Rap1 is hyper-phosphorylated in meiosis
To investigate the stability of the Rap1 protein throughout meiosis, a homozygous diploid (h
−
/h
−) temperature-sensitive pat1-114 strain carrying a mat-Pc cassette was utilized to synchronize meiosis [16, 17]. Meiosis was induced after nitrogen starvation, followed by a temperature shift from permissive (26°C) to restrictive conditions (34°C) (Fig. 1a). Progression of meiosis was monitored by assessing the number of nuclei and DNA content per cell from fractions collected at 30-minute or 1-hour intervals during the synchronization procedure (Fig. 1b). In order to assess Rap1 protein stability during meiosis, Rap1 was endogenously tagged with PK (V5) epitope peptide and detected by anti-V5 antibodies. Western blotting analysis of synchronous culture extracts showed that Rap1 protein is rather stably expressed during meiosis, although lower molecular weight, potentially truncated forms of Rap1, were observed at the end of meiosis (Fig. 1c, top panel).
Interestingly, a number of distinctly shifted bands of Rap1 were detected during meiotic prophase. Similar shifted bands of Rap1 have also been recently reported [18]. To determine if Rap1 is phosphorylated during meiosis, cell extracts were further analyzed using Phos-tag™ SDS-PAGE [19]. Phos-tag™ gel analysis revealed that the Rap1 protein is highly phosphorylated during meiosis. Strikingly, the maximum level of phosphorylation was observed at 4.5–5 hr, when almost none of the fast-migrating forms of Rap1 were detected (Fig. 1d). Phosphatase treatment confirmed that the shifted bands observed at 4.5–5 hr represented phosphorylated forms of Rap1 (Fig. 1e). Thus, our data indicates that Rap1 phosphorylation accumulates as meiotic prophase progresses, and Rap1 becomes hyper-phosphorylated at the onset of meiosis I, when the bouquet stage ends [5].
Mass spectrometry analysis of Rap1 reveals an increasing number of phosphosites detected upon completion of the bouquet stage
To determine the location of phosphorylation sites in Rap1, meiotic Rap1 was purified from fractions of the synchronized culture at 3.5 hr and 4.5 hr, and was subjected to mass spectrometry analysis. Using trypsin digestion, we covered 70–75% of the Rap1 protein sequence at 95% peptide threshold, and identified 19 and 35 phosphorylation sites from 3.5 hr and 4.5 hr, respectively (Additional file 1). Notably, all phosphorylated sites identified at 3.5 hr were also detected at 4.5 hr, suggesting that Rap1 phosphorylation accumulates with progression of meiotic prophase. Our analysis revealed several meiosis-specific phosphorylation sites in addition to those detected in mitosis-arrested cells [15]. With respect to known protein binding domains of Rap1 [15, 20], the phosphorylated sites at 3.5 hr (early prophase) fell into three clusters, whereas at 4.5 hr phosphosites were fairly evenly distributed across Rap1 (Fig. 2). Interestingly, two and six phosphosites were identified in the Bqt1-2 binding area (311–370 amino acids) at 3.5 hr and 4.5 hr, respectively. Notably, although phosphorylations were detected within the Bqt4 and Poz1 binding regions, we did not detect any phosphorylation within known structural domains of Rap1. Altogether, our mass spectrometry data suggest that the number of phosphorylated residues of Rap1 increases with the progression of meiosis, which is in agreement with our Phos-tag™ gel analysis (Fig. 1d).
Hyper-phosphorylation of Rap1 in meiosis is dispensable for telomere bouquet formation and dissociation
Since we observed that Rap1 phosphorylation peaks at meiosis I, we speculated that the resulting highly negative charge of Rap1 is responsible for the change in its affinity to the Bqt1-2 complex. In order to mimic hyper-phosphorylated Rap1, all validated phosphorylation sites from S-212 to S-562 were substituted with negatively charged glutamate residues (rap1-32E) (Fig. 3a). To monitor telomeres and the SPB through meiosis, endogenous Taz1 and Sid4 were tagged with YFP and mCherry, respectively. To our surprise, the phosphomimetic rap1-32E mutants did not exhibit any detectable meiotic defects and their telomeres clustered and dissociated from the SPB in a timely manner very similar to that of the wild-type (Fig. 3b). Accordingly, rap1-32E mutants exhibited no sporulation defects (Fig. 3c). The corresponding non-phosphorylatable mutant form of Rap1 (rap1-32A) also did not cause defects in meiotic progression and telomere bouquet behaviour (Fig. 3a,b,c). Western blot analysis from meiotic cell extracts confirmed that the mutant forms of Rap1 were stably expressed, and the phospho-modification of Rap1-32A was significantly reduced (Additional file 2). Finally, our yeast two-hybrid assay confirmed that the Bqt1/2 binding domain of Rap1 falls within 216–388 amino acids, and introduced cluster mutations did not affect its interaction with the Bqt1-2 complex (Fig. 3d).
Suspecting that some phospho-modifications might remain unidentified in our study, five additional serine and threonine residues (S-317, T-321, S-322, T-328 and S-364), along with 12 detected phosphosites within and adjacent to the Bqt1/2 binding domain, were all substituted to glutamate (rap1-17E) or alanine (rap1-17A) (Fig. 3a). However, these mutations also did not cause any defects in meiosis (Fig. 3b,c). Thus, we conclude that accumulation of negative charge at the Bqt1-2 binding domain of Rap1 does not affect its ability to form the bouquet.
Because rap1-32A and rap1-32E bear mutations within the binding domain of the telomerase negative regulator Poz1 [21], we checked whether telomere length regulation was impaired in the rap1 phospho-mutants. Since C-terminus tagging of Rap1 impaired telomere length homeostasis (Fig. 3e), the PK epitope tag was fused to the N-terminus. Although phosphomimetic forms of the Rap1 protein (Rap1-32E and 17E) migrate slower than wild-type Rap1, none of the cluster mutations affected protein stability (Fig. 3f). The strains carrying mutant Rap1 maintained their telomere length comparable to that of wild-type (Fig. 3e). Accordingly, all mutants retained their ability to interact with Poz1 by the yeast two-hybrid assay (Fig. 3g). Additionally, both 32A and 32E mutant forms of Rap1 retained the ability to interact with Bqt4 (Fig. 3h). Indeed, telomere localization to the nuclear periphery in interphase was not impaired in rap1-32A and 32E mutants (Additional file 3). Thus, hyper-phosphorylation of Rap1 observed in meiosis does not appear to have a role in telomere bouquet regulation. Furthermore, our mutagenesis analysis suggests that Rap1 is able to resist high negative charge changes without affecting its function in meiosis or telomere length homeostasis.
Intrinsic negative charge of Bqt1/2 binding domain of Rap1 is crucial for telomere bouquet formation
Rap1 protein is negatively charged, and the Bqt1/2 binding region is particularly rich in hydrophobic and negatively charged amino acid residues. Some of these negatively charged residues (D-335, D-337, D-338 and E-342) are well-conserved among fission yeast species (Fig. 4a). Importantly, mutation analysis indicated that Rap1-DD337AA (D337A and D338A mutations) no longer interacts with the Bqt1-2 complex, but retains its ability to interact with Bqt4 and Poz1 in yeast two-hybrid assay (Fig. 3d,g,h).
To study the function of Rap1-DD337AA, endogenous rap1 was mutated and fused to YFP. Accordingly, rap1-DD337AA mutants were defective in sporulation (Figs. 3c and 4b). Live cell imaging of the mutant showed that Rap1-DD337AA localized to telomeres, as determined by co-localization to Taz1 (Fig. 4c), but did not cluster at the SPB in meiotic prophase (Fig. 4d). Furthermore, in many cases the SPB was destabilized and detached from the nucleus and, as a consequence, aberrant chromosome segregation was observed (Fig. 4d). These meiotic phenotypes are characteristic of rap1∆ mutants as well as the bouquet-defective mutants [5]. However, Rap1-DD337AA was stably expressed and telomere length of the rap1-DD337AA mutant was the same as that of wild-type (Fig. 3e,f). Additionally, telomeres of the mutant cells were retained at the nuclear periphery in interphase (Additional file 3). Thus, rap1-DD337AA is a meiosis-specific loss-of-function mutation, and negatively charged aspartates at positions 337 and 338 are crucial for bouquet formation.