Abstract
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 [3H](dihydro)ceramide synthesis from l-[3-3H]serine and the increase was not blocked by the (dihydro)ceramide synthase inhibitor, fumonisin B1 (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.
Highlights
Ceramide is an important intracellular second messenger involved in a number of regulatory processes [1]
We subsequently demonstrated that overexpression of a mammalian homolog of these proteins, upstream of growth and differentiation factor 1 (UOG1), conferred fumonisin B1 (FB1)-resistance in mammalian cells, and unexpectedly, regulated the synthesis of stearoylcontaining sphingolipids (SLs) [14]
[3H]globoside labeling were increased in TRH1- and to a lesser extent in TRH4-overexpressing cells, and not inhibited by FB1. [3H]Ganglioside labeling was much lower in 293T cells than neutral [3H]GSL labeling and only moderately affected by overexpression, and showed FB1 resistance
Summary
Materials—L-[3-3H]serine (specific activity of 26 or 34 Ci/mmol) and 1-[14C]-palmitoyl-CoA (specific activity of 55 mCi/mmol) were from Amersham Biosciences, 1-[14C]-stearoyl-CoA (specific activity of 54 mCi/ mmol) was from American Radiolabeled Chemicals (St. Louis, MO) and
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