Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity

Background

Serine palmitoyltranferase (SPT) is a multi-subunit enzyme that is widely expressed in eukaryotes and some prokaryotes (Hanada et al., 1997; Ikushiro et al., 2001; Hornemann et al., 2007). The first and rate-determining step of the sphingolipid biosynthetic pathway is catalyzed by SPT, producing 3-keto dihydrosphingosine from the condensation of serine and palmitoyl-CoA (Williams et al., 1984; Hanada, 2003). The mammalian SPT complex is composed of two large subunits and one small subunit. The large subunits are termed SPTLC1 and SPTLC2 and the small subunits are termed ssSPT (Nagiec et al., 1994; Lowther et al., 2012). SPT requires all three subunits to form a functional protein complex (Han et al., 2009). In some circumstances mammalian SPT is also composed of another large subunit termed SPTLC3 (Hornemann et al., 2009), which substitutes for SPTLC2. The functional SPT complex is formed by pairing SPTLC1 with either SPTCL2 or SPTLC3 and ssSPT. The active site of SPT resides in the SPTLC2 or SPTLC3 subunit. The substrate preference between the complex incorporating SPTLC2 and SPTLC3 differs. SPTLC2 prefers palmitoyl CoA. SPTLC3 prefers myristoyl-CoA (Hornemann et al., 2009). In certain situations SPT also incorporates alanine or glycine rather than serine into the precursor sphingolipid to generate a deoxysphingolipid base (Gable et al., 2010; Ferreira et al., 2018). SPT forms a stable complex with another ER resident protein termed ORMDL. This SPT-ORMDL interaction is crucial to maintain the homeostatic regulation of de novo biosynthesis of sphingolipids (Siow and Wattenberg, 2012; Davis et al., 2018). There are three closely related ORMDL family members found in mammals (Breslow et al., 2010; Siow and Wattenberg, 2012). We previously showed that increased levels of cellular ceramide inhibit SPT activity in an ORMDL-dependent manner in HeLa cells (Davis et al., 2019). The detailed mechanism behind the homeostatic regulation of SPT activity by the ORMDLs is largely unknown. SPT forms a stable complex with ORMDL, irrespective of cellular levels of ceramide. We propose that ceramides bind to either the ORMDLs or to the SPT-ORMDL complex to trigger the inhibition of SPT activity. We recently developed a cell free system in which membranes were isolated from HeLa cells and used to test the response of SPT activity to elevated sphingolipid. This system measures SPT activity in the presence or absence of ceramides with artificially short and natural chain lengths (Davis et al., 2019). We established the assay of ORMDL dependent lipid inhibition of SPT activity as a platform to test the functional interaction of SPT and ORMDL. This SPT assay procedure was developed from the procedure of Rutti et al. (2009). The cell free in vitro system has many advantageous over in vivo systems and both systems yield similar results (Davis et al., 2018). Using this cell free reconstitution system, we have determined that ORMDL dependent regulation of SPT activity is not mediated by post transcriptional or post translational mechanisms (Davis et al., 2018). This cell free system is a powerful tool to explore the detailed mechanism behind the homeostatic regulation of SPT by ORMDLs.