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• W2068688570 abstract "How endoplasmic reticulum (ER) proteins that are substrates for the ER-associated degradation (ERAD) pathway are recognized for polyubiquitination and proteasomal degradation is largely unresolved. Inositol 1,4,5-trisphosphate receptors (IP3Rs) form tetrameric calcium channels in ER membranes, whose primary role is to control the release of ER calcium stores, but whose levels are also regulated, in an activation-dependent manner, by the ERAD pathway. Here we report that the ER membrane protein SPFH1 and its homolog SPFH2 form a heteromeric ∼2 MDa complex that binds to IP3R tetramers immediately after their activation and is required for their processing. The complex is ring-shaped (diameter ∼250Å), and RNA interference-mediated depletion of SPFH1 and SPFH2 blocks IP3R polyubiquitination and degradation. We propose that this novel SPFH1/2 complex is a recognition factor that targets IP3Rs and perhaps other substrates for ERAD. How endoplasmic reticulum (ER) proteins that are substrates for the ER-associated degradation (ERAD) pathway are recognized for polyubiquitination and proteasomal degradation is largely unresolved. Inositol 1,4,5-trisphosphate receptors (IP3Rs) form tetrameric calcium channels in ER membranes, whose primary role is to control the release of ER calcium stores, but whose levels are also regulated, in an activation-dependent manner, by the ERAD pathway. Here we report that the ER membrane protein SPFH1 and its homolog SPFH2 form a heteromeric ∼2 MDa complex that binds to IP3R tetramers immediately after their activation and is required for their processing. The complex is ring-shaped (diameter ∼250Å), and RNA interference-mediated depletion of SPFH1 and SPFH2 blocks IP3R polyubiquitination and degradation. We propose that this novel SPFH1/2 complex is a recognition factor that targets IP3Rs and perhaps other substrates for ERAD. The endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; TM, transmembrane; DRM, detergent-resistant membrane; TEM, transmission electron microscopy; HA, hemagglutinin; GnRH, gonadotropin-releasing hormone; ET1, endothelin 1; BN, blue native; shRNA, short hairpin RNA; CC, coiled-coil; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.-associated degradation (ERAD) pathway targets aberrant proteins, including irreversibly misfolded proteins and unassembled subunits of multiprotein complexes, for degradation by the ubiquitin-proteasome system (1Vembar S.S. Brodsky J.L. Nat. Rev. Mol. Cell Biol... 2008; 9: 944-957Google Scholar). Intriguingly, several ER-resident proteins that are stable under normal conditions are also processed by the ERAD pathway. For example, 3-hydroxy-3-methylglutaryl CoA-reductase, the rate-limiting enzyme in sterol synthesis, is targeted for ERAD when sterols are in excess (2DeBose-Boyd R.A. Cell Res... 2008; 18: 609-621Google Scholar), and inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which form tetrameric, IP3- and Ca2+-gated Ca2+ channels in mammalian ER membranes (3Foskett J.K. White C. Cheung K.H. Mak D.D. Physiol. Rev... 2007; 87: 593-658Google Scholar), are degraded by ERAD when persistently activated (4Wojcikiewicz R.J.H. Trends Pharmacol. Sci... 2004; 25: 35-41Google Scholar). ERAD substrates appear to be processed via four steps: recognition, retrotranslocation, polyubiquitination, and proteasomal degradation. Recognition can be prompted in various ways, either by generic signals (e.g. surface-exposed hydrophobic patches) or by specific recognition factors (e.g. Insigs, which target mammalian 3-hydroxy-3-methylglutaryl CoA-reductase for ERAD) (2DeBose-Boyd R.A. Cell Res... 2008; 18: 609-621Google Scholar, 5Ravid T. Hochstrasser M. Nat. Rev. Mol. Cell Biol... 2008; 9: 679-689Google Scholar). Following recognition, ERAD substrates are retrotranslocated to the cytosol through an as yet unidentified pore, apparently in concert with the cytosolic AAA ATPase p97, which couples ATP hydrolysis to extraction (6Ye Y. Meyer H.H. Rapoport T.A. J. Cell Biol... 2003; 162: 71-84Google Scholar). Once exposed to the cytosol, substrates are polyubiquitinated. E2s (ubiquitin-conjugating enzymes) and E3s (ubiquitin-protein ligases) impart selectivity to substrate ubiquitination, and several are known to be involved in the ERAD pathway, including the E2s Ubc6 and Ubc7, and the E3s yeast Hrd1p and mammalian Hrd1 and Gp78 (7Kostova Z. Tsai Y.C. Weissman A.M. Semin. Cell Dev. Biol... 2007; 18: 770-779Google Scholar). Finally, polyubiquitinated substrates are delivered to the 26 S proteasome either by shuttle proteins that bind both ubiquitin and the 19 S regulatory cap of the proteasome (8Raasi S. Wolf D.H. Semin. Cell Dev. Biol... 2007; 18: 780-791Google Scholar), or by directly interacting with intrinsic subunits of the 19 S cap that contain ubiquitin-binding motifs (9Husnjak K. Elsasser S. Zhang N. Chen X. Randles L. Shi Y. Hofmann K. Walters K.J. Finley D. Dikic I. Nature.. 2008; 453: 481-488Google Scholar). It appears that some of the aforementioned steps are integrated, because multiprotein complexes that can carry out more than one step are being defined. For example, a complex centered around Hrd1 contains proteins that recognize, polyubiquitinate, and perhaps even retrotranslocate ERAD substrates (10Carvalho P. Goder V. Rapoport T.A. Cell.. 2006; 126: 361-373Google Scholar, 11Christianson J.C. Shaler T.A. Tyler R.E. Kopito R.R. Nat. Cell Biol... 2008; 10: 272-282Google Scholar, 12Lilley B.N. Ploegh H.L. Proc. Natl. Acad. Sci. U. S. A... 2005; 102: 14296-14301Google Scholar-13Ye Y. Shibata Y. Kikkert M. van Voorden S. Wiertz E. Rapoport T.A. Proc. Natl. Acad. Sci. U. S. A... 2005; 102: 14132-14138Google Scholar). IP3Rs participate in a wide range of cellular processes, e.g. fertilization, secretion, apoptosis, and development (3Foskett J.K. White C. Cheung K.H. Mak D.D. Physiol. Rev... 2007; 87: 593-658Google Scholar). The three mammalian IP3R subtypes (IP3R1, IP3R2, and IP3R3) are each ∼2700 amino acids in length, are tethered to the ER membrane by 6 transmembrane (TM) domains, assemble into homo- and heterotetramers, and are expressed in varying proportions in different tissues (3Foskett J.K. White C. Cheung K.H. Mak D.D. Physiol. Rev... 2007; 87: 593-658Google Scholar). They are activated by cell surface receptors that generate IP3, with binding of IP3 and the co-agonist Ca2+ inducing a conformational change that causes channel opening (3Foskett J.K. White C. Cheung K.H. Mak D.D. Physiol. Rev... 2007; 87: 593-658Google Scholar). Because persistent activation of IP3Rs leads to their degradation, this conformational change also likely serves as a recognition signal for ERAD (4Wojcikiewicz R.J.H. Trends Pharmacol. Sci... 2004; 25: 35-41Google Scholar). This feature makes IP3Rs particularly valuable for studying ERAD, because activation almost instantaneously converts them from stable proteins into ERAD substrates. Thus, we have identified several mediators of IP3R ERAD, notably mammalian Ubc7 (14Webster J.M. Tiwari S. Weissman A.M. Wojcikiewicz R.J.H. J. Biol. Chem... 2003; 278: 38238-38246Google Scholar), the p97-Ufd1-Npl4 complex (15Alzayady K.J. Panning M.M. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2005; 280: 34530-34537Google Scholar), and most recently, SPFH2 (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar). SPFH2, also known as erlin-2 (17Browman D.T. Resek M.E. Zajchowski L.D. Robbins S.M. J. Cell Sci... 2006; 119: 3149-3160Google Scholar), belongs to a family of ∼100 mammalian proteins (“SPFH proteins”) that contain an SPFH domain, an ∼250-amino acid motif named because of minor sequence similarities in the proteins stomatin, prohibitin, flotillin, and HflC/K (18Browman D.T. Hoegg M.B. Robbins S.M. Trends Cell Biol... 2007; 17: 394-402Google Scholar). SPFH proteins tend to have similar properties, including localization to cholesterol-rich, detergent-resistant membranes (DRMs) and assembly into large oligomeric structures (18Browman D.T. Hoegg M.B. Robbins S.M. Trends Cell Biol... 2007; 17: 394-402Google Scholar). However, no universal function has yet been attributed to the SPFH domain, and SPFH proteins have distinct subcellular localizations and roles. For example, stomatin, a plasma membrane protein, binds to and regulates acid-sensing ion channels (19Price M.P. Thompson R.J. Eshcol J.O. Wemmie J.A. Benson C.J. J. Biol. Chem... 2004; 279: 53886-53891Google Scholar), and prohibitins-1 and -2 are found primarily in the inner mitochondrial membrane, where they carry out a variety of functions (20Merkwirth C. Langer T. Biochim. Biophys. Acta.. 2009; 1793: 27-32Google Scholar). Intriguingly, two plasma membrane SPFH proteins, MEC-2 from Caenorhabditis elegans and the mammalian stomatin-like protein, podocin, directly bind cholesterol via their SPFH domains, and it is likely that all SPFH proteins have this property (21Huber T.B. Schermer B. Müller R.U. Höhne M. Bartram M. Calixto A. Hagmann H. Reinhardt C. Koos F. Kunzelmann K. Shirokova E. Krautwurst D. Harteneck C. Simons M. Pavenstädt H. Kerjaschki D. Thiele C. Walz G. Chalfie M. Benzing T. Proc. Natl. Acad. Sci. U. S. A... 2006; 103: 17079-17086Google Scholar). Binding to cholesterol undoubtedly relates to the localization of SPFH proteins in DRMs and suggests that SPFH proteins either recruit cholesterol to membrane microdomains or are restricted by cholesterol to DRMs, where they play other roles. Although the cholesterol content of intracellular membranes is minimal (22Prinz W. Semin. Cell Dev. Biol... 2002; 13: 197-203Google Scholar), SPFH2 and a closely related protein, SPFH1, also known as erlin-1, localize to putative ER-derived DRMs in a cholesterol-dependent manner (17Browman D.T. Resek M.E. Zajchowski L.D. Robbins S.M. J. Cell Sci... 2006; 119: 3149-3160Google Scholar). Here, we report that SPFH1 and SPFH2 form an ∼2-MDa ER membrane complex that binds to IP3Rs immediately following their activation and is required for their ERAD. Transmission electron microscopy (TEM) of endogenous SPFH1/2 complexes reveals a double ring-shaped structure with an overall diameter of ∼250 Å. We propose that the SPFH1/2 complex plays a role in the recognition and targeting of IP3Rs and perhaps other substrates for ERAD. Materials—αT3-1, HeLa, and Rat-1 cells were cultured as described (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar). Already available antibodies used were: rabbit polyclonal anti-IP3R1 (23Wojcikiewicz R.J. J. Biol. Chem... 1995; 270: 11678-11683Google Scholar), anti-Hrd1, anti-SPFH2 (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar), and anti-α-transaldolase (a kind gift from Dr. A. Perl, SUNY Upstate Medical University, Syracuse, NY); mouse monoclonal anti-ubiquitin clone FK2 (Bio-Mol International), anti-hemagglutinin (HA) epitope clone HA11 (Covance), anti-p97 (Research Diagnostics, Inc.), anti-FLAG epitope clone M5 (Sigma), anti-IP3R3 (BD Transduction Laboratories), and anti-human SPFH1 (a kind gift from Dr. S. M. Robbins, University of Calgary, Calgary, Alberta); rat monoclonal anti-grp94 (Stress-Gen); and horseradish peroxidase- and fluorophore-conjugated secondary antibodies (Sigma). Rabbit polyclonal anti-SPFH1 (which recognizes mouse and rat but not human SPFH1) and anti-FLAG were generated against synthetic peptides corresponding to the rodent SPFH1 C terminus (EPSGESPIQNKENAG) and the FLAG epitope (DYKDDDDK), respectively, and were affinity purified as described (23Wojcikiewicz R.J. J. Biol. Chem... 1995; 270: 11678-11683Google Scholar). Coomassie G-250, digitonin, Fura2-AM, gonadotropin-releasing hormone (GnRH), hygromycin B, N-ethylmaleimide, Polybrene, protease inhibitors, puromycin, Triton X-100, and urea were purchased from Sigma; endothelin 1 (ET1) was from Calbiochem; endoglycosidase H was from New England Biolabs; bisacrylamide, dithiothreitol, Precision Plus™ Protein standards, and SDS were from Bio-Rad; doxycycline was from Clontech; Protein A-Sepharose CL-4B was from Amersham Biosciences; Lipofectamine 2000 and NativeMark Unstained Protein Standard were from Invitrogen; and bortezomib (PS-341) was a kind gift from Millennium Pharmaceuticals. Cell Lysis, Immunoprecipitation, and SPFH1/2 Complex Immunopurification—Cells grown in monolayer were harvested with lysis buffer (150 mm NaCl, 50 mm Tris-HCl, 1 mm EDTA, 1% Triton X-100 or 1% CHAPS, pH 8.0) supplemented with protease inhibitors (10 μm leupeptin, 10 μm pepstatin, 0.2 mm phenylmethylsulfonyl fluoride, and 0.2 μm soybean trypsin inhibitor) and 1 mm dithiothreitol. When IP3R polyubiquitination was to be measured, cells were harvested with dithiothreitol-free lysis buffer, and 2.5 mm N-ethylmaleimide was added to the lysates for 1 min, followed by 5 mm dithiothreitol. Lysates were incubated on ice for 30 min and clarified by centrifugation at 16,000 × g for 10 min at 4 °C. To immunoprecipitate specific proteins, clarified lysates were incubated with antisera and Protein A-Sepharose CL-4B for 4–16 h at 4 °C, and washed thoroughly with lysis buffer. To elute immunopurified SPFH1/2 complexes for subsequent blue native (BN)-polyacrylamide gel electrophoresis, anti-SPFH1 or anti-SPFH2 immunoprecipitates in 1% Triton X-100-containing lysis buffer were incubated with 100 μg/ml of SPFH1 or SPFH2 peptide antigens used to generate antisera for 48–72 h at 4 °C. To dissociate SPFH1/2 complexes from IP3R1, anti-IP3R1 immunoprecipitates in 1% Triton X-100-containing lysis buffer were incubated for 48 h at 4 °C, which allowed for dissociation of the majority of the co-purifying SPFH1/2 complexes. To elute immunopurified SPFH1/2 complexes for subsequent TEM, anti-SPFH1 immunoprecipitates prepared in 1% Triton X-100-containing lysis buffer were washed and then incubated in either detergent-free lysis buffer or 0.1% Triton X-100-containing lysis buffer with 100 μg/ml SPFH1 peptide for 72 h at 4 °C. Sample Preparation, PAGE, Immunoblotting, and Mass Spectral Analysis—For SDS-PAGE, samples were boiled in gel loading buffer (24Oberdorf J. Webster J.M. Zhu C.C. Luo S.G. Wojcikiewicz R.J. Biochem. J... 1999; 339: 453-461Google Scholar) for 3 min prior to loading. For BN-PAGE, samples prepared in NativePAGE sample buffer (Invitrogen) were supplemented with 0.25% Coomassie G-250 and loaded onto 3–12 or 4–16% gels and run at 4 °C. After electrophoresis, proteins were either stained with Coomassie Blue, or transferred to nitrocellulose (for SDS-PAGE) or polyvinylidene fluoride (for BN-PAGE) membranes and probed. Immunoreactivity was detected using Pierce chemiluminescence substrates and a Genegnome Imager (Syngene Bio Imaging). To identify proteins by matrix-assisted laser desorption ionization time-of-flight, protein bands were excised from SDS-PAGE gels and subjected to in-gel trypsinization and mass spectral analysis at the Molecular Biology Core Facilities at Dana Farber Cancer Institute (Boston, MA). The MS-Fit data base (University of California San Francisco Mass Spectrometry Facility) was used to provide possible identities for the peptides generated from each gel band. Plasmids and Short Hairpin RNA (shRNA) Constructs—To make deletion and truncation mutants, the sequence encoding mouse SPFH2-HA in pcDNA3 (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar) was mutated by inverse PCR and blunt-end ligated. An expressed sequence tag containing mouse SPFH1 cDNA was purchased from ATCC (clone BC011220), and a FLAG or HA epitope sequence was fused to the 3′ end of the open reading frame and inserted into pcDNA3 to generate SPFH1-FLAG or SPFH1-HA. SPFH1-Flag-N108Q was generated using the Stratagene QuikChange PCR mutagenesis kit. cDNAs encoding SPFH1, SPFH2, SPFH1-HA, SPFH2-HA, and SPFH2-HA mutants were also inserted into the pQCXIH vector (BD Biosciences) to generate cDNA-containing retroviruses. Sequences encoding shRNAs were inserted into 6OH1O-pSUPER.retro.puro (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar) such that short interfering RNAs were generated in cells. The short interfering RNA-encoding sequences were as follows: Random (actgtcacaagtacctaca), SPFH1si4 (gtaccaggccattgcttct), and SPFH2si6 (gtacaaggctattgcttcc). Retrovirus-mediated RNA Interference and cDNA Expression in Rat-1 Cells—Retroviruses containing either shRNA-encoding 6OH1O-pSUPER.retro.puro vectors or cDNA-encoding pQCXIH vectors were generated in HEK293T cells, titered, and Rat-1 cells stably expressing tTS were transduced as previously described (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar). To deplete either SPFH1 or SPFH2, cells were incubated with SPFH1si4- or SPFH2si6-encoding retrovirus twice, and control cells for these single knockdown cells were twice incubated with Random shRNA-encoding virus and named Random-s. To deplete both SPFH1 and SPFH2, SPFH2 knockdown cells were incubated twice with SPFH1si4-encoding retrovirus, and control cells for these double knockdown cells were generated by incubating Random-s cells with Random shRNA-encoding retrovirus two more times and were named Random-d. shRNA expression was induced by treating virally transduced cells with 500 ng/ml doxycycline for 48 h, followed by selection of shRNA-expressing cells with 2.5 μg/ml puromycin for an additional 24–48 h. For stable cDNA expression, Rat-1 cells were transduced with pQCXIH-containing retrovirus for ∼8 h and supplemented with 8 μg/ml Polybrene, followed by selection of transduced cells with 200 μg/ml hygromycin B for 8 days. TEM—SPFH1/2 complexes eluted in the absence of detergent were used without further processing, whereas complexes eluted in 0.1% Triton X-100 were first subjected to 15–50% glycerol density gradient centrifugation at 180,000 × g for 6 h at 4 °C in lysis buffer containing 0.05% Triton X-100 (SPFH1/2 complexes were typically found in the 35–45% fractions). 5 μl of SPFH1/2 complex-containing samples were applied to glow-discharged, carbon-coated copper grids were incubated for 1 min at room temperature or for >1 h at 4 °C, grids were washed once with water, and were finally stained for 1 min with 1% uranyl acetate. Samples were visualized using a 200 kV transmission electron microscope (Jeol JEM-2100) and images were recorded under minimum dose conditions using a 4096 × 4096 pixel CCD camera (TVIPS F415MP) at a magnification of ×40,000 with an underfocus of ∼1500 nm. The pixel size on the specimen level was calibrated to be 2.82 Å using catalase crystals as a standard. CCD frames were displayed with the Boxer program in the EMAN package (25Lüdtke S.J. Baldwin P.R. Chiu W. J. Struct. Biol... 1999; 128: 82-97Google Scholar), and single molecule images were selected manually. Data sets of ∼3800 particles were collected for both the detergent-free and detergent-containing samples. All subsequent image analysis was performed with the Imagic 5 software package (26van Heel M. Harauz G. Orlova E.V. Schmidt R. Schatz M. J. Struct. Biol... 1996; 116: 17-24Google Scholar). The data sets were normalized, band-pass filtered to remove high (>0.14 Å–1) and low (<0.0066 Å–1) spatial frequencies, and a circular mask was applied. The data sets were treated by reference free alignment (27Dube P. Tavares P. Lurz R. van Heel M. EMBO J... 1993; 12: 1303-1309Google Scholar), and the best averages were used as references for multireference alignment as implemented in Imagic 5. At this stage, very similar projection averages were obtained for both data sets. After several rounds of multireference alignment, the data set of the purified sample was sorted into 64 classes and angles were determined for 24 of the best averages using the Imagic command C1 start-up/new projection. The resulting three-dimensional model was forward-projected along 48 directions and the resulting images served as references for a new round of multireference alignment. The projection matching refinement was iterated until no further improvement was observed, using 83 references in the final alignment. At this stage, the two data sets were combined and subjected to two more rounds of refinement by projection matching using 131 references for the final alignment. The resolution of the final model was estimated via the Fourier shell correlation method using 0.5 as cut-off (28Böttcher B. Wynne S.A. Crowther R.A. Nature.. 1997; 386: 88-91Google Scholar). For surface display, the final model was masked using the Imagic command threed-automatic-masking using standard parameters and filtered to remove spatial frequencies above 0.05 Å–1. The model was displayed using Chimera (29Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. J. Comput. Chem... 2004; 25: 1605-1612Google Scholar). Data Analysis—All experiments were repeated at least once, and representative images of gels or micrographs are shown. Quantitated data are graphed as mean ± S.E. of n independent experiments, with paired Student's t test used to obtain p values. SPFH1 and SPFH2 Co-purify with Activated IP3Rs—We reported previously that SPFH2 is the most abundant protein that co-immunoprecipitates with activated IP3Rs (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar). Subsequent analysis of Coomassie Blue-stained anti-IP3R1 immunoprecipitates from control and GnRH-stimulated αT3-1 cells revealed that in addition to the SPFH2 band at an apparent molecular mass of ∼43 kDa, a fainter band at ∼41 kDa was also visible under stimulated conditions (Fig. 1A, lane 2). Mass spectral analysis identified the ∼41-kDa protein as SPFH1, a close homolog of SPFH2, and we validated the association of SPFH1 and SPFH2 with activated IP3R1 using antisera specific for each protein (Fig. 1A, lanes 3–6). Because SPFH2 was shown to associate with IP3Rs immediately following their activation (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar), we tested whether SPFH1 associated in a similar manner. As shown in Fig. 1B, the peak association of SPFH1 and SPFH2 with IP3R1 in GnRH-stimulated αT3-1 cells occurred at 3 min, prior to peak IP3R1 polyubiquitination and the association of p97, which binds to ERAD substrates at least in part via attached polyubiquitin chains (6Ye Y. Meyer H.H. Rapoport T.A. J. Cell Biol... 2003; 162: 71-84Google Scholar). Because association of SPFH1 and SPFH2 precedes substantial IP3R1 polyubiquitination, their interaction with activated IP3Rs is not likely dependent on polyubiquitin chain formation. Indeed, in αT3-1 cells preincubated with the proteasome inhibitor bortezomib to deplete intracellular free ubiquitin (30Xu Q. Farah M. Webster J.M. Wojcikiewicz R.J.H. Mol. Cancer Ther... 2004; 3: 1263-1269Google Scholar), the rate of GnRH-induced IP3R1 polyubiquitination was slowed, but the association of SPFH2 (Fig. 1C) and SPFH1 (not shown) with activated IP3R1 was unaffected. Thus, SPFH1 and SPFH2 bind to activated IP3R1 prior to and independently of IP3R1 polyubiquitination. Molecular Properties of SPFH1 and SPFH2—SPFH1 and SPFH2 are very closely related proteins, sharing ∼70% identity and ∼80% similarity at the amino acid level (supplemental Fig. S1A and Fig. 4A). Their SPFH domains, which account for ∼80% of each protein (residues 27–300 in SPFH1 and 25–298 in SPFH2), share ∼90% identity. In addition to the SPFH domain, SPFH1 and SPFH2 contain several identifiable features: a hydrophobic stretch at their N termini (residues 6–26 in SPFH1 and 4–24 in SPFH2), an N-linked glycosylation site (Asn108 in SPFH1 and Asn106 in SPFH2), and an α-helical stretch predicted to form coiled-coil motifs (residues 179–276 in SPFH1 and 177–274 in SPFH2). The alignment in supplemental Fig. S1A depicts the secondary structure predictions for SPFH1 and SPFH2, emphasizing their similarities. Immunofluorescence microscopy of endogenous SPFH1 and SPFH2 showed that both proteins co-localize with DsRed2-ER (Fig. 2A), suggestive of an ER localization for these proteins (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar, 17Browman D.T. Resek M.E. Zajchowski L.D. Robbins S.M. J. Cell Sci... 2006; 119: 3149-3160Google Scholar). SPFH1, like SPFH2 (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar), was microsome-associated and, like other integral membrane proteins (SPFH2, IP3R1, and Hrd1), was only solubilized by detergents (Fig. 2B). Interestingly, IP3R1, SPFH1, and SPFH2 were only partially solubilized by Triton X-100 (Fig. 2B, lanes 5 and 6), consistent with the previously reported association of these proteins with DRMs (17Browman D.T. Resek M.E. Zajchowski L.D. Robbins S.M. J. Cell Sci... 2006; 119: 3149-3160Google Scholar, 31Weerth S.H. Holtzclaw L.A. Russell J.T. Cell Calcium.. 2007; 41: 155-167Google Scholar). Similar to SPFH2, SPFH1 was resistant to Proteinase K in cells permeabilized with digitonin, but was susceptible in cells treated with Triton X-100 (Fig. 2C), indicating that the majority of SPFH1 mass is located in the ER lumen. Endogenous SPFH1 and exogenous FLAG-tagged SPFH1 (SPFH1-FLAG) were sensitive to endoglycosidase H unless asparagine 108 was mutated to glutamine (Fig. 2D), indicating that SPFH1, like SPFH2, is glycosylated. SPFH1 and SPFH2 are predicted to contain a TM domain at their N termini, and Edman degradation of endogenous SPFH1 and SPFH2 confirmed that these hydrophobic regions are not cleaved signal sequences (not shown). Overall, these data indicate that like SPFH2, SPFH1 is a type II membrane protein anchored to the ER membrane by its N terminus and with the rest of the protein, including an N-linked glycosylation site, protruding into the ER lumen (supplemental Fig. S1B). SPFH1 and SPFH2 Form a Complex That Is >1 MDa in Size—Because SPFH1 and SPFH2 co-purify with activated IP3Rs with essentially identical kinetics, we wondered whether they might be physically associated. To test this possibility, we examined Coomassie Blue-stained anti-SPFH1 or anti-SPFH2 immunoprecipitates from αT3-1 cells and observed ∼41- and ∼43-kDa bands, in an ∼1:2 ratio, in both immunoprecipitates (Fig. 3A, lanes 1 and 2). Immunoblots confirmed that these protein bands corresponded to SPFH1 and SPFH2, respectively (Fig. 3A, lower panels), and either immunoprecipitation depleted both proteins from cell lysates (Fig. 3B). Thus, SPFH1 and SPFH2 form a complex. Interestingly, no other protein bands were detectable in the immunoprecipitates (Fig. 3A), indicating that the SPFH1/2 complex is largely devoid of other proteins. In αT3-1 cell lysates, the SPFH1/2 complex was ∼1 MDa in size, as estimated by BN-PAGE, which allows for the separation of proteins while still in complexes (Fig. 3C, lanes 1 and 4). Likewise, SPFH1/2 complexes immunopurified with either anti-SPFH1 or anti-SPFH2 migrated at ∼1 MDa (Fig. 3C, lanes 2 and 5), as did urea-washed, immunopurified complexes (Fig. 3C, lanes 3 and 6), indicating that the large size of the SPFH1/2 complex is not due to loosely associated proteins. In addition, SPFH1 and SPFH2 co-purifying with IP3R1 from stimulated cells migrated at ∼1 MDa, indicating that the SPFH1/2 complex, and not the individual proteins, associates with activated IP3Rs (Fig. 3D). Interestingly, size exclusion column chromatography of αT3-1 cell lysates yielded a value of ∼2 MDa for the size of the SPFH1/2 complex (not shown), and a similar discrepancy between BN-PAGE and gel filtration has been noted for the prohibitin 1/2 complex (32Steglich G. Neupert W. Langer T. Mol. Cell. Biol... 1999; 19: 3435-3442Google Scholar, 33Tatsuta T. Model K. Langer T. Mol. Biol. Cell.. 2005; 16: 248-259Google Scholar), suggesting that BN-PAGE may underestimate the size of high molecular mass complexes. As subsequent TEM yielded a size estimate of ∼2 MDa for the SPFH1/2 complex (Fig. 8), we conclude that the SPFH1/2 complex is ∼2 MDa in size and is composed of a total of ∼50 SPFH1 and SPFH2 molecules in an ∼1:2 ratio. The C-terminal Half of SPFH2 Mediates Its Interactions with SPFH1 and the Formation of ∼2-MDa Complexes—To determine which regions of SPFH1 and SPFH2 mediate their association, we generated constructs encoding wild-type SPFH1-FLAG and either wild-type or mutant SPFH2-HA (Fig. 4A). Initially, four SPFH2-HA internal deletion mutants (SPFH2-HAΔ26–58, Δ59–100, Δ101–176, and Δ177–274) and two C-terminal truncations (SPFH2-HAΔ275–340 and Δ177–340) were generated. Residues 1–25 were preserved in each of these SPFH2-HA constructs, as this region anchors SPFH2 to the ER membrane (17Browman D.T. Resek M.E. Zajchowski L.D. Robbins S.M. J. Cell Sci... 2006; 119: 3149-3160Google Scholar). When SPFH1-FLAG was co-expressed with SPFH2-HA, SPFH2-HAΔ26–58, Δ59–100, or Δ101–176, each construct co-immunoprecipitated strongly with SPFH1-FLAG (Fig. 4B, lanes 3–6). In contrast, SPFH2-HAΔ275–340 bound less well (Fig. 4B, lane 10), and SPFH2-HAΔ177–274 and SPFH2-HAΔ177–340 bound hardly at all (Fig. 4B, lanes 7 and 11). Thus, regions near the C terminus of SPFH2, including the predicted coiled-coil (CC) region from 177–274 and another region C-terminal to 275, mediate binding to SPFH1. The region spanning residues 177–274 in SPFH2 is predicted to form two adjacent CC motifs (supplemental Fig. S1A and Fig. 4A), one from 177–230 (CC#1) and another from 231–274 (CC#2). SPFH2-HAΔ177" @default.
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• W2068688570 title "An Endoplasmic Reticulum (ER) Membrane Complex Composed of SPFH1 and SPFH2 Mediates the ER-associated Degradation of Inositol 1,4,5-Trisphosphate Receptors" @default.
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