Degradation of a Cytosolic Protein Requires Endoplasmic Reticulum-associated Degradation Machinery

Metzger, Meredith B.; Maurer, Matthew J.; Dancy, Beverley M.; Michaelis, Susan

doi:10.1074/jbc.m806424200

Cited by 128 publications

(179 citation statements)

References 77 publications

(117 reference statements)

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“…On the other hand, the ERAD ligase Doa10, known mainly to facilitate degradation of ERAD substrates with a misfolded cytosolic domain, also ubiquitinates cytosolic N-acetylated substrates or substrates containing specific C-terminal appendages and targets them for degradation (49)(50)(51). Our findings on Ubr1 participation in ERAD open opportunities for future studies to elucidate the molecular interplay of ubiquitin ligases.…”

Section: Discussionmentioning

confidence: 97%

Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation

Stolz

Besser

Hottmann

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Quality control and degradation of misfolded proteins are essential processes of all cells. The endoplasmic reticulum (ER) is the entry site of proteins into the secretory pathway in which protein folding occurs and terminally misfolded proteins are recognized and retrotranslocated across the ER membrane into the cytosol. Here, proteins undergo polyubiquitination by one of the membrane-embedded ubiquitin ligases, in yeast Hrd1/Der3 (HMG-CoA reductase degradation/degradation of the ER) and Doa10 (degradation of alpha), and are degraded by the proteasome. In this study, we identify cytosolic Ubr1 (E3 ubiquitin ligase, N-recognin) as an additional ubiquitin ligase that can participate in ER-associated protein degradation (ERAD) in yeast. We show that two polytopic ERAD substrates, mutated transporter of the mating type a pheromone, Ste6* (sterile), and cystic fibrosis transmembrane conductance regulator, undergo Ubr1-dependent degradation in the presence and absence of the canonical ER ubiquitin ligases. Whereas in the case of Ste6* Ubr1 is specifically required under stress conditions such as heat or ethanol or in the absence of the canonical ER ligases, efficient degradation of human cystic fibrosis transmembrane conductance regulator requires function of Ubr1 already in wild-type cells under standard growth conditions. Together with the Hsp70 (heat shock protein) chaperone Ssa1 (stress-seventy subfamily A) and the AAA-type ATPase Cdc48 (cell division cycle), Ubr1 directs the substrate to proteasomal degradation. These data unravel another layer of complexity in ERAD.protein quality control | stress response | heat shock C onstantly occurring statistic folding errors, as well as misfolding due to stress such as heat, heavy metal ions, or oxygen require a rigorous protein quality control system in all cellular compartments. Irreversibly misfolded proteins are degraded by a selective proteolysis machinery, the ubiquitin proteasome system. In humans, impairment of the protein quality control and elimination system contributes to several severe diseases, including Parkinson disease, Alzheimer's disease, and CreutzfeldtJakob disease (1, 2), underscoring the importance of these quality control mechanisms. About one third of the cellular proteome consists of proteins passing the secretory pathway. Most of them are translocated into the endoplasmic reticulum (ER), where they are folded and permanently scanned for their functional structure. Only properly folded proteins are allowed to exit the ER and pass on to their site of action (3). Proteins that cannot fold properly are withdrawn from the secretory pathway, retrotranslocated across the ER membrane into the cytosol, polyubiquitinated and degraded by the 26S proteasome in a process termed ER-associated protein degradation (ERAD) (4).The eukaryotic model organism Saccharomyces cerevisiae has been a driving force in the discovery and elucidation of ERAD (4-10). It has been known for years that two polytopic ligases located in the ER membrane, Hrd1/Der3 (HMG-CoA reductas...

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Section: Discussionmentioning

confidence: 97%

Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation

Stolz

Besser

Hottmann

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…A subset of Doa10-dependent substrates are fully stabilized by loss of Ubc6 or Ubc7, such as Deg1-protein fusions (13), whereas others, such as Ste6*, are only mildly impaired upon loss of the Ubc6 E2 (16,18). Ubc6 itself is relatively short lived and targeted by Doa10 in a way that depends on the conserved TEB4-Doa10 (TD) domain (23).…”

Section: Doa10 Substrates With Different Degronsmentioning

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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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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“…To determine whether ERdj3 could substitute for Ydj1, we first asked whether ERdj3 could stimulate the ATPase activity of Ssa1, an essential cytosolic Hsp70 that interacts with Ydj1 to execute key cellular functions (14,26,34,(45)(46)(47)(48)(49). We discovered that ERdj3 proficiently stimulated the ATP hydrolysis rate of Ssa1, to a similar extent as Ydj1 or Hlj1, another J domain containing Ssa1 cofactor ( Fig.…”

Section: Erdj3 Rescues Yeast Hsp40-regulated Functionsmentioning

confidence: 99%

The Mammalian Hsp40 ERdj3 Requires Its Hsp70 Interaction and Substrate-binding Properties to Complement Various Yeast Hsp40-dependent Functions

Vembar

Jin

Brodsky

et al. 2009

Journal of Biological Chemistry

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Heat shock proteins of 70 kDa (Hsp70s) and their J domaincontaining Hsp40 cofactors are highly conserved chaperone pairs that facilitate a large number of cellular processes. The observation that each Hsp70 partners with many J domain-containing proteins (JDPs) has led to the hypothesis that Hsp70 function is dictated by cognate JDPs. If this is true, one might expect highly divergent Hsp70-JDP pairs to be unable to function in vivo. However, we discovered that, when a yeast cytosolic JDP, Ydj1, was targeted to the mammalian endoplasmic reticulum (ER), it interacted with the ER-lumenal Hsp70, BiP, and bound to BiP substrates. Conversely, when a mammalian ERlumenal JDP, ERdj3, was directed to the yeast cytosol, it rescued the temperature-sensitive growth phenotype of yeast-containing mutant alleles in two cytosolic JDPs, HLJ1 and YDJ1, and activated the ATP hydrolysis rate of Ssa1, the yeast cytosolic Hsp70 that partners with Hlj1 and Ydj1. Surprisingly, ERdj3 mutants that were compromised for substrate binding were unable to rescue the hlj1ydj1 growth defect even though they stimulated the ATPase activity of Ssa1. Yet, J domain mutants of ERdj3 that were defective for interaction with Ssa1 restored the growth of hlj1ydj1 yeast. Taken together, these data suggest that the substrate binding properties of certain JDPs, not simply the formation of unique Hsp70-JDP pairs, are critical to specify in vivo function. Hsp70s3 constitute a highly conserved family of molecular chaperones that are found in all organisms and in all cellular organelles. Due to their ability to bind to unfolded regions on nascent polypeptides or unassembled subunits of heteromeric complexes in a nucleotide-dependent manner, these chaperones play critical roles in diverse cellular processes (1, 2). Two distinct sets of cofactors tightly monitor Hsp70 action by regulating ATPase activity (3, 4): J domain-containing proteins (JDPs) of the Hsp40/DnaJ family and nucleotide exchange factors. The highly conserved ϳ70-amino acid J domain of JDPs contacts the nucleotide-binding domain of Hsp70 and enhances ATPase activity by inducing a conformational change (5, 6). This leads to enhanced binding of Hsp70s to substrates. Moreover, some JDPs directly bind to unfolded regions on substrate proteins through their substrate-binding domain and deliver the unfolded protein to the ATP-bound form of their Hsp70 partner (7, 8), whereas others contain atypical domains that specify exclusive functions (9, 10). The nucleotide exchange factors, on the other hand, release bound ADP, which triggers ATP rebinding and subsequent substrate release from the Hsp70.The JDPs can be classified into three groups (11, 12): (i) type I JDPs are most similar to DnaJ and contain a J domain followed by a glycine/phenylalanine-rich region and a cysteine-rich region with four repeats of a CXXCXGXG-type zinc finger; (ii) type II JDPs lack the cysteine-rich region and are unable to coordinate Zn 2ϩ ; and (iii) type III JDPs only have the J domain in common with DnaJ. Notably, the number o...

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Degradation of a Cytosolic Protein Requires Endoplasmic Reticulum-associated Degradation Machinery

Cited by 128 publications

References 77 publications

Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation

Previously unknown role for the ubiquitin ligase Ubr1 in endoplasmic reticulum-associated protein degradation

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

The Mammalian Hsp40 ERdj3 Requires Its Hsp70 Interaction and Substrate-binding Properties to Complement Various Yeast Hsp40-dependent Functions

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