The homeodomain of the <em>Drosophila</em> Ceramide Synthase Schlank confers nuclear import information and DNA binding capabilities

Abstract

Ceramide Synthases (CerS) are membrane bound enzymes at the center of the sphingolipid synthesis cascade. A sub-class of CerS also codes for a homeodomain of unknown function.<br /> We previously identified the single<i>Drosophila</i> Ceramide Synthase Schlank (type II CerS) as essential regulator of fat stores in the larval fat body by modifying the transcription of two key regulators of lipolysis, <i>lipase 3</i> (<i>lip3</i>) and <i>adipokinetic hormone</i> (<i>akh</i>). Data on a point mutation that disables CerS enzymatic activity suggested that these transcriptional regulations would be independent of the catalytic activity but rather depend on another activity of the protein.<br /> To assess a potential role of the protein in transcriptional regulation, gene and protein expression patterns and subcellular localizations were determined. While the majority of the protein is localized in cytoplasmic organelles in neuronal and gut cells, there is strong enrichment of Schlank in fat body cells. In fat body nuclei, Schlank is present at type I indentations of the nuclear envelope, which form tubular structures in the nucleoplasm. As a transmembrane protein, Schlank is thought to remain within the inner nuclear membrane part of those indentations. Protein structure predictions show that the homeodomain would face the cytosol and nucleosol, respectively. Motif recognition algorithms predicted the presence of two nuclear localization sequences (NLS) within the homeodomain. N-terminal eGFP fusion constructs were used to verify the functionality of nuclear targeting sequences in the homeodomain and deletion constructs of the NLS proved them to be essential for nuclear import. NLS sequences are known to be bound by the Importin family of nuclear importers. Most nuclear import mechanisms rely on the ß-Importin variant. Using a clonal UAS-GAL4-based RNAi system it was shown that Schlank is imported to the nucleus via a Importin-ß-dependent mechanism.<br /> To assess a potential nuclear function of Schlank, properties of the homeodomain were studied in great detail – phylogenetic analysis showed that it was taken up early during evolution and remains conserved on the structural level to classical homeodomains. A solid-phase DNA-binding assay with randomized DNA and electromobility shift assays with<i>lip3</i> and <i>akh</i> promoter fragments containing the previously identified Schlank consensus binding site proved that the Schlank homeodomain is capable of binding DNA.<br /> Thus, the data on the Ceramide Synthase Schlank summarized in this thesis challenge two central dogmas in current Ceramide Synthase research: 1) Ceramide Synthases are not confined to the endoplasmic reticulum, as previously thought. 2) Ceramide Synthase homeodomains are not degenerate in structure and keep their DNA binding potential, a notion that is strongly debated in literature.<br /> In light of these data, it seems necessary for the research field in general to step back from the sphingolipid-centered viewpoint and reach a more balanced standpoint. Functions of Ceramide Synthases themselves independent of the mere enzymatic activity have to be considered. Both aspects and potential links between them should be addressed in the future.