Ceramide signaling targets the PP2A-like protein phosphatase Sit4p to impair vacuolar function, vesicular trafficking and autophagy in Isc1p deficient cells

• The impact of ceramide signaling on vacuolar function and autophagy was studied.
• Isc1p (sphingomyelinase) deficiency impairs vacuolar structure and functions.
• The isc1Δ cells exhibit defective vesicular trafficking and autophagy.
• These phenotypes are mediated by the ceramide-activated protein phosphatase Sit4p.
• Downregulation of TORC1–Sch9p signaling also suppresses isc1Δ phenotypes.

The vacuoles play important roles in cellular homeostasis and their functions include the digestion of cytoplasmic material and organelles derived from autophagy. Conserved nutrient signaling pathways regulate vacuolar function and autophagy, ensuring normal cell and organismal development and aging. Recent evidence implicates sphingolipids in the modulation of these processes, but the impact of ceramide signaling on vacuolar dynamics and autophagy remains largely unknown. Here, we show that yeast cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase type 2, exhibit vacuolar fragmentation and dysfunctions, namely decreased Pep4p-mediated proteolysis and V-ATPase activity, which impairs vacuolar acidification. Moreover, these phenotypes are suppressed by downregulation of the ceramide-activated protein phosphatase Sit4p. The isc1Δ cells also exhibit defective Cvt and vesicular trafficking in a Sit4p-dependent manner, ultimately contributing to a reduced autophagic flux. Importantly, these phenotypes are also suppressed by downregulation of the nutrient signaling kinase TORC1, which is known to inhibit Sit4p and autophagy, or Sch9p. These results support a model in which Sit4p functions downstream of Isc1p in a TORC1-independent, ceramide-dependent signaling branch that impairs vacuolar function and vesicular trafficking, leading to autophagic defects in yeast.