(F-225) SPNS1 mediates lysophosphatidylcholine transport that enables cell survival under choline limitation

Introduction:: Lysosomes degrade macromolecules and recycle their nutrient content to support cell function and survival. However, the machineries involved in lysosomal recycling of many nutrients remain to be discovered, with a notable example being choline, an essential metabolite liberated via lipid 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.


Materials and Methods:: PaTu-8988T, A549 cells and NOD.Cg-prkdscid/J mice were used in this study.

An endolysosomal CRISPR-Cas9 screen was performed using A list of lysosomal, endocytic, and autophagy-related genes in addition to a selective set of metabolism and signaling genes (1061 genes), for which 10 targeting sgRNAs at 1,000× library coverage were selected per gene. PaTu-8988T cells were propagated in −choline or +choline medium for 14 doublings and the genomic DNA was isolated and amplified for Illumina NextSeq 550 sequencing.

Lysosome purification by LysoIP was adapted from the method previously described (DOI: dx.doi.org/10.17504/protocols.io.bybjpskn). Briefly, cells were homogenized by glass Douncers and prepared for binding with anti-HA beads in the presence of KPBS. Metabolites and lipids were extracted by 80% methanol containing 500 nM isotope-labeled amino acids and chloroform:methanol at ratio of 2:1 (v/v) containing SPLASH LipodoMix internal standard mix, respectively.

Untargeted metabolomics was performed on an Orbitrap ID-X Tribrid mass spectrometer with an electrospray ionization (ESI) probe. A SeQuant ZIC-pHILIC column was used to carry out hydrophilic interaction chromatography (HILIC) for metabolite separation.

Untargeted lipidomics was performed on an Orbitrap ID-X Tribrid mass spectrometer with a heated ESI probe. An Ascentis Express C18 column was used to carry out lipid separation.


Results, Conclusions, and Discussions:: 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. Last, we establish that LPC efflux by SPNS1 is required for cell survival under choline limitation.



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. SPNS1-transported LPCs and LPEs support phospholipid biosynthesis in the cytosol and become essential when exogenous choline for de novo synthesis of phospholipids is limited.



While we initially set out to identify the elusive phosphocholine transporter, our data provide strong evidence that an active machinery to transport LPC, and potentially the closely related lipid LPE, from lysosomes also exists. Lysophospholipid export from the lysosome prevents phospholipid catabolism to the terminal glycerophosphodiester catabolites, salvaging lysophospholipids intermediates for direct reacylation to various lipid species—a stratagem that is easily rationalized as conserving energy equivalents that would otherwise be consumed in a futile deacylation-reacylation cycle. Moreover, although prior studies have variably suggested SPNS1 involvement in lysosomal transport of carbohydrates or sphingolipids, we find that SPNS1 is a pH-dependent lysosomal transporter of lysophospholipids.

Advancing our understanding of SPNS1 function could ultimately have important implications for human disease. Defects in lysosomal lipid catabolic pathways and lysosomal transporters are known causes of severe, early-onset, and age-related neurodegenerative diseases. Our in vivo xenograft results also provide a promising cancer therapy that combines choline deprivation and SPNS1 antagonization. SPNS1 was additionally found to be a host factor facilitating severe acute respiratory syndrome coronavirus 2 virus entry into lung tissue. SPNS1 could therefore be a candidate therapeutic target in the treatment of viral illnesses.


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