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Neutral sphingomyelinase 1 (nSMase1) belongs to the sphingomyelinase enzyme family that hydrolyzes sphingomyelin to produce signaling active lipid ceramide and phosphorylcholine. The molecular characterization and biological function of nSMase1 remain poorly studied. Here, we report that nSMase1 (gene name: SMPD2) knockdown reduces LAMP1 at the mRNA levels and is required for initiating a full-potential unfolded protein response under ER stress. Additionally, SMPD2 KD dramatically reduces the global protein translation rate. We further show that SMPD2 KD cells are arrested in the G1 phase of the cell cycle and that two important cell cycle regulating processes - PI3K/Akt pathway and Wnt signaling pathway are altered. Taken together, we propose a role for nSMase1 in buffering ER stress and modulating cellular fitness via cell cycle regulation.

Protein trafficking in the secretory pathway, for example the secretion of Wnt proteins, requires tight regulation. These ligands activate Wnt signaling pathways and are crucially involved in development and disease. Wnt is transported to the plasma membrane by its cargo receptor Evi, where Wnt/Evi complexes are endocytosed and sorted onto exosomes for long-range secretion. However, the trafficking steps within the endosomal compartment are not fully understood. The promiscuous SNARE Ykt6 folds into an auto-inhibiting conformation in the cytosol, but a portion associates with membranes by its farnesylated and palmitoylated C-terminus. Here, we demonstrate that membrane detachment of Ykt6 is essential for exosomal Wnt secretion. We identified conserved phosphorylation sites within the SNARE domain of Ykt6, which block Ykt6 cycling from the membrane to the cytosol. In Drosophila, Ykt6-RNAi mediated block of Wg secretion is rescued by wildtype but not phosphomimicking Ykt6. The latter accumulates at membranes, while wildtype Ykt6 regulates Wnt trafficking between the plasma membrane and multivesicular bodies. Taken together, we show that a regulatory switch in Ykt6 fine-tunes sorting of Wnts in endosomes.