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 (IP 3 Rs) 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 ϳ2 MDa complex that binds to IP 3 R tetramers immediately after their activation and is required for their processing. The complex is ring-shaped (diameter ϳ250 Å ), and RNA interference-mediated depletion of SPFH1 and SPFH2 blocks IP 3 R polyubiquitination and degradation. We propose that this novel SPFH1/2 complex is a recognition factor that targets IP 3 Rs and perhaps other substrates for ERAD. The endoplasmic reticulum (ER)2 -associated degradation (ERAD) pathway targets aberrant proteins, including irreversibly misfolded proteins and unassembled subunits of multiprotein complexes, for degradation by the ubiquitin-proteasome system (1). 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 (2), and inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs), which form tetrameric, IP 3 -and Ca 2ϩ

RNF170 Protein, an Endoplasmic Reticulum Membrane Ubiquitin Ligase, Mediates Inositol 1,4,5-Trisphosphate Receptor Ubiquitination and Degradation

“…1A). In addition to erlin1 (41 kDa) and erlin2 (43 kDa), which we characterized previously (14,15), one of the most abundant proteins that associated with IP 3 R1 in a stimulus-dependent manner migrated as a band at 21.5 kDa (lane 2), and excision of this band followed by mass spectral analysis revealed it to be an uncharacterized protein termed RING finger protein 170 (RNF170). Database searching and sequence analysis (Fig.…”

“…Already available antibodies used were rabbit polyclonal anti-IP 3 R1 (16), anti-erlin2 (14), anti-FLAG epitope (15), anti-␣-transaldolase (a kind gift from Dr. A. Perl, State University of New York Upstate Medical University, Syracuse, NY), mouse monoclonal anti-p97 (Research Diagnostics, Inc.), anti-ubiquitin clone FK2 (BioMol International), anti-FLAG epitope clone M2 or M5 (Sigma), anti-HA epitope clone HA11 (Covance), and horseradish peroxidase-and fluorophore-conjugated secondary antibodies raised in goat (Sigma). Rabbit polyclonal anti-RNF170 and anti-HA epitope were raised against peptides NIHPENQELVRVLREQ and YPYDVPDYA, respectively, as described (16).…”

“…The binding of erlin2 (which provides a measure of erlin1/2 complex binding (15)) and RNF170 was more rapid than both the accumulation of polyubiquitinated IP 3 R1 and the binding of p97 (Fig. 1, E and F), which associates with IP 3 receptors after they have been polyubiquitinated (4,14,15). To assess the universality of RNF170 binding, we also examined SH-SY5Y cells in which carbachol has been shown previously to induce IP 3 receptor ubiquitination and down-regulation (5).…”

“…This p97-Ufd1-Npl4 complex participates in the delivery of polyubiquitinated IP 3 receptors to the proteasome (4). Additionally, we have recently found that the ER membrane-located erlin1/2 (SPFH1/2) complex associates rapidly with activated IP 3 receptors and appears to be the element that selects them for ERAD (14,15). However, the E3 responsible for IP 3 receptor ubiquitination has remained elusive (2).…”

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).…”