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1.
Nat Rev Mol Cell Biol ; 24(11): 777-796, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37528230

ABSTRACT

Maintaining proteome integrity is essential for long-term viability of all organisms and is overseen by intrinsic quality control mechanisms. The secretory pathway of eukaryotes poses a challenge for such quality assurance as proteins destined for secretion enter the endoplasmic reticulum (ER) and become spatially segregated from the cytosolic machinery responsible for disposal of aberrant (misfolded or otherwise damaged) or superfluous polypeptides. The elegant solution provided by evolution is ER-membrane-bound ubiquitylation machinery that recognizes misfolded or surplus proteins or by-products of protein biosynthesis in the ER and delivers them to 26S proteasomes for degradation. ER-associated protein degradation (ERAD) collectively describes this specialized arm of protein quality control via the ubiquitin-proteasome system. But, instead of providing a single strategy to remove defective or unwanted proteins, ERAD represents a collection of independent processes that exhibit distinct yet overlapping selectivity for a wide range of substrates. Not surprisingly, ER-membrane-embedded ubiquitin ligases (ER-E3s) act as central hubs for each of these separate ERAD disposal routes. In these processes, ER-E3s cooperate with a plethora of specialized factors, coordinating recognition, transport and ubiquitylation of undesirable secretory, membrane and cytoplasmic proteins. In this Review, we focus on substrate processing during ERAD, highlighting common threads as well as differences between the many routes via ERAD.

2.
Methods Mol Biol ; 2602: 3-18, 2023.
Article in English | MEDLINE | ID: mdl-36446963

ABSTRACT

The traditional textbook describes ubiquitylation as the conjugation of ubiquitin to a target by forming a covalent bond connecting ubiquitin's carboxy-terminal glycine residue with an acceptor amino acid like lysine or amino-terminal methionine in the substrate protein. While this adequately depicts a significant fraction of cellular ubiquitylation processes, a growing number of ubiquitin modifications do not follow this rule. Recent data demonstrate that ubiquitin can also be efficiently attached to other amino acids, such as cysteine, serine, and threonine, via ester bonding. Initially observed for a virus-encoded ubiquitin ligase, which targets a cysteine residue in a host protein to initiate its degradation, ester-linked ubiquitylation is now shown to also drive regular cellular processes. These ubiquitylation events expand the complexity and diversity of ubiquitin signaling and broaden the capability of cellular messages in the so-called ubiquitin code. Still, questions on the prevalence, relevance, and involvement in physiological and cellular functions await clearing. In this review, we aim to summarize our knowledge on ester-linked ubiquitylation and introduce experimental strategies to circumvent technical issues that complicate analysis of this uncommon posttranslational modification.


Subject(s)
Cysteine , Ubiquitin , Ubiquitination , Protein Processing, Post-Translational , Amino Acids , Esters
3.
EMBO J ; 40(6): e106094, 2021 03 15.
Article in English | MEDLINE | ID: mdl-33576509

ABSTRACT

The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin-conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin-binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.


Subject(s)
Polyubiquitin/biosynthesis , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitination/physiology , Computer Simulation , Models, Structural , Protein Domains , Proteomics , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction/physiology , Ubiquitin-Conjugating Enzymes/genetics
4.
J Cachexia Sarcopenia Muscle ; 11(1): 103-119, 2020 02.
Article in English | MEDLINE | ID: mdl-31441598

ABSTRACT

BACKGROUND: Critically ill patients frequently develop muscle atrophy and weakness in the intensive-care-unit setting [intensive care unit-acquired weakness (ICUAW)]. Sepsis, systemic inflammation, and acute-phase response are major risk factors. We reported earlier that the acute-phase protein serum amyloid A1 (SAA1) is increased and accumulates in muscle of ICUAW patients, but its relevance was unknown. Our objectives were to identify SAA1 receptors and their downstream signalling pathways in myocytes and skeletal muscle and to investigate the role of SAA1 in inflammation-induced muscle atrophy. METHODS: We performed cell-based in vitro and animal in vivo experiments. The atrophic effect of SAA1 on differentiated C2C12 myotubes was investigated by analysing gene expression, protein content, and the atrophy phenotype. We used the cecal ligation and puncture model to induce polymicrobial sepsis in wild type mice, which were treated with the IкB kinase inhibitor Bristol-Myers Squibb (BMS)-345541 or vehicle. Morphological and molecular analyses were used to investigate the phenotype of inflammation-induced muscle atrophy and the effects of BMS-345541 treatment. RESULTS: The SAA1 receptors Tlr2, Tlr4, Cd36, P2rx7, Vimp, and Scarb1 were all expressed in myocytes and skeletal muscle. Treatment of differentiated C2C12 myotubes with recombinant SAA1 caused myotube atrophy and increased interleukin 6 (Il6) gene expression. These effects were mediated by Toll-like receptors (TLR) 2 and 4. SAA1 increased the phosphorylation and activity of the transcription factor nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-κB) p65 via TLR2 and TLR4 leading to an increased binding of NF-κB to NF-κB response elements in the promoter region of its target genes resulting in an increased expression of NF-κB target genes. In polymicrobial sepsis, skeletal muscle mass, tissue morphology, gene expression, and protein content were associated with the atrophy response. Inhibition of NF-κB signalling by BMS-345541 increased survival (28.6% vs. 91.7%, P < 0.01). BMS-345541 diminished inflammation-induced atrophy as shown by a reduced weight loss of the gastrocnemius/plantaris (vehicle: -21.2% and BMS-345541: -10.4%; P < 0.05), tibialis anterior (vehicle: -22.7% and BMS-345541: -17.1%; P < 0.05) and soleus (vehicle: -21.1% and BMS-345541: -11.3%; P < 0.05) in septic mice. Analysis of the fiber type specific myocyte cross-sectional area showed that BMS-345541 reduced inflammation-induced atrophy of slow/type I and fast/type II myofibers compared with vehicle-treated septic mice. BMS-345541 reversed the inflammation-induced atrophy program as indicated by a reduced expression of the atrogenes Trim63/MuRF1, Fbxo32/Atrogin1, and Fbxo30/MuSA1. CONCLUSIONS: SAA1 activates the TLR2/TLR4//NF-κB p65 signalling pathway to cause myocyte atrophy. Systemic inhibition of the NF-κB pathway reduced muscle atrophy and increased survival of septic mice. The SAA1/TLR2/TLR4//NF-κB p65 atrophy pathway could have utility in combatting ICUAW.


Subject(s)
Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/physiology , Muscular Atrophy/metabolism , Serum Amyloid A Protein/metabolism , Toll-Like Receptors/metabolism , Animals , Disease Models, Animal , Humans , Male , Mice
5.
J Biomol NMR ; 72(1-2): 1-10, 2018 Oct.
Article in English | MEDLINE | ID: mdl-30066206

ABSTRACT

Yos9 is an essential component of the endoplasmic reticulum associated protein degradation (ERAD) system that is responsible for removing terminally misfolded proteins from the ER lumen and mediating proteasomal degradation in the cytosol. Glycoproteins that fail to attain their native conformation in the ER expose a distinct oligosaccharide structure, a terminal α1,6-linked mannose residue, that is specifically recognized by the mannose 6-phoshate receptor homology (MRH) domain of Yos9. We have determined the structure of the MRH domain of Yos9 in its free form and complexed with 3α, 6α-mannopentaose. We show that binding is achieved by loops between ß-strands performing an inward movement and that this movement also affects the entire ß-barrel leading to a twist. These rearrangements may facilitate the processing of client proteins by downstream acting factors. In contrast, other oligosaccharides such as 2α-mannobiose bind weakly with only locally occurring chemical shift changes underscoring the specificity of this substrate selection process within ERAD.


Subject(s)
Carrier Proteins/physiology , Protein Folding , Saccharomyces cerevisiae Proteins/physiology , Endoplasmic Reticulum-Associated Degradation/physiology , Glycoproteins/chemistry , Lectins/chemistry , Oligosaccharides/chemistry , Polysaccharides , Protein Binding , Protein Conformation , Substrate Specificity
6.
Mol Cell ; 63(5): 827-39, 2016 09 01.
Article in English | MEDLINE | ID: mdl-27570077

ABSTRACT

The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system.


Subject(s)
Gene Expression Regulation, Fungal , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Humans , Hydroxylation , Lysine/metabolism , Polyubiquitin/genetics , Polyubiquitin/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Substrate Specificity , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitination
7.
J Biol Chem ; 291(23): 12195-207, 2016 Jun 03.
Article in English | MEDLINE | ID: mdl-27053108

ABSTRACT

A quality control system in the endoplasmic reticulum (ER) efficiently discriminates polypeptides that are in the process of productive folding from conformers that are trapped in an aberrant state. Only the latter are transported into the cytoplasm and degraded in a process termed ER-associated protein degradation (ERAD). In the ER, an enzymatic cascade generates a specific N-glycan structure of seven mannosyl and two N-acetylglucosamine residues (Man7GlcNAc2) on misfolded glycoproteins to facilitate their disposal. We show that a complex encompassing the yeast lectin-like protein Htm1 and the oxidoreductase Pdi1 converts Man8GlcNAc2 on glycoproteins into the Man7GlcNAc2 signal. In vitro the Htm1-Pdi1 complex processes both unfolded and native proteins albeit with a preference for the former. In vivo, elevated expression of HTM1 causes glycan trimming on misfolded and folded proteins, but only degradation of the non-native species is accelerated. Thus, modification with a Man7GlcNAc2 structure does not inevitably commit a protein for ER-associated protein degradation. The function of Htm1 in ERAD relies on its association with Pdi1, which appears to regulate the access to substrates. Our data support a model in which the balanced activities of Pdi1 and Htm1 are crucial determinants for the efficient removal of misfolded secretory glycoproteins.


Subject(s)
Endoplasmic Reticulum-Associated Degradation , Glycoproteins/metabolism , Mannosidases/metabolism , Protein Disulfide-Isomerases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Endoplasmic Reticulum/metabolism , Glycoproteins/chemistry , Glycoproteins/genetics , Immunoblotting , Mannosidases/chemistry , Mannosidases/genetics , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Mutation , Polysaccharides/chemistry , Polysaccharides/metabolism , Protein Binding , Protein Disulfide-Isomerases/chemistry , Protein Disulfide-Isomerases/genetics , Protein Folding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics
8.
Mol Biol Cell ; 26(2): 185-94, 2015 Jan 15.
Article in English | MEDLINE | ID: mdl-25428985

ABSTRACT

Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream-acting components like Der1.


Subject(s)
Endoplasmic Reticulum-Associated Degradation , Membrane Glycoproteins/metabolism , Molecular Chaperones/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Endoplasmic Reticulum/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Immunoblotting , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Mutation , Protein Binding , Protein Folding , Protein Structure, Tertiary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Unfolded Protein Response
9.
Nat Cell Biol ; 16(12): 1130-2, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25434464

ABSTRACT

Protein quality control systems protect cells from proteotoxicity caused by the accumulation of aberrantly folded polypeptides. The Rsp5 ubiquitin ligase (mammalian homologue Nedd4) is now identified as a major constituent of a clearance pathway that degrades misfolded cytosolic proteins after exposure to heat.


Subject(s)
Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , HSP40 Heat-Shock Proteins/metabolism , Heat-Shock Response/physiology , Protein Folding , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Ubiquitin-Protein Ligase Complexes/genetics , Ubiquitin-Protein Ligases/metabolism , Animals , Humans , Nedd4 Ubiquitin Protein Ligases
10.
Cell ; 156(6): 1167-1178, 2014 Mar 13.
Article in English | MEDLINE | ID: mdl-24630720

ABSTRACT

Aging entails a progressive decline in protein homeostasis, which often leads to age-related diseases. The endoplasmic reticulum (ER) is the site of protein synthesis and maturation for secreted and membrane proteins. Correct folding of ER proteins requires covalent attachment of N-linked glycan oligosaccharides. Here, we report that increased synthesis of N-glycan precursors in the hexosamine pathway improves ER protein homeostasis and extends lifespan in C. elegans. Addition of the N-glycan precursor N-acetylglucosamine to the growth medium slows aging in wild-type animals and alleviates pathology of distinct neurotoxic disease models. Our data suggest that reduced aggregation of metastable proteins and lifespan extension depend on enhanced ER-associated protein degradation, proteasomal activity, and autophagy. Evidently, hexosamine pathway activation or N-acetylglucosamine supplementation induces distinct protein quality control mechanisms, which may allow therapeutic intervention against age-related and proteotoxic diseases.


Subject(s)
Biosynthetic Pathways , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/metabolism , Hexosamines/metabolism , Longevity , Proteins/metabolism , Amino Acid Sequence , Animals , Autophagy , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)/genetics , Humans , Molecular Sequence Data , Mutation , Protein Biosynthesis , Sequence Alignment , Tunicamycin/pharmacology
11.
Nat Cell Biol ; 16(1): 77-86, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24292014

ABSTRACT

Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD ligase) and degraded by cytosolic 26S proteasomes. The movement of these proteins through the lipid bilayer is assumed to occur via a protein-conducting channel of unknown nature. We show that the integral membrane protein Der1 oligomerizes, which relies on its interaction with the scaffolding protein Usa1. Mutations in the transmembrane domains of Der1 block the passage of soluble proteins across the ER membrane. As determined by site-specific photocrosslinking, the ER-luminal exposed parts of Der1 are in spatial proximity to the substrate receptor Hrd3, whereas the membrane-embedded domains reside adjacent to the ubiquitin ligase Hrd1. Intriguingly, both regions also form crosslinks to client proteins. Our data imply that Der1 initiates the export of aberrant polypeptides from the ER lumen by threading such molecules into the ER membrane and routing them to Hrd1 for ubiquitylation.


Subject(s)
Endoplasmic Reticulum/metabolism , Intracellular Membranes/metabolism , Membrane Proteins/metabolism , Protein Folding , Saccharomyces cerevisiae Proteins/metabolism , Benzophenones/metabolism , Conserved Sequence/genetics , Cross-Linking Reagents/metabolism , Endoplasmic Reticulum-Associated Degradation , Membrane Proteins/chemistry , Models, Biological , Mutation/genetics , Phenylalanine/analogs & derivatives , Phenylalanine/metabolism , Protein Multimerization , Protein Structure, Tertiary , Protein Transport , Saccharomyces cerevisiae Proteins/chemistry , Substrate Specificity
12.
Biochim Biophys Acta ; 1808(3): 925-36, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20599420

ABSTRACT

Protein folding within the endoplasmic reticulum (ER) of eukaryotic cells is erroneous and often results in the formation of terminally malfolded species. A quality control system retards such molecules in the ER and eventually initiates their dislocation into the cytosol for proteolysis by 26S proteasomes. This process is termed ER associated protein degradation (ERAD). The spatial separation of ER based quality control and cytosolic proteolysis poses the need for a machinery that promotes the extraction of substrates from the ER. Due to the heterogeneous nature of the client proteins this transport system displays several unique features. Selective recognition of ERAD substrates does not involve transferable transport signals in the primary sequence and thus must follow other principles than established for proteins designated for the import into organelles. Moreover, an ER dislocation system must be capable to ship polypeptides, which may be at least partly folded and are in most cases covalently modified with bulky and hydrophilic glycans, through a membrane without disrupting the integrity of the ER. In this review we present current ideas on the highly dynamic and flexible nature of the dislocation apparatus and speculate on the mechanism that removes aberrant polypeptides from the ER in the course of ERAD. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.


Subject(s)
Endoplasmic Reticulum/metabolism , Intracellular Membranes/metabolism , Animals , Humans , Protein Transport
13.
Bioessays ; 32(10): 905-13, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20806269

ABSTRACT

In eukaryotic cells terminally misfolded proteins of the secretory pathway are retarded in the endoplasmic reticulum (ER) and subsequently degraded in a ubiquitin-proteasome-dependent manner. This highly conserved process termed ER-associated protein degradation (ERAD) ensures homeostasis in the secretory pathway by disposing faulty polypeptides and preventing their deleterious accumulation and eventual aggregation in the cell. The focus of this paper is the functional description of membrane-bound ubiquitin ligases, which are involved in all critical steps of ERAD. In the end we want to speculate on how the modular architecture of these entities ensures the specificity of substrate selection and possibly accomplishes the transport of misfolded polypeptides from the ER into the cytoplasm.


Subject(s)
Endoplasmic Reticulum/metabolism , Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Folding , Protein Processing, Post-Translational , Quality Control , Ubiquitin-Protein Ligases/genetics
14.
Mol Cell ; 36(5): 782-93, 2009 Dec 11.
Article in English | MEDLINE | ID: mdl-20005842

ABSTRACT

Protein quality control in the endoplasmic reticulum is of central importance for cellular homeostasis in eukaryotes. Crucial for this process is the HRD-ubiquitin ligase (HMG-CoA reductase degradation), which singles out terminally misfolded proteins and routes them for degradation to cytoplasmic 26S-proteasomes. Certain functions of this enzyme complex are allocated to defined subunits. However, it remains unclear how these components act in a concerted manner. Here, we show that Usa1 functions as a major scaffold protein of the HRD-ligase. For the turnover of soluble substrates, Der1 binding to the C terminus of Usa1 is required. The N terminus of Usa1 associates with Hrd1 and thus bridges Der1 to Hrd1. Strikingly, the Usa1 N terminus also induces oligomerization of the HRD complex, which is an exclusive prerequisite for the degradation of membrane proteins. Our data demonstrate that scaffold proteins are required to adapt ubiquitin ligase activities toward different classes of substrates.


Subject(s)
Fungal Proteins/physiology , Ubiquitin-Protein Ligases/metabolism , Yeasts/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Protein Interaction Mapping
15.
Nat Cell Biol ; 8(8): 849-54, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16845381

ABSTRACT

A quality-control system surveys the lumen of the endoplasmic reticulum for terminally misfolded proteins. Polypeptides singled out by this system are ultimately degraded by the cytosolic ubiquitin-proteasome pathway. Key components of both the endoplasmic reticulum quality-control system and the degradation machinery have been identified, but a connection between the two systems has remained elusive. Here, we report an association between the endoplasmic reticulum quality-control lectin Yos9p and Hrd3p, a component of the ubiquitin-proteasome system that links these pathways. We identify designated regions in the luminal domain of Hrd3p that interact with Yos9p and the ubiquitin ligase Hrd1p. Binding of misfolded proteins occurs through Hrd3p, suggesting that Hrd3p recognises proteins that deviate from their native conformation, whereas Yos9p ensures that only terminally misfolded polypeptides are degraded.


Subject(s)
Carrier Proteins/metabolism , Endoplasmic Reticulum/metabolism , Membrane Glycoproteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Blotting, Western , Carrier Proteins/chemistry , Carrier Proteins/genetics , Hydrolysis , Immunoprecipitation , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Plasmids/genetics , Protein Binding , Protein Conformation , Protein Folding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics
16.
EMBO J ; 25(9): 1827-35, 2006 May 03.
Article in English | MEDLINE | ID: mdl-16619026

ABSTRACT

Misfolded proteins of the endoplasmic reticulum (ER) are targeted to the cytoplasm for proteasomal degradation. Key components of this process are ER membrane-bound ubiquitin ligases. These ligases associate with the cytoplasmic AAA-ATPase Cdc48p/p97, which is thought to support the release of malfolded proteins from the ER. Here, we characterize a yeast protein complex containing the ubiquitin ligase Hrd1p and the ER membrane proteins Hrd3p and Der1p. Hrd3p binds malfolded proteins in the ER lumen enabling their delivery to downstream components. Therefore, we propose that Hrd3p acts as a substrate recruitment factor for the Hrd1p ligase complex. Hrd3p function is also required for the association of Cdc48p with Hrd1p. Moreover, our data demonstrate that recruitment of Cdc48p depends on substrate processing by the Hrd1p ligase complex. Thus, the Hrd1p ligase complex unites substrate selection in the ER lumen and polyubiquitination in the cytoplasm and links these processes to the release of ER proteins via the Cdc48p complex.


Subject(s)
Cell Cycle Proteins/metabolism , Endoplasmic Reticulum/enzymology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Ubiquitin-Protein Ligases/metabolism , Adenosine Triphosphatases , Catalysis , Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Protein Folding , Substrate Specificity , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Valosin Containing Protein
17.
Nat Cell Biol ; 7(10): 993-8, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16179953

ABSTRACT

Endoplasmic reticulum (ER)-associated protein degradation requires the dislocation of selected substrates from the ER to the cytosol for proteolysis via the ubiquitin-proteasome system. The AAA ATPase Cdc48 (known as p97 or VCP in mammals) has a crucial, but poorly understood role in this transport step. Here, we show that Ubx2 (Sel1) mediates interaction of the Cdc48 complex with the ER membrane-bound ubiquitin ligases Hrd1 (Der3) and Doa10. The membrane protein Ubx2 contains a UBX domain that interacts with Cdc48 and an additional UBA domain. Absence of Ubx2 abrogates breakdown of ER proteins but also that of a cytosolic protein, which is ubiquitinated by Doa10. Intriguingly, our results suggest that recruitment of Cdc48 by Ubx2 is essential for turnover of both ER and non-ER substrates, whereas the UBA domain of Ubx2 is specifically required for ER proteins only. Thus, a complex comprising the AAA ATPase, a ubiquitin ligase and the recruitment factor Ubx2 has a central role in ER-associated proteolysis.


Subject(s)
Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Cytosol/metabolism , Endoplasmic Reticulum/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Adenosine Triphosphatases , Cell Membrane/metabolism , Proteasome Endopeptidase Complex/metabolism , Saccharomyces cerevisiae/metabolism , Valosin Containing Protein
18.
Nat Cell Biol ; 7(8): 766-72, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16056268

ABSTRACT

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) eliminates misfolded or unassembled proteins from the ER. ERAD targets are selected by a quality control system within the ER lumen and are ultimately destroyed by the cytoplasmic ubiquitin-proteasome system (UPS). The spatial separation between substrate selection and degradation in ERAD requires substrate transport from the ER to the cytoplasm by a process termed dislocation. In this review, we will summarize advances in various aspects of ERAD and discuss new findings on how substrate dislocation is achieved.


Subject(s)
Endoplasmic Reticulum/metabolism , Proteasome Endopeptidase Complex/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Animals , Humans , Membrane Transport Proteins/metabolism , Models, Biological , Protein Folding , Protein Transport , Proteins/chemistry , Proteins/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/physiology , Yeasts/metabolism
19.
EMBO J ; 24(5): 875-84, 2005 Mar 09.
Article in English | MEDLINE | ID: mdl-15692564

ABSTRACT

BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis-Golgi membrane. The crystal structure of human BET3 has been determined to 1.55-A resolution. BET3 adopts an alpha/beta-plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane-associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein-protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions.


Subject(s)
Membrane Proteins/chemistry , Vesicular Transport Proteins/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Dimerization , Humans , In Vitro Techniques , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , Mutagenesis, Site-Directed , Palmitic Acid/chemistry , Protein Conformation , Protein Structure, Quaternary , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Homology, Amino Acid , Static Electricity , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism
20.
Dev Cell ; 8(1): 4-5, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15621524

ABSTRACT

Spatial separation of ubiquitin conjugation pathways contributes to target-specific ubiquitination. Recently, Plafker et al. reported that importin 11-dependent nuclear import of the ubiquitin-conjugating enzyme UbcM2 occurs only if the latter is charged with ubiquitin. This interesting finding describes a link between nuclear transport pathways and ubiquitin and reveals a novel mechanism for localizing components of the ubiquitin system within the cell.


Subject(s)
Active Transport, Cell Nucleus/physiology , Signal Transduction/physiology , Ubiquitin/physiology , Animals , Karyopherins/metabolism , Ubiquitin-Conjugating Enzymes/metabolism
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