CRISPR/Cas9 screen for essential endolysosomal genes under free choline limitation

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.