Aberrant substrate engagement of the ER translocon triggers degradation by the Hrd1 ubiquitin ligase

Little is known about quality control of proteins that aberrantly or persistently engage the endoplasmic reticulum (ER)-localized translocon en route to membrane localization or the secretory pathway. Hrd1 and Doa10, the primary ubiquitin ligases that function in ER-associated degradation (ERAD) in yeast, target distinct subsets of misfolded or otherwise abnormal proteins based primarily on degradation signal (degron) location. We report the surprising observation that fusing Deg1, a cytoplasmic degron normally recognized by Doa10, to the Sec62 membrane protein rendered the protein a Hrd1 substrate. Hrd1-dependent degradation occurred when Deg1-Sec62 aberrantly engaged the Sec61 translocon channel and underwent topological rearrangement. Mutations that prevent translocon engagement caused a reversion to Doa10-dependent degradation. Similarly, a variant of apolipoprotein B, a protein known to be cotranslocationally targeted for proteasomal degradation, was also a Hrd1 substrate. Hrd1 therefore likely plays a general role in targeting proteins that persistently associate with and potentially obstruct the translocon

HRD4/NPL4 Is Required for the Proteasomal Processing of Ubiquitinated ER Proteins

We isolated a temperature-sensitive mutant, hrd4–1, deficient in ER-associated degradation (ERAD). The HRD4 gene was identical to NPL4, a gene previously implicated in nuclear transport. Using a diverse set of substrates and direct ubiquitination assays, our analysis revealed that HRD4/NPL4 is required for a poorly characterized step in ERAD after ubiquitination of target proteins but before their recognition by the 26S proteasome. Our data indicate that this lack of proteasomal processing of ubiquitinated proteins constitutes the primary defect in hrd4/npl4 mutant cells and explains the diverse set of hrd4/npl4 phenotypes. We also found that each member of the Cdc48p-Ufd1p-Npl4p complex is individually required for ERAD

Lipid-mediated, reversible misfolding of a sterol-sensing domain protein

Cellular quality control requires recognition of common features of misfolding, and so is not typically associated with the specific targeting of individual proteins. However, physiologically regulated degradation of yeast HMG-CoA reductase (Hmg2p) occurs by the HRD endoplasmic reticulum quality control pathway, implying that Hmg2p undergoes a regulated transition to a quality control substrate in response to a sterol pathway molecule. Using in vitro structural assays, we now show that the pathway derivative farnesol causes Hmg2p to undergo a change to a less folded structure. The effect is reversible, biologically relevant by numerous criteria, highly specific for farnesol structure, and requires an intact Hmg2p sterol-sensing domain. This represents a distinct lipid-sensing function for this highly conserved motif that suggests novel approaches to cholesterol management. More generally, our observation of reversible small-molecule-mediated misfolding may herald numerous examples of regulated quality control to be discovered in biology or applied in the clinic

Nrdp1/FLRF is a ubiquitin ligase promoting ubiquitination and degradation of the epidermal growth factor receptor family member, ErbB3

The epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases plays fundamental roles in the regulation of cell survival, proliferation, and differentiation. Here, we present evidence that ErbB3 is degraded by proteasomes, and that Nrdp1 (referred to as FLRF in mice) associates with ErbB3 and stimulates its ubiquitination and degradation by proteasomes. Nrdp1 mRNAs are expressed in a variety of human tissues. The N-terminal half of Nrdp1 possesses an atypical RING finger domain, which is required for enhancing ErbB3 degradation. Its C-terminal half by itself associates with ErbB3 and raises ErbB3 levels in cells, probably by acting as a dominant–negative form of Nrdp1. In cell-free systems, Nrdp1 has ubiquitin ligase (E3) activity and ubiquitinates ErbB3, as well as itself, in the presence of the ubiquitin-carrier protein (E2), UbcH5. These data indicate that Nrdp1 is a RING finger-type of ubiquitin ligase, which promotes degradation of ErbB3 by proteasomes and, thus, may be an important factor influencing cell growth

Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane

Misfolded proteins are eliminated from the endoplasmic reticulum (ER) by retrotranslocation into the cytosol, a pathway hijacked by certain viruses to destroy MHC class I heavy chains. The translocation of polypeptides across the ER membrane requires their polyubiquitination and subsequent extraction from the membrane by the p97 ATPase [also called valosin-containing protein (VCP) or, in yeast, Cdc48]. In higher eukaryotes, p97 is bound to the ER membrane by a membrane protein complex containing Derlin-1 and VCP-interacting membrane protein (VIMP). How the ubiquitination machinery is recruited to the p97/Derlin/VIMP complex is unclear. Here, we report that p97 interacts directly with several ubiquitin ligases and facilitates their recruitment to Derlin-1. During retrotranslocation, a substrate first interacts with Derlin-1 before p97 and other factors join the complex. These data, together with the fact that Derlin-1 is a multispanning membrane protein forming homo-oligomers, support the idea that Derlin-1 is part of a retrotranslocation channel that is associated with both the polyubiquitination and p97-ATPase machineries

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