Expression and characterization of Drosophila signal peptide peptidase-like (sppL), a gene that encodes an intramembrane protease

Abstract

Intramembrane proteases of the Signal Peptide Peptidase (SPP) family play important roles in developmental, metabolic and signaling pathways. Although vertebrates have one SPP and four SPP-like (SPPL) genes, we found that insect genomes encode one Spp and one SppL. Characterization of the Drosophila sppL gene revealed that the predicted SppL protein is a highly conserved structural homolog of the vertebrate SPPL3 proteases, with a predicted nine-transmembrane topology, an active site containing aspartyl residues within a transmembrane region, and a carboxy-terminal PAL domain. SppL protein localized to both the Golgi and ER. Whereas spp is an essential gene that is required during early larval stages and whereas spp loss-of-function reduced the unfolded protein response (UPR), sppL loss of function had no apparent phenotype. This was unexpected given that genetic knockdown phenotypes in other organisms suggested significant roles for Spp-related proteases.

Figure 1. Sequence similarity of the SppL protein to D. melanogaster Spp and human SPPL3.

Three sequences are shown: Drosophila Spp (Dm Spp), Drosophila SppL (Dm SppL), and human SPPL3 (Hs SPPL3). Homologies between Dm Spp and Dm SppL, and between presumptive Dm SppL and Hs SPPL3 are indicated: for identity, by a letter; or for similarity, by a colon. Predicted transmembrane domains are highlighted in blue boxes and numbered TM1-TM9. The catalytic regions including the aspartyl diad and PAL motif are shown in red boxes. Dashed lines (—) indicate the extent of the sppL^24J and sppL^57D deletions.

Figure 2. Sequence comparisons of Spp and SppL proteins.

Pair-wise comparisons of amino acid identity (%) are plotted for each of the nine predicted transmembrane (TM) domains. Comparisons between Drosophila SppL and Drosophila Spp are in blue; comparisons of Drosophila SppL and human SPPL3 are in red. Whereas strong identity exists between Drosophila SppL and human SPPL3 in all transmembrane domains (red), the region of strong identity between Spp and SppL (blue) is limited to the C-terminal four transmembrane domains (TM6-TM9) that include the catalytic domains .

Figure 3. Phylogram of Spp and SppL ortholog sequences.

Sequences are marked with an abbreviation for the species (i.e., D. melanogaster SppL, Dmel-SppL; see Table 1 for a list of species); accession identifiers for each sequence are in Table 1 or in METHODS. The magenta box groups the Drosophila Spp orthologs; light pink, other insect Spp orthologs; white, human SPP, SPPL2a, SPPL2b, SPPL2c, SPPL3; blue, Drosophila SppL orthologs; and light blue, other insect SppL orthologs. SppL proteins are more closely related to human SPPL3 than to SPP or SPPL2a, b, or c; and the SppL sequences retain a higher interspecies conservation than Spp sequences. (The apparently truncated sequence of the D. virilis SppL ortholog is not included in this analysis.).

Figure 4. Expression of sppL in Drosophila embryos.

(A) A uniform distribution of sppL transcripts was detected near the surface of embryos by in situ hybridization at the cellular blastoderm stage. (B) At early germ band extension (stage 7), mesodermal expression is apparent. (C) At late germ band extension (stage 9), strong expression of sppL is seen in the developing midgut. (D) At germ band retraction (stage 12) and (E) dorsal closure (stage 14), midgut expression remains strong, while mesodermal expression is beginning to fade. (F) By late embryogenesis (stage 16), expression of sppL is no longer detectable.

Figure 5. SppL localization to the Golgi and ER.

S2 cells were transfected two express either (A–C) MYC-SppL and Crc-GFP-KDEL marking ER, or (D–F) MYC-SppL and KDEL Receptor-GFP marking Golgi and ER. (C, F) In the merged images, MYC-SppL is in red the GFP fusion proteins are in green. Hoechst staining of nuclei is included in blue (note that only two cells in each frame were transfected). While some colocalization of SppL and the ER marker can be seen in C, extensive colocalization with the ER and Golgi marker is evident in F.

Figure 6. The sppL locus.

This cartoon of 9.5 kb of chromosome III at cytological band 96F5-6 depicts the sppL gene and the ends of the adjacent Tsp96 (pink) and Lnk (blue) genes. Colored boxes indicate the sppL exon structure: coding regions (green) and non-coding 5′ and 3′ UTRs (yellow). The predicted “start” and “stop” codons of sppL are indicated. Exons N1-N3 are entirely non-coding, while exons C1–C6 contain the sppL open reading frame. The insertion sites of transposons P{lacW}sppL^SH116 (also known as P{lacW}l(3)SH116^sh116), PBac{XP}Lnk^d07478, and PBac{RB}CG17370^e00372 are indicated with red triangles. Imprecise excision of P{lacW}sppL^SH116 generated the deletion alleles sppL^24J and sppL^57D. Recombination between the two PBac insertions was used to generate deletion Df(3R)sppL. The extent of these deletions is indicated within parentheses. The sppL^57D deletion (not shown) is similar to sppL^24J. Black triangles indicate the positions of proximal (▸) and distal (◂) primers used to screen for excision mutants, denoting the positions of the following oligo sites: osppL-2000s, osppL-4000a, osppL-5000a, osppL-6000a, and osppL-7250a.

Figure 7. The unfolded protein response in spp and sppL mutant larvae.

A 77 base pair alternative splice product of the Xbp I cDNA is made after induction of the UPR by DTT. Genotypes are w (w¹¹¹⁸), spp (w¹¹⁸, Df(2L)lwr¹⁴ p(lwr⁺)), sppL (w¹¹⁸, Df(3R)sppL), and spp sppL (w¹¹⁸, Df(2L)lwr¹⁴, p(lwr ⁺), sppL^BW1). Samples of RNA were prepared from freshly dissected larvae (0 hour) or from larvae incubated in media for 2 hours with or without DTT. Relative intensities of the upper (100 bp) and lower (77 bp) bands in the experimental samples were calculated from the total intensity in each band measured with ImageJ.