Abstract
Serine palmitoyltranferase (SPT) is a pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane proteins termed the ORMDLs. Here we describe a procedure to assay ORMDL dependent lipid inhibition of SPT activity. The assay of SPT activity using radiolabeled L-serine was developed from the procedure established by the Hornemann laboratory. The activity of SPT can also be measured using deuterated L-serine but it requires mass spectrometry, which consumes money, time and instrumentation. The ORMDL dependent lipid inhibition of SPT activity can be studied in both cells and in a cell free system. This assay procedure is applicable to any type of mammalian cell. Here we provide the detailed protocol to measure SPT activity in the presence of either short chain (C8-ceramide) or long chain ceramide (C24-ceramide). One of the greatest advantages of this protocol is the ability to test insoluble long chain ceramides. We accomplished this by generating long chain ceramide through endogenous ceramide synthase by providing exogenous sphingosine and 24:1 acyl CoA in HeLa cell membranes. This SPT assay procedure is simple and easy to perform and does not require sophisticated instruments.
Keywords: Serine palmitoyltranferase, Ceramide synthases, Sphingolipids, ORMDL, Ceramide, Endoplasmic reticulum, Homeostasis
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.
Materials and Reagents
Equipment
Procedure
Data analysis
Recipes
Acknowledgments
This assay protocol is developed from the method of Rutti et al. (2009). This work was supported by funding from National Institute of Health grant RO1HL131340 to B.W.
Competing interests
Authors have no conflicts of interest to disclose
References
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