title: Topological and functional characterization of the ssSPTs, small activating subunits of serine palmitoyltransferase.
abstract: 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 (Met(25) in ssSPTa and Val(25) 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, hLCB1(S331F)/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.
authors: Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
authors: Jeffrey M
authors: JM
authors: Harmon
authors: Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
authors: Dagmar
authors: D
authors: Bacikova
authors: Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
authors: Kenneth
authors: K
authors: Gable
authors: Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
authors: Sita D
authors: SD
authors: Gupta
authors: Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
authors: Gongshe
authors: G
authors: Han
authors: Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
authors: Nivedita
authors: N
authors: Sengupta
authors: Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
authors: Niranjanakumari
authors: N
authors: Somashekarappa
authors: Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799. Electronic address: tdunn@usuhs.edu.
authors: Teresa M
authors: TM
authors: Dunn
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation; D005800:Genes, Fungal; D006031:Glycosylation; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D051102:Serine C-Palmitoyltransferase / Q000737:chemistry / Q000502:physiology*; D013107:Sphingolipids / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D008055:Lipids; D013107:Sphingolipids; D051102:Serine C-Palmitoyltransferase