Lipidomic profiling of SPNS1-KO lysosomes

Lysosomes degrade macromolecules and recycle their nutrient content to support cell function and survival. Yet, the machineries involved in lysosomal recycling of many nutrients remain to be discovered, with a notable example being choline, an essential metabolite liberated via phospholipid degradation. Here, we engineered metabolic dependency on lysosome-derived choline in pancreatic cancer cells to perform an endolysosome-focused CRISPR-Cas9 screen for genes mediating lysosomal choline recycling. We identified the orphan lysosomal transmembrane protein SPNS1 as critical for cell survival under choline limitation. SPNS1 loss leads to intralysosomal accumulation of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Mechanistically, we reveal that SPNS1 is a proton gradient-dependent transporter of LPC species from the lysosome for their re-esterification into phosphatidylcholine in the cytosol. Finally, we establish that LPC efflux by SPNS1 is required for cell surviv..., Isolation of lysosomes by LysoIP: 
Lysosome purification by LysoIP was adapted from the following protocol: DOI: dx.doi.org/10.17504/protocols.io.bybjpskn) and optimized for the PaTu-8988T cell line. Because we observed better enrichment post-optimization in SPNS1-KO clone #2 compared to clone #1, we used #2 for all main figure LysoIP and tracing experiments. Similar LysoIP results were obtained with the other clone. Briefly, cells were grown to 95% confluence in 15 cm cell culture dishes. If lysosomal isolates were to be used for downstream metabolomics analysis, 4 μM Lysotracker Red DND-99 (Invitrogen) was added to each plate 1 hour prior to cell harvest. At the time of harvest, each plate was washed 2x with 5 mL of ice-cold PBS and cells were scraped in 1 mL ice-cold KPBS (136 mM KCl, 10 mM KH2PO4, pH 7.25 in Optima LC–MS water) and transferred to a 2 mL tube. Cells were pelleted by centrifugation at 1000xg for 2 minutes at 4 °C. Following centrifugation, cells were resuspended in 50...,