Ceramidsynthese in Saccharomyces cerevisiae
The work presented in this dissertation gave the following results: (i) Sequence homologous of the membrane proteins Lag1p and Dgt1p exist all over the kingdom of eucaryotes. All of them share a highly conserved motif with two hydrophilic residues within a transmembrane domain. A limited proteolytic digestion showed, that the N-terminus of Dgt1p is orientated to the cytosolic side. Together with the in silicio analysis, the proteins of the LAG family have six transmembrane domains, a cytosolic C-terminus and an ER lumenal loop of ca. 45 amino acids. (ii) Different mutants of Dgt1p showed, that the C-terminus is essential for the function of the protein. The soluble N-terminus or its dilysine motif, normally mediating retrieval of ER resident proteins that escaped to the Golgi, have not obvious importance for Dgt1p. (iii) Lag1p and Dgt1p fulfill their function in the membrane of the ER and are no constitutive parts of COPII transport vesicles. Moreover, both proteins interact with each other and therefore appear to form a heteropolymeric complex in the ER membrane. (iv) The synthesis rates of complex sphingolipids are drastically reduced in the absence of LAG1 and DGT1. Consequently, lag1Ddgt1D cells have a decreased portion of sphingolipids compared to the wild type. The lipid defekt is not due to lacking phosphoinositolation of ceramide, since the catalytic activity of the corresponding enzyme was proven in vivo.(v) The synthesis block in lag1Ddgt1D cells leads to an accumulation of sphingoid bases. In this connection it was shown, that Lag1p and Dgt1p are essential for the incorporation of exogenous dihydrosphingosine into ceramide, whereas in their absence endogenous dihydrosphingosine is converted to ceramide with a merely decreased rate. The elongation of fatty acids to C26 is not reduced in lag1Ddgt1D cells. Lipid extracts of fumonisin B1 treated wild type cells resemble that of the double mutant, whereas the latter is not influenced by a toxin based inhibition of ceramide synthase. Overexpression of LAG1 and DGT1 does not lead to an overproduction of ceramide or sphingolipids. However, a direct in vitro assay for ceramide synthase activity finally proved the essential requirement of the reaction for Lag1p and Dgt1p. (vi) The overproduction of the ceramidases Ypc1p and Ydc1p in lag1Ddgt1D cells increases the synthesis rates as well as the effective amounts of complex sphingolipids. The defective incorporation of exogenous dihydrosphingosine into ceramide was, however, not reverted. The additional deletion of YPC1 and YDC1 in the absence of LAG1 and DGT1 decreased the amounts of inositol containing sphingolipids beyond detectable levels and, therefore, confirmed the essential requirement of Lag1p and Dgt1p for the ceramide synthase reaction. (vii) The survival of lag1Ddgt1D cells compared to wild type cells after short heat shock is drastically impaired. Moreover, the ability of yeast to form single cells on agar plates directly contributes to the temperature and the amount of ceramide available. As a consequence of the data obtained, the proteins Lag1p and Dgt1p are the first to be characterized as essential parts of the ceramide synthase reaction.
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