ABSTRACT
Defects in the spindle assembly checkpoint (SAC) can lead to aneuploidy and cancer. Sphingolipids have important roles in many cellular functions, including cell cycle regulation and apoptosis. However, the specific mechanisms and functions of sphingolipids in cell cycle regulation have not been elucidated. Using analysis of concordance for synthetic lethality for the yeast sphingolipid phospholipase ISC1, we identified two groups of genes. The first comprises genes involved in chromosome segregation and stability (CSM3, CTF4, YKE2, DCC1, and GIM4) as synthetically lethal with ISC1. The second group, to which ISC1 belongs, comprises genes involved in the spindle checkpoint (BUB1, MAD1, BIM1, and KAR3), and they all share the same synthetic lethality with the first group. We demonstrate that spindle checkpoint genes act upstream of Isc1, and their deletion phenocopies that of ISC1. Reciprocally, ISC1 deletion mutants were sensitive to benomyl, indicating a SAC defect. Similar to BUB1 deletion, ISC1 deletion prevents spindle elongation in hydroxyurea-treated cells. Mechanistically, PP2A-Cdc55 ceramide-activated phosphatase was found to act downstream of Isc1, thus coupling the spindle checkpoint genes and Isc1 to CDC55-mediated nuclear functions.
INTRODUCTION
The survival of cells and organisms requires accurate chromosome segregation. Chromosomal loss or missegregation can lead to aneuploidy and cancer development (1, 2). The spindle assembly checkpoint detects defects in spindle structure or in the alignment of chromosomes on the spindle and delays anaphase onset until these defects are corrected. More subtle defects, such as the presence of a single kinetochore that is not attached to spindle microtubules, can also activate the checkpoint (3). When the mitotic spindle assembly is impaired by microtubule-depolymerizing agents, such as nocodazole or benomyl, spindle checkpoint mutants fail to delay their cell cycle progression and die quickly because of chromosome loss (4).
In recent years, ceramide, sphingosine 1-phosphate, sphingosine, and ceramide 1-phosphate have emerged as bioactive sphingolipids implicated in many important cellular functions, such us cell growth, inflammation, and response to stress stimuli (5–7). Sphingolipid metabolism is conserved, for the most part, from budding yeasts to mammals (8), and ceramide has been implicated as a transducer for stress stimuli in mammalian systems and in yeast (9–11).
The yeast sphingolipid phospholipase ISC1 is the main enzyme involved in the catabolism of complex sphingolipids in Saccharomyces cerevisiae; it hydrolyzes inositol phosphoceramides and related yeast complex sphingolipids to generate bioactive ceramide molecules (12, 13). However, it is now increasingly appreciated that ceramides represent a family of structurally and metabolically related molecules that may be differentially regulated and may mediate distinct signals (14). As a result, it is becoming increasingly important to define the biological processes affected by specific mechanisms and pathways of ceramide generation.
We showed previously that when cells lacking ISC1 are treated with hydroxyurea (HU), they acquire an additional G2/M block, which follows the S phase block normally seen in wild-type (WT) cells upon HU treatment (15). This result raised several intriguing questions as to how a sphingolipid enzyme can be involved in cell cycle regulation, the pathways implicated, and the underlying mechanisms involved.
In an effort to address these questions, we initially adopted a data-driven bioinformatics approach (16) using available genetic interaction databases. Extensive work has been done to infer functions from genetic interaction networks (17–20). In an elaboration of these approaches, one study showed that defining shared synthetic lethal partners may serve as an indication of functional relevance that can be used to infer gene functions (21).
In this study, synthetic lethality analysis identified a set of genes (CSM3, CTF4, YKE2, DCC1, and GIM4) involved in chromatid cohesion and chromosome segregation (22, 23), being synthetic lethal partners of ISC1. The analysis also identified another set of genes involved in the spindle assembly checkpoint that share these synthetic lethal partners with ISC1. We herein provide biochemical and biological evidence that ISC1 functions in the spindle assembly checkpoint. We used quantitative phosphoproteomics analysis to identify potential phosphotargets downstream of Isc1, and this analysis revealed several nuclear phosphoproteins involved in cell division, spindle assembly, and kinetochore-microtubule attachment. We implicate a Cdc55-dependent mechanism in these actions. To our knowledge, this is the first report implicating a sphingolipid enzyme and consequently ceramides in the regulation of the spindle assembly checkpoint. They also provide a mechanism connecting Bub1 and other checkpoint regulators to activation of CDC55 phosphatase.
RESULTS
Defining functional partners of Isc1 using synthetic lethality and genetic interaction network analysis.To better understand the unexpected role of Isc1 in the response to HU and DNA damage, we resorted to synthetic lethality analysis. We examined published databases to find synthetic lethal partners of Isc1 in S. cerevisiae. We then searched for genes that share the maximum number of synthetic lethal partners with ISC1. This approach identified four genes (MAD1, BUB1, BIM1, and KAR3) sharing the highest number of synthetically lethal genes with ISC1. Importantly, they all share the same 5 synthetic lethal partners (CSM3, YKE2, CTF4, GIM4, and DCC1) (Fig. 1A). Interestingly, CSM3, YKE2, CTF4, GIM4, and DCC1 are all involved in sister chromatid cohesion, chromosomal segregation, and maintenance of telomere length. In contrast, the set of genes, namely, MAD1, BUB1, BIM1, and KAR3, showing synthetic lethality similar to that of ISC1, are mostly involved in spindle checkpoint. MAD1 encodes a coiled-coil protein that is involved in the spindle assembly checkpoint. BUB1 encodes a protein kinase that forms a complex with Mad1 and Bub3, which is crucial in the checkpoint mechanism required to prevent cell cycle progression into anaphase in the presence of spindle damage. BIM1 produces a microtubule-binding protein that together with Kar9 makes up the cortical microtubule capture site and delays the exit from mitosis when the spindle is oriented abnormally. The KAR3 gene encodes the minus-end-directed microtubule motor that functions in mitosis and meiosis, and it localizes to the spindle pole body. This protein is required for nuclear fusion during mating and is a potential Cdc28 substrate. Notably, the annotation of these genes indicates that most of them are related to cell cycle and chromosome segregation, consistent with the experimental data connecting ISC1 to cell cycle regulation (15).
Identification of the SLSP genes through network analysis of synthetically lethality. (A) ISC1 and its synthetically lethal partners. Genes on the left, SLSP genes, are the genes that share the highest number of synthetically lethal genes with ISC1, and the genes on the right are the synthetically lethal partners common with ISC1. (B) Cell cycle genes sharing at least one synthetic lethal partner with ISC1. Genes are colored by the number of synthetic lethal partners they share with ISC1: red, 5 shared partners; orange, 4 shared partners; yellow, 3, shared partners. Note that a majority of genes that share the maximum number of synthetic lethal partners with ISC1 (in red) are clustered around the G2/M phase, consistent with the experimental results showing that deletion of ISC1 resulted in G2/M arrest in HU-treated cells. Modified from original source, this panel originated from KEGG/GenomeNet (https://www.genome.jp/kegg/kegg1.html). (C) Elevated HU sensitivity of genes sharing synthetic lethal partners with ISC1 (SLSP genes). The haploid wild-type (WT) strain was used, along with individual gene deletion mutants—isc1Δ, bub1Δ, mad1Δ, bim1Δ, and kar3Δ mutants—all derived from the WT strain. Spot testing was performed on YPD plates as described in Materials and Methods. This experiment was repeated three independent times, with similar results.
In addition to these four genes that share 5 synthetic lethal partners with ISC1 (for ease of designation, we refer to this group of genes, on the left side of Fig. 1A, as ISC1 synthetic lethal shared partner [SLSP] genes), we also identified 272 additional genes that share at least one of these synthetic lethal partners. Many of these genes are cell cycle genes, which can be mapped to the cell cycle pathway (Fig. 1B). Notably, genes sharing a majority of synthetic lethal partners with ISC1 (colored in red and orange in Fig. 1B) all function at the G2/M phase, consistent with the observation that deletion of ISC1 resulted in the G2/M arrest in HU-treated cells (15). These findings indicate that genetic interaction network is a powerful tool to dissect the function of the sphingolipid pathway in cell cycle progression and genome instability.
The synthetic lethality analysis suggested that the SLSP genes most likely participate in a common pathway with Isc1. Accordingly, we hypothesized that functional loss of these four genes should result in phenotypes resembling isc1Δ cells.
Since the ISC1 deletion strain exhibits elevated HU sensitivity, we examined the ability of each of these four genes to phenocopy the deletion of ISC1. Figure 1C shows that deletion of any one of these four genes resulted in enhanced HU sensitivity.
Participation of Isc1 in a pathway regulated by SLSP genes.The above results suggested a close functional relationship between Isc1 and its partners. Since HU was shown to activate Isc1, we next sought to determine if Isc1 activity was regulated by Bub1, Mad1, Bim1, and Kar3, acting upstream of Isc1. The results showed that deletion of each of these genes abrogated the increase in Isc1 activity in response to HU (Fig. 2A). To determine if Isc1 activation by HU is influenced by the Csm3-related group (CSM3, CTF4, YKE2, DCC1, and GIM4), the activity of Isc1 was determined in deletion mutants of these genes in the presence and absence of HU. The results shown in Fig. 2B indicate that HU treatment activated Isc1 in each of these deletions. Taken together, these results demonstrate that activation of Isc1 depends on the function of Bub1, Mad1, Bim1, and Kar3 but not on its synthetic lethal partners and thus place Isc1 downstream of the SLSP group.
SLSP genes act upstream of ISC1. (A) Enzymatic activity of ISC1. Activity assays were performed on lysates of WT, bub1Δ, mad1Δ, bim1Δ, and kar3Δ cells in response to HU treatment. All cells were transformed with a vector containing ISC1 under the control of the galactose promoter. For statistical analysis, the Student t test for paired sample means was used. *, P ≤ 0.05. (B) Activity assay of ISC1 and its synthetic lethal genes. The same activity assay was performed with the deletion mutants that are synthetically lethal with isc1Δ, csm3Δ, ctf4Δ, ykeΔ, dcc1Δ, and gim4Δ mutants. The ISC1 gene was overexpressed under the control of the galactose promoter in all strains as described for panel A. For statistical analysis, the Student t test for paired sample means was used. *, P ≤ 0.05. (C) C18:1 phytoceramide profile in ISC1 and its SLSP genes. Levels of C18:1 phytoceramides were determined using mass spectrometry in WT cells, isc1Δ cells, and cells of SLSP gene deletion mutants. The data show fold changes between untreated cells and cells treated with HU. The data represent the means from three independent experiments; the bars indicate standard errors of the means (SEM). For statistical analysis, the Student t test for paired sample means was used. *, P ≤ 0.05; **, P ≤ 0.01. (D) Effects of overexpression of ISC1 on sensitivity of SLSP gene deletion strains to HU. A spot test was done to see if overexpression of ISC1 is able to correct the SLSP gene sensitivity to HU. This experiment was repeated three independent times, with the same results. (E) Effects of oleate on sensitivity of SLSP gene deletion strains to HU. The WT, isc1Δ mutant, and all SLSP gene deletion mutant strains were spotted onto YPD plates containing 7.5 mg/ml of HU and 0.005% of oleate. This experiment was repeated three independent times, with similar results.
Curiously, deletion of each of CSM3, CTF4, YKE2, DCC1, and GIM4 resulted in higher “basal” activity of Isc1. This may suggest that Isc1 participates in a checkpoint that is activated by problems in sister chromatid exchange and chromosome segregation.
To further support these results, we evaluated the sphingolipid response to HU. Phytoceramides, especially C18:1 phytoceramide, have been shown to increase after treatment of WT cells with HU but fail to do so in isc1Δ cells (24). The phytoceramide profiles of bub1Δ, mad1Δ, bim1Δ, and kar3Δ cells following HU treatment were very similar to that of isc1Δ cells (Fig. 2C).
A third and functional approach to define whether Isc1 acts downstream of SLSP was to evaluate if Isc1 can rescue the phenotype of deletions of the SLSP group in response to HU. The results (Fig. 2D) show that Isc1 overexpression was indeed sufficient to protect bub1Δ, mad1Δ, bim1Δ, and kar3Δ cells from HU.
Finally, and based on our previous study showing that oleate (C18:1 fatty acid) was the predominant fatty acid capable of protecting isc1Δ cells from HU toxicity because of its incorporation into C18:1 phytoceramide (24), the effects of oleate on the growth of bub1Δ, mad1Δ, bim1Δ, and kar3Δ cells in response to HU were evaluated. The results showed that addition of oleate protected, at least partially, the mutants from HU toxicity (Fig. 2E). Taken together, these results demonstrate that Isc1 functions downstream of SLSP genes.
Noticeably, increase of other phytoceramide chain length species, such as C16, C18, C20, C20:1, C22:1, C24:1, C26, C26:1, sphingosine (SPH), and sphingosine-1-phosphate (SPH-1P), were found in WT cells following HU treatment, and these species failed to increase in isc1Δ cells. Similarly, bub1Δ, mad1Δ, bim1Δ, and kar3Δ cells had phytoceramide levels comparable to those in isc1Δ cells (Fig. 3).
Phytoceramide level changes in the WT, isc1Δ mutant, and in all SLSP mutants upon HU treatment. Phytoceramide species changes are represented in each panel, showing the fold change of lipids following HU treatment for each chain length in the WT and in each of the isc1Δ, bub1Δ, mad1Δ, bim1Δ, and kar3Δ deletion mutants. The data represent the means from three independent experiments; the bars indicate SEM.
Involvement of Isc1 in spindle checkpoint.The above results raised the possibility of a previously unappreciated role for Isc1 and its phytoceramide products in the regulation of the spindle checkpoint. Since sensitivity to benomyl indicates dysregulation of the spindle checkpoint, we evaluated the sensitivity of isc1Δ cells to this reagent. The results in Fig. 4A show that ISC1 deletion rendered cells more sensitive to benomyl; however, the sensitivity of isc1Δ cells was less severe than that of bub1Δ cells but similar to those of mad1Δ, bim1Δ, and kar3Δ cells. Functionally, the results showed that overexpression of ISC1 protected from the severe growth defect of bub1Δ cells and the slow growth phenotype of mad1Δ, bim1Δ, and kar3Δ strains in response to benomyl (Fig. 4B). To rule out the possibility of Isc1 acting upstream of Bub1, BUB1 was overexpressed in an isc1Δ strain, the results showed that overexpression of BUB1 had no protective effect on the isc1Δ strains in its response to HU or benomyl (Fig. 4C). Taken together, these results provide further evidence that Isc1 acts downstream of the SLSP group and is indeed a key mediator of their functions.
ISC1 is involved in the spindle assembly checkpoint. (A) Sensitivity to benomyl. Spot tests were performed comparing growth on YPD plates containing 24 μg/ml of benomyl for WT, isc1Δ, and SLSP gene deletion strains. This experiment was performed three times, with the same results. (B) Overexpression of ISC1 in benomyl-sensitive SLSP gene mutants. ISC1 was overexpressed in the SLSP gene deletion mutants, and sensitivity to 12 μg/ml of benomyl was determined. The images of spot test plates were taken after 3 days of incubation at 30°C. (C) Overexpression of BUB1 in the isc1Δ mutant. BUB1 was cloned into a multicopy YEp24 vector (2μ vector), and spot tests were done on URA− plates with 7.5 mg/ml of HU or with 12 μg/ml of benomyl. This experiment was performed three independent times, with similar results. Results are expressed as means ± SD. For statistical analysis, the Student t test for paired sample means was used. *, P ≤ 0.05; **, P ≤ 0.01.
The sensitivity to benomyl and the ability of Isc1 to overcome the defects of Bub1 and partners suggested a role for Isc1 in spindle elongation. When WT cells were treated with HU, spindles became elongated (Fig. 5A, top). However, spindles were not elongated in isc1Δ or bub1Δ cells (Fig. 5A, middle and bottom, respectively). Another indicator of a spindle checkpoint deficiency is sensitivity to nocodazole (25). A spot test assay showed that deletion of ISC1 rendered cells more sensitive to nocodazole (data not shown). Checkpoint mutants tend to have a premature sister chromatid separation in the presence of nocodazole. We used a strain expressing a Tet-green fluorescent protein (GFP) fusion and containing tandem repeats of the tet operator inserted into the chromosomes in WT and isc1Δ cells. The results shown in Fig. 5B, left side, indicate that the isc1Δ strain had more sister chromatid separation than the WT at 3 and 6 h in the presence of nocodazole. Separation of sister chromatids was assayed as the percentage of cells with two separated fluorescent dots as seen in Fig. 5B, right side.
(A) Spindle elongation in WT and isc1Δ cells and in bub1Δ cells in the presence of HU. Immunofluorescence was used to determine the elongation of tubulin using anti-alpha-tubulin antibody in WT, isc1Δ, and bub1Δ cells, all treated with HU. This experiment was performed three times, and all results were the same. (B) Sister chromatid separation assay was done in WT and isc1Δ cells in the presence of nocodazole at time zero and at 3 and 6 h. Experiments were performed three independent times. (C) Rebudding assay in WT, isc1Δ, and mad1Δ cells in the presence of nocodazole for 6 h. Rebudded cells, dumbbell cells, and cells in G1 were quantified. (D) Overexpression of ISC1 was done in WT, isc1Δ, bub1Δ, mad1Δ, bim1Δ, and kar3Δ cells to show if it can stop or decrease the budding. Experiments were performed three independent times. Results are expressed as means ± SD. For statistical analysis, the Student t test for paired sample means was used. *, P ≤ 0.05; **, P ≤ 0.01.
Additionally, we performed a budding assay to establish if the isc1Δ strain followed a WT budding pattern or spindle checkpoint mutant pattern. Checkpoint mutants tend to continue to bud in the presence of nocodazole, whereas WT cells tend to have fewer buds as they arrest. Our results indicated that isc1Δ cells had higher budding pattern than the WT, but they had fewer buds than the mad1Δ spindle checkpoint mutant (Fig. 5C). Clearly, this result indicates that a higher proportion of isc1Δ cells than of WT cells continued to bud. Overexpression of ISC1 was able to reduce the budding patterns of isc1Δ, bub1Δ, mad1Δ, bim1Δ, and kar3Δ cells to WT levels. This experiment showed that overexpression of ISC1 is sufficient to halt budding increase in SLSP mutants in the presence of nocodazole. This result is additional evidence of the downstream role of Isc1 in the SLSP gene pathway.
These phenotypes of isc1Δ cells, including defects in spindle elongation, sensitivity to nocodazole, and the increase in budding and sister chromatid separation in the presence of nocodazole, illustrate a previously unknown link between Isc1 and spindle elongation and sister chromatid separation. These results further corroborate the results on the very close functional roles of Isc1 and Bub1 (and its related partners).
Defining roles for Swe1 and Cdc55 downstream of Bub1 in spindle checkpoint.Previously, we found that deletion of Swe1 kinase rescued isc1Δ cells from HU sensitivity (15). This prompted us to investigate whether deletion of SWE1 is able to protect bub1Δ, mad1Δ, bim1Δ, and kar3Δ cells from HU toxicity. The results showed that SWE1 deletion indeed protected bub1Δ, mad1Δ, and bim1Δ cells from HU toxicity; however, it did not have a protective effect on kar3Δ cells from the action of HU (Fig. 6A). The results also showed that deletion of SWE1 was able to correct the sensitivity of bub1Δ cells to benomyl (Fig. 6A), suggesting an interesting and previously unidentified role for Swe1 in protecting bub1Δ cells from benomyl toxicity. Deletion of SWE1 also protected all the strains from the action of benomyl.
SWE1 and CDC55 are downstream targets for ISC1. (A) SWE1 was deleted in isc1Δ cells and in all SLSP gene deletion mutants to assess sensitivity to HU and to benomyl. Cells were plated in YPD with or without 10 mg/ml of HU or 24 μg/ml of benomyl and incubated for 3 days at 30°C. (B) CDC55 was overexpressed using a multicopy YEp24 vector and transformed in isc1Δ cells and in each of the SLSP mutant. The transformants were assed for HU sensitivity on plates containing HU or vehicle, and incubated for 3 days at 30°C. Images were taken at the end of incubation. (C) Overexpressed CDC55 was assessed for its ability to protect isc1Δ and bub1Δ cells from benomyl toxicity. Spot tests were performed, and images were taken at the end of 3 days’ incubation.These experiments were performed three independent times, and the results were the same. (D) Spindle elongation in WT, isc1Δ, and bub1Δ cells. Immunofluorescence was performed with single mutants to assess the elongation of spindles in the presence of HU. The spindle elongation in isc1Δ sweΔ and bub1Δ swe1Δ double mutants with HU is shown in panel B. Spindle elongation in isc1Δ cells and in bub1Δ cells overexpressing CDC55 in the presence of HU is shown in panel C. Fixed cells were stained with anti-alpha-tubulin antibody and 4′,6-diamidino-2-phenylindole (DAPI). This experiment was performed three times, and similar results were obtained.
We had also shown that cells lacking ISC1 are able to overcome the sensitivity to HU by overexpressing Cdc55, the regulatory subunit of phosphatase PP2A and a direct downstream target for ceramide, the lipid product of Isc1 action (26). Deletion of BUB1 was rescued by the overexpression of CDC55 in the presence of HU or benomyl (Fig. 6B and C). Overexpression of CDC55 was also able to rescue the sensitivity of mad1Δ, bim1Δ, and kar3Δ cells to HU (Fig. 6B).
Considering these results, we explored if deletion of SWE1 or overexpression of CDC55 could correct the spindle elongation defect seen in isc1Δ and bub1Δ cells. To this end, we checked isc1Δ swe1Δ and bub1Δ swe1Δ double mutants for defects in spindle elongation in the presence of HU. The results in Fig. 6D show that the spindle elongation in the isc1Δ swe1Δ and bub1Δ swe1Δ mutants was very similar to that in WT cells. Similar results were obtained when the CDC55 gene was overexpressed in isc1Δ and bub1Δ strains (Fig. 6D), indicating that CDC55 is a key transducer of these effects downstream of both Bub1 and Isc1.
Identification of Isc1- and Cdc55-dependent dephosphorylations.To untangle how Isc1 and Cdc55 regulate signaling networks, we resorted to whole-cell phosphoproteomics analysis using iTRAQ labeling (isobaric tags for relative and absolute quantitation). Wild-type, isc1Δ, and cdc55Δ strains and isc1Δ cells overexpressing CDC55 (CDC55-OE cells) were treated with HU as described in Materials and Methods. Based on the preceding results, we reasoned that HU treatment induces Isc1-dependent activation of Cdc55, resulting in significant protein dephosphorylation, and that dephosphorylation of specific signaling components would be attenuated in the isc1Δ and cdc55Δ strains. Therefore, we searched for the profile in which phosphopeptide levels were decreased in WT cells as a result of HU treatment (i.e., HU-induced dephosphorylation) but not in isc1Δ and cdc55Δ mutant cells (i.e., HU-induced dephosphorylation that is dependent on either Isc1 or Cdc55). Furthermore, to further implicate Cdc55 downstream of Isc1, we evaluated for phosphopeptide levels (whose decrease is lost in the isc1Δ cells) and that are then reversed by CDC55 overexpression in the isc1Δ strain. This would establish Cdc55-dependent dephosphorylation. We identified 1,184 HU-induced dephosphorylations in WT cells, of which 545 phosphopeptides were Isc1 dependent, corresponding to 379 unique protein hits. We also identified 656 phosphopeptides that were Cdc55 dependent, corresponding to 396 unique protein hits, and 264 of these proteins (corresponding to 70% of the Isc1-dependent ones) were common to both Isc1 and Cdc55, strongly suggesting that Isc1 and Cdc55 are indeed in the same signaling pathway induced by HU. A Venn diagram is shown in Fig. 7A in which 264 proteins were common to Isc1 and Cdc55 while 115 and 132 proteins were unique to Isc1 and Cdc55, respectively. Importantly, out of the 545 Isc1-dependent phosphopeptides, 391 were overcome by CDC55 overexpression, corresponding to 278 unique protein hits (Fig. 7B). Moreover, of the 264 proteins common to the Isc1 and Cdc55 groups, 190 protein phosphorylations (72%) were reversed by CDC55 overexpression in the isc1Δ background strain. As a result, we have identified 190 proteins that were dephosphorylated in an Isc1- and Cdc55-dependent manner and whose phosphorylation levels were reversed by CDC55 overexpression. These results strongly support the results from this study and previous ones (24, 26) that Cdc55 phosphatase is a key downstream mediator of ceramide actions in yeast and, in this case, especially in response to HU. While this study was in progress, Baro et al. reported a SILAC (stable-isotope labeling by amino acids in cell culture)-based phosphoproteomics study of a PP2A-Cdc55 mutant in which they identified 62 potential Cdc55 substrates (27). By comparing their results to our list of Isc1 and Cdc55-dependent phosphoproteins, we found a significant overlap of ∼29%, strongly supporting the validity of our analysis.
Potential substrates for PP2A-cdc55 phosphatase. (A) Venn diagrams showing the results of iTRAQ quantitative phosphoproteomics analysis. The diagram shows the overlapping of dephosphorylated proteins that are both Isc1 and Cdc55 dependent. A total of 264 proteins were found in the common group, while 115 proteins were unique to Isc1 and 132 proteins were unique to Cdc55. These results are strongly indicative that Cdc55 and Isc1 are in the same HU-induced pathway. (B) The diagram shows the overlapping of dephosphorylated peptides that are Isc1 and Cdc55 dependent and overcome by CDC55 overexpression. Overexpression of CDC55 was carried out in the isc1Δ mutant background. Of the 264 proteins common to Isc1 and Cdc55, 190 were reversed by CDC55 overexpression while 74 proteins were not. Of the 115 proteins unique to the Isc1 group, 88 were reversed by CDC55 overexpression while 27 were not. (C) Elevated HU sensitivity for the sli15Δ mutant strain. To test for HU sensitivity, haploid WT strains were used along with cdc55Δ, isc1Δ, sli15Δ, and she1Δ individual gene deletions, in the presence or absence of 10 mg/ml of HU and incubated for 2 days at 30°C. A volume of 3 μl was spotted onto YPD plates as follows: the first spot corresponds to and OD at 600 nm of ∼0.3, and each subsequent spot is a 1:10 dilution of the preceding spot. The experiment was repeated three independent times, with similar results.
Functional annotation of the 190 proteins.We then used David 6.7 to identify the biological processes mediated by Isc1 and Cdc55. The results obtained were sorted by fold enrichment (see Table S1 in the supplemental material). Functional annotation of the selected 190 proteins showed enrichment in several biological processes, including chromatin remodeling at centromeres, transmembrane trafficking, and cytoskeleton organization. In addition, there was a 5.5-fold enrichment in spindle microtubule gene ontology (GO) term and 3.6-fold enrichment in the mitotic cell cycle checkpoint proteins (Table S1). However, David and all other GO term software did not identify all the spindle assembly proteins that were present in our list, as these genes were scattered among other categories. As a result, we sorted the 190 proteins manually, and the results are displayed in Table 1. We found 16 proteins (8.4%) that are related to spindle assembly and chromatid separation and thus were selected for further analysis. The proteins are She1, Siz1, Net1, Top2, Kcc4, Ulp2, Sli15, Swe1, Fin1, Dam1, Kin4, Gip4, Spc29, Src1, Gac1, and Sth1.
Classification of the 190 genes obtained by phosphoproteomics analysis
Isc1 and Cdc55 control the spindle checkpoint.Among the 16 proteins selected, some are known to be Cdc55 substrates, such as Swe1 and Net1. Particularly, Orc2, Kin4, Sli15 (INCEMP), Dam1, and She1 stood out since they are intimately associated with proper chromosome segregation and SAC regulation.
Orc2 is implicated in the initiation of DNA replication during S phase but is located on kinetochores during mitosis. In addition, Orc2 in yeast was implicated in sister chromatid cohesion, and its removal was found to induce SAC activation (28). Thus, Orc2 appears to function as a mediator of the actions of Isc1 and Cdc55 on the spindle checkpoint.
In yeast, Sli15-Ipl1-Bir1 form a complex called the chromosomal passenger complex (CPC) which associates with kinetochores in metaphase and with spindle midzone microtubules in anaphase. The CPC regulates key mitotic events: correction of chromosome microtubule attachment errors, activation of the spindle assembly checkpoint, and construction and regulation of the contractile apparatus that drives cytokinesis (29). As a result, the CPC is one of the main controllers of mitosis. Cdc14 dephosphorylates Sli15 and thereby directs the CPC to the spindle midzone and away from kinetochores, which contributes to mitotic exit. As a result, the CPC appears to be an important target for Cdc55 in this pathway. This could also explain the abundance of protein hits involved in budding and cytoskeleton organization that were obtained from phosphoproteomics analysis given the important role for CPC in cytokinesis (Table 1). Aurora-B/Ipl1 kinase detects and destabilizes incorrect sister chromatid attachments through control of kinesin MCAK, the DASH complex, and the Hec1 complex at the microtubule-kinetochore interface (30, 31). Dam1 is part of the DASH complex, as it couples the kinetochores to spindle microtubules. Dam1 is directly activated by Ipl1-mediated phosphorylation on S292 and is essential for proper chromosome segregation. Interestingly, the Dam1 dephosphorylation site in our phosphoproteomics data was found to be on the same S292 residue (the Ipl1 phosphorylation site). She1 is also a mitotic spindle protein that interacts with the Dam1 (DASH) complex, Sli15, and Bim1. In our phosphoproteomics data, the She1 dephosphorylation site was found to be on S165. Thus, Dam1 and its interacting partner She1 appear to be important targets for Isc1/Cdc55 in this HU-induced pathway.
Kin4 is a serine/threonine protein kinase which inhibits the mitotic exit network (MEN). It is activated by Elm1 kinase and prevents cell cycle progression when the spindle position checkpoint (SPOC) is activated. From our phosphoproteomics data, Cdc55 appears to dephosphorylate Kin4 on its T209 residue (the same Elm1 kinase phosphorylation site). As a result, Kin4 and the SPOC appear to be targeted by Cdc55 and Isc1 in this pathway. More importantly, Sli15 (INCEMP) is hyperphosphorylated on S421, S425, and S427 in an Isc1- and Cdc55-dependent manner (Table 2). To determine whether Sli15 or any of the selected proteins are involved in this signaling pathway, we tested the sensitivity of several knockout mutants to hydroxyurea and compared it to those of cdc55Δ and isc1Δ mutants (Fig. 7C). The results indicate that the sli15Δ mutant is sensitive to hydroxyurea treatment and that its sensitivity is comparable to that of the isc1Δ mutant. On the other hand, a she1Δ knockout mutant showed no significant hydroxyurea sensitivity (Fig. 7C). This result corroborates the phosphoproteomics data and indicates that Sli15 is involved in this pathway downstream of Isc1 and Cdc55.
Summary of Isc1- and Cdc55-dependent dephosphorylations of selected proteins involved in the spindle assembly checkpoint found among the selected group of 190 proteins
At the metaphase-anaphase transition, APC/cdc20 polyubiquitinates securin and causes separase activation. In turn, separase promotes sister chromatid separation by cleaving the cohesion complex. Separase can also regulate INCENP (Sli15)-Aurora B (Ipl1) anaphase spindle function through Cdc14 released from Net1 entrapment in the nucleolus (Fig. 8).
Proposed model for the role of Isc1 and Cdc55 in the HU-induced signaling pathway. When cells are treated with HU, a checkpoint signal would be activated through the SLPS genes. SLPS genes transmit the signal to Isc1, which produces C18:1 phytoceramides, which functions as a signal to directly activate Cdc55 and thus its downstream targets Cdc16, Cdc23, Cdc27, Cdc14, Net1, Clb2, and Clb5 or inhibit SWE1 kinase and its target Cdc28 kinase. Cdc55 phosphatase activity controls Cdc28 and APC/cdc20 activity through its multiple downstream targets. Swe1 can act directly on the activation of Cdc28, which then acts on APC/cdc20, with the latter triggering chromatid separation through pds1 (securin), Esp1 (separase), and cohesin cleavage. Separase can also regulate INCENP (Sli15)-Aurora B (Ipl1) anaphase spindle function through Cdc14 released from Net1 entrapment in the nucleolus. Cdc14 dephosphorylates Sli15 and thereby directs the chromosomal passenger complex (CPC) to the spindle midzone and away from kinetochores. Dam1, part of the DASH complex, couples the kinetochores to spindle microtubules. Dam1 is directly activated by Ipl1 phosphorylation and is essential for proper chromosome segregation. Dam1 interacts with another mitotic spindle protein, She1. Kin4 is activated by Elm1 kinase, it inhibits the mitotic exit network (MEN), and it prevents cell cycle progression when the spindle position checkpoint (SPOC) is activated. SLSP proteins are highlighted in blue, Csm3-related proteins in red. Potential Cdc55 substrates, identified in our phosphoproteomics study, are highlighted in green.
DISCUSSION
Overall, these new findings define a novel and unexpected role of an enzyme of sphingolipid metabolism, ISC1, in the spindle checkpoint. Specifically, the results strongly assign the action of ISC1 to the function of BUB1, MAD1, BIM1, and KAR3. The identification of this group of genes was the result of an informatic approach aimed at matching the largest group of ISC1’s synthetically lethal partners (YKE2, CSM3, CTF4, GIM4, and DCC1) to genes that, like ISC1, are synthetically lethal to the same gene group. Both groups of genes are involved in chromatid separation and checkpoint operation. The first group is intimately involved in chromatin remodeling, chromosome segregation, and sister chromatid cohesion (22, 32). In contrast to this “mechanical’” function, the second group (the SLSP group) is involved in the G2/M transition and the spindle checkpoints (25, 33).
Interestingly, these results offer a rather unappreciated explanation of synthetic lethality. Classically, synthetic lethality has been assumed to indicate the operation of two redundant pathways. The current results demonstrate that the two arms (Fig. 1A) have distinct functions. Defects in the chromatid separation machinery (right-side genes in Fig. 1A) are not lethal as long as the checkpoint machinery is intact. Likewise, defects in the checkpoint machinery would not be expected to be lethal unless challenged by complications in sister chromatid separation. Thus, synthetic lethality is generated when there is a combination of a defect in the machinery along with a defect in the checkpoint.
From a “guilt by association” standpoint, these results suggested a specific role for ISC1 in the physiology of chromosome separation, and by belonging to the SLSP group, more specifically in the checkpoints involved.
Our previous studies clearly suggested the involvement of Isc1 in the response to HU; however, it was not clear how Isc1 fits in the overall DNA damage response and checkpoint operations (15, 24). The results from this study resolve that issue and further identify Isc1 partners that act upstream of Isc1 and downstream of it in the genotoxic effects of HU.
Our approach assigned ISC1 to the SLSP group of genes, suggesting they belong to the same pathway in the response to chromatid separation defects and spindle point activation. Experimentally, the results showed that deletion of BUB1, MAD1, BIM1, and KAR3 phenocopies the deletion of ISC1 in their sensitivity to HU. In addition, our data showed that deletion of ISC1 gene yielded a phenotype similar to that yielded by the deletion of MAD1, known to play a role in spindle checkpoint and a member of the SLSP group of genes. In fact, isc1Δ cells, like mad1Δ cells, were more sensitive to nocodazole, had more budded cells, and showed a higher percentage of sister chromatid separation than the WT in the presence of nocodazole. Mechanistically, deletion of SLSP genes obliterated the increase in Isc1 activity in response to HU treatment and also abrogated the increase in C18:1 phytoceramides down to levels shown in the absence of treatment. Other chain length phytoceramides (C14, C14:1, C16, C16:1, C18, C20, C20:1, C22, C22:1, C24, C24:1, C26, and C26:1) also increased following HU treatment. Among all chain length species, it was shown that C18:1 phytoceramides were the most likely lipid mediator to protect cells from HU toxicity (24). Taken together, these results indicate that ISC1 is regulated downstream of the SLSP genes. This was further corroborated by demonstrating that overexpression of the ISC1 gene in the SLSP deletion mutants corrected their HU sensitivity phenotype. ISC1 overexpression also corrected the sensitivity to benomyl of the SLSP gene deletion, further cementing this connection downstream of the SLSP genes (Fig. 8). Although the isc1Δ mutant did not show strong sensitivity to benomyl, additional experiments indicated that the isc1Δ mutant had a spindle elongation defect similar to that of the bub1Δ mutant when treated with HU. In the chromatid separation assay, we also found that the isc1Δ mutant had earlier chromatid separation than did the WT in the presence of nocodazole. This experiment provided further evidence implicating ISC1 in the spindle checkpoint. In addition, an isc1Δ rebudding assay showed a higher percentage of budding cells than for the WT when treated with nocodazole. This assay showed the isc1Δ mutant to follow a spindle checkpoint mutant pattern of budding, although not as highly as the mad1Δ mutant. ISC1 overexpression was able to correct the growth defect in all SLSP mutants. Furthermore, deletion of the SWE1 gene did rescue growth on benomyl similarly in isc1Δ and SLSP mutants. Like in the isc1Δ mutant, overexpression of CDC55 rescued growth on benomyl in bub1Δ. These data strongly link ISC1 to the SLSP genes.
Importantly, this intimate connection of ISC1 to the SLSP genes suggested that ISC1 participates in key regulatory events mediated by Bub1 and Mad1. Indeed, Bub1 has important roles in the spindle checkpoint whereby it responds to many physical or mechanical events that occur in response to upstream alterations in chromosome separation (34). Bub1 and Mad1 are both members of the spindle assembly checkpoint, consisting of three functional units: a sensor, a signal transducer, and an effector. Importantly, while spindle elongation was detected in WT cells treated with HU, indicating that elongation mechanism was intact, isc1Δ and bub1Δ cells demonstrated significant defects in spindle elongation. This result suggested that ISC1 and BUB1 genes play a crucial role in spindle elongation during mitosis. Previous studies had suggested a role for Bub3 in spindle elongation in the context of deletion of CLB5 (35). The current results define a role for Bub1 in the spindle elongation in response to DNA damage. Importantly, the results now implicate sphingolipid metabolism in this process and specifically downstream of Bub1.
The results from this study specifically implicate bioactive lipids in a process that heretofore has not been appreciated to involve lipids or enzymes of lipid metabolism. Our previous results showed that ISC1 activity was required for the HU response, and they further “triangulated” the lipid mediator of the action of ISC1 in response to HU specifically to C18:1 phytoceramide (24), one of at least 30 distinct ceramides present in yeast, thus indicating the operation of a very specific (and probably localized) sphingolipid pathway. The current results demonstrate that the C18:1 fatty acid oleate, which is incorporated into C18:1 ceramides (24), corrected the defect of growth on HU of the SLSP deletion mutants. These results provide further evidence of a role for sphingolipid metabolism downstream of the spindle checkpoint, demonstrating the essential role of one specific bioactive sphingolipid in the HU response involving Bub1, Mad1, Bim1, and Kar3.
A major mechanistic question to arise from these results concerns identification of downstream targets of ISC1 and its lipid mediator C18:1 phytoceramide. Our results clearly implicate both the Swe1 kinase and the Cdc55-containing protein phosphatase (of the PP2A family). Deletion of SWE1 in bub1Δ, mad1Δ, and bim1Δ cells protected these mutants from HU toxicity, in a manner similar to protection seen in isc1Δ cells. Curiously, deletion of SWE1 had no protective effect in kar3Δ cells. However, deletion of SWE1 protected all mutants from the toxicity associated with benomyl.
Overexpression of CDC55 rescued growth of isc1Δ, bub1Δ, mad1Δ, bim1Δ, and kar3Δ cells. At this time, we cannot explain why SWE1 deletion failed to protect kar3Δ cells from HU, but the results suggest that the Kar3 gene has additional roles outside this pathway.
The emerging role of CDC55 significantly builds on prior results with ceramide and protein phosphatases. A permeable form of ceramide, C2-ceramide, induced cell growth arrest in yeast cells, which were shown to exhibit a ceramide-activated phosphatase (36). Studies by Nickels and Broach identified the relevant phosphatase as the one involving the Sit4 catalytic subunit and the Cdc55 regulatory subunit (26). Interestingly, other studies have implicated PP2A-CDC55 in spindle checkpoint adaptation (37, 38) and in the regulation of mitotic exit (39). In those studies, PP2A-CDC55 is described as a negative regulator of anaphase onset, by preventing Net1 phosphorylation and Cdc14 activation and thus APC-cdc20 activation. Thus, taken together, the results from this study demonstrate the involvement of a phosphatase downstream of ISC1 and C18:1 phytoceramide leading to the execution component of the spindle checkpoint (Fig. 8).
Interestingly, SWE1 has been shown to be essential in anaphase onset (40). During spindle checkpoint operation, anaphase onset is regulated by phosphorylation of cyclin-dependent kinase Cdc28. This kinase controls the metaphase anaphase transition, and mutations affecting Cdc28 or the two mitotic cyclins Clb1 and Clb2 delay anaphase initiation (41, 42). Swe1 is the kinase that phosphorylates Cdc28 and controls its activity and thus metaphase anaphase transition (40). More recently, Swe1 was shown to promote the G0-S transition in a lipid-dependent manner; furthermore, Swe1 was found to be a level sensor and activator of sphingolipids (43, 44).
These results, revealing an intimate connection between CDC55 and SWE1, suggest that Cdc55 can act in different possibilities that are not necessarily mutually exclusive (Fig. 8): (i) by regulating the levels of Swe1, thus influencing the phosphorylation of the downstream target Cdc28; (ii) by acting directly on Cdc28 and dephosphorylating the kinase to permit the metaphase-to-anaphase transition in the presence of benomyl or prolonged HU treatment; and/or (iii) by modulating the phosphorylation of Net1 and other possible substrates. Indeed, the phosphoproteomics of WT, isc1Δ, and cdc55Δ strains identified important additional substrates. Starting with 1184 HU-induced dephosphorylations, 264 proteins were identified as such in isc1Δ and cdc55Δ strains. Out of the 190 proteins dephosphorylated in an Isc1/Cdc55-dependent manner, 16 are involved in spindle assembly and chromatid separation. Within the 16 proteins, substrates of Cdc55 were identified: Orc2, Kin4, Sli15, Dam1, and She1, which connect the Isc1/phytoceramide/Cdc55 pathway to specific downstream cell processes (Fig. 8). The Kin4 protein is inactivated by dephosphorylation by Cdc55; this leads to Cdc14 activation through the mitotic exit network. On the other hand, Dam1, which was identified in this study as a new target of Cdc55, interacts with She1 to regulate the SAC. Indeed, the Dam1/DASH complex forms a ring around the attached microtubule and acts as a barrier between two proteins: Mps1 and its substrate, Spc105. This mechanical separation prevents interactions between Mps1 and Spc105 and thus inhibits the SAC signaling pathway (45).
We should note that in our hands, overexpression of the MIH1 phosphatase, which is known to be involved in the G2/M transition, did not protect cells from HU toxicity (data not shown), suggesting that the phosphatase that regulates anaphase onset, especially in the context of activation of Isc1, involves Cdc55 but not Mih1.
Interestingly, preliminary data showed that HU selectively induces an increase in the levels of the Isc1 protein in the nuclear fraction, thus placing all the components of this novel pathway (Bub1 and the SPLS proteins, Cdc55 and Swe1) in the nucleus (data not shown). These findings raise a number of intriguing questions on the localization and physical organization of lipids in the nucleus; however, an increasing body of literature points to the presence of specific bioactive lipids in the nucleus (46–49). Moreover, studies with mammals have also detected neutral sphingomyelinase (the mammalian counterpart of ISC1) (50–54) and sphingomyelin in the nucleus (55).
Taken together, the results from this study suggest that the lipid product of Isc1, C18:1 phytoceramide, functions as an intermediate molecule between the upstream SLPS genes, which constitute core components of G2/M and spindle checkpoints, and the signal transmission downstream involving Swe1 and Cdc55 (Fig. 8). According to this hypothesis, when cells are treated with HU, a latent spindle checkpoint is activated. However, WT cells resume cycling by reentering into mitosis starting from spindle elongation and anaphase onset, resolving the spindle checkpoint blockage. To achieve the goal of resolving or overcoming the spindle checkpoint blockage, SLPS genes, in response to HU-induced damage, act on Isc1, increasing its activity to produce the signaling molecule C18:1 phytoceramide. This molecule serves as an activator of Cdc55 and then Swe1 downstream, leading Cdc28, Kin4, Dam1, She1, and APC to permit the onset of anaphase. This study implicates another set of proteins, Siz1, Gac1, Ulp2, Top2, Sth1, Gip4, Spc29, Src1, and Fin1, as substrates of Cdc55. In conclusion, the results provide a very unexpected role for a nuclear pathway of sphingolipid metabolism mediated by Isc1 in the regulation of the spindle checkpoint, with Isc1 being necessary for signal transmission from Bub1, Mad1, and Bim1 to downstream targets Cdc55, Swe1 and Cdc28, Net1, Kin4, Dam1, and She1. The use of budding yeast as a model system with a combination of bioinformatic, biochemical, molecular, and phosphoproteomics approaches has provided unexpected and important biological insights that can be used as a valuable map to investigate the involvement of bioactive sphingolipids in eukaryotic cells.
MATERIALS AND METHODS
Strains used in this study are listed in Table 3.
Strains used in this study
Media.Standard yeast media—yeast extract, peptone, dextrose, and adenine (YPDA) medium, synthetic complete (SC) medium, and SD dropout medium lacking uracil (URA−)—were used in this study. Minimal SD base with galactose and raffinose URA− dropout medium was used to overexpress genes under the control of the GAL promoter, purchased from Clontech (no. 630420). HU was purchased from U.S. Biological and was employed at 7.5 mg/ml and 10 mg/ml in liquid and solid SC and YPDA media, respectively. Benomyl was purchased from U.S. Biological and used at 15 μg/ml and 24 μg/ml and in SC and YPDA media, respectively. Oleate was purchased from Sigma and used in solid agar medium at 0.003%.
Plasmids.YEp24 is a URA3 2μm autonomously replicating sequence (ARS) with multiple cloning sites. For YEp24 plus CDC55, the CDC55 open reading frame was cloned into the YEp24 vector; for YEp24 plus ISC1, the ISC1 open reading frame was cloned into the YEp24 vector); and for YEp24 plus BUB1, the BUB1 open reading frame was cloned into the YEp24 vector. The PCR product was cloned using a PCR 2.1-TOPO vector from Invitrogen (catalog no. K4560-40; TOP10 competent bacterial cells purchased from Invitrogen were transformed with the made plasmid. Bacterial colonies grown on plates containing 50 μg/ml of carbenicillin were screened for positive clones containing the CDC55 or the BUB1 insert by digesting plasmid recovered by a standard Qiagen miniprep using BamHI and XhoI restriction enzymes. The positive clones were extracted from the gel and cloned into YEP24 vector. pYES2, a vector containing a galactose-inducible promoter, was purchased from Invitrogen. The ISC1 open reading frame was cloned into pYES2 (pYES2 + ISC1 [56]).
Spot test assays.Spot test assays were described previously (15). Briefly, cells were grown to stationary phase overnight, diluted to an optical density at 600 nm (OD600) of 0.1, and then grown to an OD600 of 0.8. The cultures were diluted to an OD600 of 0.3 for the first spot on the left in all figures with spot tests. A series of 1:10 dilutions were spotted on YPD plates containing 10 mg/ml of HU or on SC plates containing 7.5 mg/ml of HU. YPD plates were incubated at 30°C for 3 days; SC or URA− plates were incubated at 30°C for 4 to 5 days.
Protein preparation and Western blotting.Western blotting was performed as described previously (15). An anti-Myc tag was used to detect Swe1-Myc; anti-Swe1 from Santa Cruz Biotechnology (catalogue no. sc7171) was also used.
Antibody against Flag tag was used to detect ISC1-Flag. Antibody against PSTAIRE (PSTAIRE domain of Cdc2 p34; Santa Cruz Biotechnology [catalogue no. sc-53]) was used as a loading control.
Enzymatic activity assays.The assay for Isc1 activity was conducted as described previously (12). Briefly, sphingomyelin labeled with 14C on the choline moiety was used as a substrate, and the hydrolysis of choline-methyl-[14C]sphingomyelin was determined by liquid scintillation counting.
Lipid extraction and analysis.Lipid extraction and analysis have been described in detail previously (24).
Lipid analyses by HPLC-MS/MS.Levels of dihydroceramide, dihydroceramide, phytosphingosine, phytosphingosine 1-phosphate, dihydrosphingosine, and dihydrosphingosine 1-phosphate were measured by high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) as previously described (57). Analytical results of lipids were expressed as lipid level/total cell number.
Lipid determination.Cells were grown overnight and then resuspended in the morning to an A600 of 0.15 in 50 ml of medium. Cells were grown to reach an A600 of 0.7 and then were treated with either HU or a vehicle. Cells were harvested after 3 and 20 h of treatment with HU. A600 was measured; all the cultures were diluted to an A600 of 0.7. Cells were then centrifuged at 3,000 × g, and the pellets were resuspended in 1 ml of a lipid extraction solvent containing isopropanol (50%), diethyl ether (10%), pyridine (2%), ammonia (25%), and water (15%). Acid-washed glass beads of 425 to 600 μm were added to a 200-μl volume. Tubes were placed on a Destroyer vortex for 5 repeats of a 3-min-on, 1-min-off regimen. The extracts were then transferred into 15-ml tubes, and the Eppendorf tubes used for lysis were washed with an additional 1 ml of extraction solvent and combined with the samples. The tubes containing approximately 2 ml of solvent, cells, and glass beads were used for lipid analysis (performed at the Lipidomic Core at the Medical University of South Carolina).
Microscopy.Briefly, cells (5 × 106 and 5 × 107) were harvested from logarithmic growth and resuspended in 5 ml of buffer. Then 0.5 ml of 37% formaldehyde was added, and cells were kept for at least 4 h at 4°C (for details, see reference 58). Slides were stored at −20°C and the cells were examined on a Nikon high-resolution microscope (simultaneous imaging) at the Stony Brook University Microscopy core.
Rebudding and sister chromatid separation assays.Wild-type cells treated with nocodazole activate the spindle assembly checkpoint and arrest as large budded cells in mitosis. However, checkpoint mutants continue through the cell cycle and pass through the subsequent G1, generating a new bud. Overnight cultures in YPD medium were diluted 1:25. When cells reached an OD of ∼0.3, they were treated with hydroxyurea for 4 h, washed twice, and then released into medium containing nocodazole (15 μg/ml). At each desired time point, cells were fixed in 4% paraformaldehyde at room temperature for 15 min, washed, and resuspended in a small volume of KPO4-sorbitol solution. Cells were vortexed vigorously and examined by bright-field microscopy and categorized into G1 (unbudded), dumbbell (large budded), and rebudded cells (more than one bud) as described before (38).
Strain K6745, originally from Koshland Lab at University of California (UC), Berkley, was a gift from Hollingsworth’s Lab at Stony Brook University. Sister chromatid separation was monitored by using strain K6745 containing a Tet-GFP fusion and a tandem repeats of the tet operator inserted into the chromosome. Cells were treated as described for the rebudding assay, then fixed with paraformaldehyde for 10 min, washed, and resuspended in a small volume of KPO4-sorbitol solution. Cells were analyzed using a Zeiss fluorescent microscope for the existence of one or two dots, corresponding to paired or separated sister chromatids, respectively. At least 200 cells were counted for each data point.
Data analysis.The genetic interaction data set was downloaded from the BioGRID database (http://thebiogrid.org), from which the yeast gene pairs that are either labeled as “synthetic lethality” or “synthetic growth defect” were extracted. We used these interaction pairs to first identify the partners of the ISC1 genes. These genes’ genetic interaction partners were then extracted. A bipartite subgraph containing ISC1 was identified from these genes. The number of genetic interaction partners shared by ISC1 and each of the other members of the group in the bipartite graph was calculated. These genes were then mapped to the KEGG pathways to obtain the insight of their functions.
iTRAQ phosphoproteomics sample preparation.Jk9-3dα/vector, Jk9-3dα-isc1Δ/vector, Jk9-3dα-cdc55Δ/vector, and Jk9-3dα-isc1Δ/pYEP24-Cdc55 were cultured in yeast synthetic medium (SD-Ura) to logarithmic phase. Each culture was divided into two equal samples. Cultures were grown for another 3 h with one sample in the presence of 10 mg/ml of hydroxyurea, while the other was left untreated. A total of ∼25 OD units of cells (1 OD unit ≈ 2 × 107 to 4 × 107 yeast cells) were collected from each sample by centrifugation at 3,000 rpm for 5 min. The cell pellets were frozen in liquid nitrogen immediately before being sent on dry ice to the phosphoproteomic facility.
iTRAQ labeling procedure.Samples were normalized to 160 μg of protein for each sample and were processed utilizing the standard ABSciex iTRAQ protocol (https://sciex.com/Documents/Downloads/Literature/mass-spectrometry-4375249C.pdf) utilizing all reagents included in the ABSciex 8-plex kit. The recommended 80 μg of peptide per sample was doubled to 160 μg to account for the low abundance of phosphopeptides. Therefore, two 8-Plex kits were utilized, with each sample requiring double the label. The nonmodified peptides and the phosphopeptides were all derived from the same iTRAQ samples. Prior to MS analysis, the iTRAQ-labeled peptides were separated into two pools of peptides. One of them were fractionated by hydrophilic interaction liquid chromatography (HILIC) and analyzed by MS (nonmodified), and the other pools were enriched for phosphopeptides by a TiO2-SIMAC-HILIC (where SIMAC is sequential elution from immobilized metal affinity chromatography) procedure (https://www.ncbi.nlm.nih.gov/pubmed/22906719).
Capillary LC-mass spectrometry.An Orbitrap Velos Pro mass spectrometer (Thermo Scientific) equipped with a nano-ion spray source was coupled to an EASY-nLC system (Thermo Scientific). The nanoflow LC system was configured with a 180-μm (inside diameter) fused silica capillary trap column containing 3 cm of Aqua 5-μm C18 material (Phenomenex), and a self-pack PicoFrit 100-μm analytical column with an 8-μm emitter (New Objective, Woburn, MA) packed to 15 cm with Aqua 3-μm C18 material (Phenomenex). Mobile phase A consisted of 2% acetonitrile-0.1% formic acid, and mobile phase B consisted of 90% acetonitrile-0.1% formic acid. An aliquot of 3 μl of each sample dissolved in mobile phase A was injected through the autosampler onto the trap column. Peptides were then separated using the following linear gradient steps at a flow rate of 400 nl/min: 5% mobile phase B for 1 min, 5% mobile phase B to 35% mobile phase B over 70 min, 35% B to 75% mobile phase B over 15 min, holding at 75% mobile phase B for 8 min, 75% mobile phase B to 8% mobile phase B over 1 min, and holding at 8% mobile phase B for the final 5 min.
Eluted peptides were directly electrosprayed into the Orbitrap Velos Pro mass spectrometer with the application of a distal 2.3-kV spray voltage and a capillary temperature of 275°C. Each full-scan mass spectrum (resolution = 60,000; 380 to 1,700 m/z) was followed by MS/MS spectra for the top 12 masses. High-energy collisional dissociation (HCD) was used with the normalized collision energy set to 35 for fragmentation, the isolation width set to 1.2, and an activation time of 0.1. A duration of 30 s was set for the dynamic exclusion with an exclusion list size of 500, repeat count of 1, and exclusion mass width of 10 ppm. We used monoisotopic precursor selection for charge states 2+ and greater, and all data were acquired in profile mode.
Database Searching-Peaklist files were generated by Mascot Distiller (Matrix Science). Protein identification and quantification were carried using Mascot 2.2 (59) against the Uniprot Human sequence database (89,706 sequences; 35,609,686 residues). Secondary searches were performed utilizing Thermo Proteome Discoverer. Methylthiolation of cysteine and N-terminal and lysine iTRAQ modifications were set as fixed modifications, methionine oxidation and deamidation (NQ) as variable. Trypsin was used as a cleavage enzyme, with one missed cleavage allowed. Mass tolerance was set at 30 ppm for intact peptide mass and 0.3 Da for fragment ions. Search results were rescored to give a final 1% false-discovery rate (FDR) using a randomized version of the same Uniprot human database. Protein-level iTRAQ ratios were calculated as intensity weighted, using only peptides with expectation values of <0.05. Global ratio normalization (summed) was applied across all iTRAQ channels. Phosphopeptides were normalized to their corresponding unmodified proteins when possible. Protein enrichment was then calculated by dividing sample protein ratios by the corresponding control sample channel.
Phosphoproteomics data analysis.Samples of wild-type Jk9-3dα, Jk9-3dα-isc1Δ mutant, Jk9-3dα-cdc55Δ mutant, and Jk9-3dα-isc1Δ mutant cells overexpressing Cdc55 (Cdc55-OE) were treated with HU or left untreated. In total, eight conditions were analyzed by iTRAQ, resulting in a total of 20,758 phosphopeptide hits. Phosphopeptide abundances were normalized for protein concentration before further analysis and sorted as follows: WT plus HU phosphopeptide abundances were normalized to the WT, and the ratios of (WT plus HU)/WT were calculated and sorted using Excel. A total of 1,184 peptides which displayed a ratio R of <0.6 (a decrease in phosphorylation levels as a result of HU treatment) were selected for further analysis. Starting with the 1,184 selected peptides, Isc1 plus HU phosphopeptide abundances were normalized to Isc1 and sorted using Excel. Isc1 plus HU phosphopeptides with 0.7 < R < 1.3 were selected and considered Isc1-dependent dephosphorylations (545 phosphopeptides). Like Isc1 selection, starting with the 1,184 peptides, Cdc55 plus HU phosphopeptide abundances were normalized to Cdc55 and sorted using Excel. Cdc55 plus HU phosphopeptides with 0.7 < R < 1.3 were selected and considered Cdc55-dependent dephosphorylations (656 phosphopeptides). Starting with the Isc1-dependent dephosphorylation hits we obtained as described above (545 phosphopeptides), Cdc55-OE plus HU phosphopeptide abundances were normalized to Cdc55-OE and sorted using Excel. Cdc55-OE plus HU phosphopeptides with 0 < R < 0.75 were selected and considered Isc1-dependent dephosphorylations reversed by Cdc55 overexpression (190 proteins).
ACKNOWLEDGMENTS
This work was supported by NIH grant GM118128 to Y.A.H. and grants P20 RR017696, P20 RR017677, CPRIT RP170668, and LM 010680 to W.J.Z.
Microscopy was performed at the Central Microscopy Imaging Center (CMIC) at Stony Brook University. We thank Guowei Tian for his help and support in microscopy and Nancy Hollingsworth at Stony Brook University, Department of Biochemistry and Cell Biology, for providing us with the K6745 strain. We also thank the lipidomic core at Stony Brook University for the lipid analysis. We thank the KEGG/GenomeNet team for granting copyright permission to publish Fig. 1B.
Author contributions are as follows: conceptualization, N.M., B.H.H., W.J.Z., and Y.A.H.; methodology and investigation, N.M., B.H.H., A.A.S., E.J., B.S., and G.C.; genetic interaction analysis, W.J.Z.; phosphoproteomics analysis, N.M., B.H.H., J.R., A.A.S., J.S., S.V.R., M.R.L., and N.J.F.; writing of original draft, N.M. and Y.A.H.; writing, review, and editing, N.M., B.H.H., J.R., B.K.M., W.J.Z., N.J.F., L.M.O., and Y.A.H.; and funding acquisition, Y.A.H.
We declare no competing interests.
FOOTNOTES
- Received 25 July 2019.
- Returned for modification 19 August 2019.
- Accepted 18 March 2020.
- Accepted manuscript posted online 23 March 2020.
- Copyright © 2020 American Society for Microbiology.
