SPFH1 and SPFH2 are type II ER membrane glycoproteins. A, Rat-1 cells plated on lysine-coated coverslips were transfected with cDNA encoding DsRed2-ER, a red fluorescent protein targeted to the ER by the calreticulin ER-targeting and KDEL ER-retention sequences. The cells were fixed and stained with anti-SPFH1 (panels a and b) or anti-SPFH2 (panels c and d), followed by fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibodies as described (16). Images were acquired with a Zeiss LSM510 confocal microscope equipped with a ×63 oil immersion objective. B, essentially as described (16), αT3-1 cells were harvested in hypotonic buffer, sonicated, and fractionated into supernatant (S) and pellet (P) fractions by centrifugation at 50,000 × g for 1 h at 4 °C (lanes 1 and 2). The cytosolic protein α-transaldolase was found in the supernatant, whereas the integral membrane proteins IP₃R1, Hrd1, SPFH1, and SPFH2 were found in the pellet. The peripheral membrane protein p97 and the ER luminal protein grp94 were found in both the supernatant and pellet. The 50,000 × g pellets were then resuspended in hypotonic buffer, 1% Triton X-100, 0.5% SDS, or 0.1 m Na₂CO₃, pH 11.2, and re-centrifuged at 100,000 × g for 1 h at 4 °C (lanes 3–10). Fractions from each centrifugation were then probed for the indicated proteins. Integral membrane proteins were released from the pellet only by detergent (lanes 5–8), whereas peripheral membrane and ER luminal proteins were released from the pellet by detergent or by Na₂CO₃ treatment (lanes 5–10). C, HeLa cells were treated with the indicated detergents for 10 min, followed by incubation with 1 μg/ml Proteinase K for 30 min. The reactions were quenched with 1 mm phenylmethylsulfonyl fluoride, and samples were probed for the ER luminal protein grp94, the ER membrane protein Hrd1, whose C-terminal epitope is exposed to the cytosol, SPFH1, and SPFH2. D, HeLa cells were transfected with the indicated cDNAs, and cell lysates prepared in 1% Triton X-100-containing lysis buffer were incubated without or with endoglycosidase H for 3 h to cleave N-linked glycans (CHO) and were probed with anti-SPFH1 (lanes 1 and 2) or anti-FLAG (lanes 3–6).

Regions within the C-terminal half of SPFH2 mediate interactions with SPFH1 and complex formation. A, diagram of the SPFH1-FLAG and SPFH2-HA constructs used in B and C, together with averaged estimates of the extent of binding of SPFH2-HA and its mutants to SPFH1-FLAG (n ≥ 5), and the extent to which SPFH2-HA and its mutants assemble into ∼2-MDa complexes. The SPFH domain, CC#1 and CC#2 in both SPFH1-FLAG and SPFH2-HA, and the α/β domain and assembly domain (AD) in SPFH2-HA are indicated. B, HeLa cells were co-transfected with cDNAs encoding wild-type SPFH1-FLAG and either wild-type SPFH2-HA or the indicated SPFH2-HA deletion mutants. Cell lysates prepared in 1% CHAPS-containing lysis buffer were incubated with rabbit polyclonal anti-FLAG, and IPs (top panels) and lysates (bottom panels) were probed with mouse monoclonal anti-FLAG or anti-HA. C, lysates from B were subjected to BN-PAGE and probed with anti-HA. IP, immunoprecipitate; IB, immunoblot.

Complex assembly is required for IP₃R ERAD. A, diagram of the constructs used in B–D, together with their overall effects on IP₃R1 polyubiquitination. B, Rat-1 cells expressing either no cDNA, SPFH1-HA, SPFH2-HA, or the indicated HA-tagged SPFH1-SPFH2 chimeras were serum starved, treated with 10 nm ET1 for 10 min, cell lysates were prepared in 1% CHAPS-containing lysis buffer, and anti-IP₃R1 IPs and lysates were probed for the indicated proteins. C, Rat-1 cells expressing either no cDNA, or the indicated SPFH2-HA constructs were serum starved, treated with 10 nm ET1 for 10 min, and anti-IP₃R1 IPs and lysates were probed for the indicated proteins. D, lysates from non-stimulated cells in C were subjected to SDS-PAGE or BN-PAGE, and were probed with anti-HA. IB, immunoblot.

TEM and image analysis of the SPFH1/2 complex. A, a representative micrograph of immunopurified endogenous SPFH1/2 complexes visualized at ×40,000 (scale bar = 400 Å). Putative “top” and “side” views are circled and boxed, respectively. B, 24 class averages, representing ∼50 particles each, resulting from several iterations of multireference alignment and selected for three-dimensional reconstruction are shown, with top and side views indicated as in A. C, final three-dimensional model of the SPFH1/2 complex, determined at a resolution of ∼33 Å and contoured at a volume corresponding to a calculated molecular mass of ∼2 MDa (scale bar = 100 Å). Putative side (1), top (2), and bottom views (3) are shown, with membrane and luminal domains indicated by arrows and arrowheads, respectively. D, four models for how the SPFH1/2 complex (dark gray) might interact with an activated IP₃Rs tetramer (light gray). See text for description.

SPFH1 and SPFH2 associate with activated IP₃R1 independently of polyubiquitination. A, αT3-1 cells were treated without or with 100 nm GnRH for 3 min, and cell lysates prepared in 1% Triton X-100-containing lysis buffer were incubated with anti-IP₃R1 to immunoprecipitate (IP) IP₃R1. IPs were subjected to SDS-PAGE and either Coomassie Blue stained (lanes 1–2) or probed in immunoblots (IB) for SPFH1 and/or SPFH2 as indicated (lanes 3–6). Arrows indicate the migration positions of IP₃R1, IgG heavy chain, SPFH1, and SPFH2. B, αT3-1 cells were treated with 100 nm GnRH for the times indicated, and anti-IP₃R1 IPs and control samples were probed for the indicated proteins. Quantitated data for IP₃R1 polyubiquitination and p97, SPFH2, and SPFH1 co-immunoprecipitation are graphed (n = 4). C, αT3-1 cells were incubated with or without 10 μm bortezomib (Bz) for 30 min, followed by treatment with 100 nm GnRH for 0–20 min, and anti-IP₃R1 IPs were probed for ubiquitin, SPFH1, and SPFH2. Quantitated data for IP₃R1 polyubiquitination and SPFH2 co-immunoprecipitation are graphed (n = 3). Data for SPFH1 were almost identical to that for SPFH2 (not shown).

SPFH1 and SPFH2 exist as a complex. A, anti-SPFH1 (lane 1) or anti-SPFH2 (lane 2) IPs fromαT3-1 cell lysates prepared in 1% Triton X-100-containing lysis buffer were subjected to SDS-PAGE and then Coomassie Blue stained (upper panel) or probed for SPFH2 and SPFH1 (lower panels). Controls with either no antibody (lane 3) or no cell lysate (lanes 4 and 5) were also analyzed. B, αT3-1 cell lysates were incubated without or with anti-SPFH1 or anti-SPFH2, and the immunodepleted lysates were subjected to SDS-PAGE and probed for SPFH1 and SPFH2. C, αT3-1 cell lysates (lanes 1 and 4) and immunopurified (IP) SPFH1/2 complex (lanes 2–3 and 5–6) were subjected to BN-PAGE and probed for SPFH1 or SPFH2. The IPs were either not washed (lanes 2 and 5) or were washed (lanes 3 and 6) with 1 m urea prior to elution of SPFH1/2 complexes. D, αT3-1 cells were treated with 100 nm GnRH for 3 min, anti-IP₃R1 IPs were prepared, co-purifying SPFH1 and SPFH2 were allowed to dissociate from the IPs, and the eluate was subjected to BN-PAGE and probed for SPFH2. IB, immunoblot.

SPFH1 and SPFH2 are required for the polyubiquitination and degradation of activated IP₃Rs. A, Rat-1 cells were incubated without or with 10 nm ET1 for 10 min, cell lysates were prepared in 1% Triton X-100-containing lysis buffer, and anti-IP₃R1 IPs were probed for ubiquitin, IP₃R1, p97, SPFH1, and SPFH2. B, lysates from Rat-1 cells expressing either Random shRNA, SPFH1si4 and/or SPFH2si6 were probed for SPFH1 and SPFH2. C, Rat-1 cells expressing the indicated shRNAs were serum starved, treated with 10 nm ET1 for the indicated times, and anti-IP₃R1 IPs were probed for ubiquitin and IP₃R1. Quantitated data for IP₃R1 polyubiquitination are graphed (n = 6; *, denotes p < 0.001 comparing SPFH1si4, SPFH2si6, or SPFH2si6 + SPFH1si4 cells to Random-s or Random-d cells). D, Rat-1 cells expressing the indicated shRNAs were serum starved and treated with ET1 as in C, and lysates were probed for IP₃R1 and IP₃R3. Quantitated data for IP₃R1 degradation are graphed (n = 8; * denotes p < 0.05 comparing SPFH1si4, SPFH2si6, or SPFH2si6 + SPFH1si4 cells to Random-s or Random-d cells). E, Rat-1 cells expressing the indicated shRNAs and serum starved were loaded with 10 μm Fura2-AM, and 10 nm ET1-induced Ca²⁺ mobilization was measured as described (n = 6) (15). F, Rat-1 cells expressing the indicated shRNAs and either no cDNA, or mouse SPFH1 or SPFH2 cDNA (which were resistant to the rat-specific shRNAs) were serum starved and treated with 10 nm ET1 for 10 min. Anti-IP₃R1 IPs (upper panels) and lysates (lower panels) were probed for the indicated proteins. IP, immunoprecipitate.

The ratio of SPFH1 to SPFH2 in the SPFH1/2 complex is flexible. A, lysates from Rat-1 cells expressing the indicated shRNAs were prepared in 1% Triton X-100-containing lysis buffer, were subjected to BN-PAGE, and were probed for SPFH1 or SPFH2. B, anti-SPFH1 or anti-SPFH2 IPs from Rat-1 cells expressing the indicated shRNAs were Coomassie Blue stained, and bands corresponding to SPFH1 and SPFH2 are marked. C, HeLa cells were transfected with varying amounts of SPFH1-FLAG and SPFH2-HA cDNA, anti-HA IPs were Coomassie Blue stained, and bands corresponding to SPFH1-FLAG and SPFH2-HA are marked. IP, immunoprecipitate; IB, immunoblot.