Sterol and Ceramide Glucosides: Cloning of Enzymes contributing to their Biosynthesis in

Sterol glucosides and ceramide glucosides are the predominant (easily extractable) glycolipids in membranes of the nucleocytoplasmic compartment of plant cells. Apart from their contribution to low temperature adaptation, hardly any precise functional implication can be ascribed to these compounds. For a functional assignment via a genetic approach, we have started to clone enzymes involved in their biosynthesis. In this context we have cloned UDP-glucose:sterol glucosyltransferases from various organisms, including plants, fungi and yeasts. Knock-out mutants of microbial organisms had lost the capacity to synthesize these glycolipids, but otherwise did not show any phenotype under normal growth conditions. Efforts in our and other labs have resulted in cloning of most of the enzymes catalyzing the biosynthesis of the predominating species of cerebrosides in plants, which are composed of the ß-glucosyl residue, a 4,8-sphingadienine or phytosphinganine and a N-linked 2-hydroxy-acyl group. The cloned enzymes include the serine palmitoyltransferase, various desaturases and hydroxylases acting on sphinganine and N-acyl moieties as well as ceramide glucosyltransferases. In plants, this glucosyltransferase has been suggested to use sterol glucoside as donor, which can be checked in appropriately engineered yeasts expressing the plant enzyme in the absence of sterol glucoside. Overexpression of ceramide glucosyltransferases from various organisms in yeasts results in the appearance of several new molecular species of cerebrosides normally not found in these hosts. Detailed structural analysis reveals unexpected complications, which necessitate additional assumptions regarding subcellular targetting of enzymes or membrane-bound substrates involved in sphingolipid biosynthesis