Drosophila KDEL Receptor Function in the Embryonic Salivary Gland and Epidermis

Abstract

Core components of the secretory pathway have largely been identified and studied in single cell systems such as the budding yeast S. cerevisiae or in mammalian tissue culture. These studies provide details on the molecular functions of the secretory machinery; they fail, however, to provide insight into the role of these proteins in the context of specialized organs of higher eukaryotes. Here, we identify and characterize the first loss-of-function mutations in a KDEL receptor gene from higher eukaryotes. Transcripts from the Drosophila KDEL receptor gene KdelR – formerly known as dmErd2 – are provided maternally and, at later stages, are at elevated levels in several embryonic cell types, including the salivary gland secretory cells, the fat body and the epidermis. We show that, unlike Saccharomyces cerevisiae Erd2 mutants, which are viable, KdelR mutations are early larval lethal, with homozygous mutant animals dying as first instar larvae. KdelR mutants have larval cuticle defects similar to those observed with loss-of-function mutations in other core secretory pathway genes and with mutations in CrebA, which encodes a bZip transcription factor that coordinately upregulates secretory pathway component genes in specialized secretory cell types. Using the salivary gland, we demonstrate a requirement for KdelR in maintaining the ER pool of a subset of soluble resident ER proteins. These studies underscore the utility of the Drosophila salivary gland as a unique system for studying the molecular machinery of the secretory pathway in vivo in a complex eukaryote.

Isolation of secreted proteins from Drosophila ovaries and embryos through in vivo BirA-mediated biotinylation

“…A PCR based approach was used to generate a BirA construct comprising the amino terminal 20 amino acids of the Drosophila secreted serine protease Easter [54, 55] corresponding to its signal peptide, followed by the entirety of the BirA open reading frame, with the addition of the four amino acids lysine-glutamic acid-glutamic acid-leucine (KEEL) at the C-terminus of the fusion protein. The Easter signal peptide has been shown to direct the efficient secretion of heterologous proteins to which it has been fused [56, 57], while the amino acid sequence KEEL is required for correct localization in some Drosophila ER proteins [58, 59]. A DNA fragment that encodes the resulting fusion protein, secBirA, was then introduced into pUAST [60] and pUASp [61], which are P-element based expression vectors that can be expressed under the control of the yeast transcriptional activator Gal4 in somatic and germline-derived tissues of Drosophila , respectively.…”

“…The four amino acids KDEL, or some close variant of this sequence, present at the carboxy terminus of proteins translated into the secretory compartment, is the canonical target of the KDEL receptor, which is responsible for retrieval of ER proteins that have trafficked to the Golgi Apparatus [85, 86]. In Drosophila , the presence of the amino acid sequence KEEL at the carboxy terminus is also a signal for retrieval of proteins from the Golgi [59]. Among ER-localized proteins bearing C-terminal KEEL sequences are Windbeutel [87] and Seele [88, 89], which participate in embryonic patterning and whose functions require that they be expressed in the ovarian follicle cells [58, 87] and in the female germline [88, 89], respectively.…”

Extracellular Mipp1 Activity Confers Migratory Advantage to Epithelial Cells during Collective Migration

“…Western blot for embryos and S2 cells were performed as described (Abrams et al, 2013; Ismat et al, 2013). Samples were separated on an 8% SDS-PAGE gel.…”

Isolation of secreted proteins fromDrosophilaovaries and embryos throughin vivoBirA-mediated biotinylation

“…secBirA, a secreted derivative of the E. coli biotin ligase BirA, carries at its amino terminus the N-terminal 22 amino acids of the Easter protease, including the Easter secretory signal peptide, followed by the full-length BirA protein, with the 4-amino acid sequence KEEL, which has been shown to act as an ER retention signal in Drosophila 56 Transgenic lines bearing the constructs described above were generated by conventional P-element mediate transformation 93 into a strain homozygous for w 1118 , with DNA microinjection carried out at Rainbow Transgenic Flies, Inc.…”

“…The Easter signal peptide has been shown to direct the efficient secretion of heterologous proteins to which it has been fused 53,54 , while the amino acid sequence KEEL is required for correct localization in some Drosophila ER proteins 55,56 . A DNA fragment that encodes the resulting fusion protein, secBirA, was then introduced into pUAST 57 and pUASp 58 , which are Pelement based expression vectors that can be expressed under the control of the yeast transcriptional activator Gal4 in somatic and germline-derived tissues of Drosophila, respectively.…”