Structural duality enables a single protein to act as a toxin-antidote pair for meiotic drive

Abstract

In sexual reproduction, selfish genetic elements known as killer meiotic drivers (KMDs) bias inheritance by eliminating gametes that do not carry them. The selective killing behavior of most KMDs can be explained by a toxin-antidote model, where a toxin harms all gametes while an antidote provides resistance to the toxin in carriers. This study investigates whether and how the KMD element tdk1 in the fission yeast Schizosaccharomyces pombe deploys this strategy. Intriguingly, tdk1 relies on a single protein product, Tdk1, for both killing and resistance. We show that Tdk1 exists in a nontoxic tetrameric form during vegetative growth and meiosis but transforms into a distinct toxic form in spores. This toxic form acquires the ability to interact with the histone reader Bdf1 and assembles into supramolecular foci that disrupt mitosis in noncarriers after spore germination. In contrast, Tdk1 synthesized during germination of carrier spores is nontoxic and acts as an antidote, dismantling the preformed toxic Tdk1 assemblies. Replacement of the N-terminal region of Tdk1 with a tetramer-forming peptide reveals its dual roles in imposing an autoinhibited tetrameric conformation and facilitating the assembly of supramolecular foci when autoinhibition is released. Moreover, we successfully reconstituted a functional KMD element by combining a construct that exclusively expresses Tdk1 during meiosis ("toxin-only") with another construct that expresses Tdk1 specifically during germination ("antidote-only"). This work uncovers a remarkable example of a single protein employing structural duality to form a toxin-antidote pair, expanding our understanding of the mechanisms underlying toxin-antidote systems.

Keywords: killer meiotic driver; structural duality; supramolecular assembly; toxin–antidote system.

Fig. 1.

A single protein product of tdk1 is responsible for meiotic drive. (A) Schematic illustrating the toxin–antidote model for KMDs. KMD elements produce a toxin harming all gametes and an antidote that neutralizes the toxin only in carriers. This ensures selective elimination of gametes lacking the KMD element. In the single-gene KMD tdk1, toxic Bdf1-binding Tdk1 foci in spores likely represent the toxin (39). However, in what form the antidote to this toxin may exist remains unknown. (B) Schematic representation of the genomic location of tdk1 in the reference genome, along with the positions of ura4, ade6, and leu1. The 1.8-kb tdk1 fragment used for ectopic integration in (C) is shown at the bottom. (C) Tetrad analyses demonstrating tdk1’s meiotic drive activity at ectopic genomic loci. A 1.8-kb fragment containing the tdk1 coding sequence along with its flanking regions was ectopically integrated in a tdk1Δ strain at either the ade6 or leu1 locus. P values were calculated using Fisher’s exact test, comparing the viability of progeny from two crosses (null hypothesis: equal viability between crosses). (D) Tetrad analyses revealing that a start codon mutation in tdk1 (tdk1-M1A) abolishes both its killing and resistance activities. A schematic illustrating the tetrad results is shown at the bottom. tdk1-M1A and wild-type tdk1 were integrated at the ade6 locus in a tdk1Δ strain. P values were calculated using the exact binomial test, comparing the observed counts of viable progeny with indicated genotypes against the expected Mendelian segregation ratio of 1:1. (E) Immunoblotting (IB) showing a single Tdk1 band in both spores and vegetative cells (Veg.). Histone H3 blotting and Coomassie Brilliant Blue (CBB) staining serve as loading controls. (F) Schematic representation of the Pmug24-tdk1-SCR-T.ADH1 construct. The tdk1 coding sequence is scrambled to alter the nucleotide sequence while keeping the amino acid sequence unchanged (DNA sequence shown in SI Appendix, Fig S2). The expression of tdk1-SCR is controlled by the mug24 promoter (~1 kb upstream of the start codon) and the ADH1 terminator from budding yeast. (G) Tetrad analysis showing that Pmug24-tdk1-SCR-T.ADH1 is a functional KMD. Pmug24-tdk1-SCR-T.ADH1 was integrated at the ade6 locus in a tdk1Δ strain. P value was calculated using the exact binomial test on counts of viable progeny with indicated genotypes. n in (C, D, and G), total number of progeny analyzed.

Fig. 2.

Tdk1 adopts a nontoxic tetrameric conformation in vegetative cells. (A) Fluorescence micrographs showing localization of Tdk1, Tdk1(1–228), and Tdk1C in vegetative cells. mECitrine-tagged proteins were expressed from the PtetO7 promoter (43). DIC, differential interference contrast. (Scale bars, 3 μm.) (B) Cotethering assay showing the recruitment of Tdk1C, but not Tdk1 or Tdk1(1–228), to the nuclear periphery in vegetative cells coexpressing mTur-Bdf1(524–554)-INM under the constitutive Pact1 promoter. mTur, mTurquoise2. (Scale bars, 3 μm.) (C) Mass photometry analysis of Tdk1 purified from vegetative S. pombe cells revealing a predominant tetrameric state. Left panel: histogram displaying particle counts at different molecular masses. The black curve represents a Gaussian fit, with its peak position closely aligning with the calculated molecular weight of a Tdk1 tetramer (175.56 kDa). This molecular weight accounts for the additional 7 C-terminal residues remaining after protease cleavage. Right panel: Coomassie Brilliant Blue (CBB) stained SDS-PAGE gel of purified Tdk1. (D) Schematic representation of the Tdk1* construct used for cryo-EM analysis. To alleviate orientation bias, residues 130–219 within the repeat region of the stalk domain were deleted. The repeat region (residues 120–226) is dispensable for KMD activity (SI Appendix, Fig. S5A). To reduce conformational heterogeneity, mutations were introduced at the trimer–trimer interface (SI Appendix, Fig. S5B). These mutations, A276R and E346A, abolish killing while preserving resistance activity (SI Appendix, Fig. S5C). (E and F) Cryo-EM density map (E) and structural model (F) of Tdk1*, with individual molecules of the Tdk1* tetramer colored differently. (G) Superimposition of the Tdk1* structure (gray) and the Tdk1–Bdf1 complex structure. For clarity, only one Tdk1 trimer unit of the Tdk1–Bdf1 complex is shown. The structures were aligned using the Bdf1-interacting region of one Tdk1 subunit. (H) Close-up of superimposed structures, highlighting clashes between Bdf1 and the Tdk1* tetramer. (I) Schematic diagram illustrating the contrasting behaviors of Tdk1 in spores and vegetative cells.

Fig. 3.

Replacement using a tetramer-forming peptide reveals dual roles of the N-terminal region of Tdk1. (A) Yeast two-hybrid (Y2H) assays showing that a direct CCtet fusion to Tdk1C disrupts its Bdf1-binding ability, unlike a direct CCtri fusion or fusions with a linker. AD and BD represent prey and bait constructs, respectively. −LW denotes the SD/−Leu/−Trp medium; −LWHA denotes the SD/−Leu/−Trp/−His /−Ade medium. A schematic of the AD constructs used for Y2H assays is shown in the Right panel. (B) Fluorescence micrographs showing localization of Tdk1 truncations and Tdk1C fusions with either CCtri or CCtet in vegetative cells. Only the CCtet-linker^NLS-Tdk1 construct forms nuclear foci. (Scale bars, 3 μm.) (C and D) Schematic illustrating the assembly of supramolecular structure by the CCtet-linker^NLS-Tdk1 fusion, but not by the CCtet-Tdk1, CCtri-Tdk1, or CCtri-linker^NLS-Tdk1 fusions. (E) Spot assays showing that among the Tdk1 truncations and fusions presented in (B), only the CCtet-linker^NLS-Tdk1 fusion exhibits toxicity. In (B and E), different Tdk1 forms were expressed from the inducible P41nmt1 promoter (49).

Fig. 4.

Tdk1 forms a toxin in spores. (A) Time-lapse fluorescence microscopy showing the formation of Tdk1 nuclear foci in spores. A diploid strain (pat1-114/pat1-114 ura4+::tdk1-mTurquoise2/ura4+ ade6::Pact1-pus1-mECitrine/ade6) was used for this experiment. tdk1-mTurquoise2 was integrated at the ura4 locus, closely linked to the wild-type tdk1 gene, in a diploid strain homozygous for the temperature-sensitive pat1-114 mutation. This mutation allows for synchronization of meiosis initiation (54, 55). Pus1 serves as a nucleoplasm marker. (Scale bar, 3 μm.) (B) Time-lapse fluorescence imaging showing the persistence of Tdk1 foci after germination in tdk1Δ progeny from a ura4-D18::tdk1-mTurquoise2 × tdk1Δ::ura4 cross. tdk1-mTurquoise2 was integrated at the ura4 locus, closely linked to the wild-type tdk1 gene. In this cross, only tdk1Δ::ura4 progeny can germinate in media lacking uracil. (Scale bar, 3 μm.) (C) Time-lapse fluorescence imaging showing the efficient dissipation of Tdk1 foci during germination in ura4+::tdk1-mTurquoise2 progeny from a ura4+::tdk1-mTurquoise2 × tdk1Δ cross. In this cross, only ura4+::tdk1-mTurquoise2 progeny can germinate in media lacking uracil. (Scale bar, 3 μm.) (D) Quantification of Tdk1 localization patterns in tdk1Δ and ura4::tdk1-mTurquoise2 progeny. Spores from a ura4-D18::tdk1-mTurquoise2 × tdk1Δ::ura4 cross or a ura4+::tdk1-mTurquoise2 × tdk1Δ cross were selectively germinated for 8 h in media lacking uracil. n, number of mitotic cells analyzed.

Fig. 5.

Tdk1 synthesized in germinating spores functions as an antidote. (A) Fluorescence micrographs showing the localization of Tdk1, Tdk1(1–228), and Tdk1C in germinating spores without (Left) or with (Right) mTur-Bdf1(524–554)-INM. mTur-Bdf1(524–554)-INM was expressed under the constitutive Pact1 promoter. Different Tdk1 forms were expressed from the inducible PtetO7 promoter. The induction was achieved by transferring spores to a germination medium containing ahTet. Images were captured after a 6-h incubation. (Scale bars, 3 μm.) (B) Tetrad analyses showing that inducing Tdk1 expression in germinating spores using the PtetO7 promoter prevents Tdk1-mediated progeny killing. P values were calculated using the exact binomial test on counts of viable progeny with indicated genotypes. n, total number of progeny analyzed. (C) Fluorescence micrographs showing colocalization of Tdk1-mECitrine (green pseudocolor), expressed from the PtetO7 promoter during spore germination with preformed Tdk1-mTurquoise2 (magenta pseudocolor) foci. Images were captured 6 h after transferring spores to germination medium containing ahTet but lacking adenine. (Scale bars, 3 μm.) (D and E) Fluorescence imaging showing that Tdk1-mECitrine expressed in germinating spores from the PtetO7 promoter causes the dissolution of preformed Tdk1-mTurquoise2 foci. (Scale bars, 3 μm.) Representative micrographs of spores after 8 h in germination medium are displayed in (D). (E) Quantification of germinated cells with different localization patterns. n, number of germinated cells.

Fig. 6.

Combining meiosis- and germination-expressed Tdk1 reconstitutes a toxin–antidote system. (A) Tetrad analysis showing lethality of all progeny from a tdk1Δ ade6::Pspo4-tdk1 × tdk1Δ cross. n, total number of progeny analyzed. The tetrad result for tdk1Δ/tdk1Δ ade6::tdk1/ade6 was identical to that shown in Fig. 1D. (B) Schematic diagram contrasting the expression patterns of the Pspo4 and Ptdk1 promoters. Pspo4 drives expression specifically during meiosis, whereas Ptdk1 enables expression in both meiotic and vegetative cells. When Tdk1 is expressed under the control of Pspo4, all progeny are indiscriminately killed. In contrast, under the control of its native promoter, Tdk1 selectively kills noncarrier progeny. (C) Schematic representation of the FLEX motif in the tdk1 promoter. A mutated version of this motif, designated as FLEXm, is also depicted. This mutation has been previously shown to abolish Mei4-dependent transcription (59). (D) Tetrad analyses showing that mutating the FLEX motif (P.FLEXm-tdk1) significantly reduces Tdk1’s killing activity while preserving resistance activity. P values were calculated using the exact binomial test on counts of viable progeny with indicated genotypes. n, total number of progeny analyzed. (E and F) Combining meiosis-expressed Tdk1 (Pspo4-tdk1) and germination-expressed Tdk1 (PtetO7-tdk1) reconstitutes a functional toxin–antidote KMD. Six representative tetrads on plates with or without ahTet are shown in (E). (F) Quantifications of progeny viability in the presence or absence of ahTet, the inducer of the PtetO7 promoter. n, total number of progeny analyzed. (G) Fluorescence micrographs showing the localization patterns of Tdk1-mTurquoise2 and Tdk1-mECitrine in germinated spores after a 9-h incubation in rich media (YES) with or without ahTet. Spores were derived from a tdk1Δ ade6::Pspo4-tdk1-mTurquoise2:PtetO7-tdk1-mECitrine × tdk1Δ cross. (Scale bars, 3 μm.) (H) A working model illustrating that structural duality enables Tdk1 to constitute a toxin–antidote pair for meiotic drive. In spores, Tdk1 assumes a toxic conformation (Tdk1 toxin) that interacts with the histone reader Bdf1 and forms supramolecular assemblies (39). These toxic assemblies persist in tdk1Δ progeny, disrupting mitotic chromosome segregation and causing lethality. Conversely, Tdk1 synthesized in tdk1+ progeny during germination adopts a nontoxic conformation (Tdk1 antidote). This form does not bind Bdf1 and dismantles preformed toxic Tdk1 assemblies, protecting tdk1+ progeny from being killed.