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

Rubenstein, Eric M.; Kreft, Stefan G.; Greenblatt, Wesley; Swanson, Robert; Hochstrasser, Mark

doi:10.1083/jcb.201203061

Cited by 58 publications

(113 citation statements)

References 70 publications

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“…Pulse-Chase Assays-Pulse-chase analysis was performed as described (13,16,20,35). Briefly, log phase yeast cultures grown in SD medium (plus 200 M CuSO 4 for pOC9-URA3-HA-CL1-transformed strains) were labeled with ϳ20 Ci of Tran 35 Slabel (MP Biomedicals, GE Healthcare) per A 600 unit of cells for 5-10 min in SD medium lacking methionine and cysteine.…”

Section: Methodsmentioning

confidence: 99%

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A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation

Zattas

Berk

Kreft

et al. 2016

Journal of Biological Chemistry

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Specific proteins are modified by ubiquitin at the endoplasmic reticulum (ER) and are degraded by the proteasome, a process referred to as ER-associated protein degradation. In Saccharomyces cerevisiae, two principal ER-associated protein degradation ubiquitin ligases (E3s) reside in the ER membrane, Doa10 and Hrd1. The membrane-embedded Doa10 functions in the degradation of substrates in the ER membrane, nuclear envelope, cytoplasm, and nucleoplasm. How most E3 ligases, including Doa10, recognize their protein substrates remains poorly understood. Here we describe a previously unappreciated but highly conserved C-terminal element (CTE) in Doa10; this cytosolically disposed 16-residue motif follows the final transmembrane helix. A conserved CTE asparagine residue is required for ubiquitylation and degradation of a subset of Doa10 substrates. Such selectivity suggests that the Doa10 CTE is involved in substrate discrimination and not general ligase function. Functional conservation of the CTE was investigated in the human ortholog of Doa10, MARCH6 (TEB4), by analyzing MARCH6 autoregulation of its own degradation. Mutation of the conserved Asn residue (N890A) in the MARCH6 CTE stabilized the normally short lived enzyme to the same degree as a catalytically inactivating mutation (C9A). We also report the localization of endogenous MARCH6 to the ER using epitope tagging of the genomic MARCH6 locus by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome editing. These localization and CTE analyses support the inference that MARCH6 and Doa10 are functionally similar. Moreover, our results with the yeast enzyme suggest that the CTE is involved in the recognition and/or ubiquitylation of specific protein substrates.Selective protein degradation in eukaryotic cells is largely carried out by the ubiquitin-proteasome system. Proteins identified for degradation by the ubiquitin-proteasome system are covalently modified with ubiquitin, a highly conserved 76-residue protein (1, 2). In most cases, ubiquitin is conjugated to the target protein via an amide linkage between its C-terminal glycine and ⑀-amino groups on substrate lysine residues. Ubiquitylation requires an enzyme cascade beginning with an E1 ubiquitin-activating enzyme, which adenylates the ubiquitin C-terminal carboxyl group and subsequently forms a high energy thioester bond with it. Ubiquitin is then transferred from the E1 active site cysteine to a cysteine in an E2 ubiquitinconjugating enzyme. Finally, an E3 ubiquitin ligase mediates ubiquitin transfer from the E2 to the target protein. E3s come in several mechanistically distinct classes, but the most abundant are the RING E3s. The RING domain is characterized by a set of Cys and His residues that coordinate a pair of zinc ions; the RING directly contacts the E2-ubiquitin thioester-linked complex and promotes ubiquitin transfer to a substrate (3). Polyubiquitin chains are formed when the C terminus of one donor ubiquitin is conjugated to one of seven lysine residues, or the ...

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Section: Methodsmentioning

confidence: 99%

“…For all experiments, yeast was grown at 30°C. Standard methods were used for recombinant DNA manipulations (20).…”

Section: Methodsmentioning

confidence: 99%

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation

Zattas

Berk

Kreft

et al. 2016

Journal of Biological Chemistry

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“…2) (41,54). Importantly, deletion of RKR1 increased the abundance of the faster migrating (translationally paused) species of this protein (Vma12(glyc)-K12) (Fig.…”

Section: Rkr1 Regulates Abundance Of a Model Translationally Stalled mentioning

confidence: 99%

Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins

Crowder

Geigges

Gibson

et al. 2015

Journal of Biological Chemistry

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Background: Translationally stalled proteins (including those aberrantly translated beyond their stop codons) pose dangers for eukaryotic cells. Results: The ubiquitin ligase Rkr1/Ltn1 targets translationally stalled ER-associated proteins for degradation. Conclusion: Cytosolic and ER-associated translationally stalled proteins are targeted for destruction by related mechanisms. Significance: Mechanisms regulating the degradation of translationally stalled ER-associated proteins may represent therapeutic targets for human disease.

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“…It has been reported that HRD1 and gp78 are two ERAD-related E3 ubiquitin ligases that are associated with the ER membrane (Chen et al, 2006;Kostova et al, 2007). Hrd1 forms a complex with SEL1L and mainly mediates the degradation of soluble, ER-luminal substrates and integral membrane proteins (Rubenstein et al, 2012;Shmueli et al, 2009). gp78, which was the first described and is the best documented human ERAD E3, is a multi-spanning membrane protein with its catalytic domain facing the cytosol (Chen et al, 2006;Fang et al, 2001;Li et al, 2009).…”

Section: E2-e3 (Ube2g2-gp78)-complex-mediated Herp Ubiquitylationmentioning

confidence: 99%

Ube2g2-gp78-mediated HERP polyubiquitination is involved in ER stress recovery

Long

Liu

Zhang

et al. 2014

Journal of Cell Science

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A large number of studies have focused on how individual organisms respond to a stress condition, but little attention has been paid to the stress recovery process, such as the endoplasmic reticulum (ER) stress recovery. Homocysteine-induced ER protein (HERP) was originally identified as a chaperone-like protein that is strongly induced upon ER stress. Here we show that, after ER stress induction, HERP is rapidly degraded by Ube2g2-gp78-mediated ubiquitylation and proteasomal degradation. The polyubiquitylation of HERP in vitro depends on a physical interaction between the CUE domain of gp78 and the ubiquitin-like (UBL) domain of HERP, which is essential for HERP degradation in vivo during ER stress recovery. We further show that although HERP promotes cell survival under ER stress, high levels of HERP expression reduce cell viability under oxidative stress conditions, suggesting that HERP plays a dual role in cellular stress adaptation. Together, these results establish the ubiquitin-proteasome-mediated degradation of HERP as a novel mechanism that fine-tunes the stress tolerance capacity of the cell.

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

Abstract: The Hrd1 ubiquitin ligase plays a role in quality control of two substrates associated with the Sec61 translocon.

Cited by 58 publications

References 70 publications

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation

A Conserved C-terminal Element in the Yeast Doa10 and Human MARCH6 Ubiquitin Ligases Required for Selective Substrate Degradation

Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins

Ube2g2-gp78-mediated HERP polyubiquitination is involved in ER stress recovery

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