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

Casso DJ, Liu S, Biehs B, Kornberg TB - PLoS ONE (2012)

Bottom Line: Although vertebrates have one SPP and four SPP-like (SPPL) genes, we found that insect genomes encode one Spp and one SppL.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America.

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.

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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}sppLSH116 (also known as P{lacW}l(3)SH116sh116), PBac{XP}Lnkd07478, and PBac{RB}CG17370e00372 are indicated with red triangles. Imprecise excision of P{lacW}sppLSH116 generated the deletion alleles sppL24J and sppL57D. Recombination between the two PBac insertions was used to generate deletion Df(3R)sppL. The extent of these deletions is indicated within parentheses. The sppL57D deletion (not shown) is similar to sppL24J. 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.
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pone-0033827-g006: 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}sppLSH116 (also known as P{lacW}l(3)SH116sh116), PBac{XP}Lnkd07478, and PBac{RB}CG17370e00372 are indicated with red triangles. Imprecise excision of P{lacW}sppLSH116 generated the deletion alleles sppL24J and sppL57D. Recombination between the two PBac insertions was used to generate deletion Df(3R)sppL. The extent of these deletions is indicated within parentheses. The sppL57D deletion (not shown) is similar to sppL24J. 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.

Mentions: To assess sppL function by loss-of-function genetics, we made sppL deletion alleles in two ways. First, we removed a portion of the sppL transcription unit by imprecise excision of a P transposon (P{lacW}sppLSH116). sppL is predicted to produce five transcripts that are distinguished by alternate use of three non-coding exons that contribute to the 5′UTRs of all of the sppL mRNA species [39; see Fig. 6]. These five transcripts share a large 5′-proximal intron where P{lacW}sppLSH116 has inserted. P{lacW}sppLSH116 was isolated as a lethal in a screen for P element mutations [28]. We determined that recombination of the P{lacW}sppLSH116 chromosome yielded viable transposon-bearing chromosomes, indicating that the lethality of P{lacW}sppLSH116 does not reside with the insertion. We sequenced PCR products amplified with primers that flanked the published insertion site and confirmed its orientation and location 2168 bases downstream of the most 5′ sppL start site and within the large sppL intron (Fig. 6). P{lacW}sppLSH116 flies were engineered to express P element transposase, and progeny were screened to identify approximately 1000 that lacked the w+ marker of P{lacW}. Lines were created from these w− excisions, and genomic DNA from these lines was then screened in pools of ten using four PCR reactions. The positions of the proximal primer (▸ at 1.6 kb) and four distal primers (◂ at 2.6, 3.6, 4.6, and 5.7 kb) are indicated in Figure 6. Deletions resulting from imprecise excision generated PCR products that were cloned and sequenced. Ten independent deletions within sppL ranging in size from 0.8 to 2.5 kb were identified. Deletions 24J and 57D were the largest. Proximal to the transposon insertion, they eliminate the branch points for the introns between exons N2-N3 and N2-C1; distally, they remove the translation start, the first transmembrane (e.g. TM1) domain, and part of the loop between TM1 and TM2 (Figs. 1, 6).


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

Casso DJ, Liu S, Biehs B, Kornberg TB - PLoS ONE (2012)

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}sppLSH116 (also known as P{lacW}l(3)SH116sh116), PBac{XP}Lnkd07478, and PBac{RB}CG17370e00372 are indicated with red triangles. Imprecise excision of P{lacW}sppLSH116 generated the deletion alleles sppL24J and sppL57D. Recombination between the two PBac insertions was used to generate deletion Df(3R)sppL. The extent of these deletions is indicated within parentheses. The sppL57D deletion (not shown) is similar to sppL24J. 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3306293&req=5

pone-0033827-g006: 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}sppLSH116 (also known as P{lacW}l(3)SH116sh116), PBac{XP}Lnkd07478, and PBac{RB}CG17370e00372 are indicated with red triangles. Imprecise excision of P{lacW}sppLSH116 generated the deletion alleles sppL24J and sppL57D. Recombination between the two PBac insertions was used to generate deletion Df(3R)sppL. The extent of these deletions is indicated within parentheses. The sppL57D deletion (not shown) is similar to sppL24J. 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.
Mentions: To assess sppL function by loss-of-function genetics, we made sppL deletion alleles in two ways. First, we removed a portion of the sppL transcription unit by imprecise excision of a P transposon (P{lacW}sppLSH116). sppL is predicted to produce five transcripts that are distinguished by alternate use of three non-coding exons that contribute to the 5′UTRs of all of the sppL mRNA species [39; see Fig. 6]. These five transcripts share a large 5′-proximal intron where P{lacW}sppLSH116 has inserted. P{lacW}sppLSH116 was isolated as a lethal in a screen for P element mutations [28]. We determined that recombination of the P{lacW}sppLSH116 chromosome yielded viable transposon-bearing chromosomes, indicating that the lethality of P{lacW}sppLSH116 does not reside with the insertion. We sequenced PCR products amplified with primers that flanked the published insertion site and confirmed its orientation and location 2168 bases downstream of the most 5′ sppL start site and within the large sppL intron (Fig. 6). P{lacW}sppLSH116 flies were engineered to express P element transposase, and progeny were screened to identify approximately 1000 that lacked the w+ marker of P{lacW}. Lines were created from these w− excisions, and genomic DNA from these lines was then screened in pools of ten using four PCR reactions. The positions of the proximal primer (▸ at 1.6 kb) and four distal primers (◂ at 2.6, 3.6, 4.6, and 5.7 kb) are indicated in Figure 6. Deletions resulting from imprecise excision generated PCR products that were cloned and sequenced. Ten independent deletions within sppL ranging in size from 0.8 to 2.5 kb were identified. Deletions 24J and 57D were the largest. Proximal to the transposon insertion, they eliminate the branch points for the introns between exons N2-N3 and N2-C1; distally, they remove the translation start, the first transmembrane (e.g. TM1) domain, and part of the loop between TM1 and TM2 (Figs. 1, 6).

Bottom Line: Although vertebrates have one SPP and four SPP-like (SPPL) genes, we found that insect genomes encode one Spp and one SppL.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, United States of America.

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.

Show MeSH