Lac1-dependent ceramide synthesis couples septum assembly to cell separation in Schizosaccharomyces pombe

Ceramides are precursors of complex sphingolipids and bioactive membrane lipids. In the fission yeast Schizosaccharomyces pombe, they are produced by a ceramide synthase with two catalytic subunits, Lac1 and Lag1, which are redundant but not functionally equivalent: only Lac1 is required for normal growth and cell size. Why Lac1 has this unique role, and how ceramide synthesis contributes to cell morphogenesis, remain unknown. Here, we show that Lac1 couples septum assembly to cell separation. Cells lacking Lac1 die predominantly during cell separation and build septa with a thinner primary layer and markedly thicker secondary layers, accompanied by altered deposition of β-(1,3)-glucans and galactomannan. These defects are not rescued by osmotic stabilization. The essential glucan synthase Bgs4 is mislocalized to cytoplasmic granules, and total β-(1,3)-glucan synthase activity is partially reduced. Nevertheless, Ags1, Bgs1 and Bgs4 accumulate to higher levels and persist almost twice as long at the division site, indicating defective clearance rather than delivery of the septum-synthesis machinery. Plasma membrane sterol distribution remains unaffected. To accurately impair ceramide synthesis, we isolated thermosensitive lac1-ts alleles in a lag1Δ background. Their substitutions cluster in the conserved TRAM/LAG1 domain. Metabolic labeling showed that these alleles reduce ceramide synthesis at the restrictive temperature. Molecular dynamics simulations indicated that two substitutions perturb distinct contacts with C26-CoA, while Lac1-M167T loses its endoplasmic reticulum localization. Acute inactivation recapitulates the deletion phenotype, causing septum accumulation and cell death. Together, our findings establish that Lac1-dependent ceramide synthesis is required for the orderly completion of septum assembly and cell separation.