Methylation of Dam1 (^MeKDam1) is cell cycle regulated

Dam1 is a key component of the budding yeast kinetochore and plays an important role in regulating the architecture of kinetochore during the cell cycle, which is essential for high fidelity chromosome segregation [26,49,50,56,57,59–61]. A previous study reported ^MeKDam1 and showed that the lysine residue 233 (Dam1-K233) is methylated by Set1 [55]. Cells with mutation of Dam1-K233 to Dam1-K233A are inviable suggesting that the PTMs of lysine 233 in Dam1 is required for cell survival under normal physiological conditions [55]. Despite these results, the molecular role of ^MeKDam1 and its physiological significance through the cell cycle remain unexplored. To facilitate the detection of ^MeKDam1, we used a methyl-lysine specific polyclonal antibody (PA5–77770, Thermo Fisher Scientific). The specificity of the antibody for methylated lysine was examined using kinetochore protein HA-Cse4 that shows lysine methylation [25] and its variant HA-Cse4^16KR as a negative control, in which all lysines were mutated to arginine [62]. The methylated proteins were immunoprecipitated using the methyl-lysine specific antibody from whole cell extracts and analyzed by western blotting by detection with anti-HA antibodies. The methyl-lysine specific antibody detected methylated Cse4 in wild-type HA-Cse4 strain, but reactivity was not observed in HA-Cse4^16KR strain (S1 Fig). These results established that the methyl-lysine specific antibody preferentially recognizes methylated lysine thereby providing an ideal tool for studies on ^MeKDam1. To determine whether ^MeKDam1 is cell cycle regulated, we assayed lysine methylation of endogenously expressed Dam1–3HA in a wild-type strain. Cells were synchronized in G1 (α-factor treatment) and released into pheromone free media (Fig 1A). Cells were collected at different time points after G1 release. Based on the flow cytometry, nuclear position and bud morphology (Figs 1A, 1B and S2), cells were categorized as G1, S, metaphase, anaphase, and telophase as described previously [29,63,64]. Whole cell extracts were prepared, and proteins methylated at lysine residues were immuno-precipitated using methyl-lysine specific antibody (PA5–77770, Thermo Fisher Scientific). The fraction of ^MeKDam1 in immunoprecipitated samples was determined by western blotting using anti-HA antibodies. ^MeKDam1 was barely detectable in G1 and S-phase (40–60 min post release from α-factor arrest), higher levels in metaphase cells (80 min post release from α-factor arrest) and reduced to the background levels in anaphase and telophase cells (Figs 1C and S2). We quantified the fraction of ^MeKDam1 and normalized this to total Dam1 levels (Dam1–3HA) in each stage of the cell cycle as described in the Materials and Methods. The enrichment of ^MeKDam1 was significantly higher in metaphase cells than any other stage of the cell cycle (Fig 1D).

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Fig 1. Methylation of Dam1 (^MeKDam1) is cell cycle regulated.

(A) Flow cytometry analysis showing DNA content and cell cycle progression. Wild-type strain (strain# ZK1-5) with Dam1-3HA expressed from its endogenous promoter was grown in YPD to LOG phase at 25°C, synchronized in G1 with α-factor, washed and released into pheromone free media. α-factor was readded at 100 min time point to block cells in the next G1. Samples were taken at time points (min) after release from G1. (B) Bud morphology analysis show synchronization in G1 and release into the cell cycle. Cell cycle stage of samples from (A) were determined based on nuclear position and bud morphology by microscopic examination of at least 100 cells for each sample. Different stages of the cell cycle: G1, S-phase (S), metaphase (M), anaphase (A), and telophase (T). Average ± range (parenthesis) from two independent biological replicates is shown. (C) The levels of ^MeKDam1 are higher in metaphase cells. Methylated proteins were enriched by immunoprecipitation with anti-methyl lysine antibodies using whole cell extracts of wild-type strain from (A) and analyzed by western blotting using α-HA (Dam1-3HA) antibodies as described in Materials and Methods. IN, input; ^MeKIP, immunoprecipitated samples. (D) Relative enrichment of ^MeKDam1 through the cell cycle after release from G1. Ratio of ^MeKDam1 to the total Dam1 was calculated using a semi-quantitative approach in Image J [117] as described in Materials and Methods. Two independent biological replicates were done. Average ±range is shown.

https://doi.org/10.1371/journal.pgen.1011760.g001

We further confirmed the enrichment pattern of ^MeKDam1 in independent cell cycle arrest and release experiments. Cells were synchronized in S-phase (HU treatment) and released into the cell cycle (Fig 2A and 2B). Cell cycle stages were determined based on the flow cytometry profiles, nuclear position and bud morphology (Figs 2A, 2B and S3) following the approach as described previously [29,63,64]. Consistent with results from Fig 1, the highest enrichment of ^MeKDam1 was observed in metaphase cells (40–60 min post release from HU arrest), whereas it was barely detectable in anaphase, telophase or G1 cells (80–120 min post release from HU arrest) (Figs 2C, 2D and S3). Notably, slightly enhanced levels of ^MeKDam1 were detected in HU-treated cells (0 min and 140 min) (Fig 2C and 2D). Moreover, levels of ^MeKDam1 in LOG phase cells was not significantly different from metaphase cells (Figs 1 and 2). Taken together, our results indicate that ^MeKDam1 is cell cycle regulated with the highest enrichment of ^MeKDam1 observed in metaphase cells.

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Fig 2. ^MeKDam1 enrichment observed in metaphase cells.

(A) Flow cytometry profiles and DNA content analysis. Wild-type strain (strain# ZK5) with Dam1-3HA expressed from its endogenous promoter was grown in YPD to LOG phase at 25°C, synchronized in S-phase with HU, washed and released into YPD media. HU was readded at 100 min time point to block cells in the next S-phase. Samples were collected at time points (min) after release from the S-phase. (B) Composition of cells in different cell cycle stage determined based on nuclear position and bud morphology by microscopic examination of at least 100 cells for each sample. Stages of the cell cycle: G1, S-phase (S), metaphase (M), anaphase (A), and telophase (T). Average ± SD from three independent biological replicates is shown. (C) ^MeKDam1 enrichment is higher in metaphase cells. The levels of ^MeKDam1 were determined in samples from (A) by immunoprecipitation with anti-methyl lysine antibodies followed by western blotting with α-HA (Dam1-3HA) antibodies as described in Materials and Methods. IN, input; ^MeKIP, immunoprecipitated samples. (D) Relative enrichment of ^MeKDam1 after release from the S-phase. ^MeKDam1 enrichment was calculated as described in Fig 1D. Three independent biological replicates were done. Average ±SE is shown. *p value <0.05, Student’s t-test.

https://doi.org/10.1371/journal.pgen.1011760.g002

Dam1 interacts in vivo with Set1 in a cell cycle dependent manner

Previous studies have shown an interaction of Dam1 with Set1 in LOG phase cells and a role of Set1 for in vivo ^MeKDam1 [55]. Based on our results for high levels of ^MeKDam1 in metaphase cells (Figs 1 and 2), we hypothesized that the in vivo interaction of Dam1 and Set1 may be cell cycle regulated. Co-IP experiments were done using a wild-type strain carrying endogenously expressed Dam1–3HA and Set1–9Myc. Protein extracts from cells synchronized in G1 (α-factor treatment), S-phase (HU treatment) and metaphase (80 min post release from α-factor arrest) were examined for Dam1-Set1 interaction (Fig 3A). The cell cycle synchronization was confirmed by flow cytometry and examination of nuclear and bud morphology (Figs 3A, 3B and S4). IP results showed an in vivo interaction between Dam1 and Set1 in metaphase cells (Figs 3C and S4), whereas interaction of Dam1 with Set1 was not detected in G1 cells despite the expression of these two proteins (Fig 3C). Consistent with ^MeKDam1 in HU-treated S-phase cells (Fig 2C and 2D), we observed an in vivo interaction between Dam1 and Set1 in these cells (Figs 3C and S4). As expected, no signals were detected in control experiments performed with an untagged Set1 strain (Figs 3C and S4). These results suggest that Set1-mediated ^MeKDam1 initiates in S-phase and peaks during metaphase. Taken together, we found that cell cycle regulated interaction between Dam1 and Set1 in vivo is coincident with the enrichment of ^MeKDam1.

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Fig 3. Dam1 interacts in vivo with Set1 in a cell cycle dependent manner.

(A) Flow cytometry analysis showing DNA content. Wild-type strain (strain# 9MYC-SET1-#2) expressing HA-tagged Dam1 and Myc-tagged Set1 (Dam1-3HA Set1-9Myc) and a control strain (strain# ZK1-5) expressing HA-tagged Dam1 (Dam1-3HA Set1) from their native promoters were grown at 25°C to LOG phase, synchronized in G1 with α-factor, S-phase with HU and synchronized in G1 with α-factor, washed and released into pheromone free media for 80 min to capture cells in metaphase. (B) Nuclear position and bud morphology analysis show synchronization in different stages of the cell cycle. Cell cycle stage of samples from (A) were determined as described in Fig 2B. Average ± SD from three independent biological replicates for G1, S-phase (S), and metaphase (M) is shown. (C) In vivo interaction of Dam1 with Set1 in G1, S-phase and metaphase cells. IP experiments using whole cell extracts from (A) were performed with α-HA conjugated agarose beads and analyzed by western blotting with α-HA (Dam1), and α-Myc (Set1) antibodies. IN, input; IP, immunoprecipitated samples.

https://doi.org/10.1371/journal.pgen.1011760.g003

Kinetochore-microtubule interactions contribute to Dam1 methylation

Dam1 regulates the dynamics of microtubules polymerization and depolymerization and is required for kinetochores to maintain attachments to microtubules in presence of tension [65–68]. To determine whether kinetochore-microtubule attachments is linked to ^MeKDam1, we treated cells with nocodazole that causes depolymerization of microtubules, thereby relieving the tension at the kinetochore [69,70]. As control, we examined the levels of ^MeKDam1 in G1, S-phase (HU treatment) and in LOG phase cells (Figs 4A, 4B, and S5). Whole cell extracts were immunoprecipitated using methyl-lysine specific antibody and examined by western blotting using anti-HA antibodies for levels of ^MeKDam1. As observed previously (Figs 1 and 2), ^MeKDam1 was observed in LOG phase cells, barely detectible in G1, and a slightly higher levels in HU-treated S-phase cells (Fig 4C, 4D and S5). In contrast, no significant enrichment of ^MeKDam1 was observed in nocodazole treated cells (Figs 4C, 4D and S5). Notably, reduced association of Dam1 with CEN chromatin has been reported in nocodazole treated cells [71]. Taken together, these observations show that kinetochore-microtubule interactions contribute to the enrichment of ^MeKDam1.

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Fig 4. Enrichment of ^MeKDam1 requires kinetochore-microtubule interactions.

Wild-type strain (strain# ZK5) with Dam1-3HA expressed from its endogenous promoter was grown in YPD to LOG phase at 25°C, synchronized in S-phase with HU, G2/M with nocodazole, and G1 (cells collected after release from G2/M). (A) Flow cytometry showing different stages of the cell cycle and DNA content. (B) Cell cycle stages of samples from (A) were determined as described in Fig 2B. G1, S-phase (S), metaphase (M), anaphase (A), and telophase (T) are shown as average ± SD from three biological replicates. (C) The levels of ^MeKDam1 are reduced in nocodazole treated G2/M cells. Methylated proteins were enriched by IP with anti-methyl lysine antibodies using whole cell extracts from (A) and analyzed by western blotting using α-HA (Dam1-3HA) antibodies as described in Materials and Methods. IN, input; ^MeKIP, immunoprecipitated samples. (D) Relative enrichment of ^MeKDam1 in different stages of the cell cycle. ^MeKDam1 enrichment was calculated as described in Fig 1D. Three biological replicates were done. Average ±SE is shown. ***p value <0.001, Student’s t-test.

https://doi.org/10.1371/journal.pgen.1011760.g004

Histone lysine demethylase Jhd2 interacts with Dam1 complex proteins in vivo

Previous studies have shown that Set1 contributes to ^MeKDam1 [55], colocalizes with histone lysine demethylase Jhd2 [72], and that the histone H3-K4 methylated by Set1 is demethylated by Jhd2 [73,74]. Moreover, our results for cell cycle dependent oscillation of ^MeKDam1 imply a role for demethylation of Dam1 during the cell cycle. Considering these observations together with a functional relationship between Set1 and Jhd2 [72–74] and in vivo interaction between Set1 and Dam1 (Fig 3), we explored the role of Jhd2 in cell cycle dependent oscillation of ^MeKDam1. We performed affinity purification-based mass spectroscopy to identify in vivo interactors of Jhd2 in a wild-type strain with either 6HIS-HA-JHD2 expressed from the GAL1 promoter or control strain with empty vector. Affinity purified proteins were analyzed by mass spectrometry and proteins identified in samples from vector containing strain were excluded. In total, 1906 proteins were identified as Jhd2-specific interactors by mass spectrometry, of which 507 and 1396 were encoded by essential and non-essential genes, respectively (S1 Table). Gene Ontology (GO) analysis of these in vivo interactors for biological processes was performed using goSlimMapper software from Saccharomyces Genome Database (https://www.yeastgenome.org/goSlimMapper). GO analysis of Jhd2-specific interactors identified proteins for kinetochore function, protein methylation, nuclear pore complex, chromosome segregation and spindle pole body (Table 1). Interactome of Jhd2 associated proteins shows an enrichment of proteins for kinetochore function and chromosome segregation such as inner kinetochore (e.g., Cbf1, Cbf2, Cep3, Scm3), outer kinetochore (e.g., Nuf2, Spc24), mitotic exit network (e.g., Nud1, Mob1, Kin4), spindle assembly checkpoint (e.g., Mad1, Mad2), mitotic spindle and microtubule interactions (e.g., Stu2, Slk19, Bni1) (Table 1). Remarkably, we observed an enrichment of Dam1 complex, where seven of ten subunits of the Dam1 complex were identified in kinetochore and chromosome segregation components (Table 1). The in vivo interaction of Jhd2 with Dam1 was examined using Co-IP experiments using a strain that expresses HA-tagged Dam1 from its endogenous promoter, and Flag-tagged Jhd2 from the GAL1 promoter. Strains were grown to LOG phase and protein extracts were prepared for Co-IP and western blot analysis (Figs 5A, 5B and S6). Co-IP experiments performed with Dam1-HA showed Jhd2-Flag; reciprocal pull down with Jhd2-Flag showed Dam1-HA (Figs 5C, 5D and S6). No signals were detected in a control experiment with a vector alone strain (Figs 5C, 5D and S6). Taken together, these results show an in vivo interaction between Dam1 and histone lysine demethylase Jhd2.

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Table 1. Histone lysine demethylase Jhd2 interacts in vivo with Dam1 complex.

Gene Ontology (GO) based categorization of Jhd2-interactors into functional and structural protein complexes. Listed are the GO categories with protein names, and fraction of proteins from the input over the total number in a given category. Gene names in bold letters represent the component of the Dam1 complex. Statistical significance was determined from the input proteins normalized to the total number of proteins in the yeast genome, and p values were calculated based on one sample t-test with an expected population mean of 1. Categories with p values = < 0.003 are shown.

https://doi.org/10.1371/journal.pgen.1011760.t001

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Fig 5. Jhd2 interacts with Dam1 in vivo.

Wild-type strain expressing HA-tagged Dam1 (Dam1-3HA) from its endogenous promoter carrying pGAL1-URA3-vector (strain# YMB12579) or pGAL1-FLAG-JHD2-URA3 (strain# YMB12580) was grown at 25°C to LOG phase in selective media with galactose+raffinose (2% each) for 6 hours. (A) Flow cytometry showing DNA content. (B) Cell cycle stages of samples from (A) were determined as described in Fig 2B. Cell cycle stages: G1, S-phase (S), metaphase (M), anaphase (A), and telophase (T). Average ± SD from three biological replicates is shown. (C) In vivo interaction of Jhd2 with Dam1. Whole cell extracts from sample in (A) were prepared and used in IP with α-HA conjugated and α-Flag conjugated agarose beads. Eluted proteins were analyzed by western blotting with α-HA (Dam1), and α-Flag (Jhd2) antibodies. IN, input; IP, immunoprecipitated samples. Tub2 served as a control.

https://doi.org/10.1371/journal.pgen.1011760.g005

Jhd2 contributes to demethylation of ^MeKDam1 in vivo

Based on our results showing an in vivo interaction of Jhd2 and Dam1, we hypothesized that Jhd2 may regulate demethylation of Dam1. We examined levels of ^MeKDam1 using strain with Flag-tagged Jhd2 expressed from the GAL1 promoter (GALJHD2) and HA-tagged Dam1 expressed from its own promoter at its endogenous locus. Protein extracts were prepared after growth of strains in galactose medium (Figs 6A, 6B and S7) and expression of Jhd2 was confirmed by western blotting (Figs 6C and S7). Proteins methylated at lysine residues were immuno-precipitated using a methyl-lysine specific antibody from whole cell extracts and levels of ^MeKDam1 were assayed as described earlier (Fig 1). We determined that GALJHD2 strain showed significantly reduced levels of ^MeKDam1 (0.14 ± 0.037, average±standard error) when compared to the vector alone strain (0.63 ± 0.028, p-value = 0.0005) (Figs 6D, 6E and S7). As a control, we examined the levels of trimethylation of histone H3-K4 in GALJHD2 strain. In agreement with previous reports showing demethylation of histone H3-K4 trimethylation by Jhd2 [73,74], the levels of histone H3-K4 trimethylation were reduced significantly in GALJHD2 strain in comparison to the vector alone strain (Figs 6F, 6G and S7).

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Fig 6. Jhd2 contributes to demethylation of Dam1.

Wild-type strain expressing HA-tagged Dam1 (Dam1-3HA) from its endogenous promoter carrying pGAL1-URA3 vector (strain# YMB12579) or pGAL1-FLAG-JHD2-URA3 (strain# YMB12580) was grown at 25°C to LOG phase in selective media with galactose+raffinose (2% each) for 3 hours. (A) Flow cytometry showing DNA content. (B) Cell cycle stages of samples from (A) were determined as described in Fig 2B. Different stages are: G1, S-phase (S), metaphase (M), anaphase (A), and telophase (T). Average ± SD from three biological replicates is shown. (C) Western blot showing protein levels of Jhd2-Flag expressed from the GAL1 promoter. (D) Overexpression of JHD2 results in reduction in the levels of ^MeKDam1. Methylated proteins were enriched by IP with anti-methyl lysine antibodies using whole cell extracts of samples from (A) and analyzed by western blotting using α-HA (Dam1-3HA) antibodies as described in Materials and Methods. IN, input; ^MeKIP, immunoprecipitated samples. (E) Relative enrichment of ^MeKDam1 in vector and GALJHD2 strains. ^MeKDam1 enrichment was calculated as described in Fig 1D. Three biological replicates were done. Average ±SE is shown. ***p value <0.001, Student’s t-test. (F) Overexpression of JHD2 results in reduction in the levels of histone H3-K4^Me3. Western blots using samples from (D) were probed with histone H3 and histone H3-K4^Me3 antibodies. IN, input; ^MeKIP, immunoprecipitated samples. (G) Relative enrichment of histone H3-K4^Me3 in vector and GALJHD2 strains. Ratio of histone H3-K4^Me3 to the total histone H3 was calculated using Image J [117] as described in Materials and Methods. Three biological replicates were done. Average ±SE is shown. **p value <0.01, Student’s t-test.

https://doi.org/10.1371/journal.pgen.1011760.g006

Since maximum enrichment of ^MeKDam1 was observed in metaphase cells (Figs 1 and 2), we examined if overexpression of Jhd2 affects ^MeKDam1 in these cells. This was done by using cells synchronized in S-phase (HU treatment), and after release into the cell cycle for 50-min, which represents metaphase cells as determined by flow cytometry, nuclear position and bud morphology assays (Figs 7A, 7B and S8). The western blot analysis showed that the levels of ^MeKDam1 were significantly reduced in metaphase cells overexpressing Jhd2 in comparison to the levels observed in the vector strain (Figs 7C, 7D and S8).

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Fig 7. Jhd2 contributes to demethylation of Dam1 in LOG phase and metaphase cells.

Wild-type strain expressing HA-tagged Dam1 (Dam1-3HA) from its endogenous promoter carrying pGAL1-URA3 vector (strain# YMB12579) or pGAL1-FLAG-JHD2-URA3 (strain# YMB12580) was grown at 25°C to LOG phase in selective media with galactose+raffinose (2% each) for 1 hours and then HU was added to synchronize cells in S-phase for 2 hours. Cells were collected, washed and released into selective media with galactose+raffinose (2% each) for 50 min to capture cells in metaphase. (A) Flow cytometry analysis showing DNA content of LOG phase and metaphase cells. (B) Cell cycle stages of LOG phase and metaphase cells. Cell cycle stage of samples from (A) were determined as described in Fig 2B. Different stages of the cell cycle: G1, S-phase (S), metaphase (M), anaphase (A), and telophase (T). Average ± SD from three biological replicates is shown. (C) Overexpression of JHD2 results in reduced levels of ^MeKDam1 in metaphase cells. Methylated proteins were enriched by IP with anti-methyl lysine antibodies using whole cell extracts of samples from (A) and analyzed by western blotting using α-HA (Dam1-3HA) antibodies. IN, input; ^MeKIP, immunoprecipitated samples. (D) Relative enrichment of ^MeKDam1 in vector and GALJHD2 strains. Enrichment was determined as described in Fig 1D. Three biological replicates were done. Average ± SE is shown. *p value <0.05, ***p value <0.001, Student’s t-test.

https://doi.org/10.1371/journal.pgen.1011760.g007

We next determined if overexpression of JHD2 affects the cell cycle regulated enrichment profile of ^MeKDam1. We examined the ^MeKDam1 pattern in strains carrying vector and compared it with GALJHD2 strain. Cultures were grown in synthetic media, synchronized in G1 with α-factor and released into pheromone free media. Flow cytometry and bud morphology analyses revealed a delay in mitotic cell cycle progression in GALJHD2 strains (S9 Fig). The enrichment of ^MeKDam1 in vector strain occurs at 120–150 min post G1 release coincident with cells in metaphase, whereas a significant reduction in the level of ^MeKDam1 was observed in GALJHD2 strain throughout the cell cycle (S9 Fig). Taken together, these results indicate that Jhd2 contributes to cell cycle dependent in vivo demethylation of Dam1.

Overexpression of JHD2 (GALJHD2) exhibits growth defects with kinetochore mutants

Our results showing Jhd2-mediated demethylation of Dam1 allowed us to test the physiological significance of ^MeKDam1. Since Dam1 is an essential component of budding yeast kinetochore [47,50,52,75–77], we postulated that constitutive demethylation of Dam1 throughout the cell cycle by Jhd2 overexpression (GALJHD2) will weaken the kinetochore and affect the growth of kinetochore mutants. Notably, the CEN recruitment of Cse4, which is an essential component of CEN nucleosome allows for the assembly of kinetochore protein complexes such as, the COMA complex (Ctf19, Okp1, Mcm22, and Ame1) at the kinetochore for faithful chromosome segregation [78–80]. Hence, we examined the growth phenotype of wild-type and kinetochore mutants (e.g., cse4–111, ctf19Δ and ame1–4) upon JHD2 overexpression. The cse4–111 carries L194Q mutation that interferes with the interaction of Cse4 and histone H4 and exhibits growth defects at 37°C [81], whereas ctf19Δ and ame1–4 mutants exhibit chromosome segregation, cohesion and cell cycle defects [82,83]. Growth assays showed that cse4–111, ctf19Δ and ame1–4 strains exhibit growth defects with GALJHD2 on galactose plates at the permissive temperature of 25°C (Fig 8A and 8B). To exclude the possibility that demethylation of histone H3-K4 trimethylation upon JHD2 overexpression may contribute to growth defects observed in kinetochore mutants, we examined the growth phenotype of ctf19Δ in combination with a non-methylatable mutant of histone H3 (H3-K4A). The growth of ctf19Δ H3-K4A mutant was similar to the single mutant (ctf19Δ or H3-K4A) and that of a wild-type strain (Fig 8C). Taken together, these results show that overexpression of JHD2 results in growth defects with kinetochore mutants, and this is independent of methylation of histone H3-K4.

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Fig 8. Overexpression of JHD2 contributes to growth defects in kinetochore mutants.

(A) Overexpression of JHD2 contributes to growth defects in cse4-111 strain. 5 μL of cell suspension (OD₆₀₀ = 1 and its 5-fold dilutions) of cse4-111 with pGAL1-URA3 vector (strain# YMB12415) or pGAL1-6HIS-HA-JHD2-URA3 (strain# YMB12416) and wild type with pGAL1- URA3 vector (strain# YMB12411) or pGAL1-6HIS-HA-JHD2-URA3 (strain# YMB12412) were plated on SC-URA with glucose (2%) or galactose+raffinose (2% each) plates and grown at 25°C for 3 days. (B) Overexpression of JHD2 contributes to growth defects in ctf19Δ and ame1-4 strains. 5 μL of cell suspension (OD₆₀₀ = 1 and its 5-fold dilutions) of ctf19Δ with pGAL1-URA3 vector (strain# YMB12274) or pGAL1-6HIS-HA-JHD2-URA3 (strain# YMB12248); ame1-4 with pGAL1-URA3 vector (strain# YMB12307) or pGAL1-6HIS-HA-JHD2-URA3 (strain# YMB12308); and wild type with pGAL1-URA3 vector (strain# YMB12282) or pGAL1-6HIS-HA-JHD2-URA3 (strain# YMB12281) were plated on SC-URA with glucose (2%) or galactose+raffinose (2% each) plates and grown at 25°C for 3 days. (C) Non-methylatable mutant of histone H3 (H3-K4A) does not show growth defects when combined with a kinetochore mutant. 5 μL of cell suspension (OD₆₀₀ = 1 and its 5-fold dilutions) of wild-type (strain# WT-12380), ctf19Δ (strain# ctf19Δ-12381), H3-K4A (strain# H3K4A-12382), and H3-K4A ctf19Δ (strain# H3K4Actf19Δ -12383) were plated on YPD plates and grown at 25°C for 3 days.

https://doi.org/10.1371/journal.pgen.1011760.g008

A previous study has shown that deletion of UBP8, a deubiquitinating enzyme that removes ubiquitin from histone H2B [84], shows increased levels of ^MeKDam1 [85]. We hypothesized that the increased ^MeKDam1 levels upon UBP8 deletion may counteract GALJHD2 activity and this may suppress the growth defects observed in ctf19∆ GALJHD2 strain. Hence, we examined the effect of JHD2 overexpression on the growth of wild type, ubp8Δ, ctf19Δ and ubp8Δ ctf19Δ strains. Our results showed that growth defects of ctf19∆ GALJHD2 were not suppressed by ubp8∆ (S10 Fig). We reasoned that ^MeKDam1 levels may still be reduced due to overexpression of JHD2 in ubp8∆ strain. Western blot analysis confirmed that deletion of UBP8 results in accumulation of ^MeKDam1 as reported previously [85]. However, levels of ^MeKDam1 were not significantly different between wild type GALJHD2 and ubp8∆ GALJHD2 strains (S11 Fig). These results suggest that increased levels of ^MeKDam1 in ubp8∆ strain cannot counteract the effect of GALJHD2. Taken together, these results show that overexpression of JHD2 contributes to growth defects in kinetochore mutants.

The levels of kinetochore proteins at CEN chromatin are reduced in GALJHD2 strains

We reasoned that growth defects due to GALJHD2 in cse4–111 and ctf19∆ strains (Fig 8) may be due to defects in kinetochore that could potentially be exacerbated by reduction in the levels of ^MeKDam1 in these strains. Hence, we examined the levels of Cse4, Ctf19 and Cse4 interacting protein Mif2 [86–89] in wild-type strain with vector or GALJHD2 (Figs 9A, 9B and S12). In agreement with our hypothesis, Chromatin Immunoprecipitation (ChIP)-qPCR showed that the enrichment of Cse4 at CEN was reduced significantly in GALJHD2 strain when compared to the vector alone (Fig 9C). The reduced CEN association of Cse4 led us to examine if CEN localization of Mif2 is affected in GALJHD2 strain. The enrichment of Mif2 at CEN was reduced significantly in GALJHD2 strain when compared to the vector alone (Fig 9D). Similarly, CEN levels of Ctf19 were also reduced significantly in GALJHD2 strain than the levels those observed for the vector strain (Fig 9E). No significant differences in CEN levels of Ctf19 were observed between wild type and H3-K4A strains (S13 Fig). Moreover, no significant enrichment of Cse4, Mif2 or Ctf19 was detected at the non-CEN ACT1 locus used as a negative control (Figs 9 and S12). The western blot analysis showed similar levels of expression of Cse4, Mif2 and Ctf19 in strains carrying vector or GALJHD2 (Figs 9F, 9G and S12). We propose that the growth defects for GALJHD2 in kinetochore mutants (Fig 8) may be due to reduced CEN association of kinetochore proteins in GALJHD2 strain.

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Fig 9. Reduced CEN association of kinetochore proteins Cse4, Mif2 and Ctf19 in GALJHD2 strain.

Wild-type strain with endogenously expressed GFP-Cse4 (only copy in the genome) carrying pGAL1-URA3 vector (strain# YMB12364) or pGAL1-FLAG-JHD2- URA3 (strain# YMB12365) was grown at 25°C to LOG phase in selective media with galactose+raffinose (2% each) for 6 hours. (A) Flow cytometry analysis showing DNA content. (B) Cell cycle stages of samples from (A) were determined as described in Fig 2B. Cell cycle stages are: G1, S-phase (S), metaphase (M), anaphase (A), and telophase (T). Average ± SD from three biological replicates is shown. (C) Levels of Cse4 are reduced at CEN chromatin in GALJHD2 strains. ChIP was performed with α-GFP antibodies using chromatin from same strains as used in (A). Enrichment of Cse4 at CENs (CEN1 and CEN3) and a negative control (ACT1) was determined by qPCR and is presented as % input. Average from three biological replicates ±SE. **p value <0.01, Student’s t-test. (D) Levels of Mif2 are reduced at CEN chromatin in GALJHD2 strains. ChIP was performed with α-Mif2 antibodies using chromatin from same strains as in (A). Enrichment of Mif2 at CENs (CEN1 and CEN3) and a negative control (ACT1) as above. Average from three biological replicates ± SE. **p value <0.01, Student’s t-test. (E) Levels of Ctf19 are reduced at CEN chromatin in GALJHD2 strains. ChIP was performed with α-Ctf19 antibodies using chromatin from same strains as in (A). Enrichment of Ctf19 at CENs (CEN1 and CEN3) and a negative control (ACT1) was determined as above. Average from three biological replicates ± SE. **p value <0.01, Student’s t-test. (F) Expression of Cse4 in vector, and GALJHD2 strains are similar. The western blotting of protein extracts was done using α-GFP (Cse4), α-HA (Jhd2) and α-Tub2 (loading control) antibodies. (G) Expression of Mif2 and Ctf19 in vector and GALJHD2 strains are similar. The western blotting of protein extracts was done using α-Mif2, α-Ctf19, and α-Tub2 (loading control) antibodies.

https://doi.org/10.1371/journal.pgen.1011760.g009

GALJHD2 strains exhibit declustering of kinetochore and chromosome missegregation

Previous studies have shown that Dam1 complex plays a role in sister kinetochore biorientation [76]. Furthermore, reduced levels of CEN-associated Cse4 contributes to defects in spindle biorientation and errors in chromosome segregation [87,90–92]. The reduced levels of CEN-associated Cse4, Mif2 and Ctf19 prompted us to examine the structural and functional integrity of kinetochores in GALJHD2 strains. We monitored the biorientation of centromeres by examining the localization of Spc42-mCherry, a central component of spindle pole and GFP-Cse4 in mitotic cells of wild-type strain with vector or GALJHD2. The defects in Cse4 biorientation in GALJHD2 strain was significantly higher than the vector strain (p-value = 2.73x10^-7, χ²-test; Fig 10A). In the vector strain, 72% of the cells had bioriented kinetochores, 13% short spindle, and 15% declustered kinetochores. Whereas, in the GALJHD2 strain, 50% of the cells had bioriented kinetochores, 21% short spindle, and 21% declustered kinetochores (Fig 10A). The defects observed in GALJHD2 strains are similar to that observed for mutants defective in kinetochore integrity such as dam1 mutants [56,93–95]. Our results show that the perturbation of cell cycle dependent ^MeKDam1 affects the structural and functional integrity of kinetochores.

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Fig 10. Overexpression of JHD2 exhibits kinetochore declustering and CIN phenotypes.

(A) Kinetochore declustering phenotype is increased in GALJHD2 strain. Wild-type strain (GFP-Cse4 Spc42-mCherry) carrying pGAL1-URA3 vector (strain# YMB12681) or pGAL1-FLAG-JHD2-URA3 (strain# YMB12682) was grown in selective media with galactose (2%) for 6 hours. Mitotic cells based on the spindle size were examined for kinetochore biorientation, declustering and spindle phenotypes. Percentage of cells in each category are shown. n, number of the cells examined. (B) Overexpression of JHD2 exhibits errors in chromosome segregation. Frequency of CF loss in wild-type with pGAL1-URA3 vector (strain# YMB12467) or pGAL1-6HIS-HA-JHD2-URA3 (strain# YMB12468) was determined as described in Materials and Methods. At least 1000 colonies each from three independent transformants were counted. Average ± SE is shown. **p value < 0.01, Student’s t test. (C) Schematic images showing chromosome segregation errors upon overexpression of JHD2. (D) GALJHD2-induced chromosome loss is mediated by its catalytic activity. Frequency of CF loss in wild-type with pGAL1-URA3 vector (strain# YMB12467), pGAL1-6HIS-HA-JHD2-URA3 (strain# YMB12468) or a catalytically inactive pGAL1-6HIS-HA-jhd2-H427A-URA3 (strain# YMB12469) was determined as described in Materials and Methods. At least 1000 colonies each from three independent transformants were counted. Average ± SE is shown. **p value < 0.01, Student’s t test. (E) Jhd2-mediated demethylation of Dam1 is modulated by its catalytic activity. Wild-type strain expressing HA-tagged Dam1 (Dam1-3HA) from its endogenous promoter carrying pGAL1-URA3 vector (strain# YMB12850), pGAL1-6HIS-HA-JHD2-URA3 (strain# YMB12851) or pGAL1-6HIS-HA-jhd2-H427A-URA3 (strain# YMB12852) was grown at 25°C to LOG phase in selective media with galactose+raffinose (2% each) for 3 hours. Methylated proteins were enriched by IP with anti-methyl lysine antibodies using whole cell extracts and analyzed by western blotting using α-Dam1 antibodies following procedure as in Materials and Methods. IN, input; ^MeKIP, immunoprecipitated samples. (F) Relative enrichment of ^MeKDam1 in GALJHD2 and its catalytic mutant strains. ^MeKDam1 enrichment was calculated from the western blots in (E) as described in Fig 1D. Three biological replicates were done. Average ± SE is shown. ns = statistically not significant.

https://doi.org/10.1371/journal.pgen.1011760.g010

We next examined if GALJHD2 strain exhibits defects in chromosome segregation. This was done by assaying the frequency of loss of a reporter chromosome fragment (CF) in strains with vector or GALJHD2 using a colony color assay as described previously [96]. The frequency of CF loss in GALJHD2 strains was 85.6 ± 1.97% (average±SE), which was substantially higher and significantly different from the vector strain (3.7 ± 0.62%; p-value = < 0.0001; Fig 10B, 10C and S2 Table). The CF loss observed in GALJHD2 strains is similar to the CF loss reported for kinetochore mutants, such as ndc10–1, mcm21∆, and mad1∆ strains [97,98]. To determine if the increased frequency of CF loss is due to demethylase activity of JHD2, we examined CF loss in cells with catalytically inactive mutant of JHD2 (jhd2-H427A) [73,99]. The increased frequency of CF loss was significantly reduced in GALjhd2-H427A strains when compared to GALJHD2 strain (Fig 10D and S2 Table) suggesting that increased frequency of CF loss in GALJHD2 strains is due to its demethylase activity. We note that the frequency of CF loss in GALjhd2-H427A is slightly higher than the vector control, which may be due to chromatin-associated role of Jhd2 independent of its demethylase activity [100]. We next examined the levels of ^MeKDam1 in wild type strain carrying catalytically inactive GALjhd2-H427A, GALJHD2 and vector control. GALJHD2 strains showed significantly reduced levels of ^MeKDam1, whereas no significant reduction in the levels of ^MeKDam1 was observed in strains overexpressing jhd2-H427A when compared to vector alone strain (Fig 10E, 10F and S14). Based on these results, we conclude that GALJHD2 contributes to kinetochore dysfunction and chromosome missegregation.

Yeast media, strains, and plasmids

The yeast strains used in this study were grown in yeast extract peptone dextrose (YPD; 1% yeast extract, 2% Bacto-peptone, 2% glucose) or in synthetic minimal medium supplemented with the required carbon source and amino acids drop out based on the plasmid selection. The demethylation and associated experiments were carried out using plasmid-borne JHD2 expressed from GAL1 promoter (pGALJHD2). All the experiments were performed in three independent biological replicates using identical methodologies. The yeast strains along with their genotypes, and plasmids used in this investigation are listed in Table 2.

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Table 2. List of strains and plasmids used in this study.

https://doi.org/10.1371/journal.pgen.1011760.t002

Detection of methylated lysine in Dam1

The yeast strains were grown at 25ºC in YPD or in selective media to LOG phase with constant shaking (200 rpm) and were synchronized in G1 with α-factor (3 μM, RP01002, GenScript Inc.), S-phase with hydroxyurea (0.2 M HU, H8627, Sigma Aldrich) or G2/M with nocodazole (20 μg/mL NOC, M1404, Sigma Aldrich) for 2 hours. For G1 synchronization-release experiments, yeast strains were grown to LOG phase at 25ºC in YPD, synchronized in G1 with α-factor and released into pheromone-free YPD medium. α-factor was re-added to the culture at 100 min after release to block cells in next G1. Samples for flow cytometry and IP were collected at time points after release from G1. For S-phase synchronization-release experiments, strains were grown in YPD to LOG phase at 25ºC, synchronized in S-phase with HU and released into YPD medium. HU was re-added at 100 min after release to block cells in next S-phase. Samples were collected for flow cytometry and IP at time points after release from the S-phase. Flow cytometry was performed to confirm the cell cycle synchronization using Cell Quest software in a BD FACSymphony flow cytometer (BD Biosciences, Boston, MA). Based on the flow cytometry, nuclear position and bud morphology, cells were categorized as G1, S, metaphase, anaphase, and telophase as described previously [29,63,64] using the Zeiss Axioskop 2 microscope (Carl Zeiss Inc., USA). Moreover, we classified cells based on the position of the nucleus with respect to size of the bud. If the bud size is at least half the size of the mother cell and there is a single nuclear mass, we classify them at metaphase. If, however, the bud size is smaller and there is a single nucleus, we categorize these as S-phase [29,63,64].

For G1 arrest-release experiments with overexpression of JHD2, strains were grown to LOG phase in 1xSC-Ura with 2% glucose at 25ºC. Cells were collected and washed 3-times with 50 mL of sterile water. Cells were used to prepare OD₆₀₀ = 0.35 (final concentration) in 1xSC-Ura with 2% each galactose+raffinose and grown at 25ºC for 2 hours with α-factor to synchronize cells in G1. Cells were collected, washed 3-times with 50 mL of sterile water and released into pheromone free 1xSC-Ura with 2% each galactose+raffinose media at 25ºC. α-factor was re-added to the culture at 180 min after release to block cells in next G1. Samples for flow cytometry and IP were collected at different time points after release from the G1.

Proteins containing methylated lysine were enriched by immunoprecipitation experiments in three independent biological replicates following identical experimental conditions using methyl-lysine specific antibodies (PA5–77770, Thermo Fisher Scientific). Briefly, yeast cell pellets were dissolved in lysis buffer (20 mM Na₂HPO₄, 20 mM NaH₂PO₄, 5 mM tetra-sodium pyrophosphate, 50 mM NaF, 1 mM DTT, 10 mM beta-glycerolphosphate, 2 mM EDTA, 1% NP-40, 5 mM NEthylmaleimide, 1 mM PMSF) with protease inhibitor cocktail (P8215; Sigma Aldrich), and were lysed by bead beating (10 times, 40 sec each time) in a FastPrep-5G system (SKU: 116005500, MP Biomedical). Whole cell protein extracts were clarified by centrifugation (13000 rpm for 5 min), normalized for protein concentration using BioRad DC protein assay to obtain an equal value of 100 based on OD₇₅₀ measurements for each sample. Input samples (1/10 volume of IP) were collected. The normalized whole cell extracts (OD₇₅₀ = 100) were used for affinity purification with 15 μL of magnetic beads (30410, Qiagen) conjugated with methyl-lysine antibodies (~15 μg per sample, PA5–77770, Thermo Fisher Scientific) on a roller with constant rotation at 4°C for 16 hrs. Incubated magnetic bead samples were collected by centrifugation (5000 rpm for 1 min), washed once with 1 mL lysis buffer at room temperature for 10 min on a roller with constant rotation followed by one wash with 1 mL 1x TBST (25 mM Tris pH 8.0, 10 mM NEM, 0.3 M NaCl, 0.1% NP-40) as described above. Proteins bound to ^MeK-magnetic beads were eluted in 50 μL of 1 × Laemmli buffer and used in western blotting. To detect the levels of ^MeKDam1 in the IP and total Dam1 in the input samples, eluted proteins were size-fractionated along with input samples by SDS-PAGE on 4–12% Bis-TRIS SDS-polyacrylamide gels and transferred to nitrocellulose membrane using XCell II Blot Module with 35 volts for 60 min following the manufacturer’s recommendations (Invitrogen Inc.). Blots were blocked in 5% milk at room temperature for 30 min and incubated in 1/1000 dilutions of α-HA antibodies (12CA5, Roche) in 5% milk at 4°C for 16 hours on a rocker platform. Blots were washed three-times at room temperature in 1xTBST for 10 min each time and incubated in 1/7500 dilutions of secondary antibodies (HRP-conjugated α-mouse IgG, Cat# NA931V; Cytiva Life Sciences) for 2 hours. After incubation with secondary antibodies, blots were washed again three-times with 1xTBST for 10 min each time, incubated for 2 min in Super Signal West Pico PLUS reagents (Thermo Scientific) and scanned with BioRad ChemiDoc MP Imager using Optimum Auto Exposure settings that selects for full dynamic range and sensitivity required for quantitative analysis (BioRad Inc). Enrichment of ^MeKDam1 was determined using a semi-quantitative approach. Protein intensity signals from input and ^MeKIP samples were quantified from western blots to determine the enrichment of ^MeKDam1 using Image J version 1.53t software [117]. Protein bands corresponding to Dam1 were selected using rectangle tool (equal size across the samples), lanes were plotted using analyze option, and band area intensities were determined using the Wand (tracing) tool as described in Image J [117]. Relative enrichment of lysine methylation of Dam1 was measured as a ratio of band area intensities from ^MeKDam1 to input Dam1 for each sample, and statistical significance was determined by Student’s t-test.

Immunoprecipitation experiments

Whole cell extracts were prepared by bead beating in a FastPrep-5G bead beating system (SKU: 116005500, MP Biomedical) from yeast cells grown at 25ºC to LOG phase in YPD or in selective medium based on the plasmids used and the cell cycle synchronization (G1 and metaphase). IP experiments were performed with anti-HA (A2095, Sigma Aldrich) and anti-Flag (A2220, Sigma Aldrich) agarose antibodies following the approach as described previously [118] and proteins were eluted using HA peptides (I2149, Sigma Aldrich) or Flag peptides (F3290, Sigma Aldrich). Immunoprecipitated protein samples were analyzed by western blotting as described above. Primary antibodies used were α-Myc (F3165, Sigma Aldrich), α-HA (H6908, Sigma Aldrich), α-FLAG (F7425, Sigma Aldrich), and α-Tub2 [119]. Secondary antibodies were HRP-conjugated α-mouse IgG (NA931V) and HRP-conjugated α-rabbit IgG (NA934V) (Cytiva Life Sciences).

Protein extractions, and expression analysis

Total protein extracts were prepared using the Trichloroacetic Acid (TCA) protein extraction method [97], and the protein levels were quantified with Bio-Rad DC protein quantitation assay (Bio-Rad Laboratories, Hercules, CA). Protein samples were size-separated on 4–12% Bis-TRIS SDS-polyacrylamide gels by SDS-PAGE, transferred to nitrocellulose membrane, and western blot analysis was performed as described above. Primary antibodies used for western blotting were α-HA (H6908, Sigma Aldrich), α-FLAG (F7425, Sigma Aldrich), α-Myc (M4439, Sigma Aldrich), α-Mif2 (a gift from Pam Meluh), α-GFP (11814460001, Roche), α-Histone H3 (Ab1791, Abcam), α-Histone H3-K4^Me3 (39160, Active Motif), α-Ctf19 (a gift from Arshad Desai) α-Dam1 [55] and α-Tub2 [119]. Secondary antibodies used for western blotting were HRP-conjugated α-rabbit IgG (NA934V) and HRP-conjugated α-mouse IgG (NA931V) (Cytiva Life Sciences).

ChIP and qPCR experiments

ChIP was performed in three biological replications using the procedure as described [24,120]. Strains were grown in selective medium at 25˚C to LOG phase and the expression of JHD2 was induced from the GAL1 promoter. Protein-DNA complexes were captured using α-GFP (11814460001, Roche), α-Ctf19 (a gift from Arshad Desai), and α-Mif2 (a gift from Pam Meluh) antibodies following the procedure as described previously [24,120]. ChIP-qPCR was performed in a 7500 Fast Real Time PCR System using Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA) with primers for CEN1, CEN3 and ACT1 (negative control) regions as described [29]. The PCR amplification conditions were 95ºC for 20 sec, followed by 40 cycles of 95ºC for 3 sec, 60ºC for 30 sec. The enrichment was calculated from three biological replicates using the _ΔΔC_T method and is shown as percent input [121].

Cell biology and microscopic assays

A strain in which Cse4 and Spc42 have been tagged with GFP and mCherry, respectively (GFP-Cse4, Spc42-mCherry) carrying vector or GALJHD2 was grown in selective medium and expression of JHD2 was induced by the GAL1 promoter. Cells were imaged at 25°C on an Eclipse Ti wide-field inverted microscope (Nikon) with a 100 × Apo TIRF 1.49 NA objective (Nikon) and Clara charge-coupled device camera (Andor) using Nikon NIS Elements imaging software. All population imaging was performed by switching the channel prior to taking a Z-step. The filter set EGFP/DsRed Dichroic Mirror (86007bs) (480/20 × , 565/25× and 525/40 m, 620/60 m) and ECFP/EYFP/mCherry (89006) (430/24 × , 500/20 × , 572/35 × ; 470/24 m, 535/30 m, 632/60 m) from Chroma Technology, Bellows Falls, Vermont, USA were used following procedure as described previously [122].

Viability and chromosome segregation assays

For viability assays, wild-type and kinetochore mutant strains containing vector (pMB433 GAL1) or GAL1–6HIS-HA-JHD2 (Open Biosystems) were grown selectively in synthetic media at 25°C. Cells concentrations were determined and OD₆₀₀ = 1.0 cells were collected, dissolved in 1 mL of sterile water and 1:5 serial dilution were made by pipetting 40 μL of cell suspension from each row above to the row immediately below carrying 160 μL of sterile water. Growth assays were carried out by spotting 5 μL from each serial dilution representing equal numbers of cells from three independent transformants for each strain on synthetic medium containing 2% glucose or 2% each galactose+raffinose and grown for 3 days at 25°C.

The frequency of chromosome segregation errors was measured by a colony color assay developed utilizing the mutation in the ADE2 gene as described previously [96]. In this assay, we used a strain that carries a centromere 3L containing mini-chromosome fragment (CF), which has been used extensively to measure the frequency of chromosome segregation in budding yeast [96,123,124]. The loss of CF leads to the formation of red-sectored colonies instead of a white color yeast colony when cells are plated on synthetic complete media with limiting adenine (1/5^th). Strains were grown to the LOG phase in medium selecting for the CF and plated on synthetic complete with limiting adenine medium at 25ºC. About 1000 colonies were counted for each strain from three independent transformants. The frequency of CF loss was determined by counting the colonies that showed red-sectors and were normalized to the total number of colonies. Statistical significance was determined by Student’s t-test.

Mass spectrometry assay

The wild-type strain with vector (pMB433 GAL1) or GAL1–6HIS-HA-JHD2 (Open Biosystems) were grown at 25ºC in 1x SC-Ura glucose (2%) medium to LOG phase. Cells were collected, washed with water and inoculated into 1x SC-Ura galactose+raffinose (2% each) medium and grown at 25ºC for 6 hours (~2 generations) to induce the expression of 6HIS-HA-JHD2. Cells were collected and dissolved in IP lysis buffer [118] with protease inhibitors and subjected to bead-beating in a FastPrep-5G bead beating system (SKU: 116005500, MP Biomedical). Whole cell extracts were collected by centrifugation, and affinity purifications were performed with Ni-NTA agarose (30230, Qiagen Inc.) and anti-HA agarose (A2095, Sigma Aldrich) beads at 4°C for 24 hours. Beads were collected and washed sequentially for 5 min at room temperature with the IP lysis buffer (1x) [118], followed by PBS (1x), and HEPES buffer pH 8.0 (1x). The beads were resuspended in 50 mM HEPES, pH 8.0 and heated at 95°C for 5 min to denature the proteins. The samples were digested overnight with 2 ug of trypsin at 37°C. The digested peptide samples were acidified by Formic acid (FA) to a final concentration of 1% and desalted using Pierce peptide desalting columns according to manufacturer’s protocol (Thermo Fisher Scientific). Peptides were eluted from the columns using 50% AcN/ 0.1% FA, vacuum centrifuged to dryness and stored at -80°C until analyzed by mass spectrometry. The dried peptide fractions were reconstituted in 0.1% TFA and subjected to nanoflow liquid chromatography (Thermo Ultimat:M3000RSLC nano LC system, Thermo Fisher Scientific) coupled to an Orbitrap Eclipse mass spectrometer (Themo Scientific, CA). Peptides were separated using a low pH gradient using a 5–50% ACN over 120 minutes in mobile phase containing 0.1% formic acid at 300 nl/min flow rate. Full MS1 scans were performed in the Orbitrap at a resolution of 120,000 with an ion accumulation target set at 4e5 and max IT set at 50 ms over a mass range of 350–1600 m/z. lons with determined charge states between 2 and 5 were selected for MS2 scans in the orbitrap with HCD fragmentation (NCE 30%; maximum injection time 22 ms; AGC 5 × 104) at 15K resolution. Acquired MS/MS spectra ware searched against Yeast proteome fasta (version: Uniprot_yeast_aug2020_reviewed_Canonical fasta) using a SEQUEST and Percolator validator algorithms in the Proteome Discoverer 2.4 software (Thermo Fisher Scientific). The precursor ion tolerance was set at 10 ppm and the fragment ions tolerance was set at 0.02 Da along with methionine oxidation and phosphorylation of serine, threonine, and tyrosine included as dynamic modification. Dynamic modification of lysine residues for acetylation, mono-methylation, di-methylation and tri-methylation was also included. Trypsin was specified as the proteolytic enzyme, with up to 2 missed cleavage sites allowed. Searches used a reverse sequence decoy strategy to control for the false peptide discovery and identification were validated by Percolator software.