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 ...

In eukaryotes, the synthesis and uptake of sterols undergo stringent multivalent regulation. Both individual enzymes and transcriptional networks are controlled to meet changing needs of the many sterol pathway products. Regulation is tailored by evolution to match regulatory constraints, which can be very different in distinct species. Nevertheless, a broadly conserved feature of many aspects of sterol regulation is employment of proteostasis mechanisms to bring about control of individual proteins. Proteostasis is the set of processes that maintain homeostasis of a dynamic proteome. Proteostasis includes protein quality control pathways for the detection, and then the correction or destruction, of the many misfolded proteins that arise as an unavoidable feature of protein-based life. Protein quality control displays not only the remarkable breadth needed to manage the wide variety of client molecules, but also extreme specificity toward the misfolded variants of a given protein. These features are amenable to evolutionary usurpation as a means to regulate proteins, and this approach has been used in sterol regulation. We describe both well-trod and less familiar versions of the interface between proteostasis and sterol regulation and suggest some underlying ideas with broad biological and clinical applicability.

Chaperones can mediate both protein folding and degradation. This process is referred to as protein triage, which demands study to reveal mechanisms of quality control for both basic scientific and translational purposes. In yeast, many misfolded proteins undergo chaperone dependent ubiquitination by the action of E3 ligases Ubr1 and San1, allowing detailed study of protein triage. In cells, both HSP70 and HSP90 mediated substrate ubiquitination, and the canonical ATP cycle was required for HSP70’s role: we have found that ATP hydrolysis by HSP70, the nucleotide exchange activity of SSE1, and the action of J proteins are all needed for Ubr1 mediated quality control. To discern if chaperones were directly involved in Ubr1-mediated ubiquitination, we developed a bead-based assay with covalently immobilized but releasable misfolded protein to obviate possible chaperone effects on substrate physical state or transport. In this in vitro assay, only HSP70 was required, along with its ATPase cycle and relevant cochaperones, for Ubr1-mediated ubiquitination. The requirement for the HSP70 ATP cycle in ubiquitination suggests a possible model of triage, in which efficiently folded proteins are spared, while slow- or non-folding proteins are iteratively tagged with ubiquitin for subsequent degradation.

Polyvinylpyrrolidone (PVP) capped rod-shaped europium (Eu3+) doped gadolinium oxide (Gd2O3) nanoparticles (PVP@Gd2O3:Eu3+ NPs) were synthesized by a wet chemical method at 90○C. NPs were characterised by X-ray diffraction (XRD), High...

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