Direct and essential function for Hrd3 in ER-associated degradation

Abstract

The HRD (HMG-CoA reductase degradation) pathway is a conserved route of endoplasmic reticulum-associated degradation (ERAD), by which misfolded ER proteins are ubiquitinated and degraded. ERAD substrates are ubiquitinated by the action of the Hrd1 RING-H2 E3 ligase. Hrd1 is always present in a stoichiometric complex with the ER membrane protein Hrd3, which is also required for HRD-dependent degradation. Despite its conserved presence, unequivocal study of Hrd3 function has been precluded by its central role in Hrd1 stability. Loss of Hrd3 causes unrestricted self-degradation of Hrd1, resulting in significant loss of the core ligase. Accordingly, the degree to which Hrd3 functions independently of Hrd1 stabilization has remained unresolved. By capitalizing on our studies of Usa1 in Hrd1 degradation, we have devised a new approach to evaluate Hrd3 functions in ERAD. We now show that Hrd3 has a direct and critical role in ERAD in addition to Hrd1 stabilization. This direct component of Hrd3 is phenotypically as important as Hrd1 in the native HRD complex. Hrd3 was required the E3 activity of Hrd1, rather than substrate or E2 recruitment to Hrd1. Although Hrd1 can function in some circumstances independent of Hrd3, these studies show an indispensable role for Hrd3 in living cells.highly conserved quality control pathway responsible for the degradation of both misfolded and normal proteins that limits cellular stress and cytotoxicity caused by an accumulation of misfolded ER proteins (1, 2). ERAD occurs through the ubiquitin proteasome pathway, wherein ER-localized ubiquitin ligases covalently modify substrates by attaching multiple copies of the protein ubiquitin, thus tagging them for degradation by cytosolic 26S proteasome (3-5).In Saccharomyces cerevisiae, the highly conserved HRD (HMGCoA † reductase degradation) pathway degrades a variety of ERAD substrates, including misfolded luminal proteins (ERAD-L substrates) such as CPY* and integral membrane proteins (ERAD-M substrates) such as Hmg2 (6, 7). Briefly, ERAD-L substrates are ubiquitinated by the HRD complex, which includes the ubiquitin RING-H2 ligase Hrd1, complex members Hrd3, Usa1, and Der1, and luminal factors implicated in substrate selection (3,(8)(9)(10)(11). Conversely, ERAD-M substrates require only Hrd1 and Hrd3 for in vivo degradation (12, 13). Although Hrd1 functions as the core catalytic subunit of the HRD E3 ligase, Hrd3's function has remained enigmatic. Both Hrd1 and Hrd3 are highly conserved, and Hrd1 is rate limiting for degradation of misfolded substrates (3,12,14,15). In yeast, the steady-state levels of Hrd1 strongly depend on Hrd3. At native levels of the HRD components, Hrd3 is required for Hrd1 stability: In a hrd3Δ, Hrd1 half-life drops to less than 15 min, resulting in very low levels (12). This concomitant loss of Hrd1 is due to rapid autodegradation, catalyzed by the RING domain of Hrd1 (16).Because of Hrd3's strong role in maintaining Hrd1 stability, it has remained unclear whether Hrd3 has any other independent ERAD functions. A ...

SEL1L-dependent Substrates Require Derlin2/3 and Herp1/2 for Endoplasmic Reticulum-associated Degradation

“…Recently, Hampton and his colleagues succeeded in the stabilization of Hrd1p in the absence of Hrd3p by simultaneously deleting the UBL domain of Usa1p (yeast homologue of Herp1/2), which is required for autodegradation of Hrd1p in the absence of Hrd3p (Vashistha et al, 2016). Using this Usa1p-ΔUBL/ΔHrd3p strain they showed that Hrd3p is required for ubiquitination of ERAD substrate by Hrd1p.…”

The evolving role of ubiquitin modification in endoplasmic reticulum-associated degradation

“…Until recently, it was difficult to study the role of Hrd3 in ERAD due to the increased turnover of Hrd1 when HRD3 is deleted. However, when the ubiquitin-like domain of Usa1 is removed, Hrd1 remains stable and a direct role for Hrd3 in ERAD was established [106]. Der1 also binds soluble luminal substrates and interacts with Hrd1 through Usa1 [107, 108].…”

Proteostatic Tactics in the Strategy of Sterol Regulation

“…Regulated ubiquitination of HMGR was first discovered and studied in S. cerevisiae (Hampton & Bhakta 1997, Hampton et al 1996), and its study continues to provide insights into both sterol regulation and ERAD (Neal et al 2017, Sato et al 2009, Vashistha et al 2016). HMGR is essential for life in yeast, allowing synthesis of many isoprenoids and ergosterol, which is the principal sterol.…”

“…Genetic analysis of Hmg2 degradation led to the discovery of the HRD genes (which are responsible for H mg-CoA r eductase d egradation), along with many other substrates. The HRD1 and HRD3 genes encode a pair of ER-resident proteins that form the core subunits of a highly conserved E3 ubiquitin ligase, which is responsible for ubiquitination of Hmg2, leading to proteasome-mediated degradation (Bays et al 2001, Gardner et al 2000, Hampton & Garza 2009, Hampton et al 1996, Vashistha et al 2016). The Hrd1 protein is a multispanning E3 ubiquitin ligase with an N-terminal transmembrane region and a C-terminal E3 ubiquitin ligase domain.…”