DOI: 10.5061/dryad.hqbzkh1mk
title: Lipidomic profiling of SPNS1-KO lysosomes

description: 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 survival under choline limitation. Collectively, our work defines
 a lysosomal phospholipid salvage pathway that is essential under nutrient
 limitation, and more broadly, provides a robust platform to deorphan
 lysosomal gene function.
description: 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 KH<sub>2</sub>PO<sub>4</sub>, 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 500 μL fresh KPBS, and 12.5 μL of the cell suspension was taken for whole-cell fraction analysis. The remaining cell suspension was lysed by trituration using a 29.5-gauge insulin syringe (EXELINT international), then diluted with an additional 500 μL KPBS and centrifuged at 1000xg for 2 minutes at 4°C to pellet cell debris. The lysosome-containing supernatant (1mL) was added to tubes containing an equivalent of 100 μL Pierce anti-HA magnetic beads (Thermo Scientific) and rocked for 3 minutes at 4 °C. Following incubation, the bead-lysosomes complexes were washed 3x with ice cold KPBS and extracted in the appropriate buffer. For lipidomic extraction, lysosome and whole-cell fractions were extracted in 1 ml chloroform:methanol at ratio of 2:1 (v/v) containing 750 ng ml<sup>−1</sup> of SPLASH LIPIDOMIX internal standard mix (Avanti) for &gt; 10 minutes. Following extraction, HA-binding beads were removed using a rotary magnet and LysoIP and whole-cell samples were vortexed for 1 hour at 4°C. 200 µL saline was then added to each sample, and samples were vortexed for an additional 10 minutes. Vortexed samples were spun at 3000xg for 5 min to separate polar (top) and nonpolar (bottom) phases. 600 µL of the lipid-containing chloroform (nonpolar) phase was dried and reconstituted in Acetonitrile:isopropanol:water 13:6:1 (v/v/v). Lipidomic extractions were analyzed on mass spectrometer with workflow and parameters described below. Untargeted lipidomics workflow Profiling of nonpolar lipids was performed on an ID-X Tribrid mass spectrometer (Thermo Fisher Scientific) with a heated electrospray ionization (HESI) probe. An Ascentis Express C18 150 x 2.1 mm column (Millipore Sigma 53825-U) coupled with a 5 x 2.1 mm guard (Sigma-Aldrich 53500-U) was used to carry out C18-based lipid separation prior to mass spectrometry. Mobile phases: A, 10 mM ammonium formate and 0.1% formic acid dissolved in 60% and 40% LC/MS grade water and acetonitrile, respectively; B, 10 mM ammonium formate and 0.1% formic acid dissolved in 90% and 10% LC/MS grade 2-propanol and acetonitrile, respectively. Chromatographic gradient: isocratic elution at 32% B from 0−1.5 minutes; linear increase from 32–45% B from 1.5–4 minutes; linear increase from 45–52% B from 4–5 minutes; linear increase from 52–58% B from 5–8 minutes; linear increase from 58–66% B from 8–11 minutes; linear increase from 66–70% B from 11–14 minutes; linear increase from 70–75% B from 14–18 minutes; linear increase from 75–97% B from 18–21 minutes; hold at 97% B from 21–35 minutes; linear decrease from 97–32% B from 35−35.1 minutes; hold at 32% B from 35.1-40 minutes. Flow rate, 0.26 ml/minutes. Injection volume, 2-4 µL. Column temperature, 55°C. Mass spectrometer parameters: ion transfer tube temperature, 300°C; vaporizer temperature, 375°C; Orbitrap resolution MS1, 120,000, MS2, 30,000; RF lens, 40%; maximum injection time MS1, 50 ms, MS2, 54 ms; AGC target MS1, 4x10<sup>5</sup>, MS2, 5x10<sup>4</sup>; positive ion voltage, 3250 V; negative ion voltage, 3000 V; Aux gas, 10 units; sheath gas, 40 units; sweep gas, 1 unit. HCD fragmentation, stepped 15%, 25%, 35%; data-dependent tandem mass spectrometry (ddMS2) cycle time, 1.5 s; isolation window, 1 m/z; microscans, 1 unit; intensity threshold, 1.0e4; dynamic exclusion time, 2.5 s; isotope exclusion, enable. Full scan mode with ddMS2 at m/z 250–1500 was performed. EASYIC<sup>TM</sup> was used for internal calibration. LipidSearch and Compound Discoverer (Thermo Fisher Scientific) were used for unbiased differential analysis. Lipid annotation was acquired from LipidSearch with the precursor tolerance at 5 ppm and product tolerance at 8 ppm. The mass list was then exported and used in Compound Discoverer for improved alignment and quantitation.  Mass tolerance, 10 ppm; minimum and maximum precursor mass, 0–5,000 Da; retention time limit, 0.1–30 min; Peak filter signal to noise ratio, 1.5; retention time alignment maximum shift, 1 min; minimum peak intensity, 10,000; compound detection signal to noise ratio, 3. Isotope and adduct settings were kept at default values. Gap filling and background filtering were performed by default settings. The MassList Search was customized with 5 ppm mass tolerance and 1 minute retention time tolerance. Area normalization was performed by constant median after blank exclusion.