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

Abstract

Inositol 1,4,5-trisphosphate (IP 3 ) 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 IP 3 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 IP 3 receptors for ERAD. Here, we sought to identify novel proteins that associate with and mediate the ERAD of endogenous activated IP 3 receptors. SPFH2, an uncharacterized SPFH domaincontaining protein, rapidly associated with IP 3 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 IP 3 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)2 -associated degradation (ERAD) pathway is responsible for the degradation of aberrant proteins in the ER (1) and, in addition to this "quality control" function, also accounts for the degradation of several metabolically regulated, native ER proteins (2, 3). The essential features of the ERAD pathway are substrate recognition, polyubiquitination, and delivery to the 26 S proteasome, which is located in the cytosol (1). 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 (4).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 (4). Hrd3p binds to and regulates Hrd1p (5), 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 (6 -8). Retrotranslocation is facilitated by the cytosolic Cdc48p-Ufd1p-Npl4p complex, which associates with Hrd1p and Doa10p via membrane-bound Ubx2p (4, 8 -10), and likely uses ATP hydrolysis to both unfold ERAD substrates and extract them from the ER membrane (11-13). Polyubiquitination then occurs either simultaneously wit...

Activation of Ca2+‐activated Cl− channel ANO1 by localized Ca2+ signals

“…ERLIN1 (SPFH1) localizes to the ER lipid rafts and physically associates with the IP 3 R in an activity‐dependent fashion to facilitate ER‐associated degradation (ERAD) of the latter (Pearce et al . 2007, 2009; Wojcikiewicz et al . 2009).…”

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

“…Already available antibodies used were: rabbit polyclonal anti-IP 3 R1 (23), anti-Hrd1, anti-SPFH2 (16), and anti-␣-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-IP 3 R3 (BD Transduction Laboratories), and antihuman SPFH1 (a kind gift from Dr. S. M. Robbins, University of Calgary, Calgary, Alberta); rat monoclonal anti-grp94 (StressGen); 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 (EPS-GESPIQNKENAG) and the FLAG epitope (DYKDDDDK), respectively, and were affinity purified as described (23).…”

“…Materials-␣T3-1, HeLa, and Rat-1 cells were cultured as described (16). Already available antibodies used were: rabbit polyclonal anti-IP 3 R1 (23), anti-Hrd1, anti-SPFH2 (16), and anti-␣-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-IP 3 R3 (BD Transduction Laboratories), and antihuman SPFH1 (a kind gift from Dr. S. M. Robbins, University of Calgary, Calgary, Alberta); rat monoclonal anti-grp94 (StressGen); and horseradish peroxidase-and fluorophore-conjugated secondary antibodies (Sigma).…”

“…This feature makes IP 3 Rs particularly valuable for studying ERAD, because activation almost instantaneously converts them from stable proteins into ERAD substrates. Thus, we have identified several mediators of IP 3 R ERAD, notably mammalian Ubc7 (14), the p97-Ufd1-Npl4 complex (15), and most recently, SPFH2 (16). SPFH2, also known as erlin-2 (17), belongs to a family of ϳ100 mammalian proteins ("SPFH proteins") that contain an "SPFH" domain, an ϳ250-amino acid motif named because of minor sequence similarities in the proteins stomatin, prohib-itin, flotillin, and HflC/K (18).…”

RNF185 Is a Novel E3 Ligase of Endoplasmic Reticulum-associated Degradation (ERAD) That Targets Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

“…FLAG-RNF185, as well as FLAG-RNF5, also efficiently pulled down Erlin2 (Fig. 4A), a prohibitin-like scaffold protein of the ERAD that has been reported to target inositol triphosphate receptor (IP3R) (51). We next probed the RNF185 interaction with Ubc6 and Ubc7, which are the two ER membrane-associated E2 ligases of ERAD.…”