FVT-1 is a mammalian 3-ketodihydrosphingosine reductase with an active site that faces the cytosolic side of the endoplasmic reticulum membrane

Sphingolipids are essential membrane components of eukaryotic cells. Their synthesis is initiated with the condensation of L-serine with palmitoyl-CoA, producing 3-ketodihydrosphingosine (KDS), followed by a reduction to dihydrosphingosine by KDS reductase. Until now, only yeast TSC10 has been identified as a KDS reductase gene. Here, we provide evidence that the human FVT-1 (hFVT-1) and mouse FVT-1 (mFVT-1) are functional mammalian KDS reductases. The forced expression of hFVT-1 or mFVT-1 in TSC10-null yeast cells suppressed growth defects, and hFVT-1 overproduced in cultured cells exhibited KDS reductase activity in vitro. Moreover, purified recombinant hFVT-1 protein exhibited NADPH-dependent KDS reductase activity. The identification of the FVT-1 genes enabled us to characterize the mammalian KDS reductase at the molecular level. Northern blot analyses demonstrated that both hFVT-1 and mFVT-1 mRNAs are ubiquitously expressed, suggesting that FVT-1 is a major KDS reductase. We also found the presence of hFVT-1 variants, which were differentially expressed among tissues. Immunofluorescence microscopic analysis revealed that hFVT-1 is localized at the endoplasmic reticulum. Moreover, a proteinase K digestion assay revealed that the large hydrophilic domain of hFVT-1, which contains putative active site residues, faces the cytosol. These results suggest that KDS is converted to dihydrosphingosine in the cytosolic side of the endoplasmic reticulum membrane. Moreover, the topology studies provide insight into the spatial organization of the sphingolipid biosynthetic pathway.