late mitotic events. Curr. Opin. Cell Biol. 13, 762–769.

3. Adams, I.R., and Kilmartin, J.V. (2000). Spindle pole body duplication: a model for centrosome duplication? Trends Cell Biol. 10, 329–335. 4. Ganem, N.J., Godinho, S.A., and Pellman, D. (2009). A mechanism linking extra centrosomes to chromosomal instability. Nature 460, 278–282. 5. Byers, B., and Goetsch, L. (1975). Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. J. Bacteriol. 124, 511–523. 6. Byers, B., and Goetsch, L. (1974). Duplication of spindle plaques and integration of the yeast cell cycle. Cold Spring Harb. Symp. Quant. Biol. 38, 123–131. 7. Kosugi, S., Hasebe, M., Tomita, M., and Yanagawa, H. (2009). Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc. Natl. Acad. Sci. USA 106, 10171–10176. 8. Keck, J.M., Jones, M.H., Wong, C.C., Binkley, J., Chen, D., Jaspersen, S.L., Holinger, E.P., Xu, T., Niepel, M., Rout, M.P., et al. (2011). A cell cycle phosphoproteome of the yeast centrosome. Science 332, 1557– 1561. 9. Chi, A., Huttenhower, C., Geer, L.Y., Coon, J.J., Syka, J.E., Bai, D.L., Shabanowitz, J., Burke, D.J., Troyanskaya, O.G., and Hunt, D.F. (2007). Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc. Natl. Acad. Sci. USA 104, 2193–2198. 10. Eissler, C.L., Mazo´n, G., Powers, B.L., Savinov, S.N., Symington, L.S., and Hall, M.C. (2014). The Cdk/cDc14 module controls activation of the Yen1 holliday junction resolvase to promote genome stability. Mol. Cell 54, 80–93. 11. Christianson, T.W., Sikorski, R.S., Dante, M., Shero, J.H., and Hieter, P. (1992). Multifunctional yeast high-copy-number shuttle vectors. Gene 110, 119–122. 12. Crasta, K., Huang, P., Morgan, G., Winey, M., and Surana, U. (2006). Cdk1 regulates centrosome separation by restraining proteolysis of microtubule-associated proteins. EMBO J. 25, 2551–2563. 13. Crasta, K., Lim, H.H., Giddings, T.H., Jr., Winey, M., and Surana, U. (2008). Inactivation of Cdh1 by synergistic action of Cdk1 and polo kinase is necessary for proper assembly of the mitotic spindle. Nat. Cell Biol. 10, 665–675. 14. Saunders, W.S., and Hoyt, M.A. (1992). Kinesin-related proteins required for structural integrity of the mitotic spindle. Cell 70, 451–458. 15. Baum, P., Furlong, C., and Byers, B. (1986). Yeast gene required for spindle pole body duplication: homology of its product with Ca2+-binding proteins. Proc. Natl. Acad. Sci. USA 83, 5512–5516. 16. Spang, A., Courtney, I., Fackler, U., Matzner, M., and Schiebel, E. (1993). The calcium-binding protein cell division cycle 31 of Saccharomyces cerevisiae is a component of the half bridge of the spindle pole body. J. Cell Biol. 123, 405–416. 17. Spang, A., Courtney, I., Grein, K., Matzner, M., and Schiebel, E. (1995). The Cdc31p-binding protein Kar1p is a component of the half bridge of the yeast spindle pole body. J. Cell Biol. 128, 863–877. 18. Rose, M.D., and Fink, G.R. (1987). KAR1, a gene required for function of both intranuclear and extranuclear microtubules in yeast. Cell 48, 1047–1060. 19. Biggins, S., and Rose, M.D. (1994). Direct interaction between yeast spindle pole body components: Kar1p is required for Cdc31p localization to the spindle pole body. J. Cell Biol. 125, 843–852. 20. Kilmartin, J.V. (2003). Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication. J. Cell Biol. 162, 1211–1221. 21. Li, S.S.L., Sandercock, A.M., Conduit, P., Robinson, C.V., Williams, R.L., and Kilmartin, J.V. (2006). Structural role of Sfi1p-centrin filaments in budding yeast spindle pole body duplication. J. Cell Biol. 173, 867–877. 22. Jaspersen, S.L., Giddings, T.H.J., Jr., and Winey, M. (2002). Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p. J. Cell Biol. 159, 945–956. 23. Haase, S.B., Winey, M., and Reed, S.I. (2001). Multi-step control of spindle pole body duplication by cyclin-dependent kinase. Nat. Cell Biol. 3, 38–42. 24. Jaspersen, S.L., Huneycutt, B.J., Giddings, T.H., Jr., Resing, K.A., Ahn, N.G., and Winey, M. (2004). Cdc28/Cdk1 regulates spindle pole body duplication through phosphorylation of Spc42 and Mps1. Dev. Cell 7, 263–274. 25. Zachariae, W., Schwab, M., Nasmyth, K., and Seufert, W. (1998). Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex. Science 282, 1721–1724.

Please cite this article in press as: Elserafy et al., Molecular Mechanisms that Restrict Yeast Centrosome Duplication to One Event per Cell Cycle, Current Biology (2014), http://dx.doi.org/10.1016/j.cub.2014.05.032 Molecular Mechanisms of SPB Duplication 11

26. Lim, H.H., Goh, P.Y., and Surana, U. (1996). Spindle pole body separation in Saccharomyces cerevisiae requires dephosphorylation of the tyrosine 19 residue of Cdc28. Mol. Cell. Biol. 16, 6385–6397. 27. Simmons Kovacs, L.A., Nelson, C.L., and Haase, S.B. (2008). Intrinsic and cyclin-dependent kinase-dependent control of spindle pole body duplication in budding yeast. Mol. Biol. Cell 19, 3243–3253. 28. Winey, M., Goetsch, L., Baum, P., and Byers, B. (1991). MPS1 and MPS2: novel yeast genes defining distinct steps of spindle pole body duplication. J. Cell Biol. 114, 745–754. 29. Schutz, A.R., and Winey, M. (1998). New alleles of the yeast MPS1 gene reveal multiple requirements in spindle pole body duplication. Mol. Biol. Cell 9, 759–774. 30. Visintin, R., Craig, K., Hwang, E.S., Prinz, S., Tyers, M., and Amon, A. (1998). The phosphatase Cdc14 triggers mitotic exit by reversal of Cdk-dependent phosphorylation. Mol. Cell 2, 709–718. 31. Anderson, V.E., Prudden, J., Prochnik, S., Giddings, T.H., Jr., and Hardwick, K.G. (2007). Novel sfi1 alleles uncover additional functions for Sfi1p in bipolar spindle assembly and function. Mol. Biol. Cell 18, 2047–2056. 32. Loog, M., and Morgan, D.O. (2005). Cyclin specificity in the phosphorylation of cyclin-dependent kinase substrates. Nature 434, 104–108. 33. Ubersax, J.A., Woodbury, E.L., Quang, P.N., Paraz, M., Blethrow, J.D., Shah, K., Shokat, K.M., and Morgan, D.O. (2003). Targets of the cyclin-dependent kinase Cdk1. Nature 425, 859–864. 34. Bishop, A.C., Ubersax, J.A., Petsch, D.T., Matheos, D.P., Gray, N.S., Blethrow, J., Shimizu, E., Tsien, J.Z., Schultz, P.G., Rose, M.D., et al. (2000). A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407, 395–401. 35. Maekawa, H., and Schiebel, E. (2004). Cdk1-Clb4 controls the interaction of astral microtubule plus ends with subdomains of the daughter cell cortex. Genes Dev. 18, 1709–1724. 36. Maekawa, H., Usui, T., Knop, M., and Schiebel, E. (2003). Yeast Cdk1 translocates to the plus end of cytoplasmic microtubules to regulate bud cortex interactions. EMBO J. 22, 438–449. 37. Sanchez-Diaz, A., Kanemaki, M., Marchesi, V., and Labib, K. (2004). Rapid depletion of budding yeast proteins by fusion to a heat-inducible degron. Sci. STKE 2004, PL8. 38. Donaldson, A.D., and Kilmartin, J.V. (1996). Spc42p: a phosphorylated component of the S. cerevisiae spindle pole body (SPD) with an essential function during SPB duplication. J. Cell Biol. 132, 887–901. 39. Yoder, T.J., Pearson, C.G., Bloom, K., and Davis, T.N. (2003). The Saccharomyces cerevisiae spindle pole body is a dynamic structure. Mol. Biol. Cell 14, 3494–3505. 40. Tsou, M.F., Wang, W.J., George, K.A., Uryu, K., Stearns, T., and Jallepalli, P.V. (2009). Polo kinase and separase regulate the mitotic licensing of centriole duplication in human cells. Dev. Cell 17, 344–354. 41. Shirk, K., Jin, H., Giddings, T.H., Jr., Winey, M., and Yu, H.G. (2011). The Aurora kinase Ipl1 is necessary for spindle pole body cohesion during budding yeast meiosis. J. Cell Sci. 124, 2891–2896. 42. Dou, Z., von Schubert, C., Ko¨rner, R., Santamaria, A., Elowe, S., and Nigg, E.A. (2011). Quantitative mass spectrometry analysis reveals similar substrate consensus motif for human Mps1 kinase and Plk1. PLoS ONE 6, e18793. 43. Stegmeier, F., and Amon, A. (2004). Closing mitosis: the functions of the Cdc14 phosphatase and its regulation. Annu. Rev. Genet. 38, 203–232. 44. Bloom, J., Cristea, I.M., Procko, A.L., Lubkov, V., Chait, B.T., Snyder, M., and Cross, F.R. (2011). Global analysis of Cdc14 phosphatase reveals diverse roles in mitotic processes. J. Biol. Chem. 286, 5434–5445. 45. Pereira, G., Manson, C., Grindlay, J., and Schiebel, E. (2002). Regulation of the Bfa1p-Bub2p complex at spindle pole bodies by the cell cycle phosphatase Cdc14p. J. Cell Biol. 157, 367–379. 46. Palani, S., Meitinger, F., Boehm, M.E., Lehmann, W.D., and Pereira, G. (2012). Cdc14-dependent dephosphorylation of Inn1 contributes to Inn1-Cyk3 complex formation. J. Cell Sci. 125, 3091–3096. 47. Bouchoux, C., and Uhlmann, F. (2011). A quantitative model for ordered Cdk substrate dephosphorylation during mitotic exit. Cell 147, 803–814. 48. Lengronne, A., and Schwob, E. (2002). The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G(1). Mol. Cell 9, 1067–1078. 49. Araki, H., Awane, K., Ogawa, N., and Oshima, Y. (1992). The CDC26 gene of Saccharomyces cerevisiae is required for cell growth only at high temperature. Mol. Gen. Genet. 231, 329–331. 50. Pereira, G., and Schiebel, E. (2003). Separase regulates INCENP-Aurora B anaphase spindle function through Cdc14. Science 302, 2120–2124.

51. Castillo, A.R., Meehl, J.B., Morgan, G., Schutz-Geschwender, A., and Winey, M. (2002). The yeast protein kinase Mps1p is required for assembly of the integral spindle pole body component Spc42p. J. Cell Biol. 156, 453–465. 52. Jones, M.H., Huneycutt, B.J., Pearson, C.G., Zhang, C., Morgan, G., Shokat, K., Bloom, K., and Winey, M. (2005). Chemical genetics reveals a role for Mps1 kinase in kinetochore attachment during mitosis. Curr. Biol. 15, 160–165. 53. Araki, Y., Gombos, L., Migueleti, S.P., Sivashanmugam, L., Antony, C., and Schiebel, E. (2010). N-terminal regions of Mps1 kinase determine functional bifurcation. J. Cell Biol. 189, 41–56. 54. Holinger, E.P., Old, W.M., Giddings, T.H., Jr., Wong, C., Yates, J.R., 3rd, and Winey, M. (2009). Budding yeast centrosome duplication requires stabilization of Spc29 via Mps1-mediated phosphorylation. J. Biol. Chem. 284, 12949–12955. 55. Liu, X., and Winey, M. (2012). The MPS1 family of protein kinases. Annu. Rev. Biochem. 81, 561–585. 56. Palframan, W.J., Meehl, J.B., Jaspersen, S.L., Winey, M., and Murray, A.W. (2006). Anaphase inactivation of the spindle checkpoint. Science 313, 680–684. 57. Go¨nczy, P. (2012). Towards a molecular architecture of centriole assembly. Nat. Rev. Mol. Cell Biol. 13, 425–435. 58. Tsou, M.F., and Stearns, T. (2006). Mechanism limiting centrosome duplication to once per cell cycle. Nature 442, 947–951. 59. Knop, M., Siegers, K., Pereira, G., Zachariae, W., Winsor, B., Nasmyth, K., and Schiebel, E. (1999). Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines. Yeast 15 (10B), 963–972. 60. Janke, C., Magiera, M.M., Rathfelder, N., Taxis, C., Reber, S., Maekawa, H., Moreno-Borchart, A., Doenges, G., Schwob, E., Schiebel, E., and Knop, M. (2004). A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947–962. 61. Taxis, C., and Knop, M. (2006). System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae. Biotechniques 40, 73–78. 62. Schramm, C., Janke, C., and Schiebel, E. (2001). Molecular dissection of yeast spindle pole bodies by two hybrid, in vitro binding, and co-purification. Methods Cell Biol. 67, 71–94. 63. Geymonat, M., Spanos, A., Walker, P.A., Johnston, L.H., and Sedgwick, S.G. (2003). In vitro regulation of budding yeast Bfa1/Bub2 GAP activity by Cdc5. J. Biol. Chem. 278, 14591–14594. 64. Geil, C., Schwab, M., and Seufert, W. (2008). A nucleolus-localized activator of Cdc14 phosphatase supports rDNA segregation in yeast mitosis. Curr. Biol. 18, 1001–1005. 65. Giddings, T.H., Jr., O’Toole, E.T., Morphew, M., Mastronarde, D.N., McIntosh, J.R., and Winey, M. (2001). Using rapid freeze and freezesubstitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis. Methods Cell Biol. 67, 27–42. 66. Geymonat, M., Spanos, A., and Sedgwick, S.G. (2007). A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression. Gene 399, 120–128.