Randolph Y. Hampton

Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation

In eukaryotes, endoplasmic reticulum-associated degradation (ERAD) functions in cellular quality control and regulation of normal ER-resident proteins. ERAD proceeds by the ubiquitin–proteasome pathway, in which the covalent attachment of ubiquitin to proteins targets them for proteasomal degradation. Ubiquitin–protein ligases (E3s) play a crucial role in this process by recognizing target proteins and initiating their ubiquitination. Here we show that Hrd1p, which is identical to Der3p, is an E3 for ERAD. Hrd1p is required for the degradation and ubiquitination of several ERAD substrates and physically associates with relevant ubiquitin-conjugating enzymes (E2s). A soluble Hrd1 fusion protein shows E3 activity in vitro — catalysing the ubiquitination of itself and test proteins. In this capacity, Hrd1p has an apparent preference for misfolded proteins. We also show that Hrd1p functions as an E3 in vivo, using only Ubc7p or Ubc1p to specifically program the ubiquitination of ERAD substrates.

ER-associated degradation in protein quality control and cellular regulation

The ER-associated degradation (ERAD) pathway directs ubiquitin-mediated degradation of a variety of ER-associated misfolded and normal proteins. Recent studies have delineated the molecular machinery responsible for protein ubiquitination and highlighted mechanistic questions surrounding the recognition, extraction and proteasomal destruction of the diverse array of ERAD substrates. Consideration of separate lines of work on this versatile pathway now indicate that despite its central role as an avenue of cellular quality control, ERAD is also harnessed for feedback regulation of sterol synthesis, and most likely numerous other cellular processes. These studies give ERAD a larger role in cellular function, and imply that cellular quality-control pathways could be widely employed in both natural and pharmaceutical control of individual proteins.

Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1

Eukaryotic cells maintain proteostasis by quality control (QC) degradation. These pathways can specifically target a wide variety of distinct misfolded proteins, and so are important for management of cellular stress. Although a number of conserved QC pathways have been described in yeast, the E3 ligases responsible for cytoplasmic QC are unknown. We now show that Ubr1 and San1 mediate chaperone-dependent ubiquitination of numerous misfolded cytoplasmic proteins. This action of Ubr1 is distinct from its role in the “N-end rule.” In this capacity, Ubr1 functions to protect cells from proteotoxic stresses. Our phenotypic and biochemical studies of Ubr1 and San1 indicate that two strategies are employed for cytoplasmic QC: chaperone-assisted ubiquitination by Ubr1 and chaperone-dependent delivery to nuclear San1. The broad conservation of Ubr ligases and the relevant chaperones indicates that these mechanisms will be important in understanding both basic and biomedical aspects of cellular proteostasis.

Cdc48-Ufd1-Npl4: Stuck in the Middle with Ub

The ubiquitin-proteasome pathway has a well-defined beginning and end. Target proteins are initially recognized by upstream components and tagged with polyubiquitin chains. The 26S proteasome then degrades these polyubiquitinated proteins. Until recently, it was not known what, if any, steps occurred between the initial polyubiquitination of target proteins and their final degradation. Several new papers investigating the function of the Cdc48-Ufd1-Npl4 complex indicate that there is indeed a middle to the ubiquitin-proteasome pathway. The Cdc48-Ufd1-Npl4 complex functions in the recognition of several polyubiquitin-tagged proteins and facilitates their presentation to the 26S proteasome for processive degradation or even more specific processing. The elucidation of Cdc48, Ufd1 and Npl4 action not only provides long-sought functions for these specific proteins, but illuminates a poorly understood part of the ubiquitin-proteasome pathway.

Misfolded Membrane Proteins Are Specifically Recognized by the Transmembrane Domain of the Hrd1p Ubiquitin Ligase

Quality control pathways such as ER-associated degradation (ERAD) employ a small number of factors to specifically recognize a wide variety of protein substrates. Delineating the mechanisms of substrate selection is a principle goal in studying quality control. The Hrd1p ubiquitin ligase mediates ERAD of numerous misfolded proteins including soluble, lumenal ERAD-L and membrane-anchored ERAD-M substrates. We tested if the Hrd1p multispanning membrane domain was involved in ERAD-M specificity. In this work, we have identified site-directed membrane domain mutants of Hrd1p impaired only for ERAD-M and normal for ERAD-L. Furthermore, other Hrd1p variants were specifically deficient for degradation of individual ERAD-M substrates. Thus, the Hrd1p transmembrane region bears determinants of high specificity in the ERAD-M pathway. From in vitro and interaction studies, we suggest a model in which the Hrd1p membrane domain employs intramembrane residues to evaluate substrate misfolding, leading to selective ubiquitination of appropriate ERAD-M clients.

Cod1p/Spf1p is a P-type ATPase involved in ER function and Ca2+ homeostasis

The internal environment of the ER is regulated to accommodate essential cellular processes, yet our understanding of this regulation remains incomplete. Cod1p/Spf1p belongs to the widely conserved, uncharacterized type V branch of P-type ATPases, a large family of ion pumps. Our previous work suggested Cod1p may function in the ER. Consistent with this hypothesis, we localized Cod1p to the ER membrane. The cod1Δ mutant disrupted cellular calcium homeostasis, causing increased transcription of calcium-regulated genes and a synergistic increase in cellular calcium when paired with disruption of the Golgi apparatus–localized Ca2+ pump Pmr1p. Deletion of COD1 also impaired ER function, causing constitutive activation of the unfolded protein response, hypersensitivity to the glycosylation inhibitor tunicamycin, and synthetic lethality with deletion of the unfolded protein response regulator HAC1. Expression of the Drosophila melanogaster homologue of Cod1p complemented the cod1Δ mutant. Finally, we demonstrated the ATPase activity of the purified protein. This study provides the first biochemical characterization of a type V P-type ATPase, implicates Cod1p in ER function and ion homeostasis, and indicates that these functions are conserved among Cod1ps metazoan homologues.

The biology of HMG-CoA reductase: the pros of contra-regulation

Hydroxymethylglutaryl-CoA reductase (HMG-R) is a key enzyme in the mevalonate pathway, from which thousands of molecules are derived including cholesterol and prenyl moieties. The regulation of HMG-R is complex and includes feedback control, cross-regulation by independent bio-chemical processes and contra-regulation of separate isozymes. From studies in yeast, these separate modes of regulation can be considered in an integrated fashion.

A highly conserved signal controls degradation of 3-hydroxy-3- methylglutaryl-coenzyme a (HMG-CoA) reductase in eukaryotes

Sterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). In both mammals and yeast, a non-sterol isoprenoid signal positively regulates the rate of HMGR degradation. To define more precisely the molecule that serves as the source of this signal, we have conducted both pharmacological and genetic manipulations of the mevalonate pathway in yeast. We now demonstrate that farnesyl diphosphate (FPP) is the source of the positive signal for Hmg2p degradation in yeast. This FPP-derived signal does not act by altering the endoplasmic reticulum degradation machinery in general. Rather, the FPP-derived signal specifically modulates Hmg2p stability. In mammalian cells, an FPP-derived molecule also serves as a positive signal for HMGR degradation. Thus, both yeast and mammalian cells employ the same strategy for regulation of HMGR degradation, perhaps by conserved molecular processes.

In Vivo Action of the HRD Ubiquitin Ligase Complex: Mechanisms of Endoplasmic Reticulum Quality Control and Sterol Regulation

ABSTRACT Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRDcomplex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent “misfolded” state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.

Finding the will and the way of ERAD substrate retrotranslocation

ER-associated degradation (ERAD) is a mechanism by which numerous ER-localized proteins are targeted for cytosolic degradation by the ubiquitin-proteasome system. A surprising and still-cryptic requirement of this process is the energy dependent retrotranslocation of both lumenal and membrane-embedded ER proteins into the cytosol for ongoing ubiquitination and proteasomal destruction. The current understanding, results, and open questions are discussed below for this intriguing and critical process of ERAD.