Telomere protein Rap1 is a charge resistant scaffolding protein in chromosomal bouquet formation

Abstract

Background: Chromosomes reorganize in early meiotic prophase to form the so-called telomere bouquet. In fission yeast, telomeres localize to the nuclear periphery via interaction of the telomeric protein Rap1 with the membrane protein Bqt4. During meiotic prophase, the meiotic proteins Bqt1-2 bind Rap1 and tether to the spindle pole body to form the bouquet. Although it is known that this polarized chromosomal arrangement plays a crucial role in meiotic progression, the molecular mechanisms of telomere bouquet regulation are poorly understood.

Results: Here, we detected high levels of Rap1 phospho-modification throughout meiotic prophase, and identified a maximum of 35 phosphorylation sites. Concomitant phosphomimetic mutation of the modification sites suggests that Rap1 hyper-phosphorylation does not directly regulate telomere bouquet formation or dissociation. Despite the negative charge conferred by its highly phosphorylated state, Rap1 maintains interactions with its binding partners. Interestingly, mutations that change the charge of negatively charged residues within the Bqt1-2 binding site of Rap1 abolished the affinity to the Bqt1-2 complex, suggesting that the intrinsic negative charge of Rap1 is crucial for telomere bouquet formation.

Conclusions: Whereas Rap1 hyper-phosphorylation observed in meiotic prophase does not have an apparent role in bouquet formation, the intrinsic negative charge of Rap1 is important for forming interactions with its binding partners. Thus, Rap1 is able to retain bouquet formation under heavily phosphorylated status.

Fig. 1

Rap1 is hyper-phosphorylated in meiosis. a Schematic diagram of meiotic culture synchronization using homozygous diploid cells carrying the temperature-sensitive pat1-114 mutation and the mat-Pc cassette. b Distribution graph of the number of nuclei in meiocytes through meiosis (top left) and images of DAPI-stained cells from indicated fractions (bottom left). FACS analysis shows DNA duplication from 2C to 4C (right). c,d Western blot analysis of Rap1-3xPK from mitotic cycling cells (mit), G1 arrested cells (time 0) and meiotic cell fractions at indicated times. Anti-Cdc2 (CDK) and anti-Cdc13 (Cyclin B) antibodies were used as a loading control and meiosis synchronicity marker, respectively. c Separation of cell extracts on a standard gradient gel. d Separation of phosphorylated Rap1-3xPK on a Phos-tag gel. e Treatment of Rap1-3xPK from the 4.5 hr fraction with lambda-phosphatase and/or phosphatase inhibitors as a control. Note that fast-migrating bands of Rap1 are observed in this case due to the presence of endogenous phosphatases

Fig. 2

Domain organization and schematic of phosphorylation sites of Rap1 protein detected at 3.5 hr and 4.5 hr into meiosis. Phosphorylation sites are highlighted as bars with a colour code (yellow, less than 10%; orange, 10–50%; and red, over 50%). Protein interaction domains are indicated above and the structural domains are shown at the bottom. BRCT, BRCA1 C-terminus domain; Myb domain, Myb-like domain; RCT, Rap1 C-terminus domain

Fig. 3

Rap1 hyper-phosphorylation in meiosis is dispensable for telomere bouquet clustering/dissociation. a Schematic of phosphomimetic and unphosphorylatable cluster mutants of Rap1 created and analyzed in this study. Additional mutation sites introduced in 17E/A (see text) are highlighted in red. b Series of frames from films of meiosis. The SPB and telomeres were observed via endogenously tagged Sid4-mCherry and Taz1-YFP, respectively. Time count starts from the beginning of filming. Scale bar equals 2 μm. Example of defective meiotic SPB is shown in rap1∆. None of the rap1 cluster mutants exhibit defective SPB (examined cell number of indicated strains is more than 20). c Frequency of normal four-spore asci in rap1 phosphomutants. Zygotic asci generated from the indicated genotypes in an h ⁹⁰ (homothallic) background were scored by light microscopy. Two hundred asci per genotype were counted in each experiment. Data represent the average of three experiments. Error bars indicate standard deviations. d,g,h Yeast two-hybrid analysis of the interaction between mutant Rap1 and the (d) Bqt1-2 fusion protein, (g) Poz1 and (h) Bqt4. e Telomere lengths of the rap1 phosphomutants. Telomere Southern blot of genomic DNA digested with EcoRI and hybridized with a telomeric probe. A fragment of the SafeView Nucleic Acid Stain stained gel image at 2.5 kb is shown below the blots as a loading control. f Protein expression levels of the N-terminal PK-tagged mutant Rap1. SPB, spindle pole body

Fig. 4

Negative charge of Rap1 Bqt1/2 binding domain is important for functional telomere bouquet. a Alignment of Rap1 protein sequences from different fission yeast species, highlighting a highly conserved area within Rap1 (performed using Clustal Omega; EMBL-EBI, Cambridge, UK). Sp, Schizosaccharomyces pombe; Scr, S. cryophilus; So, S. octosporus; Sj, S. japonicus. b Morphology of zygotic asci of rap1-DD337AA mutant compared to cells expressing wild-type Rap1. Frequency of normal four-spore asci is shown in Fig. 3b. c Mitotic cells expressing Rap1-YFP or Rap1(DD337AA)-YFP (green in the merged pictures) and Taz1-mCherry (red in the merged pictures). Both wild-type Rap1 and Rap1-DD337AA co-localize with Taz1-mCherry. Scale bar equals 5 μm. d Series of frames from films of meiosis. The SPB, telomeres and chromosomes were observed via endogenously tagged Sid4-mCherry, Rap1-YFP and Hht1-Cerulean, respectively. Time count starts from the beginning of filming. Scale bar equals 2 μm. Top image, wild-type cells; and bottom four images, examples of defective meiosis in rap1-DD337AA. Note that rap1-DD337AA meiosis is reminiscent of rap1∆ meiosis shown in Fig. 3b