Alternative Splicing Regulates the Endoplasmic Reticulum Localization or Secretion of Soluble Secreted Endopeptidase
2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês
10.1074/jbc.m101703200
ISSN1083-351X
AutoresSunu Budhi Raharjo, Noriaki Emoto, Koji Ikeda, Ryuichiro Sato, Mitsuhiro Yokoyama, Masafumi Matsuo,
Tópico(s)Signaling Pathways in Disease
ResumoA subfamily of zinc metalloproteases, represented by Neutral endopeptidase (EC 3.4.24.11) and endothelin-converting enzyme, is involved in the metabolism of a variety of biologically active peptides. Recently, we cloned and characterized a novel member of this metalloprotease family termed soluble secreted endopeptidase (SEP), which hydrolyzes many vasoactive peptides. Here we report that alternative splicing of the mouseSEP gene generates two polypeptides, SEPΔ and SEP. After synthesis, both isoforms are inserted into the endoplasmic reticulum (ER) as type II membrane proteins. SEPΔ then becomes an ER resident, whereas SEP, which differs by only the presence of 23 residues at the beginning of its luminal domain, is proteolytically cleaved by membrane secretase(s) in the ER and transported into the extracellular compartment. An analysis of the chimeric proteins between SEPΔ and bovine endothelin-converting enzyme-1b (bECE-1b) demonstrated that the retention of SEPΔ in the ER is mediated by the luminal domain. In addition, the dissection of the chimeric bECE-1b/SEP insertion showed that its insertion domain is obviously responsible for its secretion. A series of mutagenesis in this region revealed that the minimal requirement for cleavage was found to be a WDERTVV motif. Our results suggest that the unique subcellular localization and secretion of SEP proteins provide a novel model of protein trafficking within the secretory pathway. A subfamily of zinc metalloproteases, represented by Neutral endopeptidase (EC 3.4.24.11) and endothelin-converting enzyme, is involved in the metabolism of a variety of biologically active peptides. Recently, we cloned and characterized a novel member of this metalloprotease family termed soluble secreted endopeptidase (SEP), which hydrolyzes many vasoactive peptides. Here we report that alternative splicing of the mouseSEP gene generates two polypeptides, SEPΔ and SEP. After synthesis, both isoforms are inserted into the endoplasmic reticulum (ER) as type II membrane proteins. SEPΔ then becomes an ER resident, whereas SEP, which differs by only the presence of 23 residues at the beginning of its luminal domain, is proteolytically cleaved by membrane secretase(s) in the ER and transported into the extracellular compartment. An analysis of the chimeric proteins between SEPΔ and bovine endothelin-converting enzyme-1b (bECE-1b) demonstrated that the retention of SEPΔ in the ER is mediated by the luminal domain. In addition, the dissection of the chimeric bECE-1b/SEP insertion showed that its insertion domain is obviously responsible for its secretion. A series of mutagenesis in this region revealed that the minimal requirement for cleavage was found to be a WDERTVV motif. Our results suggest that the unique subcellular localization and secretion of SEP proteins provide a novel model of protein trafficking within the secretory pathway. Neutral endopeptidase endothelin-converting enzyme bovine endothelin-converting enzyme-1b soluble secreted endopeptidase endothelin bacterial artificial chromosome polymerase chain reaction Chinese hamster ovary microsomal triglyceride transfer protein polyacrylamide gel electrophoresis endo-β-N-acetylglucosaminidase H peptide-N-glycosidase F endo-β-N-acetylglucosaminidase D N α-p-tosyl-l-lysine chloromethyl ketone endoplasmic reticulum Mammalian zinc metalloproteases have been implicated in a diversity of disease states because of their roles in the activation or inactivation of a variety of biologically active peptides. Therefore, they provide important therapeutic targets for certain diseases. Within this large group, neprilysin (M13) constitutes a subfamily in which seven members have been identified to date, such as Neutral endopeptidase (EC 3.4.24.11) (NEP),1 Kell blood group protein, two different endothelin-converting enzymes (ECE-1 and ECE-2), PEX, which has been associated with X-linked hypophosphatemic rickets, endothelin-converting enzyme-like-1, and the recently identified soluble secreted endopeptidase (SEP). All these members are type II membrane glycoproteins, which display a single transmembrane stretch separating a short N-terminal cytoplasmic tail from a large C-terminal extracellular/luminal domain. This luminal domain bears the enzyme active site, which includes HEXXH, a highly conserved pentameric consensus sequence of a zinc binding motif. NEP is a metalloprotease with wide tissue distribution and is especially abundant in the brain and kidney. This endopeptidase has been shown to hydrolyze a wide range of small peptide mediators, such as enkephalins, substance P, atrial natriuretic peptide, neurotensin, bradykinin, angiotensin I and II, and endothelins (1Roques B.P. Noble F. Dauge V. Fournie-Zaluski M.C. Beaumont A. Pharmacol. Rev. 1993; 45: 87-146PubMed Google Scholar). ECE-1 is primarily involved in the production of the vasoconstrictive peptide ET-1 by the cleavage of an inactive precursor, big ET-1. Two subisoforms of bovine ECE-1 termed ECE-1a and ECE-1b that differ from each other only in the N-terminal tip of their cytoplasmic tail showed distinct subcellular localization (2Emoto N. Nurhantari Y. Alimsardjono H. Xie J. Yamada T. Yanagisawa M. Matsuo M. J. Biol. Chem. 1999; 274: 1509-1518Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). ECE-2, which also produces ET-1, has an acidic pH optimum and may function intracellularly (3Emoto N. Yanagisawa M. J. Biol. Chem. 1995; 270: 15262-15268Crossref PubMed Scopus (430) Google Scholar). Therefore, clarifying the precise subcellular localization of the protein would be favorable to characterize its physiological roles. SEP, the most recently identified member of this family, shares higher structural and functional similarities with NEP than with other members of this metalloprotease family. Structurally, the sequence identity of SEP with respect to NEP is higher than those of the other members. Two arginine residues known to constitute the substrate binding sites in NEP (Arg102 and Arg747 in human NEP) are conserved in SEP (Arg121 and Arg764 in mouse SEP). Functionally, both SEP and NEP are promiscuous enzymes that hydrolyze a variety of physiologically active peptides. SEP has been implicated in the hydrolysis of angiotensin I, atrial natriuretic peptide, bradykinin, substance P, leucine-enkephalin, big ET-1, and ET-1. The activity of SEP is efficiently inhibited by the specific NEP inhibitor thiorphan but is not completely inhibited by the specific ECE inhibitor FR901533 (4Ikeda K. Emoto N. Raharjo S.B. Nurhantari Y. Saiki K. Yokoyama M. Matsuo M. J. Biol. Chem. 1999; 274: 32469-32477Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 5Ghaddar G. Ruchon A.F. Carpentier M. Marcinkiewicz M. Seidah N.G. Crine P. Desgroseillers L. Boileau G. Biochem. J. 2000; 347: 419-429Crossref PubMed Scopus (73) Google Scholar). Although SEP shares several important properties with other members of this metalloprotease family, it exhibits features unique to itself. First, two isoforms of SEP named SEP and SEPΔ differ from each other in the presence of a 23-amino acid insertion region flanking the transmembrane domain of SEP. This feature was not found in other members of this metalloprotease family. Second, the membrane-bound SEP seems to localize in the early secretory pathway, which is unusual for these metalloprotease family members. Third, although all the other members discovered so far are membrane-associated proteins, SEP exists not only as a membrane-bound form but also as a circulating soluble form. This observation suggests that a proteolytic cleavage event occurred during the intracellular transport of SEP. These features make SEP unique among the members of this neprilysin family. Therefore, we designed the current study to investigate these special characteristics of SEP. In this report, we present evidence that two isoforms of SEP are generated via an alternative splicing mechanism. The membrane-bound SEP localized in the endoplasmic reticulum, and this ER localization is not attributed to misfolding. In addition, by making chimeric proteins between SEP and bovine endothelin-converting enzyme-1b (bECE-1b), another member of this metalloprotease family that is normally localized in the cell surface, the luminal domain of the SEP protein was identified as being important for retention. Furthermore, we also identified a specific motif in the SEP insertion region that is necessary for the cleavage process of this protein in the ER. This unique mechanism of localization and processing of SEP defines an interesting model for protein trafficking within the secretory pathway. Enzymes used in molecular cloning were obtained from Roche Molecular Biochemicals or from New England Biolabs (Beverly, MA). Endo-β-N-acetylglucosaminidase H (Endo H) and peptide-N-glycosidase F (PNGaseF) were from Roche Molecular Biochemicals, Endo D was from Seikagaku Co. Ltd. (Tokyo), rProtein A-Sepharose Fast Flow beads were from Amersham Pharmacia Biotech AB (Uppsala, Sweden). The mouse SEP gene locus was cloned by screening a BAC (bacterial artificial chromosome) library (Genome Systems, Inc., St. Louis, MO). A polymerase chain reaction (PCR)-generated radiolabeled probe containing sequences within the mouse cDNA was used to probe a mouse genomic BAC library. The probe was generated from the following oligonucleotides, 5′-TATTTCCGGCAGGGATTCTC-3′ and 5′-CATTATCATCAAAGCCGTGT-3′, which were chosen based on their likelihood to span a region within anSEP exon according to the genomic structure ofNEP (6Chen C.Y. Salles G. Seldin M.F. Kister A.E. Reinherz E.L. Shipp M.A. J. Immunol. 1992; 148: 2817-2825PubMed Google Scholar) and ECE-1 (7Valdenaire O. Rohrbacher E. Mattei M.G. J. Biol. Chem. 1995; 270: 29794-29798Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). One positive clone, pBAC-SEP, containing ∼150 kilobase pairs of genomic DNA was obtained and subjected to sequence analysis. This clone was purified and digested with several restriction enzymes and was run using a 0.7% agarose gel. The gel was Southern blotted and probed with32P-labeled oligonucleotides, which were contained within the putative insertion exon and its immediate upstream and downstream exons. Long and accurate PCR was performed on pBAC-SEP DNA to determine the size and location of the introns using Takara long and accurate PCR kit as described by the manufacturer. Oligonucleotides were derived from the mouse SEP cDNA sequence and were designed to span the putative insertion intron and its immediate downstream intron. The primers, 5′-GGGAGCCATAGTGACTCTGGGTGTC-3′ and 5′-TCGTTTTACAACCGTCCTCTCATCC-3′, were used for the putative insertion intron amplification, and primers, 5′-GGGAAGCAGCTGCCCCTCTTAACTA-3′ and 5′-GCTATCACACAGCTTGGGGTGGTGC-3′, were used to amplify an intron immediately upstream of the insertion intron. The PCR products were digested with restriction enzyme BamHI, thus resulting in four different fragments. All four fragments were then sequenced at both the 5′ and 3′ ends to verify correct oligonucleotide priming and to deduce the correct intron-exon boundaries. Automated sequencing was performed with a Model 310 DNA sequencer (Applied Biosystems). To construct the fusion genes of SEPΔ and bECE-1b, cDNAs for mouse SEPΔ(2Emoto N. Nurhantari Y. Alimsardjono H. Xie J. Yamada T. Yanagisawa M. Matsuo M. J. Biol. Chem. 1999; 274: 1509-1518Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and bECE-1b (3Emoto N. Yanagisawa M. J. Biol. Chem. 1995; 270: 15262-15268Crossref PubMed Scopus (430) Google Scholar) were used. For the generation of the pME-S/S/E chimera, the cytoplasmic domain (amino acids 1–17) and the transmembrane helix (residues 18–40) of mouse SEPΔ were fused in frame to the entire extracellular domain of bECE-1b (residues 78–758). Initially, site-directed mutagenesis was performed to introduce Eco47III and SmaI sites at the beginning of the extracellular domain of mouse SEPΔ and bECE-1b, respectively. The resulting pME-SEPΔ plasmid was digested with Eco47III and NotI to remove its luminal domain, whereas the mutant pME-bECE-1b plasmid was digested with SmaI and NotI to only give a fragment containing its luminal domain. These two fragments were then ligated in the correct reading frame. pME-E/E/S was constructed by fusing the cytoplasmic domain (amino acids 1–52) and the transmembrane domain (residues 53–73) of bECE-1b to the whole extracellular domain of mouse SEPΔ (residues 40–742) in two steps. The beginning of the extracellular domain of pME-bECE-1b and pME-SEPΔ was first mutagenized to containEcoRV and Eco47III sites, respectively. The mutant pME-bECE-1b plasmid was digested with EcoRV andXbaI to remove its luminal domain, whereas the mutant pME-SEPΔ plasmid was digested with Eco47III and XbaI to only isolate its luminal domain. These two fragments then were ligated to yield pME-E/E/S. The plasmid expressing the E/E/α/E chimeric protein was prepared by locating the insertion region (amino acids 41–63) of mouse SEP between the transmembrane and the extracellular domain of bECE-1b as follows. First, pME-bECE-1b was mutagenized to create SalI site at the beginning of its extracellular domain. Second, PCR amplification of the insertion region of SEP was performed using a sense primer containing a SalI site (underlined), 5′-GTCGACAGGGAAGCAGCTGCC-3′, and an antisense primer, 5′-GTCGACCGTTTTACAACCGTC-3′, including a SalI site in the 5′ end. The PCR product was digested with SalI and inserted into the plasmid pME-bECE-1b digested with the same enzyme. All mutants were verified by sequencing at the level of the final plasmid. Deletion constructs and amino acid-substituted constructs were made by site-directed mutagenesis (2Emoto N. Nurhantari Y. Alimsardjono H. Xie J. Yamada T. Yanagisawa M. Matsuo M. J. Biol. Chem. 1999; 274: 1509-1518Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) using the Muta-Gene Phagemid in vitro mutagenesis version 2 kit (Bio-Rad) as described by the manufacturer. All mutants were verified by DNA sequencing using primers both upstream and downstream of the insertion region. CHO-K1 cells were cultured as described previously (4Ikeda K. Emoto N. Raharjo S.B. Nurhantari Y. Saiki K. Yokoyama M. Matsuo M. J. Biol. Chem. 1999; 274: 32469-32477Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The coding region of mouse SEP or SEPΔwas subcloned into the pME18Sf(−) expression vector under the control of the SR α promoter (4Ikeda K. Emoto N. Raharjo S.B. Nurhantari Y. Saiki K. Yokoyama M. Matsuo M. J. Biol. Chem. 1999; 274: 32469-32477Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Stable transfection of CHO cells and isolation of the transfectant clones (CHO/SEP and CHO/SEPΔ) were performed as described previously (2Emoto N. Nurhantari Y. Alimsardjono H. Xie J. Yamada T. Yanagisawa M. Matsuo M. J. Biol. Chem. 1999; 274: 1509-1518Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Transient transfections of SEP, SEPΔ, and mutant cDNA were carried out using LipofectAMINE Plus (Life Technologies, Inc.) as described by the manufacturer. The cells were cultured for 30 h after transfecting the plasmid into the cells. Conditioned medium and postnuclear lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were probed with an antibody against the C terminus of mouse SEP (4Ikeda K. Emoto N. Raharjo S.B. Nurhantari Y. Saiki K. Yokoyama M. Matsuo M. J. Biol. Chem. 1999; 274: 32469-32477Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), bovine ECE-1 (2Emoto N. Nurhantari Y. Alimsardjono H. Xie J. Yamada T. Yanagisawa M. Matsuo M. J. Biol. Chem. 1999; 274: 1509-1518Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), or human microsomal triglyceride transfer protein (MTP) (8Sato R. Miyamoto W. Inoue J. Terada T. Imanaka T. Maeda M. J. Biol. Chem. 1999; 274: 24714-24720Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) and developed using the ECL Kit (Amersham Pharmacia Biotech) as recommended by the manufacturer. Cells were seeded onto coverslips and cultured for 2 days. Fluorescent immunocytochemistry was performed as described previously (2Emoto N. Nurhantari Y. Alimsardjono H. Xie J. Yamada T. Yanagisawa M. Matsuo M. J. Biol. Chem. 1999; 274: 1509-1518Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 4Ikeda K. Emoto N. Raharjo S.B. Nurhantari Y. Saiki K. Yokoyama M. Matsuo M. J. Biol. Chem. 1999; 274: 32469-32477Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Intracellular staining was performed as follows, cells were fixed and permeabilized in methanol for 5 min at −20 °C. After washing and blocking, the cells were probed with a polyclonal antibody directed against the mouse SEP C-terminal peptide (1:100), the bovine ECE-1 C-terminal peptide (1:200), or the human MTP (1:50). The cells were again washed before incubation with normal goat serum/phosphate-buffered saline containing 7.5 μg/ml fluorescein isothiocyanate-goat anti-rabbit IgG (Zymed Laboratories, Inc.). Finally, the coverslips were mounted on microscope slides with 90% (v/v) glycerol, 50 mm Tris-HCl, pH 9.0, and 2.5% (w/v) 1,4-diazadicyclo-[2.2.2]octane. Metabolic labeling and immunoprecipitation were performed as described previously (2Emoto N. Nurhantari Y. Alimsardjono H. Xie J. Yamada T. Yanagisawa M. Matsuo M. J. Biol. Chem. 1999; 274: 1509-1518Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). However, the soluble proteins in the supernatant were precipitated by the method of Wessel and Flugge (9Wessel D. Flugge U.I. Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3082) Google Scholar). The precipitates were then dissolved in 500 μl of phosphate-buffered saline and immunoprecipitated using SEP polyclonal antibody. Immunoprecipitates were analyzed on 7% SDS-PAGE and developed using the BAS2000 system. Conditioned medium and membrane fractions were used for digestion. Endo H and PNGaseF digestions were performed as described previously (4Ikeda K. Emoto N. Raharjo S.B. Nurhantari Y. Saiki K. Yokoyama M. Matsuo M. J. Biol. Chem. 1999; 274: 32469-32477Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), and endoglycosidase D was done at 37 °C for 16 h in 0.2 m phosphate buffer, pH 6.5, containing 0.1% Nonidet P-40. The samples were then subjected to immunoblotting. Previously, we have isolated and characterized two isoforms of mouse SEP (4Ikeda K. Emoto N. Raharjo S.B. Nurhantari Y. Saiki K. Yokoyama M. Matsuo M. J. Biol. Chem. 1999; 274: 32469-32477Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). SEP (765 residues) and SEPΔ (742 residues) differ only in the absence of a 23-amino acid insertion immediately following the transmembrane domain in SEPΔ, but both share the same N termini transmembrane domain and C-terminal residues. To check whether these two isoforms were produced by the same gene via an alternative splicing mechanism, the mouse SEP gene was cloned from mouse BAC genomic library using a PCR-generated32P-labeled probe spanning a putative exon area ofSEP that we predicted based on the structure of both theNEP (6Chen C.Y. Salles G. Seldin M.F. Kister A.E. Reinherz E.L. Shipp M.A. J. Immunol. 1992; 148: 2817-2825PubMed Google Scholar) and ECE-1gene (7Valdenaire O. Rohrbacher E. Mattei M.G. J. Biol. Chem. 1995; 270: 29794-29798Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). One positive clone, pBAC-SEP containing ∼150 kilobase pairs of genomic DNA was obtained and subjected to sequence analysis. Exon-specific primers were designed based on the predicted intron-exon boundaries and used to directly sequence the BAC clone. All deduced intron-exon boundaries indicate the canonical consensus splice donor and acceptor sequences in accordance with the GT/AG rule (10Shapiro M.B. Senapathy P. Nucleic Acids Res. 1987; 15: 7155-7174Crossref PubMed Scopus (1948) Google Scholar). The sequence analysis clearly showed that the exon encoding the 23-amino acid insertion of SEP is an independent exon separated from the immediate 5′ exon by a ∼9.5-kilobase pairs intron and from the next 3′ exon by a 555-base pair intron (Fig.1A). This transcript resulted in a 765-amino acid product of SEP. However, when this insertion exon skipped making its immediate 5′ exon join directly to the next 3′ exon, the 742 amino acids of SEPΔ were produced (Fig.1B). It then became apparent that this insertion exon coincided with an intron-exon boundary. These results suggest that these two isoforms of SEP mRNA originate from a single gene of the mouse genome by an alternative splicing mechanism. To characterize the features of both SEP and SEPΔ, we generated transfectant cells CHO/SEP and CHO/SEPΔ by transfecting expression constructs driven by the SR α viral promoter. Immunoblot analysis with an anti-SEP C-terminal peptide antiserum showed that only CHO/SEP and not CHO/SEPΔ cells release its soluble form with an apparent molecular mass of ∼126 kDa into the culture medium, whereas both membrane-bound forms are expressed as an approximate 110-kDa protein in the membrane preparation of these cells (4Ikeda K. Emoto N. Raharjo S.B. Nurhantari Y. Saiki K. Yokoyama M. Matsuo M. J. Biol. Chem. 1999; 274: 32469-32477Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). To further analyze these proteins, we then examined their sensitivity to Endo H and PNGaseF. Endo H digests N-glycans of a high mannose type. Resistance to Endo H digestion indicates that a glycoprotein moved from the ER to the Golgi compartment in which further modification to complex oligosaccharides occurs, whereas PNGaseF completely removes all N-linked oligosaccharides. Treatment of solubilized membranes from both CHO/SEP and CHO/SEPΔ cells with either Endo H or PNGaseF reduced the apparent molecular mass from 110- to 89-kDa, which corresponds to the calculated molecular mass of SEP, whereas N-linked glycosylation of the SEP soluble form was removed by PNGaseF and found to be resistant to Endo H (Fig.2A). These observations confirm that the 110-kDa species observed in the membrane fraction of the cells is the partially glycosylated protein present in the early secretory pathway. To precisely investigate the subcellular localization of the membrane-bound protein, we immunostained both CHO/SEP and CHO/SEPΔ cells with antibodies that recognize the common C-terminal ectodomain of SEP. After permeabilization, both cells showed strong intracellular staining. This intracellular staining pattern is indistinguishable from the results observed when we stained the microsomal triglyceride transfer protein, an ER resident protein, using a polyclonal antibody against human MTP (8Sato R. Miyamoto W. Inoue J. Terada T. Imanaka T. Maeda M. J. Biol. Chem. 1999; 274: 24714-24720Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). These intracellular staining patterns were typical ER patterns (Fig. 2B). Taken together, these data demonstrated that the membrane-bound SEP and SEPΔ are localized in the endoplasmic reticulum. To examine whether the protein localized in the ER by a retention or a retrieval mechanism, both SEP and SEPΔ proteins were subjected to endo-β-N-acetylglucosaminidase D digestion. Retention refers to the protein never being exported out of the ER. In the retrieval mechanism, however, proteins escape from the ER to thecis-Golgi where the oligosaccharide is converted from Man8GlcNAc2 to Man5GlcNAc2 by mannosidase-I and then retrieved to the ER (11Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1052) Google Scholar). Although Endo H digests both ER andcis-Golgi types of glycosylation, Endo D uniquely hydrolyzes only N-linked oligosaccharides of the Man5GlcNAc2 (12Honsho M. Mitoma J.Y. Ito A. J. Biol. Chem. 1998; 273: 20860-20866Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 13Nohturfft A. DeBose-Boyd R.A. Scheek S. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11235-11240Crossref PubMed Scopus (189) Google Scholar). As shown in Fig.2C, both SEP and SEPΔ were resistant to Endo D. In addition, it has been accepted that the incubation of cells with the microtubular inhibitor nocodazole alters the distribution of ER proteins by recycling from the intermediate compartment (14Szczesna-Skorupa E. Chen C.D. Kemper B. Arch. Biochem. Biophys. 2000; 374: 128-136Crossref PubMed Scopus (22) Google Scholar). We observed that the distribution of both SEP and SEPΔ were not altered by this treatment (data not shown). Therefore, these results suggest that these proteins localized in the ER by a retention mechanism. To analyze the features of the membrane-bound SEP/SEPΔ responsible for ER retention, we substituted the cytoplasmic transmembrane and luminal domains of this protein with the corresponding domains of bECE-1b (Fig.3A). The bECE-1b is another member of this metalloprotease family, and this protein is normally transported to the cell surface (2Emoto N. Nurhantari Y. Alimsardjono H. Xie J. Yamada T. Yanagisawa M. Matsuo M. J. Biol. Chem. 1999; 274: 1509-1518Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Because no differences were observed in the localization and/or retention of the membrane-bound SEP/SEPΔ, only the findings with SEPΔ are presented. Several reports have shown that the cytoplasmic domain of type II membrane proteins determine ER localization (15Fu J. Kreibich G. J. Biol. Chem. 2000; 275: 3984-3990Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 16Schutze M.P. Peterson P.A. Jackson M.R. EMBO J. 1994; 13: 1696-1705Crossref PubMed Scopus (267) Google Scholar, 17Nilsson T. Jackson M. Peterson P.A. Cell. 1989; 58: 707-718Abstract Full Text PDF PubMed Scopus (365) Google Scholar). Thus, we first analyzed a chimeric protein S/S/E (SEPΔ cytoplasmic, SEPΔ transmembrane, and bECE-1b luminal, Fig.3A) in which the cytoplasmic tail and transmembrane domain of SEPΔ were fused to the luminal domain of bECE-1b. When the subcellular localization of the chimeric protein was analyzed in transfected CHO cells unexpectedly, the S/S/E chimera was expressed on the cell surface as detected by immunofluorescence in permeabilized cells (Fig. 3B). This surface labeling in permeabilized cells was also observed in the wild-type bECE-1b, suggesting that like bECE-1b this chimera exits from the ER very efficiently, which results in high concentrations of the chimera at the cell surface. Nontransfected control cells did not react with the anti-ECE-1 polyclonal antibody (data not shown), consistent with the evidence that CHO cells have little or no endogenous activity of ECE-1 (2Emoto N. Nurhantari Y. Alimsardjono H. Xie J. Yamada T. Yanagisawa M. Matsuo M. J. Biol. Chem. 1999; 274: 1509-1518Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Therefore, it appears that in the absence of the SEPΔluminal domain, the cytoplasmic SEPΔ tail in combination with its transmembrane domain is not sufficient to achieve retention. We next asked whether only the luminal domain of SEPΔ is sufficient for the correct targeting of the protein. A chimeric construct in which both the cytoplasmic tail and transmembrane domain of SEPΔ were replaced by those of bECE-1b (E/E/S, ECE-1b cytoplasmic, ECE-1b transmembrane, and SEPΔ luminal, Fig.3A) was thus created. Immunofluorescence analysis of transient transfectants expressing E/E/S chimera showed the same distribution as the wild-type SEPΔ. This chimeric protein behaved similarly to wild-type SEPΔ as indicated by its typical internal staining pattern and its lack of cell surface staining (Fig. 3B). These data indicate that the SEPΔluminal domain, independent of the transmembrane domain, determines the localization of SEPΔ protein in the ER. We then confirmed these findings by endoglycosidase digestion. CHO-K1 cells were transfected with cDNAs encoding the wild-type SEP, bECE-1b, the chimera S/S/E, and E/E/S. After 30 h, the proteins were harvested and treated with Endo H and PNGaseF. SEP and E/E/S gave a single band of 110-kDa on SDS-PAGE, were sensitive to both Endo H and PNGaseF, and were converted to a band approximately 20 kDa smaller (Fig. 3C). On the other hand, bECE-1b and S/S/E gave a higher molecular mass band (126-kDa) aside from the 110-kDa band. The higher molecular mass bands were sensitive to PNGaseF but resistant to Endo H, whereas the smaller band was sensitive to both endoglycosidases as in the case of SEP, E/S/S, and E/E/S, indicating that proteins with higher molecular mass contain complex-type oligosaccharides (Fig. 3C). Together, these results clearly demonstrate that the luminal domain of SEP determines the localization of the SEP protein in the ER. The abnormally assembled proteins (misfolded proteins) in the ER are rapidly destroyed with the half-life of less than 1 h (18Tang B.L. Low S.H. Hong W. Eur. J. Cell Biol. 1997; 73: 98-104PubMed Google Scholar). Using CHO/SEPΔ stable transfectant cells, we provide a biochemical analysis of SEPΔ trafficking and turnover by pulse-chase experiments. The CHO/SEPΔ cells were pulse-labeled with 35S-amino acids for 30 min and followed by chase periods at designated times. At specific time intervals, cell extracts were prepared and immunoprecipitated with an anti-SEP antibody. Before analysis by SDS-PAGE, half of the samples were treated with Endo H. During all the chase periods, the proteins remained fully sensitive to Endo H, suggesting that it never acquired any Golgi enzyme modifications (Fig.4A). In addition, as shown in Fig. 4B, the membrane-bound SEPΔ was retained in the ER with a half-life of more than 2 h. Therefor
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