Ceramide phosphoethanolamine (CPE), a sphingomyelin analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. In this study, mushroom-derived proteins of the aegerolysin family—pleurotolysin A2 (PlyA2; K(D) = 12 nM), ostreolysin (Oly; K(D) = 1.3 nM), and erylysin A (EryA; K(D) = 1.3 nM)—strongly associated with CPE/cholesterol (Chol)-containing membranes, whereas their low affinity to sphingomyelin/Chol precluded establishment of the binding kinetics. Binding specificity was determined by multilamellar liposome binding assays, supported bilayer assays, and solid-phase studies against a series of neutral and negatively charged lipid classes mixed 1:1 with Chol or phosphatidylcholine. No cross-reactivity was detected with phosphatidylethanolamine. Only PlyA2 also associated with CPE, independent of Chol content (K(D) = 41 μM), rendering it a suitable tool for visualizing CPE in lipid-blotting experiments and biologic samples from sterol auxotrophic organisms. Visualization of CPE enrichment in the CNS of Drosophila larvae (by PlyA2) and in the bloodstream form of the parasite Trypanosoma brucei (by EryA) by fluorescence imaging demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.

We isolated a Drosophila fickle P (fic P ) mutant with a shortened copulatory duration and reduced adult-stage life span. The reduced copulatory duration is ascribable to incomplete fusion of the left and right halves of the apodeme that holds the penis during copulation. fic P is an intronic mutation occurring in the Btk gene, a gene which encodes two forms (type 1 and type 2) of a Bruton's tyrosine kinase (Btk) family cytoplasmic tyrosine kinase as a result of alternative exon usage. The fic P mutation prevents the formation of the type 2 isoform but leaves expression of the type 1 transcript intact. Ubiquitous overexpression of the wild-type cDNA by using a heat shock 70 promoter during the late larval or pupal stages rescued the life span and genital defects in the mutant, respectively, establishing the causal relationship between the fic P phenotypes and the Btk gene mutation. The stage specificity of the rescuing ability suggests that the Btk gene is required for the development of male genitalia and substrates required for adult survival.

Edited by Dennis R. VoelkerThe ⌬9-fatty acid desaturase introduces a double bond at the ⌬9 position of the acyl moiety of acyl-CoA and regulates the cellular levels of unsaturated fatty acids. However, it is unclear how ⌬9-desaturase expression is regulated in response to changes in the levels of fatty acid desaturation. In this study, we found that the degradation of DESAT1, the sole ⌬9-desaturase in the Drosophila cell line S2, was significantly enhanced when the amounts of unsaturated acyl chains of membrane phospholipids were increased by supplementation with unsaturated fatty acids, such as oleic and linoleic acids. In contrast, inhibition of DESAT1 activity remarkably suppressed its degradation. Of note, removal of the DESAT1 N-terminal domain abolished the responsiveness of DESAT1 degradation to the level of fatty acid unsaturation. Further truncation and amino acid replacement analyses revealed that two sequential prolines, the second and third residues of DESAT1, were responsible for the unsaturated fatty acid-dependent degradation. Although degradation of mouse stearoyl-CoA desaturase 1 (SCD1) was unaffected by changes in fatty acid unsaturation, introduction of the N-terminal sequential proline residues into SCD1 conferred responsiveness to unsaturated fatty acid-dependent degradation. Furthermore, we also found that the Ca 2؉ -dependent cysteine protease calpain is involved in the sequential proline-dependent degradation of DESAT1. In light of these findings, we designated the sequential prolines at the second and third positions of DESAT1 as a "di-proline motif," which plays a crucial role in the regulation of ⌬9-desaturase expression in response to changes in the level of cellular unsaturated fatty acids.

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