Uncoupling of nutrient sensing and cell size control by specific defects in ceramide structure

Ceramides are essential structural lipids whose chemical diversity arises from variations in acyl-chain length and sphingoid base modifications, yet how these structural features couple metabolic state to growth regulation remains unclear. In Saccharomyces cerevisiae , the TORC2–Ypk1/2 signaling axis coordinates plasma membrane homeostasis with cellular growth; however, the precise lipid-derived signals that modulate this pathway remain incompletely characterized. Here, we establish that the structural integrity of very-long-chain fatty acid (VLCFA)–containing ceramides is a critical determinant of nutrient-dependent cell size modulation and TORC2 activity. Disruption of VLCFA elongation elevates Ypk1/2 pT662, consistent with increased TORC2 output, in both nutrient conditions. We further discovered that yeast mutants in VLCFA elongation ( elo3 Δ), fail to modulate their cell size in response to nutrient deprivation. To separate VLCFA elongation from ceramide production, we expressed mammalian ceramide synthases with defined acyl-chain preferences. Ceramides with C ₂₄ –C ₂₆ chains (CerS3) supported near-normal nutrient-dependent size modulation, whereas production of C ₂₂ –C ₂₄ ceramides (CerS2) phenocopied elongation mutants, leading to hyperactive TORC2 signaling and defective size regulation. Remarkably, cells producing C ₁₈ ceramides retained size control despite elevated TORC2 activity, revealing that basal signaling and size regulation can be uncoupled. Furthermore, we identify a distinct role for hydroxylation. While sur2 Δ mutants exhibited size regulation defects resembling elongation mutants, they retained normal nutrient-responsive TORC2 signaling. Conversely, scs7 Δ mutants maintained normal size regulation but displayed reduced basal TORC2 activity. This striking uncoupling suggests that while the TORC2 pathway senses acyl chain length, sphingoid base hydroxylation is biophysically required downstream for the execution of cell size remodeling. Our findings demonstrate that distinct structural features of ceramides differentially regulate nutrient sensing, signaling intensity, and the mechanical execution of cell size control.