Tsc10p and FVT1: topologically distinct short-chain reductases required for long-chain base synthesis in yeast and mammals

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.     

Vegetation, landscape and human activity in Midland Ireland: mire and lake records from the Lough Kinale-Derragh Lough area, Central Ireland

A high-resolution pollen record for the Holocene has been obtained from Derragh Bog, a small raised mire located on a peninsula in Lough Kinale-Derragh Lough, in Central Ireland as part of the Discovery Programme (Ireland) Lake Settlements Project. The data are compared with two lower resolution diagrams, one obtained from Derragh Lough and one from adjacent to a crannog in Lough Kinale. The general trends of vegetation change are similar and indicate that landscape-scale clearance did not occur until the Medieval period (ca. a.d. 800–900). There are, however, significant differences between the diagrams due primarily to core location and taphonomy, including pollen source area. Only the pollen profile from Derragh Bog reveals an unusually well represented multi-phase primary decline in Ulmus ca. 3500–3100 b.c. (4800–4750¹⁴C b.p.) which is associated with the first arable farming in the area. The pollen diagram indicates a rapid, and almost complete, clearance of a stand of Ulmus with some Quercus on the Derragh peninsula, arable cultivation in the clearing and then abandonment by mobile/shifting late Neolithic farmers. Subsequently there are a number of clearance phases which allow the colonisation of the area by Fraxinus and are probably associated with pastoral activity. The pollen sequence from adjacent to a crannog in Lough Kinale shows clear evidence of the construction and use of the crannog for the storage of crops (Hordeum and Avena) whereas the Derragh Bog diagram and the diagram from Derragh Lough reflect the growth of the mire. This study reveals that in this landscape the record from a small mire shows changes in prehistoric vegetation caused by human agriculture that are not detectable in the lake sequences. Although in part this is due to the higher temporal resolution and more consistent and complete chronology for the mire, the most important factor is the closer proximity of the raised mire sequence to the dry land. However, the pollen sequence from adjacent to a crannog does provide detailed evidence of the construction and function of the site. It is concluded that in order to ascertain a complete picture of vegetation changes in a lowland shallow lake-dominated landscape, cores from both the lake and surrounding small mires should be analysed.