title: Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals.
abstract: In yeast, Tsc10p catalyzes reduction of 3-ketosphinganine to dihydrosphingosine. In mammals, it has been proposed that this reaction is catalyzed by FVT1, which despite limited homology and a different predicted topology, can replace Tsc10p in yeast. Silencing of FVT1 revealed a direct correlation between FVT1 levels and reductase activity, showing that FVT1 is the principal 3-ketosphinganine reductase in mammalian cells. Localization and topology studies identified an N-terminal membrane-spanning domain in FVT1 (absent in Tsc10p) oriented to place it in the endoplasmic reticulum (ER) lumen. In contrast, protease digestion studies showed that the N terminus of Tsc10p is cytoplasmic. Fusion of the N-terminal domain of FVT1 to green fluorescent protein directed the fusion protein to the ER, demonstrating that it is sufficient for targeting. Although both proteins have two predicted transmembrane domains C-terminal to a cytoplasmic catalytic domain, neither had an identifiable lumenal loop. Nevertheless, both Tsc10p and the residual fragment of FVT1 produced by removal of the N-terminal domain with factor Xa protease behave as integral membrane proteins. In addition to their topological differences, mutation of conserved catalytic residues had different effects on the activities of the two enzymes. Thus, while FVT1 can replace Tsc10p in yeast, there are substantial differences between the two enzymes that may be important for regulation of sphingolipid biosynthesis in higher eukaryotes.
authors.affiliation: Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, MD 20184-4799, USA.
authors.forename: Sita D
authors.initials: SD
authors.lastname: Gupta
authors.forename: Kenneth
authors.initials: K
authors.lastname: Gable
authors.forename: Gongshe
authors.initials: G
authors.lastname: Han
authors.forename: Anna
authors.initials: A
authors.lastname: Borovitskaya
authors.forename: Luke
authors.initials: L
authors.lastname: Selby
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Dunn
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Harmon
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D020134:Catalytic Domain; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006028:Glycosphingolipids / Q000096:biosynthesis*; D006031:Glycosylation; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry / Q000201:enzymology / Q000648:ultrastructure; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D006028:Glycosphingolipids; D008565:Membrane Proteins; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000429:Alcohol Oxidoreductases; C023943:3-ketodihydrosphingosine reductase; C073900:3-ketosphinganine reductase