An Endoplasmic Reticulum (ER) Membrane Complex Composed of SPFH1 and SPFH2 Mediates the ER-associated Degradation of Inositol 1,4,5-Trisphosphate Receptors

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. and as (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar). used R.J. J. Biol. Chem... Scholar), (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar), and from A. and SPFH1 from S. M. of and and and but and to the SPFH1 and the and as R.J. J. Biol. Chem... Scholar). gonadotropin-releasing and from endothelin from from and from from from and from and a from Cell and SPFH1/2 in with or CHAPS, with and and IP3R polyubiquitination to be with and to the for by for and by for specific proteins, with and for and with SPFH1/2 complexes for blue or in with of SPFH1 or SPFH2 used to generate for SPFH1/2 complexes from in for which for of the of the SPFH1/2 SPFH1/2 complexes for TEM, in and in either or with SPFH1 for and in J. J.M. Wojcikiewicz R.J. for to For in with and or and proteins either with or to or membranes and substrates and a proteins by protein from and to and the The of used to for the from each and RNA and the sequence in (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google by and expressed sequence SPFH1 from and a or sequence to the of the and into to generate or the SPFH1, SPFH2, and also into the to generate into (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google that short in The short as and RNA and in either or in and as (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar). either SPFH1 or SPFH2, with or and control for with and named both SPFH1 and SPFH2, SPFH2 with and control for by with two more and named by with for by of with for an For stable with for and with by of with for complexes in the of used complexes in to for 6 in complexes found in the of SPFH1/2 to for or for with and for with a transmission electron and under conditions a a of with an of The the to be as a with the in the W. J. Biol... Scholar), and of for both the and with the M. G. M. J. Biol... Scholar). The to and and a The by P. P. van M. Scholar), and the used as for as in this similar for both several of the of the into and for of the the The and the as for a of The no in the this the two and to two more of by for the The of the via the as B. R.A. Nature.. Scholar). For surface the the and to The J. Chem... 2004; 25: Scholar). and of or are are as of with used to SPFH1 and SPFH2 with that SPFH2 is the most protein that with activated IP3Rs (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar). of from control and that in to the SPFH2 an of a also under conditions identified the protein as SPFH1, a homolog of SPFH2, and we the of SPFH1 and SPFH2 with activated specific for each protein Because SPFH2 to 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 SPFH1 in a similar in the of SPFH1 and SPFH2 with in to polyubiquitination and the of p97, which binds to ERAD substrates in via (6Ye Y. Meyer H.H. Rapoport T.A. J. Cell Biol... 2003; 162: 71-84Google Scholar). Because of SPFH1 and SPFH2 polyubiquitination, their with activated IP3Rs is likely in with the proteasome to intracellular ubiquitin M. J.M. Wojcikiewicz R.J.H. Mol. 2004; Scholar), the of polyubiquitination but the of SPFH2 and SPFH1 with activated Thus, SPFH1 and SPFH2 bind to activated to and of of SPFH1 and and SPFH2 are closely related proteins, and the amino acid and SPFH domains, which for of each protein in SPFH1 and in to the SPFH domain, SPFH1 and SPFH2 contain several a hydrophobic their in SPFH1 and in an in SPFH1 and in and an to form motifs in SPFH1 and in The in the structure for SPFH1 and SPFH2, their microscopy of endogenous SPFH1 and SPFH2 that both proteins with of an ER localization for proteins (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar, D.T. Resek M.E. Zajchowski L.D. Robbins S.M. J. Cell Sci... 2006; 119: 3149-3160Google Scholar). SPFH1, SPFH2 (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar), other membrane proteins and by SPFH1, and SPFH2 by and with the of proteins with DRMs (17Browman D.T. Resek M.E. Zajchowski L.D. Robbins S.M. J. Cell Sci... 2006; 119: 3149-3160Google Scholar, Cell 2007; Scholar). to SPFH2, SPFH1 to in with but in with that the of SPFH1 is in the ER SPFH1 and SPFH1 to to that SPFH1, SPFH2, is SPFH1 and SPFH2 are to contain a their and degradation of endogenous SPFH1 and SPFH2 that hydrophobic are signal that SPFH2, SPFH1 is a membrane protein to the ER membrane by its and with the of the protein, including an into the ER SPFH1 and SPFH2 a MDa in SPFH1 and SPFH2 with activated IP3Rs with we they be this we or from and and in an in both and that protein to SPFH1 and SPFH2, and either both proteins from cell Thus, SPFH1 and SPFH2 form a no other protein in the that the SPFH1/2 complex is largely of other cell the SPFH1/2 complex MDa in as by which for the of proteins in complexes and SPFH1/2 complexes with either or MDa and as complexes and that the large of the SPFH1/2 complex is to SPFH1 and SPFH2 with from that the SPFH1/2 and the proteins, with activated IP3Rs of cell a of ∼2 MDa for the of the SPFH1/2 complex and a similar and has been for the complex G. W. Langer T. Mol. Biol... Scholar, T. K. Langer T. Mol. Biol. Cell.. 2005; Scholar), that the of a of ∼2 MDa for the SPFH1/2 complex we that the SPFH1/2 complex is ∼2 MDa in and is of a of SPFH1 and SPFH2 in an The of SPFH2 with SPFH1 and the of ∼2-MDa which of SPFH1 and SPFH2 their we and either or four and and two and in each of as this 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). with or each with and and all and Thus, the of SPFH2, including the from and to binding to The in SPFH2 is to form two motifs and one from and from and both with than that both to the with and which to binding to we and both of which with as as and that the and also to the and Thus, three in SPFH2 its with and a the of the SPFH domain, which is to form an by a short domain, and We the of the to form Although MDa and of the or the as a the and that are required for the assembly of into the that with and and contain assemble into complexes is but be to from the that the of SPFH2 is for two distinct one contains motifs and that the of the complex and SPFH1 and SPFH2 for the ERAD of SPFH1 and SPFH2 play a role in we used RNA to endogenous SPFH1 or SPFH2, either or in which we have used to IP3R ERAD (15Alzayady K.J. Panning M.M. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2005; 280: 34530-34537Google Scholar, M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar). SPFH1 and SPFH2 bind to activated IP3Rs in and targeted SPFH1 or SPFH2 SPFH1 or SPFH2 by as with control of SPFH1 SPFH2 polyubiquitination, with SPFH1 to an and of either SPFH2 or SPFH1 and SPFH2 to an as with and degradation when SPFH1 SPFH2 from an of SPFH1 SPFH2 because Ca2+ by of SPFH1 and SPFH2 Thus, the SPFH1/2 complex an following IP3R channel of polyubiquitination and degradation in SPFH1 and SPFH2 out the that are to of and of SPFH1 the of SPFH1 and but of SPFH2 and and of SPFH2 the of either SPFH1 or SPFH2 and in polyubiquitination, even in and the that SPFH1 polyubiquitination, that SPFH2 has that SPFH1 of an ∼2 MDa for the of SPFH2 to polyubiquitination to the of SPFH2 We in which the amino acids of with that of to of SPFH2, which the with SPFH1, for the of SPFH2, but SPFH1, to polyubiquitination all of polyubiquitination to that the SPFH1 and SPFH2 from the minor sequence in their SPFH We also the of several Although with activated and polyubiquitination and polyubiquitination and with activated and appear to have polyubiquitination and with activated and that the assembly is required for the function of the of be as its than the other and the of in the 26 and ∼2-MDa complexes by and complexes similar to Thus, the of and is to their to form ∼2-MDa of endogenous SPFH2 with activated when and but when expressed Thus, it appears that that is expressed can endogenous SPFH2 from activated and that that assemble into ∼2 MDa complexes and IP3R by the of SPFH1 to of this ∼2-MDa complex and function we the assembly of the SPFH1 and SPFH2 that after RNA and found that in or the SPFH2 or SPFH1 MDa that they assemble However, the complexes in polyubiquitination of that in control and in polyubiquitination all that the function However, of SPFH2 polyubiquitination that can for the of SPFH2 the in complexes from or from that in e.g. and in when we the of and in the in complexes to the of each protein expressed and both proteins to MDa that of SPFH2 for assembly of ∼2 MDa complexes, an of for the role of the SPFH1/2 complex in IP3R ERAD. The SPFH1/2 a of endogenous SPFH1/2 complexes ring-shaped with a diameter of ∼250 a and two of into similar which used to a This that the SPFH1/2 complex is a double ring-shaped with a apparently diameter ∼250 inner diameter by to a (diameter It is that the is by the of SPFH1 and SPFH2, which a of the complex and Although it appears as a this also be as a putative The likely the of SPFH1 and SPFH2 and appears to have with two by a one The of a that this suggests that either the SPFH1/2 complex is one or that the structure and with the It appear that the from a because similar when SPFH1/2 complexes for in the of the in is ∼2 which is with the by but from the MDa from of activated IP3Rs by the ERAD pathway a for the levels of mediators of intracellular this we have identified a complex of ER membrane proteins SPFH1 and SPFH2 that and with activated IP3Rs and is for their ERAD. The complex also to a the degradation of a of ERAD substrates (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar), binds to ERAD substrates (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar), including X. J. P. J. C. W. J. H. Cell.. 2006; Scholar), and also with several known ERAD pathway (e.g. p97, and (16Pearce M.M.P. Wang Y. Kelley G.G. Wojcikiewicz R.J.H. J. Biol. Chem... 2007; 282: 20104-20115Google Scholar). Thus, the SPFH1/2 complex appears to the recognition and targeting of activated IP3Rs and perhaps other substrates for ERAD. We that the SPFH1/2 complex is ∼2 MDa in is of subunits of SPFH1 and SPFH2 in an and via two distinct The primary SPFH1 and SPFH2 is by three the SPFH motifs and the and the the assembly domain, to the SPFH domain, and appears to are with from of SPFH1 and SPFH2 M.B. D.T. Resek M.E. Robbins S.M. J. Biol. Chem... 2009; Scholar), and are similar to other SPFH proteins e.g. are for of prohibitins-1 and -2 and and -2 T. K. Langer T. Mol. Biol. Cell.. 2005; Scholar, Hoegg M. C. Y. E. E. 2007; Scholar), and a in the structure of from is to in with H. S. I. J. Mol. Biol... 2008; Scholar). in mammalian stomatin, a short hydrophobic to the SPFH and with DRMs E. M. J. Biol. Chem... 2006; Scholar). that all SPFH proteins assemble into complexes similar SPFH1 stable when SPFH2 and which with other SPFH proteins and -2 and and that are in the of their assembly (20Merkwirth C. Langer T. Biochim. Biophys. Acta.. 2009; 1793: 27-32Google Scholar, Hoegg M. C. Y. E. E. 2007; Scholar). This be because SPFH1 and SPFH2 the to form complexes when either protein is other SPFH proteins Although in of the SPFH1/2 complex its it its SPFH1 complexes SPFH2 complexes and the of SPFH1 to SPFH2 in the complex to the ∼2 MDa for because SPFH2 that assemble of endogenous SPFH1/2 complex a ring-shaped structure similar to the of the complex T. K. Langer T. Mol. Biol. Cell.. 2005; Scholar). structures are of the family of which with exposed hydrophobic proteins and intrinsic ATPase to protein E. Rev. Cell Dev. Biol... 2007; Scholar). Although the SPFH1/2 or complexes have been to their ring-shaped structures for binding to hydrophobic e.g. the SPFH1/2 complex with hydrophobic IP3Rs that exposed IP3R the IP3R channel is by an the and transmembrane and is to a conformational change IP3R activation (3Foskett J.K. White C. Cheung K.H. Mak D.D. Physiol. Rev... 2007; 87: 593-658Google Scholar). This is known to with T. M. T. T. T. K. Cell.. 2005; and and 2007; Scholar), and also a for the of the SPFH1/2 for the complex with activated IP3Rs are in the the opening in the an activated IP3R and in the the is more and an activated IP3R the the and it to an IP3R in this the of the SPFH1/2 complex a that opening and of the a of tetrameric suggests that its membrane and has a diameter of Proc. Natl. Acad. Sci. U. S. A... 2003; Scholar), which the in the putative of the SPFH1/2 Finally, in the the SPFH1/2 complex with activated each of of the SPFH1/2 complex targets an IP3R for ERAD, perhaps by it to other ERAD pathway that and The the SPFH and cholesterol and the of SPFH proteins to form large complexes (18Browman D.T. Hoegg M.B. Robbins S.M. Trends Cell Biol... 2007; 17: 394-402Google the that SPFH proteins either the content of membrane microdomains by or are to DRMs by the SPFH1/2 complex appears to in DRMs in the ER membrane (17Browman D.T. Resek M.E. Zajchowski L.D. Robbins S.M. J. Cell Sci... 2006; 119: 3149-3160Google and IP3Rs to DRMs of the IP3 pathway Cell 2007; Scholar), that IP3R proteins to of the ER membrane in are that ER membrane microdomains play in protein processing. For example, cellular protein is localization to DRMs in the ER, its into the D. V. S. M. L. C. Mol. Biol. Cell.. 2004; Scholar). a that the ERAD for into the cytosol, with ERAD in DRMs T. L. J.M. Mol. Biol. Cell.. 2006; 17: Scholar), and with proteasome causes the to in structures that appear similar to the control in which some ERAD substrates proteasomal S. M. S. Mol. Biol. Cell.. Scholar). that ER membrane perhaps in cholesterol and the SPFH1/2 as for ERAD substrates. We Wang for and and for with