Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System
2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês
10.1074/jbc.m509715200
ISSN1083-351X
AutoresAkiko Okumura, Kiyotaka Hatsuzawa, Taku Tamura, Hisao Nagaya, Kazuko Saeki, Fumihiko Okumura, Kenji Nagao, Mitsuo Nishikawa, Akihiko Yoshimura, Ikuo Wada,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoThe cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to α-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomal-lysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes. The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to α-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomal-lysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes. Specific fusion of biological membranes is required for many cellular processes, including membrane trafficking between different organelles and within individual organelles, and is executed in eukaryotic cells by fusogenic soluble N-ethylmaleimide-sensitive factor (NSF) 3The abbreviations used are: NSF, N-ethylmaleimide-sensitive factor; ER, endoplasmic reticulum; ERGIC, ER-Golgi intermediate compartment(s); NLS, nuclear localization signal; UPR, unfolded protein response; SNAP, N-ethylmaleimide-sensitive factor attachment protein; SNARE, SNAP receptor; FL-REX, fluorescence localization-based retrovirus-mediated expression cloning; EGFP, enhanced green fluorescent protein; siRNA, small interfering RNA; NAC, N-acetylcysteine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. attachment protein (SNAP) receptors (SNAREs) (1.Blumenthal R. Clague M.J. Durell S.R. Epand R.M. Chem. Rev. 2003; 103: 53-69Crossref PubMed Scopus (242) Google Scholar). SNAREs are a group of membrane proteins that localize to and function in the diverse endomembrane system, where docking and fusion between membranes take place. All SNAREs are characterized by homologous stretches of 60-70 amino acids referred to as SNARE motifs, which are adjacent to the membrane anchor domains. SNAREs can be classified into subgroups: the syntaxin and SNAP-25 families contain a conserved glutamine at a central position called the "0" layer of the SNARE motif and are therefore called Q-SNAREs, and the VAMP (also called synaptobrevin) family contains a conserved arginine at this position and are therefore called R-SNAREs (2.Jahn R. Lang T. Sudhof T.C. Cell. 2003; 112: 519-533Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar). SNARE motifs contribute to the formation of an extended parallel four-helix bundle, termed the SNARE core complex (3.Chen Y.A. Scheller R.H. Nat. Rev. Mol. Cell Biol. 2001; 2: 98-106Crossref PubMed Scopus (878) Google Scholar). Assembly of the SNARE core complex first leads to a tight connection between two membranes and then drives lipid mixing and subsequent opening of a fusion pore (4.McNew J.A. Weber T. Parlati F. Johnston R.J. Melia T.J. Sollner T.H. Rothman J.E. J. Cell Biol. 2000; 150: 105-117Crossref PubMed Scopus (251) Google Scholar, 5.Ungar D. Hughson F.M. Annu. Rev. Cell Dev. Biol. 2003; 19: 493-517Crossref PubMed Scopus (232) Google Scholar). Although SNARE complexes are the critical machinery of membrane fusion, their role in determining the specific sites of fusion within the endomembrane system remains to be established. The early secretory pathway, which comprises the ER, the Golgi apparatus, and the intermediate compartments, is highly dynamic and tightly regulated. Cargo proteins are efficiently exported from the ER, and the constituents of the ER are retrieved by retrograde transport from the Golgi. In yeast, a variety of SNAREs or SNARE-related proteins have been implicated in the processes of retrograde (Ufe1p, Sec22p, Sec20p, and Tip20p) and anterograde (Sed5p, Sec22p, Bos1p, and Bet1p) transport (6.Pelham H.R. Exp. Cell Res. 1999; 247: 1-8Crossref PubMed Scopus (133) Google Scholar). Recently, Use1p/Slt1p was identified as a novel SNARE located in the ER. This is an essential protein and is thought to function in retrograde transport from the Golgi to the ER, because Use1p/Slt1p is associated with the retrograde SNAREs Ufe1p, Sec22p, and Sec20p but not with the anterograde SNAREs Bos1p and Bet1p (7.Dilcher M. Veith B. Chidambaram S. Hartmann E. Schmitt H.D. Fischer von Mollard G. EMBO J. 2003; 22: 3664-3674Crossref PubMed Scopus (75) Google Scholar, 8.Burri L. Varlamov O. Doege C.A. Hofmann K. Beilharz T. Rothman J.E. Sollner T.H. Lithgow T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9873-9877Crossref PubMed Scopus (83) Google Scholar). However, it was also shown that Use1p/Slt1p interacts with the farnesylated SNARE Ykt6p (8.Burri L. Varlamov O. Doege C.A. Hofmann K. Beilharz T. Rothman J.E. Sollner T.H. Lithgow T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9873-9877Crossref PubMed Scopus (83) Google Scholar). Because Ykt6p has been implicated in multiple intracellular transport pathways, particularly in homotypic fusion with vacuoles, an alternative possibility involving the later secretory pathway cannot be excluded. Recently, the mammalian homologues of yeast genes have been identified, including syntaxin 18, Sec22b, syntaxin 5, membrin, and rbet1, which in yeast correspond to Ufe1p, Sec22p, Sed5p, Bos1p, and Bet1p, respectively (9.Bennett M.K. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2559-2563Crossref PubMed Scopus (548) Google Scholar, 10.Nagahama M. Orci L. Ravazzola M. Amherdt M. Lacomis L. Tempst P. Rothman J.E. Sollner T.H. J. 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BNIP1 forms a SNARE complex with syntaxin 18, Sec22b, and p31 and participates in the formation of the ER network structure (16.Nakajima K. Hirose H. Taniguchi M. Kurashina H. Arasaki K. Nagahama M. Tani K. Yamamoto A. Tagaya M. EMBO J. 2004; 23: 3216-3226Crossref PubMed Scopus (107) Google Scholar). Interestingly, BNIP1 appears to be involved in an apoptosis pathway through the regulation of α-SNAP function (16.Nakajima K. Hirose H. Taniguchi M. Kurashina H. Arasaki K. Nagahama M. Tani K. Yamamoto A. Tagaya M. EMBO J. 2004; 23: 3216-3226Crossref PubMed Scopus (107) Google Scholar). However, the role of p31 in membrane trafficking and apoptosis is unclear. In this paper, we report the identification and characterization of a novel SNARE, D12, from murine hematopoietic stem cells using a modified FL-REX (fluorescence localization-based retrovirus-mediated expression cloning) method (17.Misawa K. Nosaka T. Morita S. Kaneko A. Nakahata T. Asano S. Kitamura T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3062-3066Crossref PubMed Scopus (65) Google Scholar). D12 shows significant homology to Use1p/Slt1p and p31. We show here that D12 is a Q-SNARE and, whereas it is mostly localized in the ER and ERGIC, it also binds to VAMP7, a SNARE involved in endosome-lysosome transport. Our analyses indicate that D12 plays no role in membrane trafficking in the early secretory pathway but rather is involved in the maintenance of proper lysosomal function. Cell Culture and Transfection—NIH3T3 and HEK293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. PLAT-E cells, a packaging cell line (18.Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1377) Google Scholar), were maintained in Dulbecco's modified Eagle's medium with 10% fetal calf serum containing blasticidin S (10 μg/ml) and puromycin (1 μg/ml). Cells were transfected by the calcium phosphate method with Cell Phect (Amersham Biosciences) or by the lipofection method using FuGENE 6 (Roche Applied Science) or Lipofectamine 2000 (Invitrogen). For retrovirus-mediated gene expression, NIH3T3 cells were infected with recombinant retroviruses produced by PLAT-E packaging cells as previously described (19.Sasaki A. Inagaki-Ohara K. Yoshida T. Yamanaka A. Sasaki M. Yasukawa H. Koromilas A.E. Yoshimura A. J. Biol. Chem. 2003; 278: 2432-2436Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Modified FL-REX to Search for Proteins in the Secretory Pathway—The murine hematopoietic stem cell cDNA was described previously (20.Masuhara M. Sakamoto H. Matsumoto A. Suzuki R. Yasukawa H. Mitsui K. Wakioka T. Tanimura S. Sasaki A. Misawa H. Yokouchi M. Ohtsubo M. Yoshimura A. Biochem. Biophys. Res. Commun. 1997; 239: 439-446Crossref PubMed Scopus (216) Google Scholar). The 3′ region of the cDNA was deleted by ExoIII digestion and inserted into a pMX vector containing the enhanced green fluorescent protein (EGFP) sequence followed by a nuclear localization signal (NLS) and an ER retention signal (KDEL) (pMX-cDNA-EGFP-NLS-KDEL). In detail, three tandem repeats of the NLS from the SV virus large T-antigen were excised from pECFP-Nuc (BD Biosciences), and the EGFP coding sequence was excised from pEGFP-C1 (Clontech). Twenty-four pooled plasmid DNA clones were transiently expressed in HEK293 cells in a 96-well dish using the standard calcium phosphate method and were examined with immunofluorescence microscopy. The cDNA pool that gave ER- or cytosolic localization of EGFP was divided into individual clones, and HEK293 cells were similarly screened for independent clones. Positive clones were isolated, and the inserted cDNAs were examined by genomic sequencing. Expression of Proteins and Imaging Analyses—The full-length cDNA of mouse D12 was obtained by PCR using the MC9 cDNA library as a template. Full-length or C-terminal-deleted mutant D12 (D12Δtm) were subcloned into the multicloning site of the pMX (21.Nosaka T. Kawashima T. Misawa K. Ikuta K. Mui A.L. Kitamura T. EMBO J. 1999; 18: 4754-4765Crossref PubMed Scopus (438) Google Scholar) and pCMV10 (Sigma) vectors. Expression vectors for mRFP1-tagged full-length D12 or the first 80 amino acids of D12 (D12-(1-80)) were also constructed by inserting the open reading frame into appropriate sites of pmRFP1-N1, in which the EGFP open reading frame of pEGFP-N1 (BD Biosciences) was replaced with the mRFP1 open reading frame (22.Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7877-7882Crossref PubMed Scopus (2015) Google Scholar). These plasmids were introduced into cells using either FuGENE6 (Roche Applied Science) or bead loading (fluorescence correlation spectroscopy analysis) (23.Nagaya H. Wada I. Jia Y.J. Kanoh H. Mol. Biol. Cell. 2002; 13: 302-316Crossref PubMed Scopus (65) Google Scholar). For immunostaining, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin for 1 min on ice or fixed and permeabilized with methanol at -20 °C essentially as described (24.Kamada A. Nagaya H. Tamura T. Kinjo M. Jin H.Y. Yamashita T. Jimbow K. Kanoh H. Wada I. J. Biol. Chem. 2004; 279: 21533-21542Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). After staining, the specimens were observed with confocal microscopy using an LSM PASCAL or LSM510metaConfoCor2 (Carl Zeiss). For counting the number of cells with a condensed nucleus, cells were stained with Hoechst33342 (1 μg/ml) and observed on an epifluorescence microscope, Eclips TE2000-U (Nikon, Tokyo, Japan), with a filter set for 4′,6-diamidino-2-phenylindole. The images were taken using cooled CCD Cascade 512B (Photometrics) and processed with MetaMorph (Universal Imaging). The diffusion time of D12-(1-80)-mRFP1 in living cells was estimated by using fluorescence correlation spectroscopy as described previously (24.Kamada A. Nagaya H. Tamura T. Kinjo M. Jin H.Y. Yamashita T. Jimbow K. Kanoh H. Wada I. J. Biol. Chem. 2004; 279: 21533-21542Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Antibodies—The antibodies used in this study were α-FLAG M2 (Sigma), α-GM130 (BD Bioscience), α-phospho-Ser51-eIF2α (Cell Signaling Technology), α-eIF2α (Cell Signaling Technology), α-PERK (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), α-ATF6 (IMGENEX), α-XBP-1 (Santa Cruz), α-HSP70 (Stressgen), α-cleaved caspase-3 (Cell Signaling), α-active BAX (Upstate Biotechnology, Inc., Lake Placid, NY), and α-GAPDH (Ambion). Antibodies against D12, Sec22b, syntaxin 5, syntaxin 18, calnexin, ERGIC53, and GRP78 were obtained by immunizing rabbits with the bacterially expressed GST fusion of each protein with the transmembrane domain deleted. Identification of D12-binding Proteins—For the proteomic screening of D12 binding proteins, 2.4 × 108 293T cells were transfected with the plasmid containing the transmembrane domain-deleted D12 (pCMV10-D12Δtm) using calcium phosphate precipitation. Fortyeight hours after transfection, the cells were harvested and lysed in lysis buffer (40 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mm EDTA, and protease inhibitor mixture (Nacalai; Japan)). The lysates were incubated on ice for 1 h and centrifuged, and the supernatant was collected and incubated with anti-FLAG M2 Affinity Gel (Sigma) at 4 °C for 12 h. The resins were washed five times with washing buffer (20 mm Tris-HCl (pH 7.4) and 150 mm NaCl) and eluted with 3×FLAG peptide (Sigma). The protein sample was concentrated and lysed with SDS-PAGE loading buffer containing 280 mm 2-mercaptoethanol. The sample was subjected to SDS-PAGE in a 10% gel, and the proteins were visualized by silver staining. The major bands were excised and subjected to in-gel digestion with trypsin, and the eluted peptides were loaded on liquid chromatography electrospray ionization tandem mass spectrometry (FINNIGAN LC Q DECA; Finnigan). The spectrophotometry data were searched for known proteins using Sequest software (25.Saeki K. Miura Y. Aki D. Kurosaki T. Yoshimura A. EMBO J. 2003; 22: 3015-3026Crossref PubMed Scopus (99) Google Scholar). Subcellular Fractionation of NIH3T3 Cells—NIH3T3 cells cultured in two 100-mm plates were washed twice with ice-cold phosphate-buffered saline. All subsequent steps were performed at 4 °C. Cells were harvested with a cell scraper in 0.6 ml of homogenization buffer (10 mm Tris-HCl (pH 7.5), 250 mm sucrose) and homogenized by passage 10 times through a 27-gauge needle on a 1-ml syringe. Unbroken cells and nuclei were removed by centrifugation at 1,200 × g for 5 min. The postnuclear supernatant was loaded on preformed Nycodenz (Sigma) gradients, which were prepared for the HITACHI S52ST rotor from initial discontinuous gradients (24, 19, 15, and 10% Nycodenz in 10 mm Tris-HCl (pH 7.5), 3 mm KCl, and 1 mm EDTA) that were allowed to diffuse in a horizontal position for 45 min at room temperature and then centrifuged for 4 h at 37,000 rpm in a Himac CS100GXL (Hitachi) ultracentrifuge to generate a nonlinear density gradient profile. The postnuclear supernatant was loaded on top of the gradient and centrifuged for 4 h at 37,000 rpm. Fourteen fractions were collected from the top, and the proteins in aliquots of the fractions were resolved by SDS-PAGE. The distribution of D12, syntaxin 18, Sec22b, GM130 (Golgi marker), ERGIC53 (ERGIC marker), and calnexin (ER marker) in the gradients was determined by immunoblotting using ECL (Amersham Biosciences). For fractionation using Percoll, NIH3T3 cells grown in a 100-mm dish were harvested and homogenized in homogenization buffer (5 mm HEPES/KOH, 0.25 m sucrose, pH 7.2) by passage 10 times through a 27-gauge needle on a 1-ml syringe. After centrifugation (1,200 × g, 10 min) to remove cell debris and nuclei, the postnuclear supernatant was combined with 90% Percoll solution containing 250 mm sucrose and 5 mm HEPES/KOH, pH 7.2, so that the final concentration of Percoll was 17%. This mixture was layered over a 2.5 m sucrose cushion and centrifuged at 29,000 × g for 75 min in a S52ST rotor (Hitachi) at 4 °C. Gradients were fractionated from the top into 16 fractions, and the proteins in aliquots of the fractions were resolved by SDS-PAGE. The distributions of cathepsin D, D12, and Sec22b in the gradients were analyzed by immunoblotting using ECL. Down-regulation of D12 Protein Expression by RNA Interference—The RNA duplexes used for targeting were mouse D12 siRNA1 (5′-CCUGAAAGUUCAAGCAAGUAAACCGAG-3′), D12 siRNA2 (5′-CCCAGAGUGUCAUCAAGAAGGACAAAG-3′), ERp72 siRNA (5′-GGAAUCGUUGAUUACAUGAUC), VIPL siRNA (5′-GUCAAACGUUCGAGUACUUGA), and control siRNA (5′-AAUGUAUGCGAUCGCAGACUU) (RNAi Co.). Transfection into NIH3T3 and HEK293 cells was performed using X-treme (Roche Applied Science) for NIH-3T3 cells or Lipofectamin 2000 (Invitrogen) for HEK293 cells according to the manufacturers' protocols. At 24-48 h after transfection, the cells were processed for immunoblotting or immunofluorescence assays. Identification of D12 Using an FL-REX Method Modified for Signal Sequence Trapping—The FL-REX method (fluorescence localization-based retrovirus-mediated expression cloning) (17.Misawa K. Nosaka T. Morita S. Kaneko A. Nakahata T. Asano S. Kitamura T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3062-3066Crossref PubMed Scopus (65) Google Scholar) was developed to clone cDNAs based on the cellular localization of cDNAs from an EGFP-fused cDNA library. To search for novel secreted or membrane-bound proteins, we modified the FL-REX method to include a signal sequence trap. To facilitate identification of proteins actively retained in the cytoplasmic compartment, we added two targeting signals, a classical nuclear localization signal (NLS) and a C-terminal ER retention signal (KDEL). When this EGFP-NLS-KDEL vector alone was expressed in cultured cells, it was confined to the nucleus. Hence, simply by examining the intracellular localization of expressed proteins with epifluorescence microscopy, this method should permit the identification of genes containing either a signal sequence or a membrane-anchoring domain. In addition, this should allow the identification of proteins with an affinity for extranuclear structures. We screened a total of 9000 clones of a murine hematopoietic stem cell cDNA library and observed several clones expressing the EGFP signal in the ER. These included SEP15 (a 15-kDa selenoprotein) (26.Korotkov K.V. Kumaraswamy E. Zhou Y. Hatfield D.L. Gladyshev V.N. J. Biol. Chem. 2001; 276: 15330-15336Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) and KDAP (kidney-derived aspartic protease-like protein, Napsin) (27.Mori K. Shimizu H. Konno A. Iwanaga T. Arch. Histol. Cytol. 2002; 65: 359-368Crossref PubMed Scopus (28) Google Scholar), both of which contain signal sequences. We also obtained clones encoding the cytoplasmic proteins Gng10 (guanine nucleotide binding protein γ 10), whose signal was mostly in the cytosol, and SAP18 (Sin3-associated polypeptide), whose signal was found in the ER (data not shown). Interestingly, we found a clone whose signal was partially expressed in the cytoplasm (Fig. 1A). Because this clone encoded the first 80 amino acids of a protein of unknown function, D12, we focused on its characterization. To identify this protein, we isolated the full-length clone of D12 from the cDNA library of a murine mast cell-derived cell line, MC9. Characterization of D12—D12 was originally identified from a cDNA library of mouse cultured bone marrow mast cells (GenBank™ accession number AF353245). According to the report, this is a 270-amino acid protein with two domains predicted to form coiled-coils, including two putative coiled-coil heptads in regions 13-39 and 206-243 identified with a 28-residue window width (28.Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3483) Google Scholar), and a C-terminal transmembrane domain (region 241-263) (Fig. 1B). When we sequenced the full-length D12 isolated from the MC9 cDNA library, we noticed the presence of a Ser after Ala153 due to the insertion of CAG after Cys469 in AF353245. The same insertion was observed in some cDNAs in the EST data base (BU841695 or BQ287737), whereas others (CB204034 or BY708461) had no such insertion. For alignment (Fig. 1C, see below), we used the sequence we obtained experimentally from the clone in the MC9 library, and we refer to this sequence, which contains the Ser insertion after Ala153, as D12. Using the SMART data base (available on the World Wide Web at smart.embl-heidelberg.de/), we predicted two regions with weak similarity to the syntaxin N-terminal domain (residues 64-189) and the t-SNARE motif (residues 166-237) (Fig. 1B). Mouse D12 shows a low level of homology to yeast Use1p/Slt1p (20.1% identity) and a high level of homology to the human uncharacterized protein MDS032 (GenBank™ accession number AF220052), except in its N-terminal extension of 11 amino acids and an internal region (residues 139-164) (overall identity 84.9%; Fig. 1B). D12-(1-80), which we initially identified by modified FL-REX, contains the first coiled-coil but no obvious hydrophobic signal sequence. To confirm that the observed weak cytoplasmic localization indicates the presence of a positive retention signal in extranuclear structures, we used fluorescence correlation spectroscopy to analyze whether the diffusional mobility of the fragment in the cytoplasm is restricted. We constructed D12-(1-80) tagged with a monomeric red fluorescent protein (mRFP1) at its N terminus (mRFP-D12-(1-80)) and expressed the construct in NIH3T3 cells. The intracellular localization was indistinguishable from that of the original EGFP fusion. When the diffusion time of mRFP-D12-(1-80) expressed in the cytoplasm was compared with that of mRFP using the one-component diffusion model, mRFP-D12-(1-80) showed 3-5-fold slower diffusion (data not shown), confirming that the cytoplasmic retention of the N-terminal fragment was actively maintained. Subcellular Localization of D12—Use1p/Slt1p was recently identified as a SNARE that functions in retrograde protein transport from the Golgi to ER in yeast (7.Dilcher M. Veith B. Chidambaram S. Hartmann E. Schmitt H.D. Fischer von Mollard G. EMBO J. 2003; 22: 3664-3674Crossref PubMed Scopus (75) Google Scholar, 8.Burri L. Varlamov O. Doege C.A. Hofmann K. Beilharz T. Rothman J.E. Sollner T.H. Lithgow T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9873-9877Crossref PubMed Scopus (83) Google Scholar). We therefore reasoned that mouse D12 may also function as a SNARE between the ER and Golgi. As a first step to investigate the subcellular distribution of D12 in NIH3T3 cells, a polyclonal D12 antibody was raised against the bacterially expressed protein lacking the transmembrane domain (GST-D12Δtm). The postnuclear supernatant of NIH3T3 cells was separated by Nycodenz gradient centrifugation, and each fraction was subjected to immunoblotting using antibodies against either D12 or the specific marker proteins of the ER (calnexin), ERGIC (ERGIC53/p58), and Golgi (GM130). As shown in Fig. 1C, calnexin was enriched in fractions 1-6, and GM130 in fractions 7-11. ERGIC53/p58, a protein that cycles between the ER, ERGIC, and Golgi, appeared to be enriched in fractions 7 and 8. Distribution of endogenous D12 was similar to that of calnexin. In contrast, more than one-third of syntaxin 18 was recovered in the lighter fractions. Enrichment in the lighter fractions was also observed for Sec22b, which is known to associate with the vesicular structures of ERGIC (29.Zhang T. Wong S.H. Tang B.L. Xu Y. Hong W. Mol. Biol. Cell. 1999; 10: 435-453Crossref PubMed Scopus (53) Google Scholar). Because we were unable to obtain high quality immunofluorescence images of endogenous D12 using our antibody, we elected to analyze the localization by another method. When Percoll gradient fractionation was used to separate dense vesicles, mature lysosomes (as indicated by cathepsin D) were well separated from the ER-Golgi-related vesicles (Sec22b) (Fig. 1D). However, D12 appeared to be absent in lysosomes, suggesting that the D12 protein is mostly located in the early secretory pathway that includes the ER and ERGIC. D12 Is a Novel SNARE Protein—To obtain information regarding the function of D12 in cells, we attempted to identify D12-binding proteins using immunoprecipitation and proteomics. A lysate of 2.4 × 108 293T cells overexpressing FLAG-tagged D12Δtm was used for immunoprecipitation with an anti-FLAG M2-agarose affinity gel. The eluate with the FLAG peptide was resolved by SDS-PAGE (Fig. 2A), and the major bands were excised from the gel to determine their amino acid sequences by in-gel tryptic digestion followed by liquid chromatography electrospray ionization tandem mass spectrometry. As shown in Fig. 2A, D12-bound proteins appeared to be classified into two groups, the ER chaperones, including calnexin and GRP78/BiP, and the ER-Golgi membrane trafficking proteins, including NSF, α-SNAP, SNAP-29, syntaxin 18, and ZW10. The specific binding between D12 and calnexin, however, was not confirmed by Western blotting with an anti-calnexin antibody in small scale immunoprecipitation experiments (data not shown), indicating that the binding of calnexin and GRP78/BiP to D12 in the large scale experiment may be artifacts caused by their protein-mediated chaperone function (30.Ihara Y. Cohen-Doyle M.F. Saito Y. Williams D.B. Mol. Cell. 1999; 4: 331-341Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). ZW10 is a spindle checkpoint protein associated with kinetochores and has also recently been reported to be a syntaxin 18-binding protein involved in membrane trafficking between the ER and Golgi (31.Hirose H. Arasaki K. Dohmae N. Takio K. Hatsuzawa K. Nagahama M. Tani K. Yamamoto A. Tohyama M. Tagaya M. EMBO J. 2004; 23: 1267-1278Crossref PubMed Scopus (145) Google Scholar). This binding profile as well as the sequence homology of D12 to Use1p/Slt1p prompted us to examine the possibility that D12 is a novel SNARE. We prepared detergent extracts of 293T cells co-expressing FLAG-D12 with N-terminally EGFP-tagged α-SNAP (EGFP-α-SNAP) and immunoprecipitated the FLAG-D12 using an anti-FLAG antibody. We used FLAG-syntaxin 18 and FLAG-p47 (32.Hatsuzawa K. Hirose H. Tani K. Yamamoto A. Scheller R.H. Tagaya M. J. Biol. Chem. 2000; 275: 13713-13720Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 33.Nagahama M. Suzuki M. Hamada Y. Hatsuzawa K. Tani K. Yamamoto A. Tagaya M. Mol. Biol. Cell. 2003; 14: 262-273Crossref PubMed Scopus (75) Google Scholar) as positive and negative controls, respectively. The immunoblots revealed that EGFP-α-SNAP co-precipitated with FLAG-D12 as well as with FLAG-syntaxin 18 but not with FLAG-p47 (Fig. 2B). In addition, even FLAG-D12Δtm lacking the transmembrane domain co-precipitated with EGFP-α-SNAP. It has been reported that the yeast D12 homologue Use1p/Slt1p interacts with ER-located SNAREs, such as Ufe1p and Sec22p (7.Di
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