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A Selective Interaction between OS-9 and the Carboxyl-terminal Tail of Meprin β

2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês

10.1074/jbc.m203986200

1083-351X

Larisa Litovchick, Elena Friedmann, Shmuel Shaltiel,

Nuclear Structure and Function

OS-9, a protein previously uncharacterized, was shown to interact specifically with the intracellular region of the membrane proteinase meprin β found in brush border membranes of kidney and small intestine. We have shown previously that this cytoplasmic region is indispensable for the maturation of meprin β, which included an endoplasmic reticulum (ER)-to-Golgi translocation. We characterized OS-9 and found that it is associated with ER membranes and that it is exposed to the cytoplasm. Consistent with the kinetics of maturation of meprin β, OS-9 associates with meprin β only transiently, coinciding with ER-to-Golgi transport of meprin β. The OS-9-binding site in the cytoplasmic domain of meprin β overlaps the region essential for this transport. We characterized alternatively spliced forms of rat and mouse OS-9, and we found that only the non-spliced form of OS-9 binds to meprin β, implicating the spliced out segment in the binding, and suggesting the possible mechanism of the regulation of OS-9 function. Taken together, our results indicated that OS-9 may be involved in the ER-to-Golgi transport of meprin β. Ubiquitous expression of OS-9 raises the possibility that it may interact with other membrane proteins that possess the cytoplasmic moiety homologous to that of meprin β during their ER-to-Golgi transition. OS-9, a protein previously uncharacterized, was shown to interact specifically with the intracellular region of the membrane proteinase meprin β found in brush border membranes of kidney and small intestine. We have shown previously that this cytoplasmic region is indispensable for the maturation of meprin β, which included an endoplasmic reticulum (ER)-to-Golgi translocation. We characterized OS-9 and found that it is associated with ER membranes and that it is exposed to the cytoplasm. Consistent with the kinetics of maturation of meprin β, OS-9 associates with meprin β only transiently, coinciding with ER-to-Golgi transport of meprin β. The OS-9-binding site in the cytoplasmic domain of meprin β overlaps the region essential for this transport. We characterized alternatively spliced forms of rat and mouse OS-9, and we found that only the non-spliced form of OS-9 binds to meprin β, implicating the spliced out segment in the binding, and suggesting the possible mechanism of the regulation of OS-9 function. Taken together, our results indicated that OS-9 may be involved in the ER-to-Golgi transport of meprin β. Ubiquitous expression of OS-9 raises the possibility that it may interact with other membrane proteins that possess the cytoplasmic moiety homologous to that of meprin β during their ER-to-Golgi transition. endoplasmic reticulum human OS-9 mouse OS-9 proteinase K reverse transcriptase phosphate-buffered saline endo-β-N-acetyglucosaminidase H Dulbecco's modified Eagle's medium maltose-binding protein green fluorescent protein open reading frame hemagglutinin glutathione S-transferase The kinase splitting membrane proteinase was discovered as an enzyme that specifically clips and inactivates protein kinase A in the preparations of the brush border membranes of the small intestine and kidney (1Alhanaty E. Patinkin J. Tauber-Finkelstein M. Shaltiel S. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3492-3495Crossref PubMed Scopus (47) Google Scholar, 2Alhanaty E. Shaltiel S. Biochem. Biophys. Res. Commun. 1979; 89: 323-332Crossref PubMed Scopus (45) Google Scholar). Subsequently, this proteinase was shown to be identical to a β subunit of meprin (3Chestukhin A. Muradov K. Litovchick L. Shaltiel S. J. Biol. Chem. 1996; 271: 30272-30280Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), a membrane metalloendoproteinase of the astacin family (4Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (357) Google Scholar). We therefore refer to it here as meprin β. The physiological role of meprin is not established yet; however, it has been implicated in the degradation of the subset of biologically active polypeptides (4Bond J.S. Beynon R.J. Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (357) Google Scholar). Proteolytic activity of meprin β is highly specific toward substrates that contain a cluster of acidic amino acids decorated with hydrophobic residues, such as found in the peptide hormone gastrin (5Chestukhin A. Litovchick L. Muradov K. Batkin M. Shaltiel S. J. Biol. Chem. 1997; 272: 3153-3160Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 6Bertenshaw G.P. Turk B.E. Hubbard S.J. Matters G.L. Bylander J.E. Crisman J.M. Cantley L.C. Bond J.S. J. Biol. Chem. 2001; 276: 13248-13255Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Meprins (meprin A, EC 3.4.24.18, and meprin B, EC 3.4.24.63) and oligomeric proteases are composed of two types of structurally similar subunits (α and β) that are targeted to the cell surface or are secreted (7Johnson G.D. Hersh L.B. J. Biol. Chem. 1992; 267: 13505-13512Abstract Full Text PDF PubMed Google Scholar, 8Bond J.S. Beynon R.J. Curr. Top. Cell. Regul. 1986; 28: 263-290Crossref PubMed Scopus (37) Google Scholar). Meprin A is composed of the disulfide-bridged dimers of α subunits, whereas meprin B is a heterodimer of α and β subunits. Higher multimeric structures formed by a non-covalent association of functionally active dimers of mouse meprin α were recently observed (9Ishmael F.T. Norcum M.T. Benkovic S.J. Bond J.S. J. Biol. Chem. 2001; 276: 23207-23211Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Despite the high sequence homology and similar domain structure, the α and β subunits of meprin undergo different post-translational processing. Meprin α (but not meprin β) undergoes proteolysis in the endoplasmic reticulum (ER),1 which results in the removal of its short carboxyl-terminal cytoplasmic tail as well as a transmembrane segment and an epidermal growth factor-like domain (cf. Fig. 1A) (10Johnson G.D. Hersh L.B. J. Biol. Chem. 1994; 269: 7682-7688Abstract Full Text PDF PubMed Google Scholar). The transmembrane and cytoplasmic domains of the immature α subunit of the human meprin mediate the retention of this subunit in the ER through an association with chaperones (11Hahn D. Lottaz D. Sterchi E.E. Eur. J. Biochem. 1997; 247: 933-941Crossref PubMed Scopus (24) Google Scholar). Contrary to that, the cytoplasmic domain of rat meprin β is indispensable for its ER-to-Golgi transport (12Litovchick L. Chestukhin A. Shaltiel S. J. Biol. Chem. 1998; 273: 29043-29051Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). Although the cytoplasmic tail of meprin β does not contain any of the previously characterized ER export signals (13Nishimura N. Balch W.E. Science. 1997; 277: 556-558Crossref PubMed Scopus (399) Google Scholar), its removal results in the entrapment of the truncated meprin β in the ER (12Litovchick L. Chestukhin A. Shaltiel S. J. Biol. Chem. 1998; 273: 29043-29051Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). This truncation did not affect the proteolytic activity and stability of meprin β, suggesting that this retention is not due to the incorrect folding of meprin β and is not mediated by ER chaperones. Mutant of meprin β, where the basic amino acids of the juxtamembrane region (682RRKYRKK688) were substituted with alanines, demonstrates a decreased rate of the ER-to-Golgi transport. A tyrosine-to-proline substitution (Y685P) in the middle of this cluster prevents the export of corresponding mutant from the ER, suggesting that this region of the cytoplasmic tail of meprin β is important for its recruitment into the transport vesicles that depart from the ER and may possess a novel ER export signal (12Litovchick L. Chestukhin A. Shaltiel S. J. Biol. Chem. 1998; 273: 29043-29051Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). In this report we describe a protein that selectively interacts with the carboxyl-terminal tail of meprin β. This protein binds to the region 674TLISVYCTRRKYRKKA689 of rat meprin β in a yeast two-hybrid assay (14Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4880) Google Scholar) and forms a transient complex with meprin β during its export from the ER when co-expressed in mammalian cells. This protein has a high degree of sequence identity with a product of a recently cloned human gene, OS-9 (15Su Y.A. Hutter C.M. Trent J.M. Meltzer P.S. Mol. Carcinog. 1996; 15: 270-275Crossref PubMed Scopus (55) Google Scholar), and represents a rat homologue of human OS-9. The mRNA of human OS-9 (hOS-9) is ubiquitously present in human tissues and overexpressed in certain sarcomas, but to the best of our knowledge, no function has been assigned thus far to the OS-9 gene product. Moreover, OS-9 has no significant homology with any of the functionally characterized proteins, although the cysteine-rich amino-terminal domain of OS-9 is highly similar to the protein fragments predicted from genomic sequences of yeast Saccharomyces cerevisiae(YD9609.11) and Caenorhabditis elegans (F48E8.4) (15Su Y.A. Hutter C.M. Trent J.M. Meltzer P.S. Mol. Carcinog. 1996; 15: 270-275Crossref PubMed Scopus (55) Google Scholar). Three alternatively spliced isoforms of hOS-9 mRNA were described (16Kimura Y. Nakazawa M. Yamada M. J. Biochem. (Tokyo). 1998; 123: 876-882Crossref PubMed Scopus (41) Google Scholar) (Fig. 1B). We found that only the non-spliced OS-9 binds to the cytoplasmic tail of meprin β, suggesting the different functional role of the alternatively spliced isoforms. By using antibodies raised against the carboxyl terminus of rat OS-9, we demonstrated that OS-9 is a peripheral membrane protein associated with the cytoplasmic side of the ER. These results, together with the fact that OS-9 interacts with the region in meprin β that is essential for its export from the ER, raise the possibility that OS-9 may be involved into the ER-to-Golgi transport of meprin β as well as other membrane proteins containing a motif similar to the OS-9-binding site in meprin β. Polyclonal antibodies against rat meprin β were prepared as described previously (5Chestukhin A. Litovchick L. Muradov K. Batkin M. Shaltiel S. J. Biol. Chem. 1997; 272: 3153-3160Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Antibodies against the His6-tagged 339-amino acid-long carboxyl-terminal part of rat OS-9 (OS-9/L1 fragment, expressed in Escherichia coli) were raised in guinea pigs using the immunization protocol described previously (17Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988: 92-116Google Scholar). Anti-OS-9 antibodies were affinity-purified using a Sepharose column with an immobilized (His)6-OS-9/L1. Anti-GST antibodies were a gift of J. Blechman. Other antibodies were from commercial sources as follows: rabbit antibody against the cytoplasmic domain of calnexin and mouse monoclonal antibody against the immunoglobulin binding protein BiP (Stressgen Biotechnologies Corp.); rabbit antibody against Rab1 and Rab2 (Calbiochem-Novabiochem); mouse monoclonal antibodies against β-COP and the lumenal domain of calnexin (Transduction Laboratories); mouse monoclonal antibody against the Golgi 58K protein (Sigma); and mouse monoclonal antibody against the HA epitope tag YPYDVPDYA (Covance). Secondary antibodies against guinea pig, rabbit, and mouse IgG (conjugated with horseradish peroxidase, Cy3TM, or Cy2TM) were from Jackson ImmunoResearch. The cDNA of rat meprin β (coding carboxyl-terminal segment674Thr–Phe704, see Fig. 1) was amplified by PCR using 5′-CTGGAATTCACCCTTATCAGCGTCT-3′ and 5′-TGCTGGATCCAGTTAATATTCAAAACG-3′ primers (containingEcoRI and BamHI sites, respectively). The resulting fragment was cloned into the pGBT9 vector (MatchmakerTM Two-hybrid System, CLONTECH Laboratories) in-frame with the DNA-binding domain of the GAL4 transcription activator, thus generating the cyt-β/pGBT9 plasmid. This plasmid, together with the rat embryonic brain cDNA library cloned in the pACT2 vector (18Soussan L. Burakov D. Daniels M.P. Toister-Achituv M. Porat A. Yarden Y. Elazar Z. J. Cell Biol. 1999; 146: 301-311Crossref PubMed Scopus (87) Google Scholar), was used for the sequential transformation of S. cerevisiaestrain HF7c by the lithium acetate method (cf.CLONTECH protocol). As many as 2 × 106 independent cDNA clones were plated on the selective synthetic medium lacking His, Trp, and Leu. Among 63 clones which grew on this medium, 13 were found to produce β-galactosidase. The library plasmid DNA was isolated from these clones and used for co-transformation of the second yeast strain, SFY526, together with the cyt-β/pGBT9. The β-galactosidase assay was used to further select 11 clones that were negative in this assay when co-transformed with the pGBT9 vector alone or with the pGBT9 vector carrying non-relevant inserts (SNF1, supplied with the MatchmakerTM System, and the 63Asn–Leu260 fragment of rat meprin β). Of 11 sequenced clones, 10 clones contained 1.6–1.8-kb fragments of the same cDNA sequence, highly homologous to the carboxyl-terminal part (positions 329–667) of the isoform 1 of human OS-9 (hOS-9) (GenBankTM accession number U41635). The longest insert, coding for a 339-amino acid segment following the GAL4 DNA binding domain in the correct reading frame, was designated L1 and used in further experiments. Fragments of interest were subcloned into the pACT2 and pGBT9 vectors and used for co-transformation of the yeast SFY526 cells as described above. Transformants were grown on the solid synthetic medium lacking Trp and Leu. A pool of 10–15 colonies from each transformation was used for inoculation of the liquid medium lacking Trp and Leu. These cultures were grown for 24 h and used for the liquid culture assay for β-galactosidase activity (19Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar), using the 0.5 mmchlorophenol red β-d-galactopyranoside (Roche Molecular Biochemicals) as a substrate. The formation of the reaction product was monitored at a wavelength of 565 nm, and the β-galactosidase activity was expressed in units defined as shown in Equation 1, γ­D­galactosidase unit=1000×A565/(t×V×A650)Equation 1 where t indicates time of incubation (min), andV indicates volume of culture added to the assay (ml). DNA fragments corresponding to the carboxyl-terminal tail of meprin β with deletions and substitutions were generated by the PCR-directed mutagenesis using the cDNA of the rat meprin β (3Chestukhin A. Muradov K. Litovchick L. Shaltiel S. J. Biol. Chem. 1996; 271: 30272-30280Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) or the full-length meprin β mutants (12Litovchick L. Chestukhin A. Shaltiel S. J. Biol. Chem. 1998; 273: 29043-29051Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar) as a template. The resulting fragments were subcloned into the pGBT9 vector (using the sameEcoRI and BamHI restriction sites as for cloning of the wild type bait fragment), and mutations were verified by DNA sequencing. For the RT-PCR analysis of OS-9 mRNA, total RNA was prepared from various mouse (Balb/c) or rat (Wistar) tissues, using the TRI Reagent (Sigma). The protein was also extracted and saved for Western blot analysis with anti-OS-9 antibodies. The first strand cDNA was synthesized from 5 μg of total RNA using 2 pmol of the antisense primer 5′-CACACCCACAGAGTTGCCCGAGAG-3′ (annealing with 3′-untranslated region of mOS-9), and 200 units of SuperScript II RNase H-minus Reverse TranscriptaseTM(Invitrogen). PCR was carried out using DyNAzyme II DNA polymerase (Finnzymes Oy), and 5′-GCGCCCATGGGGGAACAGGACCTGAAC-3′ (direct) and 5′-AGGCTCGAGTCAGAAGTCAAATTCATCC-3′ (reverse) primers containing NcoI and XhoI sites, respectively. PCR mixture was supplemented with 5% (v/v) Me2SO because of the high GC content of OS-9 cDNA. PCR products were purified and either subcloned into pGEM®-T Easy vector (Promega) or digested with the indicated enzymes for cloning into the pACT2 vector. In order to obtain the full-length isoform 1 of mOS-9, theBspMII–AflII restriction fragment of the mouse 1050-bp non-spliced RT-PCR product (subcloned into the pGEM®-T Easy vector) was inserted into the pcDNA3 vector carrying the complete mOS-9/isoform 2 cDNA using the same unique restriction sites flanking the splice site. For cloning of the 5′-end of rat OS-9, RT-PCR was performed on total RNA isolated from rat liver, as described above. The RT primer was designed to anneal with the bp 252–274 segment of the rat OS-9/L1 clone (5′-CATCCTCTTCTTCCACGAGACCC-3′). PCR primers were designed as follows: direct, 5′-CGGGTACCGCGGAAGATGGCGGCG-3′ (with KpnI site, derived from an identical region of human and mouse OS-9 preceding the first ATG codon), and reverse primer 5′-AGACCCCGGGGTTCACCACCC-3′ (annealing with bp 248–269 in OS-9/L1 clone). The amplified product of 1.2 kb was purified, subcloned into pGEM®-T Easy vector (Promega), and sequenced. Sequence analysis confirmed that the amplified cDNA fragment indeed contained the 5′-part of the rat OS-9 homologue (on the basis of the high homology with the mouse and human OS-9, and the identity of the 250 bp 3′-sequence of this cDNA with the 5′-part of the rat OS-9/L1 clone). The 5′-end of mouse OS-9 cDNA coding for amino acids 1–300 was subcloned into pcDNA3 vector with GFP fused in-frame at the carboxyl terminus (N-OS-9/GFP) and was used for fractionation studies. The fragment of rat meprin β corresponding to the bait used in the two-hybrid screening (amino acids 674Thr–Phe704) was expressed as an MBP fusion protein (designated as MBP-cyt). The rat OS-9 fragment corresponding to the L-1 clone was expressed as a GST fusion protein (GST-OS-9/L1). The fusion proteins were affinity-purified as recommended by the manufacturers of the corresponding expression systems (New England Biolabs and Amersham Biosciences) and dialyzed against 20 mm Tris-HCl buffer, pH 7.5, containing 100 mm NaCl and 5 mm MgCl2 (binding buffer). The MBP-β-galactosidase fusion protein (MBP-gal) was used as a control for nonspecific interaction. For the binding assay, 0.5 μg of MBP-cyt and MBP-gal proteins was incubated with the 0.1 μg of purified recombinant GST-OS-9/L1 for 1 h at 4 °C. Then the amylose resin (10 μl of a 50% slurry) was added to the reaction mixture (50 μl) and further incubated for 1 h at 4 °C. The resin was intensively washed with the binding buffer, containing 1% Nonidet P-40, prior to the elution with the binding buffer containing 10 mm maltose. The eluates were then analyzed by SDS-PAGE and Western blotting with anti-GST antibodies. The DNA fragments of interest subcloned in the pcDNA3 vector (Invitrogen) were used for transient and stable transfection of HEK 293 cells by the LipofectAMINETMreagent (Invitrogen). The expression of the proteins was analyzed by Western blot with specific antibodies or as specified in the text and in the legends to the figures. For metabolic labeling, subconfluent cells were incubated in a methionine-free DMEM for 1–3 h before the assay. [35S]Methionine (50 μCi/ml, Amersham Biosciences) was added to the same medium for 1 h. The cells then were rinsed with PBS and lysed in an IP buffer containing a 50 mm Tris buffer, pH 8.0, supplemented with 1% Brij 97, 10% glycerol, 150 mm NaCl, and proteinase inhibitor mixture (Calbiochem-Novabiochem). Immunoprecipitation was carried out as described earlier (12Litovchick L. Chestukhin A. Shaltiel S. J. Biol. Chem. 1998; 273: 29043-29051Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The HEK 293 cells (stably transfected by the pcDNA3 vector carrying the full-length isoform 1 of mOS-9) were transiently transfected by meprin β, using LipofectAMINETM reagent. Twenty four hours after transfection, the cells were re-plated onto four identical 10-cm dishes and grown for an additional 48 h. Then the cells were incubated in serum-free DMEM lacking methionine for 1 h and pulse-labeled by the addition of [35S]methionine (1 mCi/ml) for 15 min. The labeling medium was then replaced with complete DMEM containing 10% fetal calf serum, and the cells were further incubated for different times prior to lysis in the IP buffer (see above). The clarified cell extracts were then subjected to immunoprecipitation as follows: 20% of each lysate was directly immunoprecipitated by anti-meprin β antibodies and used for Endo-H (Roche Molecular Biochemicals) assay, as described earlier (12Litovchick L. Chestukhin A. Shaltiel S. J. Biol. Chem. 1998; 273: 29043-29051Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). The remaining extracts were used to detect the meprin β associated with OS-9. The protein complexes were first immunoprecipitated with anti-OS-9 antibodies, washed 5 times with cold IP buffer, and released from the protein A beads by boiling for 10 min in a 10 mm Tris-HCl buffer, pH 7.5, containing 2% SDS and 30 mm dithiothreitol. Of each eluate, 10% was saved for detection of metabolically labeled OS-9. The remaining samples were diluted 10 times with IP buffer and subjected to a second immunoprecipitation using anti-meprin β antibodies. The radioactively labeled proteins were analyzed by SDS-PAGE and fluorography using an AmplifyTM solution and a HyperfilmTM MP autoradiography film (AmershamBiosciences). Staining of GH3 cells (rat pituitary epithelial cell line), and transiently transfected COS-7 cells grown on glass coverslips was performed as described previously (12Litovchick L. Chestukhin A. Shaltiel S. J. Biol. Chem. 1998; 273: 29043-29051Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). In order to study the topology of OS-9, the plasma membrane of NIH 3T3 fibroblasts was selectively permeabilized using low concentration of digitonin (20Diaz E. Pfeffer S.R. Cell. 1998; 93: 433-443Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Briefly, cells were fixed by 4% paraformaldehyde for 20 min, washed three times by PBS, and incubated for 15 min at 4 °C with a solution containing 5 μg/ml digitonin, 0.1 m KCl, 2.5 mm MgCl2, and 10 mm Hepes, pH 6.9. The cells were then washed three times with PBS and incubated with 4% normal donkey serum in PBS for 30 min at 22 °C to block the nonspecific binding sites. Control cells were treated as above, but 0.2% saponin was included to the blocking solution to achieve complete permeabilization of the cell membranes. The cells were incubated for 1 h at 22 °C with anti-OS-9 (1:200 serum or 1 μg/ml affinity purified antibodies), anti-BiP (mouse monoclonal, 1:200), and anti-calnexin cytoplasmic domain (rabbit polyclonal, 1:200) antibodies. Donkey anti-rabbit IgG-Cy2, anti-mouse IgG-Cy2, and anti-guinea pig IgG-Cy3 (all diluted 1:200 in PBS) were used to visualize cross-reacting material. The coverslips were mounted using FluorSaveTM reagent (Calbiochem-Novabiochem), observed using a microscope equipped with an epifluorescence attachment EFD-3 (Nikon) and photographed. For double-staining experiments, the colored images were prepared using the SPOT video attachment (Diagnostic Instruments, Inc.) and processed using the Adobe® Photoshop® 5.5 software (Adobe Systems Inc.). Subconfluent NIH 3T3 fibroblasts were rinsed with ice-cold PBS and scraped into a homogenization buffer containing a mixture of proteinase inhibitors (Calbiochem-Novabiochem) in PBS. Cells were homogenized by 10 passages through a 28½-gauge needle and centrifuged at 600 × g for 5 min at 4 °C in order to remove unbroken cells and nuclei. The resulting supernatant was further centrifuged at 100,000 × g for 1 h at 4 °C in a Beckman OptimaTM LE-80K ultracentrifuge (Beckman Instruments), and the supernatant (cytosol) and pellet (membranes) were collected. Extraction with precondensed Triton X-114 was carried as described earlier (21Bordier C. J. Biol. Chem. 1981; 256: 1604-1607Abstract Full Text PDF PubMed Google Scholar). Proteins from the detergent and aqueous phases were concentrated by precipitation with 5% trichloroacetic acid for 30 min (4 °C) before SDS-PAGE. For alkaline extraction, membranes were resuspended in 0.2 m Na2CO3, pH 11.5 (22Fujiki Y. Hubbard A.L. Fowler S. Lazarow P.B. J. Cell Biol. 1982; 93: 97-102Crossref PubMed Scopus (1385) Google Scholar), incubated on ice for 30 min, and centrifuged at 100,000 × g for 1 h at 4 °C. The pellets were resuspended in the SDS-PAGE loading buffer, and the proteins from the supernatants were precipitated by trichloroacetic acid as described above. Proteinase K (PK) digestion was performed as described previously (23Lin P., Le, N.H. Hofmeister R. McCaffery J.M. Jin M. Hennemann H. McQuistan T., De, V.L. Farquhar M.G. J. Cell Biol. 1998; 141: 1515-1527Crossref PubMed Scopus (140) Google Scholar) using 20 μg of membranes as starting material for incubation with 0.2 or 2 μg of PK (Roche Molecular Biochemicals). The reaction products were separated by SDS-PAGE and analyzed by immunoblotting with the appropriate antibodies. Subcellular fractionation of rat liver was performed as described previously (24Fleischer S. Kervina M. Methods Enzymol. 1974; 31: 6-41Crossref PubMed Scopus (362) Google Scholar). The rat liver was homogenized in 5 volumes (v/w) of cold homogenization buffer (buffer H), containing 0.25 m sucrose and proteinase inhibitors mixture (Calbiochem-Novabiochem) in 10 mm Hepes, pH 7.5. The homogenate was spun at 960 × g for 10 min in order to remove nuclei, mitochondria, and plasma membranes. The supernatant (S1) was centrifuged at 34,000 × g for 10 min; the pellet was discarded, and the supernatant (S2) was spun at 50,000 × g for 30 min. The resulting pellet (P3) containing the heavy microsomal fraction was collected, and the supernatant (S3) was centrifuged at 200,000 × g for 60 min. The supernatant was collected to represent the cytosol (S4) and the pellet (P4) to represent a light microsomal fraction. The heavy microsomal fraction was resuspended by gentle homogenization in 10 mm Hepes, pH 7.5, containing 52% sucrose, adjusted to a 1.22 m sucrose, and loaded under a sucrose step gradient (1.15, 0.86, and 0.25 m sucrose). The gradient was centrifuged for 3 h at 82,500 × g and carefully unloaded from the top. Protein Identification and Analysis Tools available in the ExPASy Server (Geneva University, expasy.hcuge.ch/www/tools.html) were used to characterize proteins of interest. Multiple alignments of the DNA and protein sequences were performed using the Clustal method and MegAlign software (DNAstar Inc.). The prediction of coiled-coil regions was performed using the COILS program (25Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3483) Google Scholar) available at www.ch.embnet.org (MTIDK matrix with a window width of 21). To elucidate the role of the intracellular (carboxyl terminus) moiety of meprin β, we attempted to identify proteins that might interact with this tail. Yeast two-hybrid screening of a rat cDNA library using the segment674Thr–Phe704 of rat meprin β as a bait (Fig. 1A) revealed 11 specific interacting clones, 10 of which contained overlapping fragments of the same cDNA sequence. Data base search revealed that the protein encoded by this cDNA is highly homologous to the carboxyl-terminal part (positions 329–667) of the isoform 1 of human OS-9 (hOS-9) (GenBankTM accession number U41635) (Fig. 1B). Computer analysis of the hOS-9 protein did not detect any similarity with known functional domains or characterized proteins. The cysteine-rich amino-terminal region of hOS-9 has a significant homology with the ORFs deduced from the genomic sequences of S. cerevisiae (YD9609.11), Arabidopsis thaliana(U41635), C. elegans (F48E8.4), and fruit fly (AAF53149.1). A secondary structure prediction identified a carboxyl-terminal region in OS-9 that has a high probability for a coiled-coil structure (Fig.1B). The interaction between the cytoplasmic fragment of meprin β and OS-9/L1 fragment was confirmed using an in vitro binding assay. The MBP-tagged fragment of meprin β corresponding to the bait used in the two-hybrid screening (MBP-cyt) and GST-tagged OS-9/L1 fragment were expressed in bacteria, purified, and used an in vitro binding assay (see "Experimental Procedures"). Purified MBP-gal fusion protein containing a 20-kDa fragment of β-galactosidase was used as a control. As shown in the Fig.2A, GST-OS-9/L1 specifically interacts with MBP-cyt. The beads containing the control MBP-gal protein adsorbed a negligible amount of GST-OS-9/L1. This result indicates that the rat OS-9 fragment forms a complex with the cytoplasmic tail of meprin β in vitro and that no additional components are required for this interaction to occur. To identify the region in the carboxyl-terminal tail of meprin β that is involved in binding to OS-9/L1, we prepared a series of mutant baits with deletions and alanine substitutions, and we compared them in the OS-9 binding assay. As shown in Fig. 2B, under the conditions of our experiment, the segment 674Thr–Lys688 in the meprin β tail is both necessary and sufficient for the binding of OS-9. For example the mutant obtained by truncation at Ala689(number 2) retains full (in fact somewhat enhanced) binding capacity, whereas the mutants with a truncation at Tyr679(number 5) or with a 674Thr–Lys688 deletion (number 3) have a negligible binding capacity. Furthermore, mutants in which the cluster of basic amino acids was substituted by alanines, in part (numbers 8 and 9) or completely (number 7), were found to have a reduced or a complete loss of binding capacity. Although these findings indicate an important contribution of this cluster to the binding of OS-9, they do not restrict the binding site to this segment, because the binding capacity of meprin β also is abolished upon substitution by ala