Topological and Functional Characterization of the ssSPTs, Small Activating Subunits of Serine Palmitoyltransferase

• Published in: The Journal of biological chemistry Vol. 288; no. 14; pp. 10144 - 10153
• Main Authors: Harmon, Jeffrey M., Bacikova, Dagmar, Gable, Kenneth, Gupta, Sita D., Han, Gongshe, Sengupta, Nivedita, Somashekarappa, Niranjanakumari, Dunn, Teresa M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05.04.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Amino Acids - chemistry; Animals; Cell Membrane - metabolism; Dimerization; Enzyme Activation; Enzyme Mutation; Genes, Fungal; Glycosylation; HSAN1; Humans; Lipids; Lipids - chemistry; Long Chain Bases; Membrane Enzymes; Membrane Lipids; Microsomes - metabolism; Molecular Sequence Data; Mutation; Plasmids - metabolism; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Serine C-Palmitoyltransferase - chemistry; Serine C-Palmitoyltransferase - physiology; Serine Palmitoyltransferase; Sphingolipid; Sphingolipids - chemistry; ssSPTs; Substrate Specificity; Long Chain Bases; Membrane Enzymes; HSAN1; ssSPTs; Sphingolipid; Lipids; Enzyme Mutation; Membrane Lipids; Serine Palmitoyltransferase
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M113.451526

The topological and functional organization of the two isoforms of the small subunits of human serine palmitoyltransferase (hssSPTs) that activate the catalytic hLCB1/hLCB2 heterodimer was investigated. A variety of experimental approaches placed the N termini of the ssSPTs in the cytosol, their C termini in the lumen, and showed that they contain a single transmembrane domain. Deletion analysis revealed that the ability to activate the heterodimer is contained in a conserved 33-amino acid core domain that has the same membrane topology as the full-length protein. In combination with analysis of isoform chimera and site-directed mutagenesis, a single amino acid residue in this core (Met25 in ssSPTa and Val25 in ssSPTb) was identified which confers specificity for palmitoyl- or stearoyl-CoA, respectively, in both yeast and mammalian cells. This same residue also determines which isoform is a better activator of a mutant heterodimer, hLCB1S331F/hLCB2a, which has increased basal SPT activity and decreased amino acid substrate selectivity. This suggests that the role of the ssSPTs is to increase SPT activity without compromising substrate specificity. In addition, the observation that the C-terminal domains of ssSPTa and ssSPTb, which are highly conserved within each subfamily but are the most divergent regions between isoform subfamilies, are not required for activation of the heterodimer or for acyl-CoA selectivity suggests that the ssSPTs have additional roles that remain to be discovered. Background: The ssSPTs activate serine palmitoyltransferase and specify its acyl-CoA selectivity. Results: Both properties are contained within a 33-amino acid core that spans the membrane. Conclusion: A single amino acid difference between ssSPTa and ssSPTb is responsible for the acyl-CoA preference of heterotrimers containing each isoform. Significance: The ssSPTs are critical regulatory components of the rate-limiting enzyme in sphingolipid biosynthesis.