SPFH2 Mediates the Endoplasmic Reticulum-associated Degradation of Inositol 1,4,5-Trisphosphate Receptors and Other Substrates in Mammalian Cells

Inositol 1,4,5-trisphosphate (IP3) receptors are endoplasmic reticulum (ER) membrane calcium channels that, upon activation, become substrates for the ER-associated degradation (ERAD) pathway. Although it is clear that IP3 receptors are polyubiquitinated upon activation and are transferred to the proteasome by a p97-based complex, currently nothing is known about the proteins that initially select activated IP3 receptors for ERAD. Here, we sought to identify novel proteins that associate with and mediate the ERAD of endogenous activated IP3 receptors. SPFH2, an uncharacterized SPFH domain-containing protein, rapidly associated with IP3 receptors in a manner that preceded significant polyubiquitination and the association of p97 and related proteins. SPFH2 was found to be an ER membrane protein largely residing within the ER lumen and in resting and stimulated cells was linked to ERAD pathway components, apparently via endogenous substrates undergoing degradation. Suppression of SPFH2 expression by RNA interference markedly inhibited IP3 receptor polyubiquitination and degradation and the processing of other ERAD substrates. Overall, these studies identify SPFH2 as a key ERAD pathway component and suggest that it may act as a substrate recognition factor. Inositol 1,4,5-trisphosphate (IP3) receptors are endoplasmic reticulum (ER) membrane calcium channels that, upon activation, become substrates for the ER-associated degradation (ERAD) pathway. Although it is clear that IP3 receptors are polyubiquitinated upon activation and are transferred to the proteasome by a p97-based complex, currently nothing is known about the proteins that initially select activated IP3 receptors for ERAD. Here, we sought to identify novel proteins that associate with and mediate the ERAD of endogenous activated IP3 receptors. SPFH2, an uncharacterized SPFH domain-containing protein, rapidly associated with IP3 receptors in a manner that preceded significant polyubiquitination and the association of p97 and related proteins. SPFH2 was found to be an ER membrane protein largely residing within the ER lumen and in resting and stimulated cells was linked to ERAD pathway components, apparently via endogenous substrates undergoing degradation. Suppression of SPFH2 expression by RNA interference markedly inhibited IP3 receptor polyubiquitination and degradation and the processing of other ERAD substrates. Overall, these studies identify SPFH2 as a key ERAD pathway component and suggest that it may act as a substrate recognition factor. The endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; siRNA, short interfering RNA; E3, ubiquitin-protein isopeptide ligase; HMGR, hydroxy-3-methylglutaryl-coenzyme A reductase; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; GnRH, gonadotropin-releasing hormone; ET1, endothelin 1; RNAi, RNA interference; HA, hemagglutinin; TNF, tumor necrosis factor; Endo H, endoglycosidase H; DTT, dithiothreitol; PBS, phosphate-buffered saline; DRM, detergent-resistant membrane. 2The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; siRNA, short interfering RNA; E3, ubiquitin-protein isopeptide ligase; HMGR, hydroxy-3-methylglutaryl-coenzyme A reductase; IP3, inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; GnRH, gonadotropin-releasing hormone; ET1, endothelin 1; RNAi, RNA interference; HA, hemagglutinin; TNF, tumor necrosis factor; Endo H, endoglycosidase H; DTT, dithiothreitol; PBS, phosphate-buffered saline; DRM, detergent-resistant membrane.-associated degradation (ERAD) pathway is responsible for the degradation of aberrant proteins in the ER (1Meusser B. Hirsch C. Jarosch E. Sommer T. Nat. Cell Biol. 2005; 7: 766-772Crossref PubMed Scopus (993) Google Scholar) and, in addition to this “quality control” function, also accounts for the degradation of several metabolically regulated, native ER proteins (2Hampton R.Y. Curr. Opin. Cell Biol. 2002; 14: 476-482Crossref PubMed Scopus (394) Google Scholar, 3Webster J.M. Tiwari S. Weissman A.M. Wojcikiewicz R.J.H. J. Biol. Chem. 2003; 278: 38238-38246Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The essential features of the ERAD pathway are substrate recognition, polyubiquitination, and delivery to the 26 S proteasome, which is located in the cytosol (1Meusser B. Hirsch C. Jarosch E. Sommer T. Nat. Cell Biol. 2005; 7: 766-772Crossref PubMed Scopus (993) Google Scholar). Much of our understanding of the ERAD pathway has been obtained using yeast as a model system, and although there are many parallels between the yeast and mammalian ERAD pathways, there appear to be some key differences. Most notably, mammalian cells have additional components that add diversity and complexity to substrate recognition and processing (4Carvalho P. Goder V. Rapoport T.A. Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). Quite a lot is known about how ERAD substrates are polyubiquitinated and transferred to the proteasome. Studies in yeast suggest that ER luminal substrates and membrane substrates with aberrant luminal or membrane domains are polyubiquitinated by an ER membrane protein complex containing the ubiquitin ligase (E3) Hrd1p, whereas membrane substrates with aberrant cytosolic domains are targeted by a complex containing the E3 Doa10p (4Carvalho P. Goder V. Rapoport T.A. Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar). Hrd3p binds to and regulates Hrd1p (5Gardner R.G. Swarbrick G.M. Bays N.W. Cronin S.R. Wilhovsky S. Seelig L. Kim C. Hampton R.Y. J. Cell Biol. 2000; 151: 69-82Crossref PubMed Scopus (244) Google Scholar), and together with the ER luminal lectin Yos9p, may use its large luminal domain to recruit ERAD substrates to a putative “retrotranslocation” channel in the ER membrane (6Denic V. Quan E.M. Weissman J.S. Cell. 2006; 126: 349-359Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 7Gauss R. Jarosch E. Sommer T. Hirsch C. Nat. Cell Biol. 2006; 8: 849-854Crossref PubMed Scopus (193) Google Scholar, 8Gauss R. Sommer T. Jarosch E. EMBO J. 2006; 25: 1827-1835Crossref PubMed Scopus (113) Google Scholar). Retrotranslocation is facilitated by the cytosolic Cdc48p-Ufd1p-Npl4p complex, which associates with Hrd1p and Doa10p via membrane-bound Ubx2p (4Carvalho P. Goder V. Rapoport T.A. Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, 8Gauss R. Sommer T. Jarosch E. EMBO J. 2006; 25: 1827-1835Crossref PubMed Scopus (113) Google Scholar, 9Neuber O. Jarosch E. Volkwein C. Walter J. Sommer T. Nat. Cell Biol. 2005; 7: 993-998Crossref PubMed Scopus (226) Google Scholar, 10Schuberth C. Buchberger A. Nat. Cell Biol. 2005; 7: 999-1006Crossref PubMed Scopus (242) Google Scholar), and likely uses ATP hydrolysis to both unfold ERAD substrates and extract them from the ER membrane (11Elkabetz Y. E. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. C. Volkwein C. J. Sommer T. Nat. Cell Biol. 2002; PubMed Scopus Google Scholar, E. A. S. Cell. Biol. 2002; PubMed Scopus Google Scholar). with or Rapoport T.A. J. Cell Biol. PubMed Scopus Google Scholar, Rapoport T.A. V. Biol. Cell. PubMed Scopus (113) Google Scholar). the mammalian of many of these proteins have been and appear to be (4Carvalho P. Goder V. 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A. 2005; PubMed Scopus Google Scholar, Y. T. J. Cell Biol. 2006; PubMed Scopus Google Scholar, Y. Y. S. E. Rapoport T.A. S. A. 2005; PubMed Scopus Google Scholar). how proteins are initially for ERAD is and to the of recognition and a A. Cell Biol. 14: Full Text Full Text PDF PubMed Scopus Google Scholar). in Yos9p, together with and binds to and a of luminal to the Hrd1p complex (4Carvalho P. Goder V. Rapoport T.A. Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, V. Quan E.M. Weissman J.S. Cell. 2006; 126: 349-359Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 7Gauss R. Jarosch E. Sommer T. Hirsch C. Nat. Cell Biol. 2006; 8: 849-854Crossref PubMed Scopus (193) Google Scholar). the mammalian of (11Elkabetz Y. E. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and Hrd3p B. J. Cell Biol. 2006; PubMed Scopus Google Scholar), as as the mammalian proteins J. Cell 2006; PubMed Scopus Google Scholar), appear to with for ERAD. substrate recognition be by that select or of proteins for degradation. the mammalian to A and this the E3 that are for the ERAD of Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). the diversity of proteins that are targeted for ERAD, it is likely that additional substrate recognition Inositol 1,4,5-trisphosphate (IP3) receptors and channels in ER and a key in P. Nat. Cell. Biol. 2000; PubMed Scopus Google Scholar, Cell 2005; PubMed Scopus Google Scholar). of receptors IP3 the which the cytosol and binds to P. Nat. Cell. Biol. 2000; PubMed Scopus Google Scholar). in with in the channel that to from within the ER lumen the cytosol T. PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar). are in and and although in have are and or Cell 2005; PubMed Scopus Google Scholar, R.J.H. J. Biol. Chem. 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Here, we endogenous in stimulated mammalian cells in an to identify novel proteins that be in the ERAD of and other substrates. that an uncharacterized protein, known as SPFH domain-containing protein, associates rapidly with activated in a of that a of SPFH2 is associated with several ERAD pathway components, and that RNA interference of SPFH2 polyubiquitination and degradation and the of model ERAD substrates. SPFH2 with a of proteins that an SPFH an of function, for the and R.G. 2005; PubMed Scopus Google Scholar, E. E. Cell. 2006; PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar). Overall, these studies identify SPFH2 as a key ERAD pathway component in mammalian cells and suggest that it may act as a substrate recognition factor. and cells as Wojcikiewicz R.J.H. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), and cells in with and used and R.J.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), from of and from T. and and and and and to the of protein and and as R.J.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). GnRH, and from was from and endoglycosidase from DTT, and from was from was from was from was from and was a from Cell and in or with GnRH, and cells by containing a and DTT, the cells the and for by for and and with J. J.M. Wojcikiewicz R.J.H. J. PubMed Scopus Google Scholar) for or proteins by with and for with in to and transferred to for or with the in or for and with or by by with for to by by and was in or and as from and with a and the several with and in the or with The in as R.J.H. Full Text Full Text PDF PubMed Scopus Google Scholar) and with a of and to receptor which as for in or with and cells by and the cells the with for by by and for or was as for and in J. J.M. Wojcikiewicz R.J.H. J. PubMed Scopus Google Scholar) to for to and for to protein proteins transferred to and and was with and using a protein from and for and the The of was used to for the from by and containing the SPFH2 in was from and by and the was SPFH2 was with an its by using to the with the of SPFH2 and was using the was a from R. was a from A. Weissman of and was a from J. short interfering RNA to to of and SPFH2 and of by and by found to be a by was to have to or and and the R. R. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, R. R. 2002; PubMed Scopus Google Scholar), for of these was by and was by the and of with the of from for The V. R. EMBO 2003; PubMed Scopus Google Scholar). in the of also of the from the RNA in of these short that are by the endogenous R. R. 2002; PubMed Scopus Google Scholar). cells with and SPFH2 using the cells in for in for in for in in for in in for with in for in for and with in for in PBS, the cells using to the a with a from Cell and cells in of containing and DTT, and for The for and the in of or containing or for with and for to and in of the and of was used as a cytosolic J.M. Tiwari S. Weissman A.M. Wojcikiewicz R.J.H. J. Biol. Chem. 2003; 278: 38238-38246Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). the cells and in containing or and for was the and the for an additional with and SPFH2 in by cells using calcium with together with and that for and proteins the was and the was and by cells with of and that in cells a protein of the and the by cells with containing the in and was and in a and was with of with for by in for expression of the cells and with for by in for to the of endogenous SPFH2 and other or polyubiquitination and was by the cells with as Wojcikiewicz R.J.H. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of SPFH2 was using a SPFH2 containing that the to using the or with and or using The of for the of polyubiquitination in the which was as cells and ERAD in cells in in and using with or of studies that this of and SPFH2 the was with with and cells with of or in cells with for and by in and for in and DTT, for for and in with to and and and of or are are as of with used to SPFH2 with known of proteins that mediate ERAD the to associate with activated Wojcikiewicz R.J.H. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) to novel also we cells with or for which polyubiquitination and R.J.H. J.M. C. J. Biol. 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Full Text Full Text PDF PubMed Scopus Google Scholar), and found that SPFH2 with activated of to a of proteins containing an SPFH also known as the or domain R.G. 2005; PubMed Scopus Google Scholar, E. E. Cell. 2006; PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar). The SPFH domain is and in SPFH2 with the other mammalian proteins in this SPFH2 is to the to the and to the and proteins by The of the SPFH domain is currently and although the proteins that to this are the some notably, to and via of and, in the of B. L. E. T. 2003; PubMed Scopus Google Scholar, A. Y. S. A. PubMed Scopus Google Scholar, J. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, C. B. P. S. V. R. J. PubMed Scopus Google Scholar, C. Y. E. E. J. PubMed Scopus Google Scholar, T. Cell. Biol. PubMed Google Scholar, T. T. Biol. Cell. 2005; PubMed Scopus (242) Google Scholar, E. R. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). SPFH2 is the and are and SPFH2 and with a protein from its to within the there are yeast of of SPFH2 the the is to be A and that SPFH2 is an membrane protein with domain its SPFH2 is in the of SPFH2, we be with which is the of the protein, an for endogenous SPFH2 to that of the ER a protein targeted to the ER by an ER and a ER The was also in cells SPFH2 and and for endogenous SPFH2 in and cells cells that used in the of SPFH2 was cells with or with the membrane this that the SPFH2 is located within the ER SPFH2 is an membrane protein, we cells by and and the which SPFH2 the cytosolic and in several to and membrane proteins by membrane for p97 (11Elkabetz Y. E. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), from the by in or whereas membrane proteins by the ER membrane and by and by SPFH2 was from the by that it is an membrane the membrane of SPFH2 was or in by of the membrane with Rapoport T.A. J. Cell Biol. PubMed Scopus Google Scholar, J. J. Cell Biol. PubMed Scopus Google Scholar) was with of SPFH2, its is located in the ER with the of which is located the of the ER that was in the membrane. the ER luminal protein was in cells was with that the ER membrane. SPFH2 is a ER membrane protein, with a short to the cytosol and its with the of the protein, located within the ER SPFH2 an we it is a of with Endo to a of in the of endogenous SPFH2, with the of a and we in cells SPFH2 and SPFH2 with to and Endo an with the of a and was by Endo and SPFH2 is by a and this is with the of SPFH2 located within the ER A the of SPFH2 is in SPFH2 with of the ERAD SPFH2 binds to activated which are by the ERAD we SPFH2 be in the ERAD of substrates. several proteins with in ERAD and with SPFH2 and as an of the of these an ER luminal a putative ubiquitin and a component of the ER membrane with SPFH2 resting a of SPFH2 is linked to ERAD pathway this is and the of SPFH2 is SPFH2 to associate with with from other ERAD pathway components The between SPFH2 and ERAD pathway components is likely via endogenous substrates undergoing ERAD, the of these substrates with the proteasome the of p97 that with SPFH2 with for and the of that with SPFH2, and for the of polyubiquitinated and the in SPFH2 with activated of the ERAD pathway components and with SPFH2 was by as be from the that these proteins associate with polyubiquitinated of SPFH2 the of the between SPFH2 and activated we used to endogenous used cells for these as we to p97 in these cells and its in ERAD Wojcikiewicz R.J.H. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Here, we a expression to SPFH2, to that in to SPFH2 targeted to of SPFH2 and was obtained in cells as with cells The of other related proteins and ubiquitin by SPFH2 SPFH2 is and have a the of ubiquitin to substrates. that polyubiquitination was by SPFH2 polyubiquitination was the that SPFH2 is in the of ERAD, and of SPFH2 with activated was in the that the endogenous SPFH2 was to of SPFH2 association with activated and as of p97 was markedly inhibited likely polyubiquitination was and the complex binds to polyubiquitinated proteins Y. EMBO J. 2002; PubMed Scopus Google Scholar, Y. Rapoport T.A. J. Cell Biol. 2003; PubMed Scopus Google Scholar). from these was markedly inhibited by SPFH2 that these of SPFH2 to of activation, we in to and found that it was inhibited was in the likely resting by SPFH2 the that of for the of we SPFH2 cells via an SPFH2 that an to of SPFH2 the of SPFH2 polyubiquitination and that this from of Overall, these that SPFH2 a key in the polyubiquitination and degradation of of ERAD by SPFH2 SPFH2 ERAD and with several ERAD pathway components, we to it is in the degradation of other mammalian ERAD substrates. model ERAD substrates are and is the and of mammalian HMGR, and, the protein, its degradation by the ERAD pathway is by R. R. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is a of the receptor complex and is by the ERAD pathway B. J. Weissman A.M. S. A. 2006; PubMed Scopus Google Scholar, R. R. S. S. J. V. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Weissman A.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these we used is in this and it is to endogenous proteins and proteins. SPFH2 with both ERAD substrates in cells of endogenous SPFH2 was obtained upon with and in and upon addition of the protein and with of and whereas in SPFH2 that SPFH2 is in degradation with this the of both and in SPFH2 cells as with the with that and are rapidly a endogenous also the of the of SPFH2 using a cytosolic protein that is rapidly via the of ERAD, in cells A. A. S. A.M. J.S. Y. PubMed Scopus (568) Google Scholar). to the for and for and SPFH2 with undergoing polyubiquitination and degradation polyubiquitination and degradation was inhibited by SPFH2 Overall, these that SPFH2 is in the processing of ERAD substrates. a to ERAD in mammalian activation in to receptor them from proteins ERAD substrates R.J.H. 25: Full Text Full Text PDF PubMed Scopus Google Scholar). other ERAD substrates and RNAi, we that SPFH2 an in the mammalian ERAD pathway. SPFH2 was found to associate with its activation, and SPFH2 inhibited polyubiquitination and degradation. SPFH2 associated with the model ERAD and and SPFH2 these proteins. SPFH2, likely a of the was also found to be associated with ERAD pathway components in via with endogenous substrates undergoing ERAD. SPFH2 is in ERAD, it or with undergoing degradation. SPFH2 to be in the of ERAD, it with its activation and to the association of the ERAD pathway components and of which to it has been SPFH2 inhibited polyubiquitination the the of SPFH2 within the ER it is that it with luminal of ERAD substrates to from the ER, and that it as a recognition that substrates for ERAD. SPFH2 may to other ER luminal proteins in ERAD substrate recognition, as Hrd3p and in yeast (4Carvalho P. Goder V. Rapoport T.A. Cell. 2006; 126: 361-373Abstract Full Text Full Text PDF PubMed Scopus (568) Google Scholar, V. Quan E.M. Weissman J.S. Cell. 2006; 126: 349-359Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 7Gauss R. Jarosch E. Sommer T. Hirsch C. Nat. Cell Biol. 2006; 8: 849-854Crossref PubMed Scopus (193) Google Scholar) or the proteins in J. Cell 2006; PubMed Scopus Google Scholar). it a that SPFH2 substrate polyubiquitination, by as a novel E3 this putative of SPFH2 in ERAD, it that SPFH2 the of ubiquitin in are for ERAD substrates may a of the of ubiquitin and a in the polyubiquitination of these substrates the of SPFH2 that of may be to ERAD substrates in resting cells is are ERAD substrates upon or cells are to or an we p97 by this of p97 the of ubiquitin the processing of cells stimulated Wojcikiewicz R.J.H. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). SPFH2 with activated and the of have been to the of proteins for ERAD, by for recognition by ERAD pathway components (1Meusser B. Hirsch C. Jarosch E. Sommer T. Nat. Cell Biol. 2005; 7: 766-772Crossref PubMed Scopus (993) Google Scholar, A. Cell Biol. 14: Full Text Full Text PDF PubMed Scopus Google Scholar). a in yeast by pathway to it to ERAD Hampton R.Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Hampton R.Y. EMBO J. 2005; PubMed Scopus Google Scholar), and in mammalian may for the of Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). to from within the ER lumen the an as in to IP3 and that likely a to the of the which is located in an between the and domains of Cell 2005; PubMed Scopus Google Scholar, T. PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar). luminal may the for SPFH2, the of SPFH2 in the ER and this is known to mediate between and A and J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) and T. T. T. T. 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