Signal Peptidase Complex Research Articles

Modeling Escherichia coli signal peptidase complex with bound substrate: determinants in the mature peptide influencing signal peptide cleavage

BackgroundType I signal peptidases (SPases) are essential membrane-bound serine proteases responsible for the cleavage of signal peptides from proteins that are translocated across biological membranes. The crystal structure of SPase in complex with signal peptide has not been solved and their substrate-binding site and binding specificities remain poorly understood. We report here a structure-based model for Escherichia coli DsbA 13–25 in complex with its endogenous type I SPase.ResultsThe bound structure of DsbA 13–25 in complex with its endogenous type I SPase reported here reveals the existence of an extended conformation of the precursor protein with a pronounced backbone twist between positions P3 and P1'. Residues 13–25 of DsbA occupy, and thereby define 13 subsites, S7 to S6', within the SPase substrate-binding site. The newly defined subsites, S1' to S6' play critical roles in the substrate specificities of E. coli SPase. Our results are in accord with available experimental data.ConclusionCollectively, the results of this study provide interesting new insights into the binding conformation of signal peptides and the substrate-binding site of E. coli SPase. This is the first report on the modeling of a precursor protein into the entire SPase binding site. Together with the conserved precursor protein binding conformation, the existing and newly identified substrate binding sites readily explain SPase cleavage fidelity, consistent with existing biochemical results and solution structures of inhibitors in complex with E. coli SPase. Our data suggests that both signal and mature moiety sequences play important roles and should be considered in the development of predictive tools.

A Bipartite Trigger for Dislocation Directs the Proteasomal Degradation of an Endoplasmic Reticulum Membrane Glycoprotein

Polypeptides are organized into distinct substructures, termed protein domains, that are often associated with diverse functions. These modular units can act as binding sites, areas of post-translational modification, and sites of complex multimerization. The human cytomegalovirus US2 gene product is organized into discrete domains that together catalyze the proteasome-dependent degradation of class I major histocompatibility complex heavy chains. US2 co-opts the endogenous ER quality control pathway in order to dispose of class I. The US2 endoplasmic reticulum (ER)-lumenal region is the class I binding domain, whereas the carboxyl terminus can be referred to as the degradation domain. In the present study, we examined the role of the US2 transmembrane domain in virus-mediated class I degradation. Replacement of the US2 transmembrane domain with that of the CD4 glycoprotein completely blocked the ability of US2 to induce class I destruction. A more precise mutagenesis revealed that subregions of the US2 transmembrane domain differ in their ability to trigger class I degradation. Collectively, the data support a model in which US2-mediated class I degradation occurs as a highly regulated process where the US2 transmembrane domain and cytoplasmic tail work in concert to eliminate class I molecules. Host factors, including a signal peptidase complex, probably associate with the US2 molecule in a coordinated fashion to create a predislocation complex to promote the extraction of class I out of the ER. The results imply that the ER quality control machinery may recognize and eliminate misfolded proteins using a similar multistep regulated process.

Plasmodium falciparum signal peptidase is regulated by phosphorylation and required for intra-erythrocytic growth

The human malaria parasite Plasmodium falciparum exports a variety of its proteins through its endoplasmic reticulum (ER) based secretory pathway in order to survive in the host erythrocyte. Signal peptidases are membrane-bound endopeptidases and have an important role in the transport and maturation of these parasite proteins. Prokaryotic signal peptidases are indispensable enzymes required for the removal of N-terminal signal peptide from the secretory proteins. Eukaryotic signal peptidases exist as multimeric protein complex in the ER and the catalytic subunit of this complex catalyzes removal of the N-terminal signal peptide from preproteins. All the signal peptidases contain five regions of high-sequence similarity referred to as boxes A–E. Here we report characterization of the catalytic subunit of signal peptidase complex (SPC) from P. falciparum. This protein designated as PfSP21 shows homology with the similar subunit from other sources and contains all the conserved boxes A–E. PfSP21 is able to cleave the peptide substrate containing the signal peptidase cleavage site. PfSP21 is phosphorylated by protein kinase C and its enzyme activity was upregulated after this phosphorylation. Immunofluorescence assay studies revealed that PfSP21 is localized in the ER of P. falciparum. PfSP21 dsRNA specifically inhibits the growth of P. falciparum in culture and this inhibition is most likely due to the decrease in the amount of endogenous PfSP21 protein. These studies demonstrate the characterization of a functional subunit of SPC from P. falciparum and should make an important contribution in our better understanding of the complex process of protein translocation in the parasite.

SLURP1 Is a Late Marker of Epidermal Differentiation and Is Absent in Mal de Meleda

SLURP1 is a secreted member of the LY6/PLAUR protein family. Mutations in the SLURP1 gene are the cause of Mal de Meleda (MDM), a rare autosomal recessive genetic disease, characterized by inflammatory palmoplantar keratoderma. In this study, we have analyzed the expression of SLURP1 in normal and MDM skin. SLURP1 was found to be a marker of late differentiation, predominantly expressed in the granular layer of skin, notably the acrosyringium. Moreover, SLURP1 was also identified in several biological fluids such as sweat, saliva, tears, and urine from normal volunteers. In palmoplantar sections from MDM patients, as well as in their sweat, mutant SLURP1, including the new variant R71H-SLURP1, was either absent or barely detectable. Transfected human embryonic kidney 293T cells expressed the MDM mutant SLURP1 containing the single amino-acid substitution G86R but did not tolerate the MDM mutation W15R located in the signal peptide. Thus, most MDM mutations in SLURP1 affect either the expression, integrity, or stability of the protein, suggesting that a simple immunologic test could be used as a rapid screening procedure.

Biosynthesis of the dystonia-associated AAA+ ATPase torsinA at the endoplasmic reticulum

TorsinA is a widely expressed AAA(+) (ATPases associated with various cellular activities) ATPase of unknown function. Previous studies have described torsinA as a type II protein with a cleavable signal sequence, a single membrane spanning domain, and its C-terminus located in the ER (endoplasmic reticulum) lumen. However, in the present study we show that torsinA is not in fact an integral membrane protein. Instead we find that the mature protein associates peripherally with the ER membrane, most likely through an interaction with an integral membrane protein. Consistent with this model, we provide evidence that the signal peptidase complex cleaves the signal sequence of torsinA, and we show that the region previously suggested to form a transmembrane domain is translocated into the lumen of the ER. The finding that torsinA is a peripheral, and not an integral membrane protein as previously thought, has important implications for understanding the function of this novel ATPase.

Isolation and characterization of Coffea genes induced during coffee leaf miner ( Leucoptera coffeella) infestation

Coffea arabica, one of the most important breeding species in the world, is susceptible to the leaf miner Leucoptera coffeella, which causes severe damage to coffee plantations. A closely related coffee species ( Coffea racemosa) resistant to this insect was crossed with C. arabica and resulted in segregating progenies with resistance or susceptibility to coffee leaf miner. The aim of this work was to isolate and characterize the genes involved in coffee resistance to this pest. Subtracted cDNA libraries enriched in genes preferentially expressed in coffee plants resistant to L. coffeella were constructed. Approximately 1500 clones were spotted on nylon membranes and hybridized to cDNA probes derived from RNA samples from infestation experiments. Several genes were differentially expressed. We selected expressed ESTs with the most interesting expression profiles and confirmed the up-regulation of five of them (Class III Chitinase PR-8, signal peptidase complex subunit SPC25, photosystem gene psaH, a putative calcium exchanger similar to CAX9 and a homeotic gene BEL) by RNA blot. The possible functions of these genes in coffee resistance and coffee development, and a hypothetical defense mechanism against L. coffeela are discussed.

Ribophorin I Associates with a Subset of Membrane Proteins after Their Integration at the Sec61 Translocon

The biosynthesis of membrane proteins at the endoplasmic reticulum (ER) involves the integration of the polypeptide at the Sec61 translocon together with a number of maturation events, such as N-glycosylation and signal sequence cleavage, that can occur both during and after synthesis. To better understand the events occurring after the release of the nascent chain from the ER translocon, we investigated the ER components adjacent to the transmembrane-spanning domain of a well characterized fragment of the amyloid precursor protein. Using individual cysteine residues as site-specific cross-linking targets, we found that several ER components can be cross-linked to the fully integrated polypeptide. We identified strong adducts with both the ribophorin I subunit of the oligosaccharyltransferase complex and the 25-kDa subunit of the signal peptidase complex. Focusing on the association with ribophorin I, we found that adduct formation occurred exclusively after the exit of the nascent chain from the Sec61 translocon and was unaffected by the N-glycosylation status of the associated precursor. Only a subset of newly made membrane proteins associated with ribophorin I in vitro, and we could recapitulate a specific association between the amyloid precursor protein fragment and ribophorin I in vivo. Taken together, our data suggest a model where ribophorin I may function to retain potential substrates in close proximity to the catalytic subunit of the oligosaccharyltransferase and thereby stochastically improve the efficiency of the N-glycosylation reaction in vivo. Alternatively ribophorin I may be multifunctional and facilitate additional processes, for example, ER quality control.

Prototype Foamy Virus Envelope Glycoprotein Leader Peptide Processing Is Mediated by a Furin-Like Cellular Protease, but Cleavage Is Not Essential for Viral Infectivity

Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.

Characterization of Candida albicans orthologue of the Saccharomyces cerevisiae signal-peptidase-subunit encoding gene SPC3.

The Candida albicans orthologue of the SPC3 gene, which encodes one of the subunits essential for the activity of the signal peptidase complex in Saccharomyces cerevisiae, was isolated by complementation of a thermosensitive mutation in the S. cerevisiae SEC61 gene. The cloned gene (CaSPC3) encodes a putative protein of 192 amino acids that contains one potential membrane-spanning region and shares significant homology with the corresponding products from mammalian (Spc22/23p) and yeast (Spc3p) cells. CaSPC3 is essential for cell viability, since a hemizygous strain containing a single copy of CaSPC3 under control of the methionine-repressible MET3 promoter did not grow in the presence of methionine and cysteine. The cloned gene could rescue the phenotype associated with a spc3 mutation in S. cerevisiae, indicating that it is the true C. albicans orthologue of SPC3. However, in contrast with results previously described for its S. cerevisiae orthologue, CaSPC3 was not able to complement the thermosensitive growth associated with a mutation in the SEC11 gene. The heterologous complementation of the sec61 mutant suggests that Spc3p could play a role in the interaction that it is known to occur between the translocon (Sec61 complex) and the signal peptidase complex, at the endoplasmic reticulum membrane.

The mitochondrial IMP peptidase of yeast: functional analysis of domains and identification of Gut2 as a new natural substrate.

The mitochondrial inner membrane peptidase IMP of Saccharomyces cerevisiae is required for proteolytic processing of certain mitochondrially and nucleus-encoded proteins during their export from the matrix into the inner membrane or the intermembrane space. The membrane-associated signal peptidase complex is composed of the two catalytic subunits, Imp1 and Imp2, and the Som1 protein. The IMP subunits are thought to function in membrane association, interaction and stabilisation of subunits, substrate specificity, and proteolysis. We have analysed inner membrane peptidase mutants and substrates to gain more insight into the functions of various domains and investigate the basis of substrate recognition. The results suggest that certain conserved glycine residues in the second and third conserved regions of Imp1 and Imp2 are important for stabilisation of the Imp complex and for the proteolytic activity of the subunits, respectively. The non-conserved C-terminal parts of the Imp subunits are important for their proteolytic activities. The C-terminal region of Imp2, comprising a predicted second transmembrane segment, is dispensable for the stability of Imp2 and Imp1, and cannot functionally substitute for the C-terminal segment of Imp1. Alteration of the Imp2 cleavage site in cytochrome c(1) (from A/M to N/D) reveals the specificity of the Imp2 peptidase. In addition, we have identified Gut2, the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase, as a new substrate for Imp1. Failure to cleave the Gut2 precursor may contribute to the pet phenotype of certain imp mutants. Gut2 is associated with the inner membrane, and is essential for growth on glycerol-containing medium. Suggested functions of the analysed residues and domains of the IMP subunits, characteristics of the cleavage sites of substrates and implications for the phenotypes of imp mutants are discussed.

Genetic Complementation in Yeast Reveals Functional Similarities between the Catalytic Subunits of Mammalian Signal Peptidase Complex

Type I signal peptidases (SPs) comprise a family of structurally related enzymes that cleave signal peptides from precursor proteins following their transport out of the cytoplasmic space in eukaryotic and prokaryotic cells. One such enzyme, the mitochondrial inner membrane peptidase, has two catalytic subunits, which recognize distinct cleavage site motifs in their signal peptide substrates. The only other known type I SP with two catalytic subunits is the signal peptidase complex (SPC) in the mammalian endoplasmic reticulum. Here, we tested the hypothesis that, as with inner membrane peptidase catalytic subunits, SPC catalytic subunits exhibit nonoverlapping substrate specificity. We constructed two yeast strains without endogenous SP, one expressing canine SPC18 and the other expressing a truncation of canine SPC21 (SPC21 Delta N), which lacks 24 N-terminal residues that prevent expression of SPC21 in yeast. By monitoring a variety of soluble and membrane-bound substrates, we find that, in contrast to the tested hypothesis, SPC catalytic subunits exhibit overlapping substrate specificity. SPC18 and SPC21 Delta N do, however, cleave some substrates with different efficiencies, although no pattern for this behavior could be discerned. In light of the functional similarities between SPC proteins, we developed a membrane protein fragmentation assay to monitor the position of the catalytic sites relative to the surface of the endoplasmic reticulum membrane. Using this assay, our results suggest that the active sites of SPC18 and SPC21 Delta N are located 4-11 A above the membrane surface. These data, thus, support a model that SPC18 and SPC21 are functionally and structurally similar to each other.

Tail-anchored and Signal-anchored Proteins Utilize Overlapping Pathways during Membrane Insertion

Tail-anchored proteins are a distinct class of membrane proteins that are characterized by a C-terminal membrane insertion sequence and a capacity for post-translational integration. Although it is now clear that tail-anchored proteins are inserted into the membrane at the endoplasmic reticulum (ER), the molecular basis for their integration is poorly understood. We have used a cross-linking approach to identify ER components that may be involved in the membrane insertion of tail-anchored proteins. We find that several newly synthesized tail-anchored proteins are transiently associated with a defined subset of cellular components. Among these, we identify several ER proteins, including subunits of the Sec61 translocon, Sec62p, Sec63p, and the 25-kDa subunit of the signal peptidase complex. When we analyze the cotranslational membrane insertion of a comparable signal-anchored protein we find the nascent polypeptide associated with a similar set of ER components. We conclude that the pathways for the integration of tail-anchored and signal-anchored membrane proteins at the ER exhibit a substantial degree of overlap, and we propose that this reflects similarities between co- and post-translational membrane insertion.

Detection of Genes with Atypical Nucleotide Sequence in Microbial Genomes

Type I signal peptidases (EC 3.4.21.89) are integral membrane proteins responsible for the removal of signal sequences from preproteins following protein translocation across a variety of membranes (Dalbey et al. 1997). Insight into the behavior of signal peptidase has come from the recent description of the crystal structure of the ectodomain of the Escherichia coli enzyme (Paetzel et al. 1998). In E. coli, signal peptidase is composed of two structural components, referred to as domain I and domain II. Corresponding to the “catalytic core” of the enzyme, domain I encompasses boxes A–E, five regions of significant sequence homology preserved throughout evolution (Fig. 1). Box B contains the nucleophilic Ser90 residue (E. coli numbering) and the conserved Met91 residue, while box D contains the proposed general base Lys145 and the conserved Arg146. Ser90 and Lys145 are believed to form the catalytic dyad responsible for enzyme activity (Paetzl et al. 2000). Domain II, corresponding to the stretch of amino acid residues between box D and box E (residues 154–271), folds as a large b-sheet. Sequence alignment studies of signal peptidases from various Gram-negative bacteria (Salmonella typhimurium, Pseudomonas fluorescens, Haemophilus influenzae, Bradyrhizobium japonicum, Borrelia burgdorferi, Phormidum laminosum, Azotobacter vinelandii, Rickettsia prowazekii, Thermotoga maritima, and Rhodobacter capsulatus) reveal the presence of domain II sequences containing between 39 and 152 residues. Examination of the corresponding regions of Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Streptococcus pneumoniae, Aquifex aeolicus, Myobacterium leprae, and Myobacterium tuberculosis) reveals the presence of smaller domain II regions. Despite their omnipresence, the role of domain II in bacterial signal peptidase activity is currently not understood. In contrast to their bacterial homologues, eukaryal signal peptidases do not contain domain II regions. Eukaryal signal peptidases also differ from their bacterial counterparts in that the eukaryal enzyme functions as part of a multisubunit signal peptidase complex (SPC). In Saccharomyces cerevisiae, this complex is comprised of the Sec11, Spc1, Spc2, and Spc3 proteins (YaDeau et al. 1991). Both Sec11 and Spc3 are required for signal sequence cleavage and indeed can be coimmunoprecipitated (Meyer and Hartmann 1997; Fang et al. 1997). While Sec11 contains the evolutionarily conserved boxes A–E sequences (Fig. 1), it has been proposed that Spc3 provides the lysine residue of the catalytic serine/lysine dyad conserved in bacteria (K145) yet absent in eukarya, where it is replaced by a conserved histidine residue. Site-directed mutagenesis of conserved and nonconserved lysine residues as well as of other selected Spc3 amino acids, however, failed to affect enzyme activity (Van Valkenburgh et al. 1999). It appears, therefore, that Correspondence to: Jerry Eichler; email: jeichler@bgumail.bgu.ac.il J Mol Evol (2002) 54:411–415 DOI: 10.1007/s00239-001-0035-8

Cover Picture

The cover picture shows two features from the complex area of protein targeting in eukaryotic cells. The upper image illustrates the recruitment of the signal peptidase complex (SPC; light blue) and the oligosaccharidyl transferase complex (OST; green) to the ribosome–nascent polypeptide chain–Sec61 complex in the endoplasmic reticulum (ER) membrane. SPC endoproteolytically removes the signal peptide, OST attaches core oligosaccharides to the translocating nascent chain. The lower image shows other systems for protein translocation across distinct cellular membranes that work on principles similar to those first established for the ER (bold blue arrow). The slim blue arrows indicate the related systems within the mitochondrial matrix and the chloroplast stroma. Bold and slim red and green arrows indicate protein translocation systems for import across the outer and inner mitochondrial or chloroplast membrane, respectively. White and black arrows indicate transport into and out of the nucleus, respectively, across the nuclear pore complex. Further details are given in the Nobel Lecture by G. Blobel on p. 86.

Cover Picture

The cover picture shows two features from the complex area of protein targeting in eukaryotic cells. The upper image illustrates the recruitment of the signal peptidase complex (SPC; light blue) and the oligosaccharidyl transferase complex (OST; green) to the ribosome–nascent polypeptide chain–Sec61 complex in the endoplasmic reticulum (ER) membrane. SPC endoproteolytically removes the signal peptide, OST attaches core oligosaccharides to the translocating nascent chain. The lower image shows other systems for protein translocation across distinct cellular membranes that work on principles similar to those first established for the ER (bold blue arrow). The slim blue arrows indicate the related systems within the mitochondrial matrix and the chloroplast stroma. Bold and slim red and green arrows indicate protein translocation systems for import across the outer and inner mitochondrial or chloroplast membrane, respectively. White and black arrows indicate transport into and out of the nucleus, respectively, across the nuclear pore complex. Further details are given in the Nobel Lecture by G. Blobel on p. 86.

The Catalytic Mechanism of Endoplasmic Reticulum Signal Peptidase Appears to Be Distinct from Most Eubacterial Signal Peptidases

Many type I signal peptidases from eubacterial cells appear to contain a serine/lysine catalytic dyad. In contrast, our data show that the signal peptidase complex from the endoplasmic reticulum lacks an apparent catalytic lysine. Instead, a serine, histidine, and two aspartic acids are important for signal peptidase activity by the Sec11p subunit of the yeast signal peptidase complex. Amino acids critical to the eubacterial signal peptidases and Sec11p are, however, positioned similarly along their primary sequences, suggesting the presence of a common structural element(s) near the catalytic sites of these enzymes.

The beta subunit of the Sec61 complex facilitates cotranslational protein transport and interacts with the signal peptidase during translocation.

The Sec61 complex is the central component of the protein translocation apparatus of the ER membrane. We have addressed the role of the beta subunit (Sec61beta) during cotranslational protein translocation. With a reconstituted system, we show that a Sec61 complex lacking Sec61beta is essentially inactive when elongation and membrane targeting of a nascent chain occur at the same time. The translocation process is perturbed at a step where the nascent chain would be inserted into the translocation channel. However, if sufficient time is given for the interaction of the nascent polypeptide with the mutant Sec61 complex, translocation is almost normal. Thus Sec61beta kinetically facilitates cotranslational translocation, but is not essential for it. Using chemical cross-linking we show that Sec61beta not only interacts with subunits of the Sec61 complex but also with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the first time a tight interaction between the SPC and the Sec61 complex. Interestingly, the cross-links between Sec61beta and SPC25 and between Sec61beta and Sec61alpha depend on the presence of membrane-bound ribosomes, suggesting that these interactions are induced when translocation is initiated. We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity.

19] Topology of P-glycoproteins

Publisher Summary This chapter describes the techniques, which have generated new insights and questions regarding P-glycoprotein (Pgp) folding, topology, and general mechanisms of polytopic protein biogenesis. For most eukaryotic polytopic proteins, transmembrane topology is established in the endoplasmic reticulum (ER) membrane through a series of programmed folding events. These events are directed through interactions between specific sequence determinants encoded in the nascent chain and their cognate cytosolic and membrane-bound receptors. Several complementary techniques are used to study Pgp biogenesis and topology in the ER membrane. These techniques exploit the physical integrity and/or asymmetric enzymatic activities of the ER membrane, which include protease protection of translocated peptide domains, cleavage of the chain by signal peptidase complex, and covalent addition of N-linked high mannose oligosaccharides.

The Yeast SPC22/23 Homolog Spc3p Is Essential for Signal Peptidase Activity

In eucaryotic cells signal sequences of secretory and membrane proteins are cleaved by the signal peptidase complex during their transport into the lumen of the endoplasmic reticulum. The signal peptidase complex in yeast consists of four subunits. To date, three of these subunits have been functionally characterized. One of them, the Sec11p, is essential for viability of yeast cells. It shows significant homology to the mammalian SPC18 and SPC21 as well as to bacterial leader peptidases. Two other subunits, Spc1p and Spc2p, have been shown to be homologous to mammalian SPC12 and SPC25, respectively, and are not essential for protein translocation or signal peptide cleavage. We have purified and analyzed the fourth subunit of yeast signal peptidase, Spc3p. The protein is essential for viability of yeast cells. Depletion of SPC3 leads to accumulation of precursors of secretory proteins in vivo and to the loss of the signal peptidase activity in vitro. Therefore, in contrast to the bacterial leader peptidases, yeast signal peptidase requires a second subunit for its function.

In Addition to SEC11, a Newly Identified Gene,SPC3, Is Essential for Signal Peptidase Activity in the Yeast Endoplasmic Reticulum

Among the three characterized subunits comprising the signal peptidase complex of the yeast Saccharomyces cerevisiae (Sec11p, Spc1p, and Spc2p), only Sec11p is essential for cell growth, signal peptide cleavage, and signal peptidase-dependent protein degradation. Here we report the cloning of the SPC3 gene encoding the homolog to mammalian signal peptidase subunit SPC22/23. We find that Spc3p is also required for cell growth and signal peptidase activity within the yeast endoplasmic reticulum.