The auxiliary ESCRT complexes provide robustness to cold in poikilothermic organisms.

The ESCRT pathway, comprising the in sequence acting ESCRT-0, -I, -II, -III and Vps4 complexes, conducts the abscission of membranes away from the cytosol. Whereas the components of the central ESCRT-III core complex have been thoroughly investigated, the function of the components of the associated two auxiliary ESCRT sub-complexes are not well-understood in metazoans, especially at the organismal level. We here present the developmental analysis of the Drosophila orthologs of the auxiliary ESCRTs Chmp5 and Ist1, DChmp5 and DIst1, which belong to the two auxiliary sub-complexes. While each single null mutant displayed mild defects in development, the Dist1 Dchmp5 double mutant displayed a severe defect, indicating that the two genes act synergistically, but in separate pathways. Moreover, the presented results indicate that the auxiliary ESCRTs provide robustness against cold during development of diverse poikilothermic organisms, probably by preventing the accumulation of the ESCRT-III core component Shrub on the endosomal membrane.

Fosmid-based structure-function analysis reveals functionally distinct domains in the cytoplasmic domain of Drosophila crumbs.

The evolutionarily conserved transmembrane protein Crumbs is required for epithelial polarity and morphogenesis in the embryo, control of tissue size in imaginal discs and morphogenesis of photoreceptor cells, and prevents light-dependent retinal degeneration. The small cytoplasmic domain contains two highly conserved regions, a FERM (i.e., protein 4.1/ezrin/radixin/moesin)-binding and a PDZ (i.e., postsynaptic density/discs large/ZO-1)-binding domain. Using a fosmid-based transgenomic approach, we analyzed the role of the two domains during invagination of the tracheae and the salivary glands in the Drosophila embryo. We provide data to show that the PDZ-binding domain is essential for the maintenance of cell polarity in both tissues. In contrast, in embryos expressing a Crumbs protein with an exchange of a conserved Tyrosine residue in the FERM-binding domain to an Alanine, both tissues are internalized, despite some initial defects in apical constriction, phospho-Moesin recruitment, and coordinated invagination movements. However, at later stages these embryos fail to undergo dorsal closure, germ band retraction, and head involution. In addition, frequent defects in tracheal fusion were observed. These results suggest stage and/or tissue specific binding partners. We discuss the power of this fosmid-based system for detailed structure-function analyses in comparison to the UAS/Gal4 system.

The Drosophila Crumbs signal peptide is unusually long and is a substrate for signal peptide peptidase.

N-terminal signal sequences mediate nascent protein targeting to and protein insertion into the membrane of the endoplasmic reticulum. They are typically 15-30 amino acid residues long with a core hydrophobic region flanked by an N-terminal (n-) and a C-terminal region. Following cleavage by signal peptidase, some of the resulting signal peptides are further processed by signal peptide peptidase (SPP) and fragments are liberated into the cytosol. Such fragments can have independent, post-targeting functions affecting diverse cellular processes. We show that Drosophila melanogaster Crumbs, a transmembrane protein controlling cell polarity and morphogenesis, is synthesized with an 83 residues-long signal sequence. To our knowledge, this is currently the longest signal sequence described for an eukaryotic protein. The unusual length is caused by an extended n-region, but the extension does neither affect protein targeting nor signal sequence cleavage. The signal sequence is cleaved off and the resulting signal peptide, SP(Crb), is proteolytically processed by SPP, thus representing the first substrate described for the Drosophila enzyme. We further show that signal peptide fragments can be degraded by the proteasome. Expression of transgenes encoding tagged variants of Crumbs in Drosophila embryos suggests that the signal peptide is short-lived in vivo. Our findings support a model suggesting that besides generating fragments with post-targeting functions, SPP-mediated processing is the first step in the degradation of signal peptides.