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. 2005 May;170(1):139-48.
doi: 10.1534/genetics.104.039933. Epub 2005 Feb 16.

Drosophila Signal Peptide Peptidase Is an Essential Protease for Larval Development

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Free PMC article

Drosophila Signal Peptide Peptidase Is an Essential Protease for Larval Development

David J Casso et al. Genetics. .
Free PMC article

Abstract

We identified the Drosophila melanogaster Signal peptide peptidase gene (Spp) that encodes a multipass transmembrane aspartyl protease. Drosophila SPP is homologous to the human signal peptide peptidase (SPP) and is distantly related to the presenilins. We show that, like human SPP, Drosophila SPP can proteolyze a model signal peptide and is sensitive to an SPP protease inhibitor and that it localizes to the endoplasmic reticulum. Expression of Drosophila SPP was first apparent at germ band extension, and in late embryos it was robust in the salivary glands, proventriculus, and tracheae. Flies bearing mutations in conserved residues or carrying deficiencies for the Spp gene had defective tracheae and died as larvae.

Figures

F<sc>igure</sc> 1.—
Figure 1.—
The Drosophila Spp gene. (A) The protein sequence of Drosophila SPP (Dm) aligned with human SPP (Hs) and an SPP family consensus sequence derived from four species [D. melanogaster (CG11840), human (NP_110416), Mus musculus (BAC25752), and Arabidopsis thaliana (NP_565294)] using the program MultAlin (Corpet 1988). Capitalized amino acids indicate absolute conservation among the four species, while lowercase characters represent amino acids conserved in three of the four. Residues mutated in alleles 5, 6, and 7 (red) and those that make up the catalytic core (green) are highlighted. Transmembrane regions (TM1–9) shown in blue are based on predictions by the algorithm TopPred II (Claros and von Heijne 1994). TM5 was predicted for Drosophila and Arabidopsis SPP, but not for the human or the mouse sequences. (B) The Spp locus and neighboring genes are shown. Predicted translational start and stop codons are shown above a diagram of the intron/exon structures. The regions used in two genomic rescue constructs P(W8, Spp+) and P(H-Pelican, lwr+) as well as the sequence deleted in Df(2L)lwr14 are shown.
F<sc>igure</sc> 2.—
Figure 2.—
Immunofluorescence of Schneider-2 cells expressing MYC-tagged Drosophila SPP. Drosophila SPP that was tagged on either the N terminus (top) or the C terminus (bottom) revealed perinuclear and reticular localization. Left, SPP (red); middle, KDEL-receptor-GFP fusion protein (top) and calreticulin-GFP-KDEL fusion protein (bottom), labeled ER + Golgi and ER, respectively (green); right, merged images.
F<sc>igure</sc> 3.—
Figure 3.—
Signal peptide peptidase activity of Drosophila SPP. In this assay, SDS-PAGE resolves the test peptide but cleavage products could not be identified. In the left six lanes, extracts of solubilized crude microsomes from S. cerevisiae expressing either human SPP or Drosophila SPP or carrying the empty expression vector (vector) were added to radiolabeled human HLA signal peptide (top band) in either the presence or the absence of the SPP inhibitor (Z-LL)2-ketone. In the right four lanes, a solubilized extract of canine pancreatic microsomes (pancreatic microsomes) or solubilization buffer (buffer) as a control were assayed with the same substrate and inhibitor. SPP activity is indicated by the (Z-LL)2-ketone-sensitive loss of the HLA signal peptide band.
F<sc>igure</sc> 4.—
Figure 4.—
Spp expression in embryos and larvae. (A–D) Expression in embryos was not detectable at cellularization (A), but at germ band extension was present (B, arrowhead) in the salivary primordium (SP) posterior to the stomodeal invagination (St). Enhanced levels of expression are seen in older embryos adjacent to the segmental involutions (C, arrowheads) and in the salivary glands (SG), proventriculus (PV), and somatic musculature (SM). (E) Expression in the tracheal branches of a late stage embryo. (F) Expression in the salivary gland duct cells (SD) of a third instar larva. Expression in Scr2 mutants (G and I) and their Scr2/+ siblings (H and J). Brown stripes indicate anti-β-galactosidase staining from a balancer chromosome. Light micrograph showing the longitudinal dorsal tracheal trunks of an Spp mutant (K) and wild-type (L) first instar larva. Incomplete tracheal air filling in the tracheal dorsal trunks in the Spp mutant is indicated with an arrow. Anterior is left except in F.
F<sc>igure</sc> 5.—
Figure 5.—
Ectopic expression of Spp in the posterior compartment of the wing. Genotypes were as follows: en-GAL4 alone (A); en-Gal4, UAS-Spp (B); en-Gal4, UAS-Spp, Spp RNAi (C); and en-Gal4, UAS-SppM (D). Numbers refer to the five wing veins.

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