Analysis of Domain Specific Functions of the Ceramide Synthase Schlank in <em>Drosophila melanogaster</em>

Abstract

Ceramide synthases (CerS) are integral membrane proteins at the center of sphingolipid (SL) biosynthesis. Their enzymatic activity is dependent on the conserved lag1 motif, but from cnidarians onwards many CerS proteins also contain a homeodomain with yet unknown function. Homeodomains are commonly known as DNA-binding domains in transcription factors regulating developmental processes. Previous studies of our group show that the only CerS ortholog in <i>Drosophila melanogaster</i> -Schlank is not only involved in the synthesis of SLs but also in the regulation of body fat. This regulation seems to be independent on the CerS activity. Lately, Schlank was shown to localize in the nucleus in an Importin dependent manner. In this thesis, the functional properties of the Schlank protein were studied in more detail. A genomic engineering strategy where specific mutations were introduced into the <i>schlank</i> sequence in it’s endogenous locus allowed the direct comparison of Schlank variants. This approach could prove that up regulation of <i>lipase 3</i> in <i>schlank</i> deficient larvae is due to the absence of the Schlank protein, and not due to absent ceramide synthesis. Mutations within the homeodomain targeting a putative nuclear localization signal (NLS) caused phenotypes in locomotion, behavior and body fat level that are also known from <i>schlank</i> P-element mutant alleles, further implying a functional relevance of the homeodomain. Mutations in this NLS reduced the relative amount of nuclear Schlank protein showing it’s functionality <i>in vivo</i>, whereas mutations in the other predicted NLS did not reduce nuclear Schlank. To address a potential nuclear function of the homeodomain Schlank transmembrane topology was investigated experimentally. Those experiments strongly suggest a cytosolic and nucleoplasmic orientation of the homeodomain. Previously, a cleavage of the Schlank protein has been proposed. As Schlank fragment bands were detected to a different extend under different conditions a regulated reasonable cleavage of the Schlank protein could be hypothesized. Unexpectedly, the site of this cleavage was localized to an amino acid stretch within the homeodomain. Altogether, with this work we gained more insight into the various roles of the Schlank protein apart from it’s catalytic activity.