Gp78, a Membrane-Anchored Ubiquitin Ligase, Associates with Insig-1 and Couples Sterol-Regulated Ubiquitination to Degradation of HMG CoA Reductase

Abstract

Sterol-regulated ubiquitination is an obligatory step in ER-associated degradation (ERAD) of HMG CoA reductase, a rate-limiting enzyme in cholesterol synthesis. Accelerated degradation of reductase, one of several strategies animal cells use to limit production of cholesterol, requires sterol-induced binding of the enzyme to ER membrane proteins called Insigs. Once formed, the reductase-Insig complex is recognized by a putative membrane-associated ubiquitin ligase (E3) that mediates the reductase ubiquitination reaction. Here, we show that gp78, a membrane bound E3, binds to Insig-1 and is required for sterol-regulated ubiquitination of reductase. In addition, gp78 couples regulated ubiquitination to degradation of reductase by binding to VCP, an ATPase that plays a key role in recognition and degradation of ERAD substrates. The current results identify gp78 as the E3 that initiates sterol-accelerated degradation of reductase, and Insig-1 as a bridge between gp78/VCP and the reductase substrate.

Dislocation of HMG-CoA Reductase and Insig-1, Two Polytopic Endoplasmic Reticulum Proteins, En Route to Proteasomal Degradation

“…This dislocation requires metabolic energy and is facilitated by the AAA-ATPase p97/VCP, probably with its ERAD partners Ufd1p and Npl4p. These results are consistent with previous findings that p97 preferentially associates with ubiquitinated HMG 350 -HA (Doolman et al, 2004) and HMGR (data not shown) and that HMGR degradation is abrogated upon siRNA-mediated p97 depletion or abolishment of the ubiquitin binding capacity of Ufd1p (Song et al, 2005;Cao et al, 2007).…”

“…Their studies, which were carried out mostly with the short-lived Insig-1, depict a model in which the membrane region of HMGR binds to Insigs in a sterolstimulated manner, akin to the association of SCAP with Insigs. Insig-1, but much less so Insig-2, interacts constitutively with gp78, a RING finger membrane E3 ubiquitin ligase (Fang et al, 2001;Kostova et al, 2007), which ubiquitinates Insig-1 and causes its rapid turnover (Song et al, 2005;Lee et al, 2006a). The sterols-stimulated association of Insig-1 with either SCAP or HMGR is mutually exclusive and leads to different consequences: although binding of SCAP displaces gp78 and thus stabilizes Insig-1 by preventing its ubiquitination, association of HMGR does not displace gp78 from Insig-1, but rather the bound HMGR becomes ubiquitinated and rapidly degraded (Lee et al, 2006a).…”

“…Together with its ERAD partners Ufd1p and Npl4p, and at the expense of ATP hydrolysis, this cytosolic complex is thought to drive the dislocation of most ERAD substrates and facilitate their delivery to the proteasome . Indeed, p97 preferentially associates with the ubiquitinated HMGR (Doolman et al, 2004) and silencing p97 expression abrogates HMGR degradation (Song et al, 2005). However, the precise details of how HMGR is dislocated from the ER membrane remain unknown.…”

Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRΔF508

“…Coexpression of wild-type gp78-Myc had no effect on the degradation of CFTR⌬F508-GFP (Supplemental Figure 3), even though it enhanced the ubiquitylation of CFTR⌬F508-GFP. Overexpression of gp78 has been shown to accelerate the degradation of other gp78 substrates Song et al, 2005;Chen et al, 2006), which suggests that additional factor(s) are required for the degradation of CFTR⌬F508. HEK293 cells transiently expressing CFTR⌬F508-GFP and HA-ubiquitin, with or without (control lane) the indicated ubiquitin ligases, were lysed and subjected to immunoprecipitation by using anti-GFP antibody, followed by immunoblot using anti-HA antibody.…”

“…Subsequently, it was shown that gp78 localizes not only to the cell surface but also to the ER (Wang et al, 1997). Similar to Hrd1p, gp78 is a RING finger-dependent ubiquitin ligase, an integral ER membrane protein, and it is involved in ERAD, in cooperation with Ube2g2/UBC7, the mammalian counterpart of yeast Ubc7p (Fang et al, 2001;Liang et al, 2003;Song et al, 2005;Lee et al, 2006). Recently, Chen et al (2006) demonstrated that both the E2 binding site and the coupling of ubiquitin to ER degradation (CUE) domain of gp78 are essential for its ubiquitin ligase activity.…”