Ceramidsynthese in Saccharomyces cerevisiae

Episode von Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 01/06 von Ludwig-Maximilians-Universität München · 2. April 2001

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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