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Fss1, a CTNS/PQ domain protein, has dual roles in promoting fertility in Schizosaccharomyces pombe

Andrew J. BestuliD, Jeffrey J. LangeiD, Stephanie H. NowotarskiiD, William B. RedwineiD, Sarah E. ZandersiD

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Abstract

Sexual reproduction is critical in most eukaryotes, but many genes required for the process remain to be identified and characterized. A previous saturation mutagenesis study in Schizosaccharomyces pombe implicated several unnamed genes in having roles in sexual reproduction. In this work, we provide an initial characterization of 13 of these genes and an in-depth characterization of one gene, fss1 (facilitates starvation sex). Fss1 has high structural homology to the human PQ-loop repeat family PQLC2 transceptor (amino acid transporter plus membrane receptor), but likely lacks the ability to transport amino acids due to loss of a critical PQ motif. fss1{Delta} mutants have no defects in meiosis but have pronounced defects in packaging the products of meiosis into spores, particularly in nutrient-poor environments. The spore formation phenotypes are recessive as fss1{Delta} spores produced by a fss1+/fss1{Delta} heterozygote develop normally. Immuno-gold electron microscopy shows Fss1 localizes to vesicles that fuse to the ephemeral outer spore membrane as the spore wall matures below. Fss1 protein is also produced within spores and this population is required for normal vacuole morphology and timely germination. We propose a model in which Fss1 is a vesicle receptor that facilitates spore wall formation from diploid-expressed protein, while spore-expressed protein acts at the vacuole to promote an internal cellular environment that facilitates germination. This work expands our catalog of genes that contribute to sexual reproduction and identifies two previously unknown tasks in spore biology that are both facilitated by Fss1.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (31 Oct 2026) · CC BY 4.0

Fission yeast Fss1, a PQ-loop membrane protein, acts in diploids to build full-thickness, chitin-containing spore walls and within spores to maintain vacuole morphology and timely germination.

Classic yeast screens were not saturating and favoured severe phenotypes, so many genes acting in sexual reproduction remain uncharacterized; a prior genome-wide transposon mutagenesis (TN-seq) in S. pombe flagged unnamed candidates. Individual deletions of 14 of them each impair at least one reproductive step, and fss1 imaging shows a late vesicle-fusion wave onto the outer spore membrane, previously only hypothesized, plus a separate spore-autonomous role.

Key results

  1. ShownFss1 is needed in the diploid, not the spore, for normal spore walls. Spores from fss1Δ homozygous diploids have thinner walls, more often lack nubs, carry less chitin and germinate less often; spores from fss1Δ/fss1+ diploids match wild-type for nubs, chitin staining variability and germination frequency.

    STEM: thinner walls (p<0.0001, t-test, n>30). SEM: more smooth, nub-less spores, most on SPA (p<0.0001, chi-squared, n>400). Calcofluor White (chitin) staining reduced (p<0.0001, t-test); Aniline Blue (β-1-3-glucan) unchanged. More spores failed to germinate within 16 h (n>200). Heterozygotes matched wild-type for smooth spores, Calcofluor White staining variability and germination. · Fss1 is needed in the diploid to form a proper…

  2. ShownFss1 has a second, spore-autonomous role. fss1Δ spores form fused vacuoles, clear a reporter that is degraded in the vacuole less well, and germinate later than fss1+ sister spores from the same heterozygous diploids.

    Cpy1-mCherry: one or two enlarged foci in fss1Δ vs several small foci in wild-type, 2:2 in heterozygote asci. From heterozygotes, fss1Δ spores divided later than fss1+ sisters (p<0.0001, t-test, n>80) and, in asci, kept more Wtf4-GFP (p<0.0001, n>60). Homozygous-mutant spores more often segregated vacuoles (FM4-64) to daughters and divided later (p=0.0012 SPAS, p<0.0001 SPA); heterozygotes intermediate. · fss1Δ mutant spores have altered vacuole…

  3. Shownfss1Δ diploids mate, fuse nuclei and complete meiosis normally. The defect begins at forespore membrane and spore formation, lowers viable spore yield recessively, is strongest on unsupplemented SPA medium and persists in a prototrophic background.

    Prototrophic fss1Δ viable spore yield fell on SPA and SPAS (p<0.005, t-test); heterozygotes did not (p>0.05). Failure to complete meiosis II not significantly higher (n=90 WT, n=88 fss1Δ); chromosome 3 disomy unchanged (n=232). Atypical asci by Isp3-GFP rose (p<0.0001, chi-squared, n>200; heterozygotes wild-type), as did abnormal mCherry-Psy1 forespore membranes (p<0.0001, n>50). · Fss1 promotes sexual reproduction, particularly…; fss1Δ diploids exhibit defects in spore…

  4. SuggestedFss1 appears to travel in cytoplasmic vesicles that fuse with the outer spore membrane after the forespore membrane closes, while the wall matures beneath it. This points to a late vesicle-fusion step in spore maturation. A separate Fss1 pool appears inside mature spores.

    Live imaging of 41nmt1-GFP-fss1 (functional but not fully wild-type): diffuse after karyogamy, then at the spore perimeter after meiosis II, brightening with maturation. Immunogold EM: cytoplasmic clusters of 2-4 particles, masses fusing with the outer spore membrane, clusters in outer spore layers, particles inside spores; secondary-only and untagged controls had very few particles. · The Fss1 protein localizes to cytoplasmic…

  5. SuggestedDeletions of 14 unnamed TN-seq hits each gave a mild-to-moderate defect in at least one reproductive assay. One, SPBC1604.03c (named sdg2), also has a density-dependent growth defect like sdg1Δ that may partly explain its screen signal.

    Most common defect, in 11 mutants: lower viable spore yield on SPA. SPBC530.11c and tlc4 reduced mating on SPA; fss1, dcw1, sdg2 and ank1 increased atypical asci. sdg2Δ often failed to grow at low density, partly rescued by conditioned media. No mutant grew markedly slowly on rich media. Table S1 p-values are uncorrected for multiple tests. · Survey of 14 candidate sexual reproduction…; section 26

Methods, data and reproducibility

Deletions of 14 unnamed TN-seq hits were scored for mating, sporulation, ascus morphology, germination and viable spore yield on two sporulation media (SPA; nutrient-supplemented SPAS). fss1 was then studied by live imaging, immunogold and other electron microscopy, cell wall dyes and heterozygote crosses separating diploid from spore genotype effects.

  • Complete coding-sequence deletions of 14 candidate genes from a prior TN-seq screen, made in an h90 leu1-32 ura4-D18 background · Schizosaccharomyces pombe · n = 14 genes; viable spore yield from 6 experiments for each of two transformants
  • Prototrophic wild-type, fss1Δ homozygous and fss1Δ/fss1+ heterozygous diploids, plus leu1-32 wild-type and fss1Δ diploids, assayed for viable spore yield · Schizosaccharomyces pombe · n = 6 assays per diploid and medium
  • Meiotic time-lapse of hht1-mRFP homozygous diploids on SPAS · Schizosaccharomyces pombe · n = 90 (wild-type), 88 (fss1Δ)
  • Chromosome 3 disomy assay among viable spores using ade6 markers · Schizosaccharomyces pombe · n = 232 per cross
  • Electron microscopy of spores: STEM wall thickness and SEM surface morphology · Schizosaccharomyces pombe · n = n>30 (STEM); n>400 (SEM)
  • Spore germination time-lapse and vacuole imaging (Cpy1-mCherry, FM4-64, Wtf4-GFP) · Schizosaccharomyces pombe · n = n>200 germination; n>110 vacuole foci; n>80 and n>60 heterozygote assays
  • Raw data and images deposited in the Stowers Original Data Repository · Schizosaccharomyces pombe · LIBPB-2656
Methods & tools
TN-seq (transposon mutagenesis with sequencing), AlphaFold, HCOP, Pombe Deletion Collection (Bioneer), pFA6a-KanMX6-P41nmt1-GFP (Addgene #39290), ImageJ, MicroManager, Immunogold scanning transmission electron microscopy, Scanning electron microscopy, Calcofluor White and Aniline Blue staining, FM4-64, Fluorescent markers hht1-mRFP, Isp3-GFP, mCherry-Psy1, Cpy1-mCherry, Wtf4-GFP
Reproducibility gaps
No code is linked. Germination in the 14-gene survey was scored computationally (loss of circularity), but no software is named for it. ImageJ and MicroManager are named without versions. Strain and oligo lists are in supplementary Tables S2 and S3.
Limitations (6)
  • The 41nmt1-driven GFP-Fss1 allele used for localization is functional but not fully wild-type: its viable spore yield on SPA lies between wild-type and fss1Δ. · stated by the authors
  • Fss1-bearing vesicle membranes were not clearly seen before fusion. At that stage the vesicle interpretation rests on predicted transmembrane domains and clustering of gold particles. · stated by the authors
  • The most malformed spores from fss1Δ diploids were probably lost during sample processing for electron microscopy and germination imaging, so those assays may understate the defect. · stated by the authors
  • How Fss1 acts in spores is not identified. The vacuole defect could be a direct function or a marker of cell stress, and the receptor models are speculative. · stated by the authors
  • In the 14-gene survey, significance is not corrected for multiple tests, and the sdg2Δ reproductive defects may stem from its low-density growth defect rather than a direct role. · stated by the authors
  • Loss of transport activity and the proposed receptor role rest on AlphaFold models and the conserved P-to-L substitution; no transport, binding or vesicle-docking assay is reported. · noted in this summary

Read this if you study fungal spore wall assembly, vacuoles in germination or PQ-loop and cystinosin-family membrane proteins, or want phenotypes for poorly characterized S. pombe sexual reproduction genes.

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