Forward genetic screens with small molecules (SMs) in C. elegans carried out in our and other laboratories have repeatedly shown that some SMs aggregate in the anterior pharynx of worms as dark objects. Our group first identified the accumulation as at least partly, if not wholly composed of SM itself manifest as birefringent crystals and/or globular spherical objects. The crystallizing molecules are all lethal to worms. Our data indicates that the process of SM crystallization begins in the pharyngeal cuticle of the anterior pharynx, and over time, extends to the plasma membrane, ultimately causing extensive cellular damage. The crystallizing molecules can be grouped into 24 distinct structurally similar families (called scaffolds), and tend to be generally more hydrophobic and polar than molecules that do not aggregate. To understand the molecular basis of crystal accumulation, we screened 1.3 million randomly mutagenized wild-type F2 genomes and isolated 46 mutants that resist the lethal effects of a crystallizing SM,
wact-190. This uncovered loss or reduction-of-function mutations in genes encoding components of a putative sphingomyelin synthesis pathway, SMS-5, TTM-5 and SPTL-2. Additionally, loss or reduction-of-function mutations in the ABC transporter PGP-14 also confer robust resistance to all crystallizing SMs.
sms-5 and
pgp-14 are moderately and highly expressed, respectively, in pharyngeal cells that line the pharynx cuticle. Both
sms-5 and
pgp-14 mutants show reduced accumulation of the hydrophobic, crystallizing SMs. Interestingly, both mutants show higher accumulation and sensitivity to a different set of lethal SMs with different physical properties relative to the wildtype. Together, these results indicate that (1) the anterior pharyngeal cuticle may be an important route of SM entry and a "sink" for many hydrophobic molecules, and (2) loss of SMS-5 or PGP-14 activity leads to altered barrier properties. Remarkably,
sms-5 mutants are hypersensitive to cholesterol-deprived conditions, suggesting
sms-5 is specifically required for accumulation of hydrophobic nutrients and other SMs in the pharynx cuticle and in turn into pharyngeal cells. PGP-14 maybe required for transporting a hydrophobic molecule related to SMS-5-catalyzed polar lipid synthesis into the developing cuticle. Taken together, our data suggest that SMS-5 and PGP-14 contribute to a lipid barrier in the anterior pharynx cuticle to limit access of small molecules to the animal.