Two mammalian longevity assurance gene (LAG1) family members, trh1 and trh4, regulate dihydroceramide synthesis using different fatty acyl-CoA donors

Overexpression of upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene (LAG1), selectively induces the synthesis of stearoyl-containing sphingolipids in mammalian cells ( Venkataraman, K., Riebeling, C., Bodennec, J., Riezman, H., Allegood, J. C., Sullards, M. C., Merrill, A. H. Jr., and Futerman, A. H. (2002) J. Biol. Chem. 277, 35642 - 35649). Gene data base analysis subsequently revealed a new subfamily of proteins containing the Lag1p motif, previously characterized as translocating chain-associating membrane ( TRAM) protein homologs (TRH). We now report that two additional members of this family regulate the synthesis of ( dihydro) ceramides with specific fatty acid(s) when overexpressed in human embryonic kidney 293T cells. TRH1 or TRH4-overexpression elevated [H-3]( dihydro) ceramide synthesis from L-[3-H-3] serine and the increase was not blocked by the ( dihydro) ceramide synthase inhibitor, fumonisin B-1 (FB1). Analysis of sphingolipids by liquid chromatography-electrospray tandem mass spectrometry revealed that TRH4-overexpression elevated mainly palmitic acid-containing sphingolipids whereas TRH1 overexpression increased mainly stearic acid and arachidic acid, which in both cases were further elevated upon incubation with FB1. A similar fatty acid specificity was obtained upon analysis of ( dihydro) ceramide synthase activity in vitro using various fatty acyl-CoA substrates, although in a FB1-sensitive manner. Moreover, in homogenates from TRH4-overexpressing cells, sphinganine, rather than sphingosine was the preferred substrate, whereas no preference was seen in homogenates from TRH1-overexpressing cells. These findings lend support to our hypothesis ( Venkataraman, K., and Futerman, A. H. (2002) FEBS Lett. 528, 3 - 4) that Lag1p family members regulate ( dihydro) ceramide synthases responsible for production of sphingolipids containing different fatty acids.