A subunit of mammalian signal peptidase is homologous to yeast SEC11 protein.

Canine signal peptidase consists of a complex of five proteins (Evans, A. E., Gilmore, R., and Blobel, G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 581-585). A cDNA encoding the 21-kDa subunit of the signal peptidase complex was isolated from a liver cDNA library using an 88-base pair probe, generated by the polymerase chain reaction. The 820-base pair cDNA was sequenced and found to encode a protein of 21,585 daltons. The deduced amino acid sequence from the canine cDNA was found to be 47% identical to the yeast SEC11 protein. SEC11 has been shown to be required for signal peptide cleavage, normal rate of secretion, and cell survival in Saccharomyces cerevisiae (Böhni, P. C., Deshaies, R. J., and Schekman, R. W. (1988) J. Cell Biol. 106, 1035-1042). It is, therefore, likely that the 21-kDa subunit of signal peptidase complex is the structural and functional homologue of the yeast SEC11 gene product.

microsomes (7). It consists of a complex of five proteins (molecular mass of 12,18,21,23, and 25 kDa) (7). The conclusion that the pancreatic enzyme is a pentameric complex rests on two lines of evidence. First, the detergentsolubilized enzyme complex remained associated throughout the purification procedure including sucrose gradient sedimentation (7). Second, the pentameric complex could be isolated by concanavalin A-Sepharose affinity chromatography, although only one of its subunits (23 kDa) is coreglycosylated (9). Recently the cDNA for the 23-kDa glycoprotein subunit of the canine SPC' has been cloned and sequenced (10). The deduced amino acid sequence showed no sequence homology to proteins in the GenBank and Dayhoff data bases.
Unlike canine signal peptidase, hen oviduct signal peptidase has been reported to consist of only two subunits, a glycoprotein subunit of 22-24 kDa and a nonglycosylated subunit of 19 kDa (11). Signal peptidase I (leader peptidase) of Escherichia coli, the functional equivalent of the eukaryotic signal peptidase consists of only one polypeptide of M , = 35,994 (12).
Here, we describe the cloning and sequencing of a cDNA encoding the 21-kDa subunit of the canine signal peptidase complex and find it to be highly homologous to the yeast SECll gene product, which has been shown genetically to be required for signal peptide cleavage and cell survival (8). Protein Sequencing-The 21-kDa protein was purified from canine pancreas as described previously (7,lO). For amino-terminal sequencing the purified protein was applied in 0.5% sodium dodecyl sulfate directly to an Applied Biosystems model 470A gas-phase sequenator (13). For internal amino acid sequence, the purified protein was first cleaved with CNBr (14) and the cleaved peptides separated by reverse phase high performance liquid chromatography using a C-4 column in an HaO/acetonitrile gradient containing 0.1% trifluoroacetic acid.

Materials
Polymerase Chain Reaction-The nucleotide probe used to isolate clones was generated by PCR (15) using Taq polymerase. Two oligonucleotides corresponding to the extreme ends of the aminoterminal protein sequence were synthesized with restriction sites using an Applied Biosystems model 380B oligonucleotide synthesizer. 0.5 PM of gel-purified oligonucleotide 1, 5"CGCCTAGGG-CCGGCGCCGTgGGXACXCA-3', and oligonucleotide 2, 5'-CXTTETACTTgTTECTTAAGCG-3', were used in the PCR with template cDNA synthesized from canine liver RNA (161, which was poly(A+)-selectedusing oligo(dT)-cellulose chromatography (17). The reaction was run in a DNA thermal cycler (Perkin-Elmer-Cetus) with the following incubation conditions: denaturation at 95 "C for 1 min, The abbreviations used are: SPC, signal peptidase complex; PCR, polymerase chain reaction; X (in nucleotide sequence), A and T and C and G. annealing at 45 "C for 2 min, and extension at 72 "C for 3 min, with reaction component concentrations as described by Perkin-Elmer-Cetus. The PCR product (20C) was cloned into the BarnHI and EcoRI cloning site of pUC 19 and sequenced.
cDNA Library Screening-The cDNA clone 4AA was isolated from an amplified hgtl0 canine liver library (10). The 20C probe was labeled with [cY-~~PI~CTP (Du Pont-New Engfand Nuclear NEG-013H) using the random-primed method (18). Plaque lifts were hybridized with the 3ZP-labeled 2OC probe, and positive plaques were isolated and placed in PCR buffer (50 mM KCl, 10 mM Tris-HCI, pH 8.3, 1.5 mM MgC12, 0.01% (w/v) gelatin). 50 p1 of eluted phage from the positive plaques was used as template in a PCR to identify inserts near the transcript size of 1 kilobase. The PCR contained oligonucleotide 1 (above) and one of two primers flanking the EcoRI restriction site of X g t l O (New England Biolabs 1231 or 1232). An amplification product of 0.85 kilobase was identified, and the corresponding plaque was selected for replating and three more rounds of filter hybridization were performed. The insert from plaque-purified clone 4AA was subcloned into the EcoRI site of pUC 19 and sequenced.
DNA Sequencing-The double-stranded template was sequenced using the dideoxy chain termination method of Sanger et al. (19) using synthetic oligonucl.eotides (20). Both strands of the doubiestranded template were sequenced with Sequenase (United States Biochemical Gorp.) using both dGTP and dITP. Searches of GenBank and Dayhoff data bases were performed on the Rockefeller University VAX computer using FASTA (21). Alignment was performed using the method of Needleman and Wunsch (22), as implemented on the DNAstar AALIGN program (DNAstar Inc., Madison, WI) on an IBM AT computer.

RESULTS AND DISCUSSION
To clone the cDNA for the 21-kDa subunit of SPC, we synthesized degenerate o~gonucleotides based on information derived from the amino acid sequence of the NH2 terminus of the 21-kDa subunit. We used these in a PCR (15) to obtain a DNA probe of 88 base pairs which was used to screen a cloned canine liver cDNA library in XgtlO. The cDNA sequence of one clone is shown in Fig. 1 electrophoresis (7). The clone begins with a 208-nucleotide 5"untranslated region. There is a Kozak consensus sequence (CCGCCATGG) surrounding the initiating ATG codon (23) of the open reading frame. This clone (4AA) contains a total of 820 base pairs, close to the transcript size of 1 kilobase determined by Northern blot analysis (data not shown). No polyadenylation signal was found, probably due to the short 3'-untranslated region (36 nucleotides) of the clone. The deduced amino acid sequence of the 21-kDa clone was confirmed by NHp-terminal and internal amino acid sequence data obtained by automated Edman degradation of purified 21-kDa protein. Met' of the cDNA deduced amino acid sequence is followed by a Val residue which is the N:H2-terminal residue of sequence derived from Edman degradation. Thus, the NHp-terminal Met in the primary translation product has been removed, as is seen for many proteins where Met is followed by Val (24). Another putative initiation codon upstream (nucleotide position -99) is out of frame. Thus, it is clear that the 21-kDa protein is not synthesized with a cleavable signal sequence.
A homology search comparing the 21-kDa deduced amino acid sequence with sequences present in the GenBank and Dayhoff data bases was negative. Comparison of the deduced amino acid sequence of the 21-kDa subunit of SPC with the recently published sequence of the yeast SECll protein (8) revealed a striking sequence similarity (Fig. 2). From Metz5 to G~Y'~' the 21-kDa protein is 47% identical to the SECll protein. A 30-residue ,stretch within this region (Thr55-Ar$5) is 81% identical. The high degree of stmctural homology in -208

A G T G T~C C A G A A G A T A~G G T C~G T C C -118 AATTTCAATCTCTGGTGGATGGAACCCTGACACGTA~AGTTCAGGAAGGCCGGGGCTC
-59 CGCAGCCGCCTGTGACACCCGGGAGCCGGCGGGCCGCCTGGGCCCCTGGAGAGCCCGCC  The average hydropathy of the deduced amino acid sequence was plotted as described by Kyte and Doolittle (25) (Fig. 3). A large hydrophobic domain is located in the first half of the protein between residues 29 and 69 (Fig. 1). 20 amino acids of this hydrophobic domain (Fig. 1, residues 29-48) are bordered by charged residued at positions 28 and 49. This region has an average hydrophobicity greater than 1.6, making it a possible candidate for a transmembrane domain (25) and an internal uncleaved signal sequence. This domain is followed by a very hydrophilic region that comprises more than a third of the protein. The last portion of the protein (Fig. 1, residues 157-168) is relatively hydrophobic but contains less than the 20 amino acids needed to span the lipid bilayer. The two hydrophobic domains of the 21-kDa protein closely correspond to those of the SECll protein (8). Interestingly, like the 21-kDa and SECll proteins, the 23-kDa protein of the SPC also contains two hydrophobic domains (10). The first is thought to be a membrane anchor while the second resides in the lumen of the endoplasmic reticulum. The fact that bacterial signal peptidase I also contains a hydrophobic domain in the periplasm (26) (which corresponds to the endoplasmic reticulum lumen) suggests that this structural motif might be involved in a common function necessary for signal peptide cleavage.

12; , ATG ATC T S A A A GGC TTG ATC GTC CTC X C -G G C -A G T~N AGC-CCG
As separation of the subunits of the SPC without denaturation has been unsuccessful (9) the protein subunit(s) responsible for signal peptide cleavage have yet to be identified. Genetic analysis of yeast mutants has clearly demonstrated that the SECll protein is required for cell survival and signal  peptide processing (8)-It is likely that the 21-kDa canine homologue of SECll protein has a similar if not identical function in the mammalian endoplasmic reticulum. Whether these two proteins are the catalytic subunit of signal peptidase remains to be determined, but the lack of sequence homology with the larger bacterial signal peptidase I (12) suggests that another protein may contain the signal sequence cleavage activity, while these proteins function in an ancillary way (8). The recent evidence that SECll protein is a member of a protein complex with signal peptidase activity' taken together with the work presented in this paper raises the intriguing possibility that, like the 21-kDa protein, other members of J. T. YaDeau and G. Blobel, unpublished result. the canine SPC may have counterparts in yeast. Expression of the canine 21-kDa protein and the other subunits of the canine SPC in yeast and in yeast secretory mutants may help to elucidate their role in signal sequence processing and protein translocation.